marisa r. nucci, md and christopher p. crum, md early... · marisa r. nucci, md and christopher p....

Redefining Early Cervical Neoplasia: Recent Progress

Marisa R. Nucci, MD and Christopher P. Crum, MD

Abstract: The classification of cervical precancers has evolved

over the past 40 years as knowledge concerning the pathogenesis

of these human papillomavirus-related lesions has expanded.

This commentary reviews the current classification scheme in

light of (1) the historical classification systems and (2) the ability

of the target epithelium, that is the transformation zone to

exhibit varied morphology depending on the cell type that is

infected with human papillomavirus. The evolution in our

understanding of preinvasive glandular neoplasia is also

summarized, with particular attention to so-called superficial

(or early) adenocarcinoma in situ. In addition, practical issues in

the diagnosis and management of squamous epithelial lesions,

particularly the recognition of nonconventional variants and the

application of biomarkers, are discussed.

Key Words: HSIL, LSIL, HPV, adenocarcinoma in situ

(Adv Anat Pathol 2007;14:1–10)

The concept that invasive squamous cell carcinoma ofthe cervix is preceded by a morphologically identifi-

able noninvasive precursor, whether identified by histo-logy or cytology, has long been established. In fact, theobservation by Dr Papanicolaou that precursor lesionscould be cytologically detected through the examinationof vaginal smears dates to the early 1940s.1 However,despite the wide acceptance of the existence of aprecancerous lesion, terminology has varied and at least3 classification systems have been proposed, including (inorder of their inception): (1) dysplasia-carcinoma in situ(CIS), (2) cervical intraepithelial neoplasia (CIN), and (3)squamous intraepithelial lesions (SILs) also known as theBethesda (binary) system.2

In the first half of the 20th century, the term CISwas widely used, because lesions classified as such wereshown to confer an increased risk of subsequent invasivecarcinoma in limited followup studies.3 The first wellestablished CIN classification in which CIS was distin-guished from dysplasia (ie, lesions with less atypia) cameto prominence in the 1960s after its proposal by theInternational Committee on Histologic Terminology forLesions of the Uterine Cervix.3 In their proposal, thecommittee offered histologic criteria for distinguishing

CIS from dysplasia, which in turn allowed clinicians totemper their management of the patient based on thepresence or absence of CIS. However, this proposal wassoon fraught with a number of concerns, most of whichchallenged the concept that dysplasia and CIS could beconsidered (and reproducibly distinguished) as separateentities. Some of the observations that led to theseconcerns included: (1) dysplasia and CIS share similargenetic abnormalities,4 (2) lesions classified as severedysplasia that have morphologic overlap with CIS arealso at risk for progression to invasive carcinoma,5 (3)patients with lesions classified as dysplasia have a 20-foldincreased risk for CIS than patients with normalPapanicolaou smears,6,7 CIS or carcinoma is detected inup to 40% of smears subsequent to a smear containingmild or moderate dysplasia6 and (5) in patients witha cytologic diagnosis of dysplasia, there is a 100-foldincreased projected incidence of invasive carcinoma ascompared to women with normal smears.7 As a directresult of these observations, the CIS-dysplasia classifica-tion was replaced by one that merged dysplasia and CISinto one category—CIN—defined as a full-thicknesspopulation of atypical cells subdivided according todegree of atypia and maturation.5

In 1955, Koss and Durfee8 observed that somesquamous atypias exhibited nuclear atypia in the surfaceepithelial cells only, and termed these lesions ‘‘koilocyto-tic atypia.’’ Twenty years later, the viral pathogenesis ofcervical precursor lesions began to gain credibility withthe direct demonstration of human papillomavirus (HPV)in koilocytotic atypia, prompting the alternate term of flator exophytic condyloma of the cervix.9,10 HPV 6 and 11,the first of the genital HPV types cloned, segregated tolower genital tract condyloma and some koilocytoticatypias, but were not associated with the higher-gradeprecursor lesions with full-thickness atypia assigned to thetraditional CIN classification.11 However, the missinglink in HPV-related cervical carcinogenesis, HPV 16, wassoon cloned, and found in a high percentage of squamouscarcinomas and CIN.12,13 Other cancer-associated HPVswere subsequently cloned, and found in flat cervicalcondyloma and CIN.14 Approximately 70% of flatcondylomas contained high-risk HPVs, but were segre-gated with greater efficiency from CIN by HPV16, whichwas associated with CIN in about 85% of infections thatproduced a morphologic abnormality, and which wasfound 4 to 5 times more frequently in CIN.15

Because HPV infections caused both CINs (astraditionally defined) and flat and exophytic condylomas,some confusion followed regarding the classification ofCopyright r 2006 by Lippincott Williams & Wilkins

From the Division of Women’s and Perinatal Pathology, Department ofPathology, Brigham and Women’s Hospital, Boston, MA.

