Cervical Squamous Intraepithelial Lesions







  • Chapter Outline



  • Introduction 200



  • Squamous Intraepithelial Lesions (SILs) 202




    • Nomenclature 202



    • Features 203



    • Disease States: Types of SIL 207



    • LSIL (Condyloma/CIN 1) 208



    • High-Grade Squamous Intraepithelial Lesions (CIN 2–3) 212



    • Areas of Diagnostic Difficulty 214



    • Biomarkers 218



    • Histologic Features of SIL (CIN) Affecting Management and Prognosis 219



    • Regression and Progression to Invasive Carcinoma 221




  • Correlation Between Cytology and Histology 223



  • Distribution and Site of Origin of SIL 224



  • Colposcopy 224




    • Normal Colposcopic Findings 225



    • Abnormal Colposcopic Findings 226





Introduction


Human papillomavirus (HPV) infection is now understood to be the underlying cause of squamous carcinogenesis in the cervix. Older diagnostic classifications of preinvasive disease based purely on descriptive correlation of histology with clinical behavior included dysplasia/carcinoma in situ (CIS; a four-grade system) and cervical intraepithelial neoplasia (CIN; a three-grade system). Each provided a convenient diagnostic spectrum against which patient samples could be matched, and both are still in use to varying degrees.


The clinical goal of segregating individual lesions into dichotomous high- and low-risk categories was furthered by evidence showing that cancer outcomes correlated predominantly with one of two HPV subtype classes. Thus, the two-class Bethesda System of low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL) was formally endorsed by the National Cancer Institute (USA) at a workshop held in December 1988. Implementation was recommended for lesions in the cervix and vagina. LSIL was generally equated with condylomata and CIN 1, and HSIL with CIN grades 2 and 3 (CIN 2, CIN 3, or CIN 2–3). This principle of a two-tier system was reaffirmed by consensus of the College of American Pathologists and American Society for Clinical Pathology (ASCP) in 2012, with the additional recommendation that it be extended to all anogenital HPV-related lesions, including those in vulvar and perianal sites.


The initial expectation of the two-tier system was that viral type would be the primary determinant of outcome, and careful correlation of intraepithelial morphology with viral type would yield diagnostic criteria for the new entities of LSIL and HSIL. However, it is now clear that LSIL is the biologic manifestation of productive HPV infection with episomal viral DNA. Therefore, all HPVs can generate low-grade morphology as this type of cellular differentiation is required for viral replication to produce viral particles (virions). These infections and their cytohistologic manifestations are almost always transient, resolving on average in less than a year.


HSIL results predominantly from genomic integration of high-risk types of HPV DNA into the host genome of replicating parabasal cells, with consequent deregulation of expression of E6 and E7 viral oncogenes. The parabasal cells become disorganized relative to the underlying basement membrane, with subsequent clonal expansion extending upward toward the epithelial surface ( Figure 10.1 ). Cells containing only integrated viral DNA are virologically noninfectious as no viral particles are produced but the viral genes drive the human (host) cells to proliferate. HSILs may, however, contain both episomal and integrated HPV DNA, which is consistent with the presence of koilocytes in some HSILs.




Figure 10.1


Morphologic features and nomenclature of preinvasive cervical disease. The morphologic changes that occur with increasing lesion grade and how the SIL (Bethesda) and CIN systems relate to one another are illustrated. Quantitative features that become increasingly more abnormal with increasing grade are listed, along with those qualitative features that differ across the LSIL–HSIL boundary. The corresponding cytologic smear appearances resulting from exfoliation of the most superficial cells are also illustrated. Note that some pathologists subdivide HSIL into HSIL (CIN 2) and HSIL (CIN 3), which are comparable to CIN 2 and CIN 3 respectively.


Of the more than 120 HPV types known to exist, only about 30–40 affect the genital tract. Probably less than 15 are oncogenic; the most common is HPV type 16 and its related viruses 31, 33, 35, and 56 from the α 9 clade (a taxonomic group derived from a common ancestor) and type 18 with its close relations such as type 45 ( Table 10.1 ). They are termed high risk or oncogenic because they are the types found in invasive tumors. Progression of high-risk viral infection to HSIL is inefficient and, although high-risk types are the most common cervical HPV infections, most result in only low-grade lesions. The low-risk group constitutes only a minority of cervical infections. The low-risk HPV types are prototypically HPV types 6 and 11 whose biology is one of transient productive infection with only extremely rare examples of progression to carcinoma. The apparent prevalence of HPV infection in cervical lesions of different types and grades depends on the technique used for detection of the virus and the criteria applied for histologic annotation.



Table 10.1

Classification of HPV Types Associated with Genital Lesions
















































Most Common Types
6,11 Condyloma acuminatum, LSIL (condyloma/CIN 1)
16 All grades of SIL, squamous cell carcinoma
18 All grades of SIL, adenocarcinoma, squamous cell carcinoma, small cell carcinoma
Less Common Types
31, 33, 35, 39, 45, 51, 52, 56 All grades of SIL, squamous cell carcinoma
30, 40, 58, 69 All grades of SIL
42, 43, 44 Condyloma acuminatum, LSIL (condyloma/CIN 1)
53 Normal cervical epithelium, LSIL (condyloma/CIN 1) and sometimes HSIL (CIN 2)
54 Condyloma acuminatum
55 Bowenoid papulosis
59 Vulvar HSIL (usual type vulvar intraepithelial neoplasia)
61, 62, 64, 67 Vaginal HSIL (vaginal intraepithelial neoplasia)
66 Squamous cell carcinoma
70 Vulvar papilloma


Irrespective of HPV type, the viral cytopathic effect (koilocytosis) and the production of viral capsid protein become progressively less frequent with increasing lesion grade ( Table 10.2 ). Indeed, the inability of most high-grade lesions to support productive HPV infection can be viewed in a biologic context as viral failure. Local immune response within the cervical microenvironment is somewhat more common with high-risk HPV infections, and this can be associated with regression of HSILs. Conversely, low-grade lesions are, in a sense, ‘successful’ infections in that they represent productive infections with little or no propensity to kill the host.



