Current Concepts in Cervical Pathology
Kay J. Park, MD; Robert A. Soslow, MD
Context.The correct diagnosis and reporting of cervical
in situ and invasive carcinoma are essential for the appropri-
ate clinical management of patients with human papilloma-
virusassociated disease.
Objectives.To review common mistakes made in the
diagnosis of cervical dysplasia and invasive carcinoma, de-
scribe variants and benign mimics of high-grade squamous
intraepithelial lesion and adenocarcinoma in situ, and dis-
cuss available ancillary studies that can be useful in making
the distinctions as well as to review important factors re-
C ervical cancer is one of the most common cancers in
women worldwide with about 493 000 new cases
each year resulting in 274 000 deaths globally.1 In the Unit-
ed States it is the 14th most common cancer in women
and affects nearly 12 000 women each year causing ap-
proximately 4000 deaths.2 Most cervical carcinomas are
etiologically related to the human papillomavirus (HPV)
and about 70% of all cervical carcinomas are caused by 2
types of high-risk HPV, 16 and 18.3 Once infected by HPV,
cervical neoplasia undergoes a stepwise progression start-
ing from preinvasive lesions that can be detected by
screening and cured with complete excision. Therefore,
the detection of such lesions is critical in the appropriate
management of patients. Although it is important not to
miss lesions, the overdiagnosis of benign atypias also has
important clinical implications and can result in undue
physical and psychologic distress, as well as waste valu-
able resources and funds. Pathologists are faced with
many diagnostic difficulties when assessing cervical tis-
sue, particularly in distinguishing benign from in situ dis-
ease and in situ from invasive carcinoma. This review fo-
cuses on variants and mimics of in situ squamous and
glandular lesions, important prognostic factors in invasive
disease that must be reported by the pathologist, and an-
cillary tests that are available to assist in the diagnosis of
HPV-related lesions.
Accepted for publication December 11, 2008.
From the Department of Pathology, Memorial Sloan-Kettering Cancer
Center, New York, New York.
The authors have no relevant financial interest in the products or
companies described in this article.
Presented in part at the Surgical Pathology of Neoplastic Diseases
course, Memorial Sloan-Kettering Cancer Center, New York, New York,
May 1216, 2008.
Reprints: Kay J. Park, MD, Department of Pathology, Memorial Sloan-
Kettering Cancer Center, 1275 York Ave, New York, NY 10065
(e-mail: parkk@mskcc.org).
Arch Pathol Lab MedVol 133, May 2009
lated to prognosis that should be included in the pathology
report.
Data Sources.Review of current literature.
Conclusions.There are many mimics and variants of
cervical squamous and glandular lesions that can be re-
solved with ancillary studies and careful histologic exami-
nation. Prognostically important features, such as tumor
size, presence of vascular invasion, and margin status,
should always be included in the pathology report.
(Arch Pathol Lab Med. 2009;133:729738)
HUMAN PAPILLOMAVIRUS
Human papillomavirus preferentially infects cells in the
cervical transformation zone, an area of active cell turn-
over. Basal cells, which feature the HPV receptor, are a
natural target for infection. The virus remains in the cell
in a latent state until activation causes viral replication and
squamous dysplasia. Low-grade squamous intraepithelial
lesion/cervical intraepithelial neoplasia 1 (LSIL/CIN 1) is
characterized by koilocytotic atypia, nuclear enlargement,
hyperchromasia, and perinuclear cytoplasmic clearing.
These features are the result of viral proteins that affect
DNA synthesis and the structure of intermediate filaments
in the host cell cytoplasm. Human papillomavirus gene
expression is tightly controlled in LSILs, which result
from productive infections of cells that have begun the
process of differentiation. High-grade squamous intraep-
ithelial lesions/cervical intraepithelial neoplasia 2 to 3
(HSILs/CIN 23) do not support HPV genomic replica-
tion and, therefore, do not usually have the nuclear or cy-
toplasmic characteristics of an active viral infection.
Traditionally, cervical squamous lesions were thought to
progress in a stepwise fashion from low grade to high
grade, the theory being that a focus of HSIL arises in,
gradually expands, and finally replaces LSIL as a mono-
clonal event. However, studies have shown that HSIL may
also develop independently without progression or trans-
formation from LSIL; instead, they likely arise de novo
from epithelium adjacent to LSIL.4
Accurate histologic and cytologic classification of HPV-
mediated lesions has important implications for guiding
patient management, but studies have shown that inter-
observer and intraobserver reproducibility of diagnoses of
cervical lesions is suboptimal. The atypical cells of unde-
termined significance (ASCUS)/LSIL Triage Study
(ALTS)5 showed that variability of histologic diagnoses on
biopsy is as great as with cytologic smears, even though
histology is still considered the gold standard. Between
the clinical center and central quality control groups, there
Current Concepts in Cervical PathologyPark & Soslow 729
was only moderate concordance, with the quality control
group more frequently rendering low-grade diagnoses.