Reprints: Marisa R. Nucci, MD, Department of Pathology, Brighamand Women’s Hospital, 75 Francis Street, Boston, MA 02445(e-mail: [email protected]).

REVIEW ARTICLE

Adv Anat Pathol � Volume 14, Number 1, January 2007 1

lesions that had a common origin but did not fall withinthe classic definitions of a cervical cancer precursor. Someauthors chose to separate flat condyloma from CIN I16;others chose to assign the term ‘‘CIN I’’ to flatcondyloma, and reserving CIN II and CIN III for thoselesions that fell into the traditional CIN classification.17,18

This evolution of terminology was not a smooth one,hampered in part by the fact that many pathologistscould not consistently distinguish CIN or condylomafrom non-neoplastic epithelial alterations.

In 1988, a conference sponsored by the NationalCancer Institute proposed classifying cervical precancersinto 2 categories in cytologic practice-low and high-gradesquamous intraepithelial lesions (HSILs)—which becameknown as the Bethesda System for classifying cervicalprecancers.19 In this classification scheme, lesions for-merly considered CIN I were merged with condylomainto the low-grade group whereas lesions formerlyclassified as CIN II or III were merged into the high-grade category. Two criticisms quickly emerged after theintroduction of this classification system: (1) althoughcondyloma and CIN I can share a similar cytologicappearance, they differ in their classic (or original)histologic description and (2) the apparent differing riskof progression to invasive carcinoma for CIN II ascompared to CIN III. This latter concern posed less of achallenge to overcome, as both CIN II and CIN III aresimilarly associated with high risk HPV. Moreover, thedistinction between these 2 grades has been shown to havepoor reproducibility among pathologists although eitherdesignation elicits the same therapeutic (ablative) out-come, rendering this separation less important.15 Byredefining CIN I as only those lesions that fulfillmorphologic criteria for flat and exophytic condyloma,the former concern could also be addressed. In doing so,this revision of the Bethesda system, which is the currentaccepted classification system, would allow a category,CIN I, to identify a process that could, with goodreproducibility, be distinguished histologically from high-er-grade lesions with a greater risk of progression toinvasive carcinoma. Based on the illustrations in anumber of textbooks and reviews, low-grade squamousintraepithelial lesions (LSILs) now typically include thoselesions left out of the traditional CIN classification, andoverlapping with some lesions traditionally classified asCIN I, whereas high grade SILs, are well within the upperpart of the spectrum of traditional CIN II or III (Table 1).

The practitioner who applies the current criteria forLSIL and HSIL must be able to navigate the variablesthat impact on morphologic presentation, including thetransformation zone and intervening conditions thatproduce abnormalities in epithelial growth and differ-entiation. These are discussed below.

THE TRANSFORMATION ZONEAlthough the revised Bethesda classification scheme

depicts a traditional pattern of squamous alterationssecondary to HPV infection, it is clear that there is a

morphologic spectrum of cervical epithelial alterationssome of which are not encompassed by this system. Thisspectrum of morphologic alterations likely reflects theplasticity of the epithelium that may be infected, as theepithelium that comprises the transformation zone canshow differences in their differentiation level and differ-ences in their differentiation pathway (Fig. 1).20 Reserve(or indifferent) cells within the transformation zone maydifferentiate toward squamous, columnar, or a combina-tion of phenotypes, which when infected by HPV canaccount for a similar morphologic range of lesionsincluding mature and immature squamous (eg, matureLSILs, immature condyloma, immature ‘‘metaplastic’’HSILs), columnar [adenocarcinoma in situ (AIS)], ora combination (stratified mucinous intraepithelial le-sions).21,22 Therefore an understanding of the range ofnormal phenotypic expression of cervical epithelium helps

FIGURE 1. A, Reserve cells in the cervical transformation zone,associated with a focus of microglandular hyperplasia (top)and established between columnar epithelium (bottom). B,Transition from reserve cells to squamous metaplasia in anevolving microglandular hyperplasia (bottom).