Table 10.2

Comparison of HPV Frequency Detected by Laboratory Techniques


































DNA Hybridization (%) Koilocytosis (%) Immunocytochemistry for Viral Capsid Protein (%)
LSIL (Condyloma) 100 80 80
LSIL (CIN 1) 100 89 61
HSIL (CIN 2) 86 57 29
HSIL (CIN 3, severe dysplasia) 100 33 17
HSIL (CIN 3, CIS) 100 20 0


Although SILs are capable of changing dynamically over time as a combined function of viral type(s), phase of viral life cycle (especially episomal vs integrated) and host factors, classification of SILs must be accomplished from the morphologic appearance of an individual HPV-infected squamous lesion. Further information about virologic and immunologic dynamics of HPV infection from individual and public health perspectives are available in Chapter 9 . Cervical biopsy and cytologic smears each have their own sampling and interpretive errors, occasionally creating discordant results. Both are equally variable and may provide valuable information on disease presence or absence. Even visually directed colposcopic biopsy is imperfect in identifying the area of most severe pathology. Every attempt should be made to reconcile discordances by a combination of interpretive re-review, or further sampling, as clinically appropriate. Evidence-based criteria-driven approaches to diagnosis remain the best way to achieve clinical utility with a minimum of interpretive variation.


In this chapter we will follow a two-class schema of cervical squamous precancers designated as LSIL (condyloma/CIN 1) and HSIL (CIN 2–3), digressing into more specialized distinctions where appropriate.




Squamous Intraepithelial Lesions (SILs)


Nomenclature


The nomenclature used to describe the precursor conditions of invasive squamous cell carcinoma continues to be a subject of some debate. In the 1950s and into the 1960s, the term ‘dysplasia’ was equated to a graded lesion something less than CIS, which was diagnosed and managed separately. Both were composed of basal-type cells extending upward in the epithelium, to almost full thickness in the case of severe dysplasia. Flattening of the topmost surface cells, construed as a sign of differentiation, was said to occur in severe dysplasia, but not CIS. These diagnoses led to widely divergent interventions: the former passively followed, and the latter often to hysterectomy. Critical examination of the data subsequently showed that distinction between severe dysplasia and CIS was not only poorly reproducible by pathologists, but, when applied, failed to stratify patients into differing risk groups for invasive carcinoma. Lack of clinical significance of the dysplasia/CIS distinction led to the simplified classification of CIN in which three grades of precancerous lesions were acknowledged, but the term ‘carcinoma’ was reserved for invasive processes. New treatment options for intraepithelial precursor lesions, intended to ablate only the cervical lining, were introduced. These included topically applied liquid nitrogen (cryosurgery), laser ablation, and local excision by electrocautery loop.


Knowledge of the role of HPV infection (see Chapter 9 ) and more specifically the roles of individual HPV types, suggested division according to the presence of the low-risk HPV types such as HPV 6 and 11 and the high-risk HPV types, most commonly HPV 16 and 18. Since the late 1980s, the Bethesda classification scheme, introduced for use in reporting cervical cytology smears, has incorporated these concepts into a bimodal classification system of LSIL/HSIL, which differ in their risk for subsequent carcinoma. HPV lesions previously considered condylomata or CIN 1 (mild dysplasia) are grouped into the single category of LSIL. These low-grade lesions have a low growth fraction, koilocytotic atypia (also called ‘HPV cytopathic effect’), and are the morphologic manifestation of productive HPV infection. They might perhaps be better called ‘low risk for the development of high-grade intraepithelial neoplasia or invasive carcinoma.’ All HPV types functionally produce LSIL at some point in their life cycle (or they would not survive). In contrast, HSILs are the approximate equivalent of CIN 2 and CIN 3 (moderate dysplasia, severe dysplasia, and CIS). HSIL is sometimes subdivided into HSIL-2 and HSIL-3 to reflect this comparison with CIN 2 and CIN 3. The relationship between these various terms is shown in Figure 10.1 . A strength of the Bethesda System is that it may be applied to both cytologic and histologic specimens. The cells from an HPV-associated lesion are all included under the umbrella of SIL, as there is no underlying biologic or diagnostic basis to maintain a separate category of non-SIL condyloma. As suggested earlier, the concept of progression is best considered probabilistically, incorporating independent assessment of the results of cytology and biopsy and, when appropriate, HPV testing.


The cytopathologist, in contrast to the histopathologist, can only examine cells that have been exfoliated, which tend to be the most superficial cells in the mucosa. Yet, in some ways, the cytologic sample may be more representative of the spectrum of pathology in the cervix because of its ability to sample a much larger surface area compared to the punch biopsy. The fact that smear and biopsy findings from samples obtained during the same patient examination show high degrees of correlation supports the concept that SIL, even when low-grade, affects all layers of the epithelium. The sections that follow, therefore, emphasize the quantitative and qualitative changes that occur throughout the epithelium in the various degrees of SIL.


Features


The histopathologic assessment of a cervical biopsy must determine whether SIL is present in a sample of epithelium and, if so, the grade of SIL ( Figure 10.2 ). Both of these decisions may be difficult to make: the former because benign and reactive changes may be mistaken for SIL; the latter because interpretation of features used for grading is complex ( Table 10.3 ).