CIN 1 had only 42% concordance, whereas the diagnostic
extremes had much better concordance, 91% for normal
and 77% for high grade. Most LSILs and even HSILs un-
dergo spontaneous regression within 6 to 12 months due
to host cell immune response. However, there is no way
to prospectively differentiate among lesions that are des-
tined to regress, persist, or progress to carcinoma. Over-
diagnosis of benign lesions as LSIL adds unnecessary cost
and wastes resources, as clinicians will often proceed di-
rectly to excisional biopsy for LSIL, especially in older pa-
tients. The patients physical and psychologic distress
should also not be underestimated.
SQUAMOUS LESIONS
The most common diagnostic challenges occur in dis-
tinguishing LSIL from benign reactive atypia, CIN 2 from
CIN 3, and CIN 3 from atrophy and immature metaplasia.
Mild nuclear cytologic changes, especially those that oc-
cur in a background of inflammation, can be misinter-
preted as LSIL. Normal squamous epithelium, with prom-
inent cytoplasmic glycogen or vacuolization, is also com-
monly mistaken for koilocytosis. But true koilocytosis is
often focal, whereas in normal epithelium there is no clear
demarcation between abnormal and normal-appearing
epithelium. Benign epithelium lacks nuclear enlargement,
multinucleation (although binucleate cells are sometimes
encountered), cellular disorganization, and maturation
disturbances. Probably the most important feature is the
lack of true nuclear atypia (Figure 1, A and B).
The distinction between CIN 2 and CIN 3 is not as crucial,
because both lesions are in the high-grade category. Studies
have shown that the subjective criteria used for separating
the different grades and the fact that the thickness of the
immature basaloid cells can vary within the same lesion lead
to failures in reproducible diagnosis of CIN 2.6
The importance of the difference between CIN 3 and
atrophy or metaplasia is obvious. Atrophy and immature
metaplasia do not have the mitotic activity, nuclear pleo-
morphism, loss of cell polarity, nuclear hyperchromasia,
or clumped chromatin of HSIL. Reparative change can
also sometimes mimic HSIL with atypical basal cells oc-
cupying the lower half of the epithelium. These cells have
regular nuclear contours, prominent nucleoli, and distinct
cell borders, often accompanied by dense acute or chronic
inflammation.
SQUAMOUS VARIANT LESIONS
Variants of squamous dysplasia that appear reactive or
metaplastic may also cause diagnostic dilemmas. These
include atypical immature metaplasia (AIM) and eosino-
philic dysplasia (ED).
Atypical immature metaplasia is a loosely defined term
for immature metaplastic cells with mild cytologic atypia,
first described by Crum et al in 1983.7 By their description,
AIM was similar to condyloma, that is, low-grade lesion. But
since then, there has been a shift in the diagnosis to include
both high-grade and low-grade lesions.810 This attests to the
fact that this entity spans a morphologic spectrum and likely
includes many diagnostically challenging cases with an im-
mature appearance. There is lack of a uniform definition,
which has resulted in inconsistencies in morphologic criteria.
Several studies have shown that AIM can be HPV positive
or negative, be variably positive for p16, and display a large
730 Arch Pathol Lab MedVol 133, May 2009
range of proliferative rates with Mib-1810 (see section on an-
cillary studies on page 735). Follow-up studies have reported
that HPV-associated AIM showing p16 expression and high
proliferative rates subsequently recur as a bone fide dys-
plastic lesion in many instances. It has been hypothesized
that AIM lesions that are HPV and p16 positive but hypo-
proliferative with Mib-1 may be early or regressing lesions.
Atypical immature metaplasia is not commonly used as a
diagnostic term in surgical pathology reports. Our group
currently diagnoses HSIL when we encounter AIM lesions
that diffusely express p16 and are hyperproliferative with
Mib-1. Immunohistochemical stains that do not support a
proliferative, HPV-associated lesion usually result in a report
of AIM with a note describing the significance of the finding.
Eosinophilic dysplasia (ED), another descriptive term, has
been used for intraepithelial lesions showing both squa-
mous metaplasia and dysplasia.11 Eosinophilic dysplasia
is commonly associated with HPV infection and adjacent
foci of classic HSIL. Most ED lesions are diffusely and
strongly positive for p16, hyperproliferative with Mib-1,
and positive for HPV DNA by polymerase chain reaction,
all high or intermediate risk. Therefore, ED likely repre-
sents a variant of HSIL with misleading morphologic fea-
tures that suggest a benign diagnosis. Eosinophilic dys-
plasia is another term that is not commonly used in sur-
gical pathology reports. Our group currently diagnoses
HSIL when we encounter ED lesions that diffusely express
p16 and are hyperproliferative with Mib-1. Immunohis-
tochemical stains that do not support a proliferative, HPV-
associated lesion result in a report of atypical metaplasia
with a note describing the significance of the finding.