TABLE 1. Categories of CIN

SIL

Classification Descriptor Other Terms HPV

LSIL Exophytic condyloma LRFlat condyloma CIN I HR/

LRImmature condyloma Papilloma LR

Transitionalpapilloma

CIN I HR/LR

HSIL Atypical condyloma Koilocytotic CIN II HRCIN II HRCIN III HREosinophilic dysplasia CIN II HRAtypical immaturemetaplasia

CIN II and III HR

SMILE Adenosquamous CIS HR

HR indicates high risk; LR, low risk.

Nucci and Crum Adv Anat Pathol � Volume 14, Number 1, January 2007

2 r 2006 Lippincott Williams & Wilkins

explain the varied differentiation level (mature vs.immature) and differentiation state (squamous vs. co-lumnar) of the diverse number of cervical precancerslesions that may occur.

SQUAMOUS LESIONS

The Threshold for LSILDistinguishing LSIL from non-HPV–related condi-

tions of the cervix has been an issue in practice since theearly 1980s when HPV was first associated with earlycervical neoplasia. The consequences of an over-diagnosisof LSIL (then referred to as condyloma or CIN I) werenot trivial, inasmuch as the patient was often informedthat she had a sexually transmitted disease and in the eyesof some practitioners, required an ablative technique,

such as cryotherapy or carbon dioxide laser, to removethe lesion. Similar lesions in the vagina were routinelymanaged with topical applications of 5-fluoro-uracil, anapproach that could lead to chronic dyspareunia ifcarried out too aggressively. Such complications man-dated a morphologically definable and reproduciblethreshold for LSIL. With the advent of LEEP excisionin the 1990s, LSIL was increasingly managed moreconservatively, and a recent study suggests that thewoman with an abnormal smear (ASCUS or LSIL) hasa similar risk of HSIL at 24 months (11% to 13%)irrespective of whether the pathologist recognizes LSIL inthe biopsy.23 Nonetheless, an accurate distinction of LSILfrom normal or reactive mucosa is important to avoidmisclassification of an sexually transmitted disease andprovide reliable diagnostic information to the clinician.

FIGURE 2. Mimics of LSIL include (A)mild disturbances in maturation asso-ciated with reactive changes, (B) cyto-plasmic halos, and (C) dyskeratosis.Panel (D) contains a subtle LSIL forcomparison.

Adv Anat Pathol � Volume 14, Number 1, January 2007 Redefining Early Cervical Neoplasia

r 2006 Lippincott Williams & Wilkins 3

The strongest correlate for the presence of HPV wassuperficial nuclear atypia in the form of koilocytoticatypia. However, other parameters are helpful and thefollowing approach to the diagnosis of LSIL facilitates itsrecognition as well as allows its separation from potentialmimics such as nonspecific cellular changes due todisturbances in maturation or inflammatory-relatedcellular changes.

One of the earliest indications that a cervicalepithelial alteration represents an LSIL is the presenceof alterations in the low power appearance of theepithelium in comparison to adjacent normal, whichmay include increased nuclear density near the surface,abnormal surface keratinization with increased eosino-philia of the cytoplasm, conspicuous superficial nuclearatypia, and alterations of the epithelial thickness (Fig. 2).Low power suspicion requires high power confirmationwith an assessment of the type, degree, and location ofnuclear atypia. LSILs are characterized by nuclear atypiain the upper portion of the epithelium with lack of a highmitotic index (and atypical mitotic figures), lack of atypiain the lower epithelial layers (basal and parabasal), andmaintenance of cellular polarity (particularly at the base).The HPV-related nuclear changes typically present inLSIL show alterations in size and staining of the affectedepithelium, with the nuclei showing at least a 3-folddifference in size and variable degrees of hyperchromasia.Other findings, though not specific, are often helpfulin confirming the diagnosis when present in associationwith the aforementioned histologic features. These includecytoplasmic halos of irregular size and shape andbinucleated cells with enlarged, hyperchromatic nuclei,the latter occurring in greater than 90% of LSIL. It isimportant to remember that binucleation is nonspecificand may occur in association with reactive epithelialchanges; however, if there are multiple binucleated cellsthat have enlarged, hyperchromatic nuclei in a given highpower field, this strongly supports the diagnosis of anLSIL. Cytoplasmic halos are also a nonspecific feature,however, those associated with LSIL are usually irregularin shape and size and surrounded by a rim ofhypereosinophilic cytoplasm that imparts a basket weaveappearance in contrast to the halos of reactive epithelium,which typically lacks these features.