Figure 10.2


Abrupt transition between normal squamous epithelium (right) and an adjacent HSIL (left).


Table 10.3

Histopathologic Features of SIL/CIN







  • 1.

    Nuclear abnormalities



    • a.

      Nuclear to cytoplasmic ratio (↑)


    • b.

      Hyperchromasia (↑)


    • c.

      Nuclear pleomorphism and anisokaryosis (↑)


    • d.

      Nuclear polarity (↑ irregular)


    • e.

      Wrinkling of nuclear membrane (↑)



  • 2.

    Mitotic activity



    • a.

      Number of mitotic figures (↑)


    • b.

      Height in epithelium (↑)


    • c.

      Abnormal mitotic figures (↑)



  • 3.

    Differentiation (maturation, stratification)



    • a.

      Proportion of epithelium showing differentiation (↓)


    • b.

      Proportion of unit area occupied by nuclei (↑)



These features become progressively more prominent with increasing grade.


SIL is divided into grades as a prognostic aid, implying it can be used as a guide in managing the patient. As a result, criteria that are purely morphologic are used to predict the clinical behavior of the abnormal epithelium. The correlation between histologic appearance and behavior is, however, imperfect. This may be in part due to diagnostic imprecision, in addition to modifying host factors not reflected in the classification system itself. An additional complication in the cervix is that the current aim of treatment is to eradicate the disease before it becomes invasive. Thus the clinical outcomes observed are rarely unaltered by therapy, especially for HSILs. True invasion is found only in a minority of SIL cases, even in untreated HSILs, not exceeding 30% after many years.


Changes of HPV Infection


In cervical epithelia, all productive HPV infections commonly manifest themselves both cytologically and histologically through a distinctive cytopathic effect. The cell in which this is found has been termed the ‘koilocyte’ ( Figure 10.3 ). This feature, first recognized more than 50 years ago, was given the term ‘koilocytotic atypia’ (some use koilocytic) as these cells histologically resembled those in skin warts. Some 20 years later it was recognized that this same cell type occurred in genital warts and was associated with both HPV infection and preinvasive squamous intraepithelial lesions.




Figure 10.3


Koilocytes (arrows) in a cervical smear.


The koilocyte is an intermediate cell that has a prominent cytoplasmic space around an atypical nucleus ( Table 10.4 ; Figures 10.3–10.8 ). Due to an extensively marginated cytoplasm, the halo has a sharp edge. The cytoplasmic change is thought to be due to abundant expression of the HPV E1∧E4 fusion protein, which binds with cytoplasmic keratin. Even in the state where the nuclei are not overtly dysplastic by size criteria, they are still, nonetheless, irregular and hyperchromatic and, if near the surface, may show a wrinkled nuclear membrane. The nuclei, which lack nucleoli, are usually two to four times larger in nuclear area than those of the adjacent, nonballooned cells. Koilocytotic atypia, which under the Bethesda classification is considered a low-grade SIL, is the most common definite abnormality in cytologically screened women and is found in up to 4% of all cervical smears.



Table 10.4

Koilocytotic Change: Diagnostic Criteria



















Well-defined and exaggerated perinuclear halo
Cytoplasmic condensation around the halo
Nuclear area at least 2–3 times that of a normal intermediate cell nucleus
Increased nuclear to cytoplasmic ratio
Mild nuclear hyperchromasia (usually)
Wrinkled nuclear membrane
Nuclear membrane chromatin condensation
Degenerative nuclear changes (variable)



Figure 10.4


HPV-associated koilocytes in an LSIL. The cells in the intermediate layers are ballooned with copious clear cytoplasm. One cell is binucleate (arrow). The lowermost layer of cells against the basement membrane is orderly.



Figure 10.5


LSIL. The basal layer is slightly thickened. Koilocytes are prominent.



Figure 10.6


LSIL (CIN 1). Several of the koilocytes show nuclear atypia, which persists in the parakeratotic scale. Figures 10.4–10.6 are all low-risk viral lesions.



Figure 10.7


The nuclei of koilocytes are wrinkled and enlarged but show neither mitotic activity nor nucleoli.



Figure 10.8


High-power magnification of koilocytes. The nuclei are wrinkled and enlarged with coarse chromatin.


While the koilocyte is often described to be pathognomonic for HPV infection, perinuclear halos that mimic koilocytotic atypia may be caused by other infectious diseases and at times may be due to artifact. Epstein–Barr virus, when present in the cervix, may be associated with koilocytic change (although this may also be due to coinfection with HPV). Likewise, when the cytoplasmic halo is less than morphologically perfect, i.e., when the borders are smooth or when the nuclei have smooth borders, trichomoniasis may be a consideration. It is therefore important that the presence of a cytoplasmic halo is not overinterpreted as due to HPV infection. Rather, it is the combination of a well-defined halo plus definite nuclear atypia as defined previously that confers specificity to the morphologic findings. Other features associated with HPV infection include binucleation ( Figure 10.4 ) and meganuclei. Both are found in the mid to superficial levels. Some investigators have suggested that the latter feature, or more severely pleomorphic koilocytes, is more associated with high-risk HPV types. However, since over 85% of cervical HPV infections are due to viruses from the high-risk group, this is not a practically useful concept and LSILs of the cervix cannot be reliably genotyped by morphology.