GLANDULAR LESIONS
Adenocarcinoma in situ (AIS) is much less common
than squamous intraepithelial lesion (SIL). Unlike squa-
mous neoplasms in which precursor lesions predominate
over invasive carcinomas, the opposite is true for glan-
dular lesions. It is more difficult to screen effectively for
AIS than for SIL, so AIS may not be detected before the
development of invasive adenocarcinoma. Histologic fea-
tures of AIS include preservation of normal glandular ar-
chitecture and involvement of part or all of the epithelium
by enlarged, hyperchromatic, stratified nuclei with coarse
chromatin, small nucleoli, mitoses, and apoptosis. The cy-
toplasm can be depleted or abundant, vacuolated, granu-
lar, and basophilic or eosinophilic (Figure 2). At least 90%
of AIS cases are associated with HPV (most commonly
HPV 18 and 16). It coexists with SIL or squamous cell
carcinoma in 50% of cases.1223 Part of the difficulty in
identifying AIS is that the lesion is not well visualized
colposcopically as it can arise high up in the endocervical
canal,17 and the cytologic criteria for identifying neoplastic
glandular lesions are not as well defined as for SIL. Ad-
enocarcinoma in situ has been described as multifocal,
though the definitions of multifocality are often unclear.
Various studies report that AIS can be multicentric (13%)19
and multiquadrant (53%).21 This is important because
multifocality may affect the meaning of a negative margin
on cone excisions. Studies have shown that cone biopsies
for AIS with negative margins result in residual disease
in a hysterectomy 6% to 44% of the time, whereas cone
biopsies for AIS with positive margins are associated with
residual disease 44% to 75% of the time.24 Therefore, the
standard treatment for AIS is simple hysterectomy or a
cold knife cone excision (not loop electrosurgical excision
Current Concepts in Cervical PathologyPark & Soslow
Figure 1. A, Benign squamous mucosa with glycogenization of cytoplasm forming pseudokoilocytes. The process involves the entire epithelium
(hematoxylin-eosin, original magnification 40). B, Low-grade squamous intraepithelial lesion (cervical intraepithelial neoplasia 1) with true
cytologic atypia, koilocytosis, and parabasal proliferation (hematoxylin-eosin, original magnification 400).
Figure 2. Adenocarcinoma in situ. The cells are pseudostratified and hyperchromatic with some cytoplasmic mucin. Note the luminal mitoses
and apoptotic bodies (hematoxylin-eosin, original magnification 200).
Figure 3. Tubal metaplasia. The cells are pseudostratified and appear somewhat atypical, but close evaluation reveals cells resembling fallopian
tube epithelium with ciliated and secretory type cells (hematoxylin-eosin, original magnification 200).
Figure 4. Tuboendometrioid metaplasia (hematoxylin-eosin, original magnification 200).
Figure 5. Tubal metaplasia with irregularly shaped glands and stromal reaction mimicking invasive adenocarcinoma (hematoxylin-eosin, original
magnification 100).

Arch Pathol Lab MedVol 133, May 2009 Current Concepts in Cervical PathologyPark & Soslow 731
procedure [LEEP]) with widely clear margins (10 mm ide-
al according to Goldstein and Mani25). Therefore, it is im-
portant to always mention the status of the margins and
give the closest distance from the lesion if possible. Some
AISs exhibit intestinal differentiation with goblet cells and
sometimes Paneth or argentaffin cells.26 Other related le-
sions include adenosquamous carcinoma in situ27 and
stratified mucin-producing intraepithelial lesion (SMILE),
described subsequently.
AIS MIMICS
Glandular lesions that can mimic AIS include reactive
glandular atypia, tubal/tuboendometrioid metaplasia,
and so-called glandular dysplasia (GD).