LSIL and its VariantsThe diagnosis of LSIL carries with it a significant

responsibility. In making this diagnosis, the pathologistis essentially stating that the patient does not require areturn visit for the next 12 months. The cytology shouldmatch the biopsy; if an HSIL is suspected, a repeatcolposcopy may be required.

The diagnosis of LSIL should be reserved for aselect group of entities within a narrow spectrum ofhistologic changes. The fundamental premise for thisdiagnosis is the absence of atypia that confers a diagnosisof HSIL (CIN II or III) or its variants. Another usefulperspective is to require the presence of cells in theepithelium which, taken in a cytologic context, merit

classification as LSIL. Given this, LSIL can be dividedinto 4 morphologic subsets: (1) condyloma acuminatum(exophytic condyloma), (2) immature condyloma (squa-mous papilloma, papillary immature metaplasia), (3) flatcondyloma (CIN I), and (4) flat lesions with surfaceatypia and inconspicuous halos (also termed CIN I).Exophytic condyloma (Fig. 3), which is highly associatedwith HPV types 6 and 11 and is relatively uncommon inthe cervix, is characterized by verruciform growth, blunt-shaped papillae, acanthosis, and superficial koilocytoticatypia (viral cytopathic effect). Immature condyloma(Fig. 3) is characterized by slender, filiform papillae linedby squamous epithelium only showing slight nuclearcrowding and mild superficial koilocytotic atypia mimick-ing immature metaplasia. The relative lack of koilocytoticatypia reflects the dependence of viral cytopathic effect onmaturation, which is limited in these lesions; however,similar to exophytic condyloma, immature condylomais associated with HPV types 6 and 11, which supportsits classification as a type of condyloma.24,25 As lesionsdescribed as papillary immature metaplasia and squa-mous papillomas are histologically identical to immaturecondyloma and also contain HPV types 6 and 11, theterm immature condyloma not only best reflects theirpathogenesis but will also result in the appropriate clinicalresponse.24,25 Flat condyloma (CIN I) lacks an exophyticgrowth pattern but is otherwise morphologically similarto condyloma acuminatum with atypia confined to theupper third of the epithelium (Fig. 3). As these lesionsare usually associated with HPV types that confer anintermediate or high risk, they are considered separatelyfrom immature and exophytic condyloma.14,26

Some LSILs have the mild parabasal hypercellular-ity of flat condyloma, and exhibit increased nucleardensity in the upper epithelial layers with anisokaryosis,yet lack the prominent cytoplasmic halos. Purists wouldnot classify these as flat condyloma because of theabsence of koilocytosis, and might prefer to use the termCIN I. Nevertheless, the appropriate diagnosis from amanagement perspective is LSIL.

Distinguishing LSIL From HSILThe distinction of LSIL from HSIL is one of the

most important decisions in the laboratory managementof precursor lesions. A verdict of LSIL will result in afollowup evaluation in 12 months. In contrast a diagnosisof HSIL will set into motion a chain of events eventuatingin (often) an appointment to day surgery, anesthesia, anda cone or LEEP excision. For this reason, the practitionershould be wary of overdiagnosing CIN II. If thepathologist is unsure whether to make a diagnosis of LSILor HSIL, a second opinion from a colleague is recom-mended, inasmuch as the decision will have significantimpact on the immediate plans of the patient in question.This is particularly important in young patients, who areat a low short-term risk for an adverse (cancer) outcome,often revert to normal, and may rarely suffer adversesequelae (cervical incompetence) from LEEP or conebiopsy. In these patients particularly, the pathologist



should be certain of his or her diagnosis; if not,uncertainty in lesion grade should be communicated tothe clinician.