Nuclear Abnormalities


The defining hallmarks of SIL are its nuclear abnormalities. These include nuclei that are enlarged, pleomorphic (irregular in size and shape), and often have a wrinkled nuclear membrane. The chromatin is increased in amount (hyperchromasia) and irregularly clumped, often condensing along the inside of the nuclear membrane. Collectively, this constellation of features is described as ‘nuclear atypia.’ These changes may reflect the polyploid and/or aneuploid DNA content of the cells induced by the action of HPV E6 and E7 on host DNA synthesis and cell cycle checkpoints (see Chapter 9 ) and are important for the diagnosis of SIL. Neoplastic atypia is distinguished from reactive changes by the heterogeneity of the nuclear changes in SIL, contrasting with the relatively homogeneous changes in reactive atypia. It is of note that nucleoli are rare in preinvasive lesions, especially in smears, but are commonly seen in reactive atypia.


Cytologic atypia must always be interpreted within the local context of level in the epithelium, and by comparison with adjacent cells. It is common for the koilocytes of LSILs, which reflect HPV DNA amplification and viral propagation, to demonstrate bizarre nuclear pleomorphism in an irregular distribution. These are generally confined to the superficial layers, have classic perinuclear halos, and may be polynucleated. Clonal HSILs may contain koilocytes, but their more characteristic feature is an increased nuclear to cytoplasmic ratio which creates a basal-type appearance to cells high in the epithelium. This is in addition to the previously mentioned pleomorphism and chromatin changes, which are also present.


If a particular epithelium is extremely thin, it may be difficult to relate changes in cytology to position within the vertical height of the epithelium, thus confounding evaluation of maturation from base to surface. In this situation, the cytology alone may provide some clues as to grade, or the specimen may be regarded as ungradeable (or the grade undetermined/indeterminate).


Mitotic Activity


Mitotic activity is the histologic hallmark of cell proliferation. The conceptual distinction between a normal epithelium and SIL has much to do with the frequency, distribution, and type of mitotic activity. HSIL has a proliferative phenotype. Though LSIL is slightly more proliferative than normal epithelium, it does not display the same viral oncogene-driven proliferation as HSIL: this is related at least in part to the switch to productive HPV infection, which occurs in the suprabasal keratinocytes of low-grade lesions.


In normal epithelium, mitotic figures are rare and are restricted to the parabasal cells. In contrast, SIL shows an increased number of mitoses, and they may be present at any level in the epithelium. The frequency of mitoses in the epithelium increases from normal epithelium to LSIL to HSIL. Moreover, the density of mitotic figures in the superficial third of the epithelium increases with the degree of abnormality. Thus, vertical position in the epithelium at which mitotic figures are found is a useful diagnostic indicator when contemplating the grade of SIL. However, not all SILs show mitoses above the parabasal layer.


Mitotic activity above the basal layer, however, is not always by itself pathognomonic for SIL. A reactive or inflamed but otherwise normal differentiated epithelium may also have increased mitotic activity, but the mitotic figures are concentrated in the basal areas. More problematic are reactive changes in metaplastic squamous areas, such as an inflamed transformation zone, as these may have full thickness mitotic activity and fail to demonstrate the polarization of differentiation. The presence of reactive cytologic features lacking the particular chromatin condensation of SIL may assist in the distinction. Special stains for p16, which is positive in epithelia infected with high-risk HPV, may also be helpful in differentiating between reactive metaplasia and SIL.


Abnormal mitotic configurations, which reflect aneuploidy, are common in HSIL, where they account for between 15% and 30% of total mitoses, but rare in LSIL. HSIL associated with HPV type 16 infection has the highest number of mitoses and the most abnormal forms. The most common of these abnormal configurations is the lag-type mitosis, which is defined as a metaphase with non­attached chromatin in the area of the mitotic figure. The ‘three-group metaphase’ ( Figure 10.9 ), which is where the main mass of the chromatin aligns along the equatorial plate and the nonattached condensed chromatin remains laterally at the two polar sites, has been found in 6% of CIN 1–2, 56% of CIN 3, and 93% of high-grade CIN lesions just adjacent to microinvasive carcinoma. Two-group metaphases (displaced chromatin at only one polar site) may also be seen. Less common than either of these forms is the multipolar mitotic figure, either as a triaster (tripolar) ( Figure 10.10 ) or as a more bizarre multipolar figure ( Figure 10.11 ). Tripolar mitoses may reflect polyploidy, rather than aneuploidy, and thus may be seen in both HSIL and LSIL.




Figure 10.9


Three-group metaphase mitosis (arrow) in HSIL.



Figure 10.10


Tripolar mitosis (arrow) in HSIL.



Figure 10.11


Multipolar mitosis in HSIL.


Differentiation, Maturation, and Stratification


‘Differentiation’ and ‘maturation’ are terms that, while having slightly different meanings, are often used synonymously and interchangeably in the cervix.


The proportion of epithelial cells showing differentiation is a useful indicator of the grade of SIL, although it must not be taken as the only criterion. For example, while SIL develops by a dysplastic process and may show little if any differentiation, it may be difficult to distinguish from an immature, but nondysplastic metaplastic squamous epithelium that also lacks any substantial degree of cytoplasmic differentiation. In this case, the distinguishing feature is the lack of nuclear atypia in the metaplastic process as well as much less evidence of mitotic activity. Nonetheless, distinction between the two conditions can be difficult and the presence of atypical changes in immature metaplastic squamous epithelium may indicate SIL, as shown by the presence of high-risk HPV types, high Ki-67 proliferation indices, p16 expression, and follow-up studies.