Reactive glandular atypias can be secondary to inflam-
mation, radiation, or viral infections. Glandular lesions
showing cytologic atypia can also be found in the context
of Arias-Stella reaction, microglandular hyperplasia, en-
dometriosis, and mesonephric remnants.28 In reactive
changes, the nuclei are typically enlarged, have prominent
nucleoli, but lack nuclear hyperchromasia. Mitotic activity
is absent or minimal, as is pseudostratification. Papillary
endocervicitis, featuring exaggerated papillary projections
and a dense infiltrate of acute and/or chronic inflamma-
tion, can also occur.29 Radiation change causes the nuclei
and cytoplasm to enlarge, often with vacuolization, but
with only a slight increase in the nuclear to cytoplasmic
ratio. The nuclei can look round, look vesicular with prom-
inent nucleoli, and rarely have a smudged hyperchromatic
appearance.30
Tubal metaplasia tends to occur in the upper endocer-
vix. It is usually not difficult to distinguish from AIS be-
cause ciliated, intercalated (peg), and secretory cells, more
characteristically found in the fallopian tube, are present
at least focally in tubal metaplasia. Atypical tubal meta-
plasia is a form of tubal metaplasia in which the glands
are lined by similar tubal type cells, but these are crowded
and have larger, more hyperchromatic nuclei and are
pseudostratified. Mitosis and apoptosis are not common
like they are in AIS, but the degree of atypia can be mis-
taken for AIS (Figure 3). Tuboendometrioid metaplasia is
essentially tubal metaplasia without ciliated cells, al-
though it commonly displays more pseudostratification
and higher nuclear to cytoplasmic ratios than other tubal
metaplasia lesions; it lacks the architectural complexity,
mitoses, apoptosis, and nuclear irregularity of AIS (Figure
4). Tubal and tuboendometrioid metaplasia can involve
the deep cervical wall with dilated irregular gland for-
mation and stromal reaction around the glands; this latter
feature, in particular, may erroneously suggest a diagnosis
of invasive adenocarcinoma31 (Figure 5). Careful exami-
nation of the nuclear features should allow one to properly
differentiate between benign and malignant.
Glandular dysplasia has been defined in a variety of
ways, but in general, GD reportedly displays a lesser de-
gree of abnormality than AIS.3235 The diagnostic criteria
remain largely subjective. The most objective criteria for
diagnosing noninvasive glandular lesions of the cervix
were presented in a report by Ioffe et al36 in which a nu-
merical scoring system was proposed. In this scoring
scheme, separate scores are assigned for the presence and
degree of (1) nuclear atypia, (2) stratification, and (3) mi-
toses/apoptosis (counted in the 2 most active glands and
the average number used). These 3 scores are then added
to result in the total score (03, benign; 45, endocervical
732 Arch Pathol Lab MedVol 133, May 2009
GD; 69, adenocarcinoma in situ). Although this method
improves interobserver discordance, the biologic and clin-
ical significance of GD remains to be determined. The as-
sociation between GD and HPV infection is variable, and,
as it only infrequently coexists with AIS, it has been sug-
gested that GD might not represent an AIS precursor at
all. It is therefore imprudent to make an unqualified di-
agnosis of GD.24 Application of ancillary tests, discussed
subsequently, can be contributory.
GLANDULAR VARIANT LESIONS
SMILE, first described by Park et al in 2000,27 is an HPV-
associated intraepithelial columnar cell neoplasm that is
thought to arise from the reserve cells of the transforma-
tion zone. Morphologically, SMILEs are composed of im-
mature stratified cells that, although appearing similar to
SILs, display intracytoplasmic mucin or cytoplasmic vac-
uoles that separate the cells in the mid to lower layers of
the epithelium (Figure 6). Overt gland formation, seen in
typical AIS, is not found in SMILE. The most consistent
feature of SMILE is the spacing of nuclei by mucin, which
can be highlighted with a mucicarmine stain. SMILEs are
commonly associated with HSIL, AIS, adenocarcinoma,
squamous carcinoma, and adenosquamous carcinoma.
The natural history of these lesions has not been fully elu-
cidated, but it has been reported that lesions may recur in
the form of SIL or AIS. As is the case with AIM and ED,
SMILE should not be used as a stand-alone term in sur-
gical pathology reports. Either AIS or adenosquamous car-
cinoma in situ can be used, depending on the context and
the pathologists preference.
INVASIVE CARCINOMA
The diagnosis of invasive squamous or adenocarcinoma
is usually not challenging. Stage is the most important
prognostic factor in cervical cancer, but unlike the practice
in other organ systems, staging is primarily assessed by
physical examination. In developed countries, imaging
modalities may also be used, but physical examination is
still the gold standard in countries where these tests are
not widely available. Grossly inapparent carcinoma (car-
cinoma detected by Papanicolaou test and biopsy not by
physical examination) is assessed on LEEP or cone biopsy,
where stage is assigned by microscopic measurements of
the extent of invasive carcinoma. Occult spread of disease
may be found at the time of surgery, but this does not
change the initial stage (though it may change the treat-
ment plan). Although various staging systems exist, the
International Federation of Gynecology and Obstetrics
(FIGO) system is almost universally used (Table). There is
a clear difference in survival depending on stage with
near 99% 5-year survival for stage IA1 to 65% for IIB and
43% for IIIB.37
In pathology reports for LEEPs and cone biopsies, the
size of the invasive tumor, including depth of stromal in-
vasion and horizontal tumor spread, should always be in-
cluded, as well as the presence of lymphovascular invasion
(LVI) and margin status. LEEP and cone specimens should
be inked with differential coloring of the endocervical
margin, if possible.