It is not uncommon for the basal and parabasal areaof an LSIL to exhibit increased cellularity and somedegree of nuclear enlargement, particularly in thoselesions that are inflamed. In such cases, distinction froman HSIL may be particularly difficult as this separationis based on the assessment of atypia and epithelialorganization in the lower epithelial layers. If there is noloss of cell polarity and the nuclei in the lower layers showonly minimal nuclear pleomorphism and lack coarsechromatin, then the lesion is best classified as an LSIL.

The diagnosis of HSIL is based on atypia in thelower epithelial layers that exceeds the threshold forLSIL/condyloma, that is the cells contain a high nuclear/cytoplasmic ratio and coarse-appearing chromatin. Le-sions with full-thickness atypia and surface maturation

(often discernible as koilocytotic atypia or abnormalkeratinization) correspond to lesions classified as CIN IIwhereas those with little to no maturation correspond toCIN III (Fig. 4). Additional features that aid indistinguishing LSIL from HSIL are: (1) lack of nuclearatypia in the lower epithelial layers, (2) fewer number ofmitoses, which when present are confined to the basal andparabasal areas, (3) maintenance of cell polarity (parti-cularly at the base of the epithelium which is oftendisorganized in high grade lesions), and generally, (4) lackof abnormal mitotic figures. As a rule of thumb, if cellsare present in the lower epithelial layers that would meritthe diagnosis of HSIL on cytology, a diagnosis of HSILshould be strongly considered.

As implied earlier, this classification system differsfrom the classic descriptions of CIN in the 1960s, whichrequired full-thickness atypia for all CIN diagnoses.However, as flat and exophytic condylomata are now

FIGURE 3. Morphologic spectrum ofLSIL, including (A) exophytic condylo-ma, (B) flat condyloma (or CIN I, and(C) immature condyloma. The imma-ture cell layers of the latter exhibituniform nuclei and small chromocen-ters (Panel D).



incorporated into the family of intraepithelial lesions, theclassification has been altered somewhat to equate flatcondyloma with CIN I. In equating the two, we now usesimilar criteria for their diagnosis, and designate alllesions with full-thickness atypia as either CIN II or III(HSIL).

Nonconventional SIL VariantsMost of the aforementioned subsets SIL fall into the

well-described continuum that is depicted in most text-books. In this system the distinction of LSIL from HSILis usually relatively straightforward and the transitionfrom the former to the latter is marked by the criteriaoutlined above. However, the reader has likely encoun-tered a wider range of abnormalities than illustratedso far in this review, because of the plasticity of thetransformation zone and the fact that both high andlow risk HPVs can infect a range of cells, including fromthe mature portio, reserve cell, columnar cell, and cells intransition between the two. The following list designateseach group by its proposed cell of origin and HPVcategory.

Immature metaplasia and low risk HPV: Thisresults in the immature condyloma (LSIL) discussedabove, by infection of immature squamous metaplasiawith HPV 6 or 11 (Fig. 3).24,25

Immature metaplasia and high risk HPV: Thisresults in HSIL with an immature metaplastic phenotypeand can be subdivided further into 3 patterns.(1) HSIL akin to atypical immature metaplasia. In most

cases, HSIL with an immature metaplastic phenotypecan be distinguished from immature squamousmetaplasia as the former shows increased nucleardensity in the upper epithelial layers and nuclearhyperchromasia whereas the latter lacks these featuresand may be associated with surface columnar cells ormucin droplets, which is uncommonly seen with HSIL(Fig. 5). The key features are an immature (and oftenrather uniform) metaplastic cell population, a rela-tively high nuclear density in all epithelial layers andlack of normal cytoplasmic maturation with discretecell borders on the surface.27,28 However, it isimportant to note that immature squamous metapla-

sia may show some variation in nuclear staining andsize, surface atypia or polynucleation, particularlywhen associated with inflammation. In these cases,distinction from an HSIL is made by the lack of amarked degree of nuclear atypia, hyperchromasia,and increased nuclear density and disorganization. Indifficult cases, application of biomarkers may behelpful (see section below).