As normal cells differentiate and mature and migrate toward the surface, stratification is observed. One means of assessing maturation is to look for a decreasing percentage of nuclear area to overall epithelial area, reflecting a decreasing nuclear to cytoplasmic ratio at increasingly more superficial levels of the epithelium. In smears, which sample superficial cell layers preferentially, this change in ratio is quite dramatic. In normal smears, the nucleus has undergone pyknosis by the time the cell reaches the surface, so that in the smear the nuclear to cytoplasmic ratio is quite low. With increasing grade of SIL, the individual cells have matured progressively less so that the amount of cytoplasm present, as well as its differentiation, is less. Thus, mildly dyskaryotic (a synonym of dysplasia used in cytologic terminology)/LSIL cells present in cervical smears/scrapes have ample cytoplasm with a well-defined polygonal squamous shape, and moderately dyskaryotic/HSIL-2 cells possess less cytoplasm and a less well-defined oval or elliptical squamous shape, whereas in severely dyskaryotic/HSIL-3 cells the rim of cytoplasm that encircles the nucleus is small. These features also occur in histopathologically defined lesions and, in SIL, the proportionally decreasing quantity of cytoplasm contributes far more to the increase in the nuclear to cytoplasmic ratio with increasing lesion grade than does the change in nuclear size. This is consistent with the persistence of nuclear abnormality throughout the epithelial thickness in all grades of SIL.


The presence of surface differentiation, which defined the difference between CIS and severe dysplasia, is poorly reproducible and of no biologic import, as mentioned previously. Thus, with the adoption of the CIN terminology and now with SIL terminology, the former class of squamous CIS is encompassed in CIN 3 and HSIL, respectively.


In smears, the Pap stain accentuates states of maturation. The color of staining, which results in either acidophilic (pink) or basophilic (blue-green) cytoplasm, usually corresponds to normal superficial and intermediate cell types, respectively. Although this helps to distinguish the various grades of SIL, it is not a reliable criterion, being influenced, for example, by stain quality control factors.


Disease States: Types of SIL


Despite the earlier critical comments about reproducibility, application of discrete diagnostic criteria allows for distinction between benign cervix and SIL, and grading of SILs. SIL is primarily divided into two major classes of LSIL (condyloma/CIN 1) and HSIL (CIN 2–3), using similar terminologies across cytologic and histologic specimens.


We have retained, in this section, separate CIN 2 and 3 descriptions under the umbrella of HSIL for several reasons. First is that specification of an HSIL diagnosis by appending its CIN equivalent, although not essential, remains a common practice. Thus a diagnosis of HSIL (CIN 2), or HSIL (CIN 3), may be rendered in an individual HSIL case. In addition, the CIN system is still used in some healthcare systems, for example the UK cervical screening program. Second, direct translation of legacy CIN 2 and 3 into HSIL remains one of the most widely used implementations of the Bethesda System today. In part this is because many pathologists assume that diagnostic criteria have not really changed since the 1960s when CIN was first defined, but rather the entities have simply been rebundled. This is not strictly true, as the LSIL–HSIL boundary has become the predominant threshold for management, and certainly some diagnostic insights have since been gained by focusing more intently at this junction. For example, disorderly versus orderly arrangement of the deepest layer of epithelial cells may be useful in, respectively, recognizing HSIL compared with LSIL. Also, atypical mitotic figures are more common in HSIL than in LSIL. Neither of these were formal components of CIN grading when first proposed. Third, since the all-important LSIL–HSIL threshold is best understood by comparison of those lesions that flank the boundary, this ends up effectively being a contrast between CIN 1 and 2.


The following discussion should be used as a general guide to the central features of the distinct grades of SIL, since examples of the same SIL grade may have varying appearances. For example, one specimen may show a lack of differentiation and stratification throughout ( Figure 10.12 ), whereas others may show more prominent mitoses, some being abnormal ( Figure 10.13 ), or bizarre nuclei located at superficial levels ( Figure 10.14 ). Many of the histologic features used in the grading of SIL may vary independently of each other, so the emphasis put on each of these criteria may vary from one specimen to another. All of this variation diminishes reproducibility of diagnosis.




Figure 10.12


HSIL (CIN 3). Maturation is lacking throughout the entire thickness of the epithelium.



Figure 10.13


HSIL (CIN 3) in which a thin residual layer of mucinous columnar cells is present on the surface.



Figure 10.14


HSIL (CIN 3). The superficial nuclei are markedly enlarged and bizarre in shape.


Like histology, assignment of an overall grade of SIL to a smear is as subjective as the assessment-based examination of the individual cells. The cervical smear will frequently contain cells showing both grades of SIL, and it is the most severe cell type that determines the grade ( Figure 10.15 ). A smear containing only a few dyskaryotic (dysplastic) cells, all of which show a marked degree of abnormality, almost certainly reflects HSIL. Likewise, a smear containing a majority of dyskaryotic (dysplastic) cells of moderate degree with only occasional severely dyskaryotic (dysplastic) cells, while seemingly suggestive of HSIL (CIN 2), will often disclose HSIL (CIN 3) on biopsy or conization.




Figure 10.15


HSIL, severe dyskaryosis, on a cervical smear. The atypical cells have a high nuclear to cytoplasmic ratio.


LSIL (Condyloma/CIN 1)


LSIL is a term that unifies entities previously referred to as condyloma acuminatum, ‘flat condyloma,’ and CIN 1. LSIL is the histopathologic presentation of productive episomal HPV propagation, essentially a field effect caused by viral activation in maturing squamous cells. Although a degree of nuclear maturation occurs, abnormal nuclei persist throughout the full thickness of the epithelium (if this were not so, a diagnosis by cytologic smear would not be possible; see earlier sections) ( Figures 10.16–10.18 ). Mitotic figures, if present, are few in number and generally confined to the basal third of the epithelium. Abnormal mitosis forms are uncommon: these usually indicate aneuploidy and thus are more specific for the diagnosis of HSIL. Characteristic LSIL changes are concentrated in the upper part of the epithelium where productive episomal virus propagation in maturing squamous cells forms characteristic koilocytes. These koilocytotic nuclei can be wildly pleomorphic, and are among the largest nuclei seen in any kind of SIL. Upon integration of viral DNA into the genome of host cells, episomal propagation, and their koilocytotic phenotype, are diminished. Thus koilocytes are less common in HSIL (CIN 2 and 3), although they do still occur in these lesions, particularly where surface epithelial maturation is retained. LSILs, on occasion, may lie adjacent to high-grade lesions, or occasionally even adjacent to carcinomas. Sometimes this reflects progression of a single viral infection from an episomal to integrated phase across one epithelium, whereas in others it may be due to infection by multiple viral types.