Lymphovascular invasion has been shown to be an in-
dependent risk factor in relation to disease-free survival
in a Gynecologic Oncology Group (GOG) prospective
study of 645 patients (disease-free survival, 89% vs 77%
without and with LVI, respectively).38 However, reproduc-
Current Concepts in Cervical PathologyPark & Soslow
Figure 6. Stratified mucin-producing intraepithelial lesion (hematoxylin-eosin, original magnification 200).
Figure 7. Superficial invasive squamous carcinoma arising from endocervical gland involved by high-grade squamous intraepithelial lesion
(cervical intraepithelial neoplasia 3) (hematoxylin-eosin, original magnification 100).
Figure 8. A, High-grade squamous intraepithelial lesion (cervical intraepithelial neoplasia 2 [CIN 2]) (hematoxylin-eosin, original magnification
40). B, p16 immunohistochemical stain of CIN 2 (original magnification 40).

ible and accurate diagnosis of LVI can be problematic. The
most difficult issue concerns artifactual retraction or
shrinkage of stroma around invasive nests that results in
spaces that resemble vessels. Immunostains for vascular
markers, such as CD31, CD34, and lymphatic marker D2-
40 (podoplanin) can help if the focus is present in sub-
sequent sections.39 Despite problems with recognizing
LVI, most studies have shown that there is a relationship
between the depth of invasion and the presence of LVI and
that LVI is a predictor of residual invasive disease in the
hysterectomy specimen.40 For some gynecologists, the
presence of LVI in the context of microinvasive carcinoma
(FIGO stage IA1) triggers radical resection instead of con-
servative management.
In a prospective study from the GOG by Sedlis et al,41
patients with stage IB cervical carcinomas were random-
ized to pelvic radiotherapy following radical hysterectomy
and lymphadenectomy if their tumors fulfilled 2 of the
following 3 criteria: large clinical tumor size, deep (greater
than one-third) stromal invasion, and LVI. They found that
patients receiving adjuvant radiotherapy suffered fewer
Arch Pathol Lab MedVol 133, May 2009
recurrences than controls. Surgical pathology reports of
radical hysterectomy specimens should therefore include
the same information as reported for LEEP and cone bi-
opsies in addition to the percentage invasion into the cer-
vical wall, the gross size of the tumor, and the status of
the parametrium.
The distinction between squamous and adenocarcinoma
might have clinical relevance, as it has been postulated
that adenocarcinomas and adenosquamous carcinomas
have a worse prognosis than pure squamous carcinomas,
with higher rates of lymph node metastases. However, a
GOG study looking at 626 patients with locally advanced
disease treated with radiation showed no difference based
on histologic cell type.42 The impact of tumor grade on
prognosis has also been debated. Another GOG study by
Zaino et al40 showed that in squamous carcinomas grade
has little prognostic value. On the other hand, tumor dif-
ferentiation may have a significant role in adenocarcino-
ma.43 There are many different types and variants of pri-
mary cervical carcinomas, but this review is limited to
commonly encountered squamous cell and adenocarcino-
Current Concepts in Cervical PathologyPark & Soslow 733
 International Federation of Gynecologists and
Obstetricians Staging
Stage Iconfined to the cervix
IAdiagnosed only by microscopy; no visible lesions
IA1stromal invasion less than 3 mm in depth and 7 mm
or less in horizontal spread
IA2stromal invasion between 3 and 5 mm with horizon-
tal spread of 7 mm or less
IBvisible lesion or a microscopic lesion with more than 5
mm of depth or horizontal spread of more than 7 mm
IB1visible lesion 4 cm or less in greatest dimension
IB2visible lesion more than 4 cm
Stage IIinvades beyond cervix
IIAwithout parametrial invasion, but involves upper two-
thirds of vagina
IIBwith parametrial invasion
Stage IIIextends to pelvic side wall or lower one-third of the
vagina, causes hydronephrosis or nonfunctioning kid-
ney
IIIAinvolves lower third of vagina
IIIBextends to pelvic wall and/or causes hydronephrosis or
nonfunctioning kidney
Stage IVextends beyond true pelvis or clinically involves the
mucosa of bladder or rectum
IVAinvades mucosa of bladder or rectum and/or extends
beyond true pelvis
IVBdistant metastasis
mas. A partial list of other carcinomas, some of which
have important clinical differences with the tumors dis-
cussed herein, include verrucous carcinoma, lymphoepi-
thelial-like carcinoma, intestinal adenocarcinoma, gastric-
like adenocarcinoma, endometrioid adenocarcinoma, clear
cell and serous adenocarcinoma, minimal deviation ade-
nocarcinoma, mesonephric adenocarcinoma, glassy cell
carcinoma, mucoepidermoid carcinoma, adenoid cystic
carcinoma, adenoid basal carcinoma, large cell and small
cell neuroendocrine carcinoma, and mixed epithelial and
mesenchymal tumors.