(2) Eosinophilic dysplasia. This relatively recently recog-nized variant of SIL is characterized by its combina-tion of both metaplastic and dysplastic epithelialfeatures.29 These lesions are expansile proliferations inwhich the cells lack normal maturation, have abun-dant eosinophilic cytoplasm with distinct cell mem-branes, distinct nucleoli, a modest increase in nuclearto cytoplasmic ratio and focal nuclear dysplasticfeatures with mild nuclear enlargement, hyperchro-masia and irregular nuclear membrane contour(Fig. 6A). As these lesions are commonly associatedwith an adjacent conventional appearing HSIL orLSIL and are commonly associated with intermediate

FIGURE 4. Morphologic patterns span-ning LSIL and HSIL (CIN II). A, LSIL withmild parabasal nuclear enlargement,(B) a problematic lesion with manyfeatures of LSIL but containing anabnormal (tripolar) mitotic figure. C,CIN II (HSIL) containing koilocytoticatypia and parabasal nuclear crowdingand atypia.

FIGURE 5. HSIL with an immature metaplastic phenotype.Note the focal basal nuclear enlargement and preservation ofcolumnar cells on the surface.



or high-risk HPV types, they likely represent a variantof HSIL. As the epithelium that comprises thetransformation zone can show differences in theirdifferentiation level and differences in their differen-tiation pathway, it is conceivable that this lesion’sunusual morphologic appearance is due to HPVinfection of target cells in the transformation zonewith a phenotype of intermediate differentiationbetween immature and mature epithelium.

(3) SIL occurring in the setting of microglandular change.This is an uncommon lesion characterized by itsclose resemblance to squamous metaplasia involvingmicroglandular change. It is composed of a lobulatedproliferation of squamous epithelial cells that are evenlyspaced with moderate nuclear density and a low mitoticrate, which may be admixed with mucin droplets butwhich also show prominent nuclear enlargement andmultinucleation (Fig. 6B). These lesions are oftenassociated with high-risk type HPV, are best classifiedas ‘‘SIL of uncertain grade,’’ and are distinguished fromearly metaplasia by conspicuous anisokaryosis, albeitwith a low mitotic index.

GLANDULAR LESIONS

Early (Superficial) AISThe majority of adenocarcinomas in situ are easily

recognized by a constellation of findings, includingdiscrete clusters of glandular epithelium with hyperchro-matic nuclei, increased mitotic index and karyorrhexis.However, a subset of AIS occurring in younger womenmay be diagnostically challenging. Witkiewicz et al20

recently described a series of these lesions that were foundon the surface mucosa or in papillae. This entity whichwas termed superficial or early AIS, presented in youngerwomen, exhibited a variable degree of hyperchromasia,mitotic activity, nuclear atypia, and karyorrhexis, yetwere strongly positive for p16 and contained HPV nucleicacids in a dot-forming or ‘‘integrated’’ localizationpattern.30 This entity underscores the subtle nature ofsome AISs yet emphasizes that these lesions share all ofthe biologic attributes of conventional AIS. For thisreason, we do not use the term ‘‘glandular dysplasia’’ forthese subtle lesions but rely more heavily on biomarkerstaining in problematic cases (Fig. 7).

Stratified Mucin-producing IntraepithelialLesions

Most HSILs exhibit minimal columnar differentia-tion and most mucin detected in these lesions is containedwithin normal columnar cells displaced to the surface ofthe lesion as it undermines normal mucosa. However, anuncommon precursor variant exhibits both the stratifica-tion of an HSIL and discrete mucin production in alllayers of the epithelium. This entity, which has beentermed ‘‘stratified mucin-producing intraepithelial lesion’’or SMILE, frequently coexists with both HSIL and AISand immunophenotypically is more consistent with astratified variant of AIS. In limited numbers of cases, ithas been found to frequently coexist with invasivecarcinoma, usually with an ‘‘adenosquamous’’ patternof differentiation (Fig. 7). Our approach in practice is toclassify SMILE as such, specifying that it is most likely avariant of AIS. The management of SMILE is identical toAIS, which is cone biopsy to ensure complete removaland exclude invasive carcinoma.22