Figure 10.16


LSIL (CIN 1). Cytologic atypia extends throughout the epithelium but there is cytoplasmic maturation and the superficial-most epithelial cells display marked koilocytosis.



Figure 10.17


LSIL (CIN 1).



Figure 10.18


LSIL (CIN 1). There is prominent nuclear pleomorphism in koilocytes with extensive cytoplasmic maturation.


In the past, much attention was paid to distinguishing isolated ‘koilocytosis’ from ‘dysplasia.’ These were pressing issues in a premolecular world where histologic–viral correlations were not yet available, and the functional significance of HPV infection was unknown. From a pathologist’s perspective, there was no objective gold standard to establish what was actually a koilocyte, or its diagnostic and clinical relationship to what had previously, and separately, been identified as a precancerous process (dysplasia). One solution was to diagnose HPV effects and dysplasia separately, so it was frequent to see a diagnosis of ‘koilocytotic change without dysplasia/CIN.’ Many of these ‘dysplasia-free’ koilocytes were overdiagnosed binucleate but otherwise normal-appearing nuclei in a glycogen-rich cell. This has been remedied by more specific criteria for histologic diagnosis of HPV-related koilocytes, which include nuclear size variation, wrinkled contours, and coarse chromatin as defined elsewhere. Other ‘dysplasia-free koilocytes’ described diagnoses separately applied to the superficial (koilocyte-containing) and basal (dysplasia) zones of one epithelium. This is no longer strictly necessary, as examination of all features of the epithelium must be considered in rendering a comprehensive diagnosis. Thus, using the diagnostic criteria described later, the distinction of whether an LSIL does or does not have a coexisting dysplasia is no longer clinically or scientifically significant. Rather, distinction of no-SIL from LSIL, and LSIL from HSIL are the key clinical management thresholds.


The basal area of the majority of LSILs lacks any notable architectural changes, more resembling the aligned uniform basal cells of a normal epithelium. When the basal area is affected, and this can be difficult to assess reliably in an inflamed or disoriented specimen, the pleomorphism is minimal, and confined to the deepest aspects. Usually the basal-most cells retain their alignment with the basement membrane in LSILs, a feature that changes with the clonal outgrowth of HSILs. LSILs lack the degree and extent of basal disorganization, which can be seen to creep upward in the epithelium of HSILs.


Several other features are found with low-grade lesions. Nuclei that are binucleate ( Figure 10.4 ), or even sometimes multinucleate, are found in 95% of HPV infections. Occasional cells may show individual cell keratinization (dyskeratosis) ( Figures 10.19 and 10.20 ). In smears, LSIL (mildly dyskaryotic) cells have ample cytoplasm with a well-defined squamous shape ( Figure 10.21 ). Anucleate and nucleate keratinized cells are commonly present in sheets or plaques with poorly defined cell borders.




Figure 10.19


Focal individual cell dyskeratosis in LSIL (condyloma).



Figure 10.20


Focal individual cell dyskeratosis in LSIL (condyloma), detail.



Figure 10.21


LSIL, mild dyskaryosis. Cells show dyskaryotic nuclei with abundant cytoplasm.


On a cellular basis, the koilocyte in histologic specimens is usually distinctive although the number of cells with koilocytotic change ranges from few to many ( Figure 10.22 ). Typically, the nucleus is 3–4 times enlarged in area compared to a normal intermediate cell, uniform in size and shape, and has a halo with smooth outer borders. HPV immunostains have shown that typical koilocytes usually react with antibodies to the HPV L1 group-specific capsid protein ( Figures 10.23 and 10.24 ). Since capsid/virion production is a temporally controlled phenomenon linked to both differentiation and lesional age, which likely explains the variability in koilocyte number between lesions, not all cells containing HPV will necessarily stain. Cells less typical for koilocytes are less likely to stain. Because of the potential social stigma often attached to a smear or biopsy specimen that is diagnosed as harboring HPV, prudence dictates that no specimen should be diagnosed as LSIL unless the overall microscopic picture is distinctive.




Figure 10.22


Koilocytes in LSIL (CIN 1).



Figure 10.23


LSIL with minimal parabasal hyperplasia.



Figure 10.24


LSIL. Immunocytochemical staining shows viral capsid protein in koilocytes.


If left untreated, most LSILs regress but laser vaporization or diathermy also easily eradicates them, although recurrence is common. Condylomata acuminata are a subtype of LSIL, so the patient with a cervical wart must have continued cytologic surveillance. Furthermore, histologic examination of LSIL should, of course, include an assessment of atypia throughout the epithelial thickness, as well as the surrounding epithelium. Sometimes a lesion with prominent superficial koilocytes suggestive of an LSIL contains cells that have sufficient atypia or basal changes to warrant a diagnosis of HSIL.