Correctly measuring the extent of invasion is crucial for
staging cervical cancer, especially in early-stage (micro-
scopically detected) disease because stage dictates treat-
ment and prognosis. However, superficial invasion is fre-
quently overcalled. Of 265 purported cases of microinva-
sion submitted to a group of reference pathologists of the
GOG, about 50% were rejected.44 Pitfalls of misdiagnosing
SIL with microinvasion include SIL involving endocervical
glands and areas of prior biopsy site with entrapped SIL.
Areas of prior biopsy will have associated acute and
chronic inflammatory infiltrate and may have entrapped
neoplastic epithelium buried within the stroma as a result
of disrupted SIL from a punch biopsy. Definitive examples
of invasive squamous carcinoma should demonstrate stro-
mal desmoplasia, irregular or scalloped margins, and/or
abundant, paradoxically mature eosinophilic cytoplasm.
Once the presence of invasion is confirmed, measuring
the depth of invasive squamous carcinoma is the next im-
portant step. For consistency, the depth of stromal invasion
should be measured following these guidelines: the depth
of neoplastic projections should be measured from the ini-
tial site of invasion, either from the basal lamina of the
surface epithelium or from the endocervical glands re-
placed by intraepithelial lesions (Figure 7); in cases in
which a direct histologic continuum between invasive foci
and SIL cannot be demonstrated, it is assumed that the
734 Arch Pathol Lab MedVol 133, May 2009
invasion originated from the basal cells of the overlying
SIL. Horizontal extent of the invasive carcinoma only
needs to be measured in cases in which the tumor depth
is 5 mm or less because once the tumor invades greater
than 5 mm, it is stage IB regardless of the extent of hori-
zontal spread. FIGO includes horizontal measurement in
staging but does not delineate how to measure it. When
there is only unifocal invasion, measuring horizontal ex-
tent is not difficult. However, invasive carcinoma can often
be multifocal. There is no absolute method for how to best
measure the horizontal extent of invasion in these circum-
stances, but because tumor volume is thought to be an
important factor in prognosis45,46 with higher rates of
lymph node metastases and recurrence in women with
greater tumor volume, it may be best to measure in a way
that most accurately approximates volume. In an article
by Reich and Pickel,47 they describe 3 patterns of micro-
invasion and how each of those should be measured:
Type Imultiple discrete foci of invasion are contiguous
with surface epithelium and the origin of invasion can
be detected. In this case, measure each invasive focus
separately and then add together for a total horizontal
length. The HSIL in between the invasive foci is not
measured.
Type IIthe origin of invasion cannot be seen in all of the
early invasive foci but the greatest lateral extent can be
seen in a single section. The breadth of the lesion is
considered to lie between the 2 most lateral foci count-
ing everything, including normal and HSIL, in between.
Type IIIlike type II, except that the lesion is present in
multiple contiguous sequential sections; the greatest
horizontal length cannot be measured in a single slide.
In this case, the width must be calculated by measuring
the distance between the sections; this is usually as-
sumed to measure 2 to 3 mm per section.
Therefore, the growth pattern of the invasive foci is im-
portant and the entire cervix must be embedded and eval-
uated when the lesion is microscopic. Examining multiple
levels may also be useful.
The nomenclature used to describe bona fide examples
of superficially invasive carcinomas is, unfortunately, not
standardized. Proposals for definitions of microinvasion
or microinvasive carcinoma include examples showing in-
vasion less than 3 mm deep or those with invasion less
than 5 mm deep, with or without LVI. The horizontal ex-
tent of the invasive front is also inconsistently mentioned
as a criterion. The term has also been used, historically,
more frequently in reference to squamous cell carcinoma
than adenocarcinoma, as discussed subsequently. In prac-
tice, therefore, we do not advocate the use of the term
microinvasive carcinoma. Instead, we report the depth of
invasion, measure the horizontal extent of the invasive
component, and mention whether LVI is present. If it is a
pathologists practice to use the term microinvasive carci-
noma it is recommended that the term only be used when
definitive measurements can be made (and surgical mar-
gins are negative for invasive carcinoma) and the patients
gynecologist is informed about the criteria the pathologist
uses for such tumors.
Although the FIGO staging system for cervical cancer ap-
plies to both squamous and adenocarcinoma, there is con-
siderable controversy as to whether microinvasive adenocar-
cinoma exists as a histologically recognizable entity. One rea-
son is that the irregular distribution and architecture of the
Current Concepts in Cervical PathologyPark & Soslow
normal endocervical crypts in the cervical stroma make it
difficult to differentiate between early stromal invasion and
AIS. There are 2 unequivocal features of invasion in adeno-
carcinoma: individual cells/fragmented glands/incomplete
glands lined by malignant cells at a stromal interface and
malignant glands with desmoplastic stromal response. Other
less definitive soft features that are suggestive of early in-
vasion include complex architectural growth with papillae,
cribriforming and irregular glands growing in a confluent
or labyrinthine pattern, malignant glands adjacent to medi-
um- or thick-walled vessels, and the presence of malignant
glands below the level of normal glands. Normal cervical
glands extend to the inner one-third of the cervical wall and
can measure up to 1 cm. Nabothian cysts, tunnel clusters,
endocervical hyperplasia, deep endocervical glands, and me-
sonephric duct remnants can all extend to the outer third of
the cervical wall.