BIOMARKERS FOR DIAGNOSISRecognition and correct classification of cervical

precancers is clinically relevant as management is basedupon both the presence and type of cervical SIL.Although in most cases the diagnosis is straightforward,some inflammatory-related atypias and disturbances inmaturation may mimic a SIL. Conversely, as discussedabove, some SILs may be subtle and less conspicuous onhistologic examination rendering them difficult to con-firm. In these instances, application of certain biomarkersmay be useful in supporting or refuting one’s histologicimpression. The biomarkers that are most useful in dailypractice are proliferation marker Ki-67 and cell cyclemarker p16ink4, the latter acting as a surrogate markerof HPV infection as HPV oncoproteins produce

FIGURE 6. A, Eosinophilic dysplasia is another subtle form ofSIL consisting of a uniform population of stratified, immaturemetaplastic cells with minimal reduction in nuclear density onthe surface. B, SIL occurring in the setting of microglandularchange is distinguished by a greater degree of anisokaryosisthan seen in typical reserve cells.



disturbances in the cell cycle that result in overexpressionof this protein.31,32 As a general rule, these biomarkersare useful in identifying whether or not a SIL is presentand are not useful in assigning a grade, high versus low,for cervical precancer disease. In contrast to normal,atrophic, or reactive squamous epithelium, Ki-67 willshow positive nuclear staining in the upper epitheliallayers of a SIL, typically in greater than 30% of nuclei(Fig. 8). p16, which is strongly expressed in lesionsassociated with intermediate and high risk HPV willexhibit diffuse nuclear and cytoplasmic reactivity in bothLSIL and HSIL in contrast to reactive or immaturemetaplastic proliferations (Fig. 8). Therefore, confirma-tion of a SIL can be made when both Ki-67 showsincreased staining in the upper epithelial layers and thereis diffuse p16 positivity throughout the epithelium. Thesame markers are useful for confirming AIS, taking intoaccount the comments below.

The practitioner who uses these biomarkers must becognizant of the following:

(1) p16 will stain columnar epithelium, including endo-metrium and lower uterine segment.

(2) Ki-67 is particularly difficult to interpret in the settingof partially denuded inflamed epithelium.

(3) Neither marker should be used routinely to distinguishLSIL from reactive squamous epithelium. The dis-

tinction of the two is not usually clinically relevant,not withstanding the importance of precise diagnosisin general.

(4) p16 is most useful for distinguishing immature meta-plasia from HSIL; Ki-67 for distinguishing atrophicchanges from HSIL.

(5) Both markers should be used concurrently until thepractitioner is comfortable with one or the other.

FUTURE CONSIDERATIONS IN PRACTICEManagement of CIN II in young women and the

role of HPV: There is increasing emphasis on avoidingunnecessary ablative procedures, particularly in youngwomen, and the target of these efforts will be the changingclinical approach to CIN II. In our experience, CIN IIis more likely to harbor so-called ‘‘intermediate risk’’ (orthose on the lower end of the high risk spectrum) HPVs.In view of the much weaker association between theseHPVs and the development of CIN III and theirassociation with cancers of lower mortality, there is arationale for following young women with CIN II ratherthan resorting to cone biopsy. This applies in particular towomen under age 20. It is conceivable that biopsies withthis diagnosis (or their concurrent smears) could be testedfor HPV and managed with LEEP or followup accordingto HPV type.

FIGURE 7. Variants of preinvasiveglandular neoplasia in the cervix in-clude (A and B) superficial (early) AISwith more subtle atypia, and (C)stratified mucin-producing intraepithe-lial lesions that have features of bothAIS and HSIL and are diffusely mucinpositive (D).



CIN in the vaccine era: The widespread use ofvaccines will theoretically reinforce the above approach asthe percentage of CINs with a higher rate of progressiondecrease and efforts are increased to reduce the propor-tion of CINs that require ablation. This process willchange the relative roles of HPV testing, cytologicscreening, and biopsy-directed management. Althoughthe impact of vaccines will take years to be realized, theprospect and anticipation of their success will very likelyinspire novel approaches to management designed toenrich for both patients at risk (who will be treated) andthose with minimal risk (who will be followed). Underideal conditions, the latter will expand over time and withit, a progressive reduction in biopsies and ablativeprocedures.

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FIGURE 8. SIL with a metaplastic growth pattern (A) AIS (B) and immature condyloma (LSIL; C), respectively stained for p16(D–F) and MiB1 (G–I).



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