LSIL (Condyloma Acuminatum)


Condylomata acuminata of the cervix, exophytic papillary lesions caused by HPV ( Figure 10.25 ), are much less common than those with flat architecture. This fact is consistent with the knowledge that the acuminate architecture is somewhat more frequently associated with HPV types 6 and 11 and some other low-risk types, and that these make up only 10–15% of cervical infections. Yet HPV 6/11 account for around 95% of cutaneous genital condylomata. Larger condylomata can be seen with the naked eye, and they may initially be mistaken for carcinoma ( Figure 10.26 ).




Figure 10.25


LSIL (condyloma acuminatum). The condyloma is exophytic but very small in size. It turned white when 3% acetic acid was applied.



Figure 10.26


LSIL (condyloma acuminatum). This exophytic condyloma acuminatum is large and cauliflower-like.


The histology of exophytic, or acuminate, LSIL (condyloma) shows papillomatosis, acanthosis, parakeratosis, and hyperkeratosis. At higher magnification, each asperity, i.e., each papillary frond, has a tiny blood vessel at its core. Koilocytotic atypia is usually a prominent feature, with individual cell keratinization (dyskeratosis) and multinucleation. There is often a chronic inflammatory infiltrate in the underlying cervical stroma ( Figures 10.27–10.30 ).




Figure 10.27


LSIL (condyloma acuminatum) that is sessile and broad.



Figure 10.28


LSIL (condyloma acuminatum). Medium magnification of a sessile and broad condyloma.



Figure 10.29


LSIL (condyloma) with asperities.



Figure 10.30


LSIL (condyloma) with asperities. High magnification of asperity showing both koilocytes and a fibrovascular core containing a central blood vessel.


LSIL (‘Flat Condyloma’/CIN 1)


Flat LSILs that lack the architecture of condyloma acuminatum are recognizable colposcopically, cytologically, and histologically but cannot usually be seen with the naked eye (not to be confused with condylomata lata or flat warts of secondary syphilis, which are external genital lesions) ( Figures 10.4–10.6, 10.16–10.18, 10.22–10.24 ). The colposcopic features are fully described later, but they are not altogether diagnostic. Based on histology, several features in addition to the presence of koilocytes are useful in the detection of HPV infection. On low-power magnification, large areas may be composed of squamous epithelial cells lacking glycogen. While suggestive, this feature is nonspecific. The cytoplasm from the basal-most cells to the surface is eosinophilic. Commonly a sharp boundary demarcates the epithelium that is glycogen rich (normal) and glycogen poor (HPV). In addition, the superficial cells in the glycogen-poor zones commonly show acanthosis, parakeratosis, and sometimes even hyperkeratosis. The last, rarely, may be quite striking ( Figure 10.31 ). The term flat condyloma, originally used to describe lesions containing koilocytes but without a condylomatous architecture or CIN 1, is a contradiction and is no longer used, and is now replaced by LSIL. Whether flat lesions with and without CIN 1 can be separated reproducibly, and whether their separation has any clinical meaning, is debatable but some, particularly those using the CIN system, attempt to make this distinction.




Figure 10.31


LSIL (condyloma) with marked hyperkeratosis. The lesion grossly had the appearance of a cutaneous horn.


High-Grade Squamous Intraepithelial Lesions (CIN 2–3)


HSIL is a neoplastic process composed of a clonal outgrowth of cells with genomically integrated HPV. Its characteristic histologic features include jumbled or irregular arrangements of epithelial cells against the basement membrane, and extension of basaloid atypical cells above the lower third of the epithelial thickness with an accompanying reduction in cytoplasmic maturation. These distinguish HSIL from LSIL, which must be diagnosed separately as they often lead to differing patient management. Koilocytotic change may or may not be present within HSIL, and some HSILs coexist with LSIL. The appearance of abnormal mitotic configurations is common, reflecting aneuploidy induced by high-risk viruses, and when present should be considered a basis for upgrading to HSIL.


The category of HSIL encompasses both CIN 2 and 3 as carried forward from the cervical intraepithelial schema. In creating HSIL, the Bethesda System unified high-risk lesions to promote uniform management of these patients, and today these are minimally diagnosed simply as HSIL, or HSIL with a parenthetic reference to the combined CIN 2 and 3 categories (HSIL, CIN 2–3). Nonetheless, there is persistent clinical interest in substratifying HSIL into risk subgroups. This clinical goal has remained somewhat elusive. Not only is there no consensus on precise criteria for this distinction in a given patient, but the magnitude of risk substratification required to justify triaging into differing management options is unclear. Furthermore, there is confusion and disagreement among pathologists regarding whether a reintroduced category of CIN 2 should be defined by diagnostic imprecision in labeling a lesion as LSIL or HSIL (essentially a SIL that is difficult to grade), or whether it is a biologically intermediate entity between LSIL and HSIL. The latter is perhaps a more proactive stance, as true diagnostic improvements might be realized through revised histologic criteria, perhaps integrated with biomarkers or host parameters, to guide management.


HSIL (CIN 2)


In HSIL (CIN 2), the upper two-thirds of the epithelium shows some differentiation and maturation, with, as in LSIL, nuclear atypia persisting to the surface ( Figures 10.32–10.38 ). Nuclear abnormalities are more marked than in LSIL, and more nuclei with greater degrees of abnormality are found high in the epithelium. Examination of the lower­most epithelial layer, abutting the basement membrane, is an important aspect of segregating LSIL from HSIL (CIN 2), with irregular nuclear placement in HSIL (CIN 2) creating a ‘jumbled’ look. Mitotic figures are present in the basal two-thirds of the epithelium. If attention is focused upon the findings in the upper portion of the epithelium, the changes would be similar qualitatively, but more advanced quantitatively, in comparison to LSIL. More nuclei are pleomorphic in relation to neighboring nuclei, lack polarity to various degrees, have wrinkled nuclear membranes, and show various degrees of hyperchromasia. Overall, the percentage of nuclear area to total epithelial area is roughly 40–60% in the upper half of the epithelium.