Admittedly, in about 20% of the cases, it is impossible
to distinguish between AIS and early invasive adenocar-
cinoma.48 Because there is no overlying basement mem-
brane from which to measure the starting point of inva-
sion, as there is in squamous carcinoma, measuring the
exact depth of invasion of cervical adenocarcinoma is
problematic. Most sources, including the World Health Or-
ganization,49 recommend measuring the thickness of the
tumor from the mucosal surface, rather than depth of in-
vasion as defined previously.
Some cervical adenocarcinomas, particularly villoglan-
dular adenocarcinomas, and many papillary squamous
cell carcinomas can grow as exophytic, polypoid masses
that are obvious at physical examination. Substantial ar-
chitectural complexity and the presence of a gross, mass-
forming lesion qualify for invasive carcinoma (as opposed
to HSIL or AIS), but measurement of the depth of invasion
can be problematic because these lesions frequently grow
up into the endocervical canal, instead of down into the
cervical stroma. The depth of stromal invasion here should
not reflect thickness, rather it should be measured from a
plane that underlies the exophytic tumor. Regardless of
this measurement, these tumors are regarded as at least
FIGO stage IB because they are easily detected on physical
examination.
ANCILLARY STUDIES
Various immunohistochemical and molecular assays are
available as ancillary studies in the workup of difficult
squamous and glandular lesions.
p16INK4a is a tumor suppressor gene that encodes a pro-
tein involved in cell cycle regulation. It acts in a negative
feedback loop with another tumor suppressor, retinoblasto-
ma protein, so that cell proliferation is held in check. When
high-risk HPV DNA integrates into the host cell genome, the
viral oncoprotein E7 binds to retinoblastoma protein render-
ing it inactive. The feedback loop is lost and p16 is overex- Figure 9. Tubal metaplasia showing focal patchy staining with p16.
pressed as a result. This manifests as diffuse, strong, cyto- This is not the diffuse staining pattern seen in true adenocarcinoma in
plasmic and/or nuclear staining in squamous and glandular situ (original magnification 200).
lesions associated with high-risk HPV infection (Figure 8, A Figure 10. A, Reactive squamous epithelium with inflammatory cells
and B).5053 Interpreting p16 immunostaining, however, can and pseudokoilocytes (hematoxylin-eosin, original magnification 40).
be complicated, as both the context in which p16 is applied B, Mib-1 stain in reactive epithelium. Notice staining of lymphocytes
and parabasal cells that extend to the surface of the epithelium. This
and also the distribution and intensity of its staining pattern should not be interpreted as evidence of dysplasia (original magnifi-
must be considered before using the stain to confirm the cation 40).
presence of an HPV-associated neoplasm. A variety of le-
sions unassociated with HPV can show focal or patchy p16
staining, including tubal metaplasia, squamous metaplasia,
and endometrioid adenocarcinoma (Figure 9). Serous carci-
Arch Pathol Lab MedVol 133, May 2009 Current Concepts in Cervical PathologyPark & Soslow 735
nomas can show diffuse, strong staining as well.51,54,55 Atyp-
ical immature metaplasia lesions that are p16 positive have
been shown to have a higher incidence of subsequent HSILs
and are likely HSIL lesions that have an immature meta-
plastic appearance.9 Eosinophilic dysplasias are also usually
p16 positive, supporting the contention that these are vari-
ants of dysplastic lesions arising from cervical reserve cells.11
Atypical, proliferative glandular lesions that are diffusely
and strongly p16 positive are also likely HPV-associated ad-
enocarcinomas as opposed to one of the reactive lesions dis-
cussed earlier. The diffuse staining pattern of HPV-associated
glandular lesions can be especially helpful in curettage spec-
imens where the differential is often with endometrial en-
dometrioid adenocarcinomas. A useful panel in this situation
includes p16, estrogen and progesterone receptors, monoclo-
nal carcinoembryonic antigen (CEAM), and vimentin.56,57 Al-
though overlapping patterns are seen, the general trend is
for cervical adenocarcinomas to be positive for CEA(m) and
p16 and negative for estrogen and progesterone receptors
and vimentin, whereas the opposite is likelier in endometrial
endometrioid adenocarcinomas.
Mib-1 is a monoclonal antibody that reacts with Ki-67, a
nuclear cell proliferationassociated antigen that is expressed
in all active parts of the cell cycle (G1, S, G2/M). Positive
staining in parabasal cells is found under normal conditions,
so interpreting the results of this stain rests on examination
of the middle and upper thirds of the squamous epithelium.
High-grade squamous intraepithelial lesion (CIN 2 and CIN
3) usually shows diffuse nuclear positivity scattered
throughout all layers of the epithelium,58 whereas the distri-
bution of Mib-1 staining in CIN 1 can be less diffuse with
only small clusters of squamous cells staining in the upper
two-thirds of the epithelium. One should be careful not to
overinterpret positive staining in the presence of inflamma-
tion because intraepithelial lymphocytes will be positive for
Mib-1 and inflammation can also cause reactive proliferation.
It should also be noted that tangential sectioning sometimes
results in the impression that Mib-1positive parabasal cells
are superficially placed, leading to confusion with a SIL59,60
(Figure 10, A and B). Adenocarcinoma and AIS should show
strong diffuse staining with Mib-1, whereas benign mimics
such as tubal metaplasia, radiation change, and reactive atyp-
ia should show only focal or patchy staining at most.61,62
SMILE should also show diffuse positivity of the entire
thickness of the lesion, including the mucin-producing cells.27
Atypical immature metaplasia, when diffusely positive for
Mib-1, is likely an immature variant of HSIL.9
ProEx C is a recently developed immunohistochemical
assay that targets the expression of topoisomerase II- and
minichromosome maintenance protein-2, two genes that
have been shown to be overexpressed in cervical can-
cers.6370 The assay is a nuclear stain that is positive in
cervical dysplasia and has been validated in cytologic
specimens for the detection of HSIL.66,67 Studies have also
shown that ProEx C is comparable to p16 and Mib-1 in
the detection of high-grade lesions in formalin-fixed tissue
sections and in distinguishing them from benign mim-
ics.6870 The staining pattern of ProEx C in histologic sec-
tions is similar to that of Mib-1 in that only nuclear pos-
itivity above the basal one-third layer is considered posi-
tive. In a recent study by Pinto et al70 it was shown that
the combination of p16 and ProEx C predicted more non-
SILs than p16 combined with Mib-1. This suggests that
ProEx C may be more efficient than Mib-1 for distinguish-
ing reactive epithelial changes from squamous lesions.
736 Arch Pathol Lab MedVol 133, May 2009
In situ hybridization (ISH) is a direct signal detection as-
say that allows visualization of HPV DNA within infected
cells. Unlike other molecular detection methods (eg, poly-
merase chain reaction), the ISH assay can be automated and
does not require the skills of highly trained laboratory per-
sonnel or the stringent laboratory setup required to prevent
contamination. The interpretation of ISH is similar to that of
immunohistochemistry staining in tissue sections (look for
positive nuclear staining). The ISH signal patterns of HPV
DNA have been associated with the physical status of HPV
in infected cells, that is, episomal or integrated forms.71,72 Epi-
somal forms result in blocklike nuclear labeling, whereas in-
tegrated forms result in punctate, nuclear signals. Punctate
signals are most frequently found in HSILs and invasive car-
cinomas but not LSILs. Because the integration of oncogenic
HPV into the human genome is a critical step for cervical
cancer carcinogenesis, the signal pattern could be a useful
marker for predicting precancerous lesion progression. In a
recent study by Guo et al,73 the authors showed that the
Inform HPV III (Ventana Medical Systems, Tuscon, Arizona)
in situ hybridization assay is comparable to polymerase
chain reaction and may be a useful adjunct in detecting
HPV-positive lesions and carcinomas. However, ISH has also
been shown to have low sensitivity so its utility has been
questioned.74
CONCLUSIONS
There are many common difficulties that arise in the
daily assessment of cervical specimens. Practicing pathol-
ogists should not overcall reactive squamous atypia as
squamous dysplasia and they should be aware of variants
of squamous lesions and AIS that may appear metaplastic
or reactive (AIM, ED, SMILE). Stage is the most important
prognostic factor for cervical cancer, but other pathologic
indices are also important. One should include all prog-
nostically relevant information in pathology reports for
best patient care, including the extent of invasion, margin
status, and the presence of LVI. The depth of invasion
should be measured from the nearest overlying basement
membrane in squamous carcinoma, but the thickness of
tumor (rather than depth of invasion) should be reported
in adenocarcinoma. Radical hysterectomy reports should
also contain information about the greatest tumor dimen-
sion and the status of the parametria. It may be difficult
to distinguish AIS from early invasive adenocarcinoma,
but using the criteria discussed previously should be help-
ful. Finally, ancillary studies (p16, MIB-1, ProEx C, HPV
in situ) can be useful in difficult cases, but they must be
interpreted in the proper context.
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