Figure 10.32


HSIL (CIN 2). There are several atypical mitoses in a lesion that exhibits suprabasal cytoplasmic maturation.



Figure 10.33


HSIL (CIN 2). Cytoplasmic maturation is less than that seen in LSIL, and is largely confined to the upper third of the epithelium. Basal cells are not oriented against the basement membrane.



Figure 10.34


HSIL (CIN 2) with abnormal mitotic figures in the middle third of the epithelium.



Figure 10.35


HSIL (CIN 2). Many of the abnormal nuclei in the upper epithelium are larger than those seen in the basal epithelium.



Figure 10.36


HSIL (CIN 2) with koilocytosis. Although cut tangentially, the cells show cytoplasmic maturation and koilocytosis toward the surface.



Figure 10.37


HSIL, moderate dyskaryosis. The nuclei are large, occupying more than half the total cell size.



Figure 10.38


HSIL, moderate dyskaryosis. A sheet of cells with atypical nuclei and reduced amounts of cytoplasm showing some differentiation.


The diagnosis of CIN 2 is the least reproducible form of CIN. It is viewed by some as an equivocal diagnostic interpretation, by others as a distinctive intermediate biologic state. Across the spectrum of individual examples encountered, it is probably both. By forcing diagnostic reassignment of HSIL (CIN 2) into LSIL and HSIL (CIN 3) categories, approximately one-third are the former and two thirds the latter.


HSIL (CIN 3)


In HSIL (CIN 3), any maturation, if present, is confined to the superficial third of the epithelium. Generally, it is minimal to completely absent. Nuclear abnormalities are marked throughout the whole thickness of the epithelium. Similarly, mitotic figures are found at all levels of the epithelium and may be numerous. The findings in the upper portion of the epithelium include more extensive nuclear changes and the proportion of the lesional area that consists of nuclear material can exceed 60% in the upper half of the epithelium ( Figure 10.39 ). In smears, the rim of cytoplasm is thin, and the nucleus occupies virtually the entire cell ( Figure 10.15 ). As with HSIL (CIN 2), the basal-most layer of cells is disorderly in HSIL (CIN 3).




Figure 10.39


HSIL (CIN 3). There is full thickness involvement of the epithelium by undifferentiated epithelial cells.


In some cases of HSIL (CIN 3) the entire epithelium is occupied by monomorphic basal-like cells, and these can easily be mistaken for atrophy, squamous metaplasia, or reactive change. Careful examination of chromatin texture, which is coarse in HSIL, the assessment of mitotic figures, and immunostains for p16 and Ki-67 are useful in this differential diagnosis.


Given the previous discussion, SIL (CIN) terminology will be used from this point onward, except where specific studies using either the dysplasia/CIS or CIN terminologies are cited.


Areas of Diagnostic Difficulty


The Scant Endocervical Curettage


In the absence of any generally accepted criteria for what constitutes either an adequate or scant specimen, pathologists frequently encounter the problem of what language to use when reporting a curettage specimen where the diagnostic tissue is less than sufficient. This is also true for endometrial samples where only endocervical tissue is present. Many specimens, even with copious amounts of material, consist largely of mucus, which itself is not of diagnostic value. Often the only diagnostic cellular component present consists of little more than a few small strips of mucinous columnar epithelium devoid of any underlying stroma. To diagnose a specimen as ‘unsatisfactory’ would generally require that the clinician repeat a painful procedure. The recommended approach is to report endocervical curettages (ECCs) descriptively (mucus, mucinous columnar cells, fragments of endocervix, fragments of exocervix) and when necessary with the qualifiers ‘scant, rare, miniscule quantities of, etc.’ A typical example is ‘scant mucus and mucinous columnar epithelium.’ When the specimen consists of just a few exfoliated mucinous cells, some use the phraseology ‘rare exfoliated mucinous cells, inadequate (or insufficient or suboptimal) for further diagnosis,’ which serves as a trigger for the clinician to rethink the issue. A distinction should be made between strips of mucinous columnar cells and rare isolated exfoliated mucinous cells as the former likely reflects the curettage while the latter may represent little more than cells already exfoliated into the mucus. Although the ECC is primarily used to assess the extent of squamous neoplasia, it also helps in evaluating the presence or absence of glandular tumors and their precursors that may involve the endocervix.


Lesions Suspicious for, but Subdiagnostic of, SIL


Conceptually, this is the histologic equivalent of the cytologic diagnosis of atypical squamous cells of undetermined significance (ASCUS/borderline change). Since ASCUS/borderline change is a cytologic equivocation and a risk assessment rather than a biologic entity, there has been no formal translation to a histologic diagnostic entity. Rather, if features are present that raise the possibility of SIL (CIN) but this cannot be diagnosed, the findings should be described. Samples may be subdiagnostic because of adequacy (technical condition or abundance) or interpretive difficulties. Where appropriate, some indication of the basis of diagnostic uncertainty, and recommendation for clinical correlation and follow-up, can be made.


Basal Cell Hyperplasia


Basal cell hyperplasia shows regular replication of basal layers with nuclear enlargement. However, nuclear pleomorphism and hyperchromasia are absent. Differentiation is relatively normal in the upper half of the epithelium. While the significance of basal cell hyperplasia and its long-term implications are unknown, it may reflect the early stages of dysplasia occurring in the ‘original’ (native rather than metaplastic) squamous epithelium.


In some cases, the distinction between basal cell hyperplasia and HSIL (CIN 2-3) can be difficult to make. Cases exist where the basal and parabasal cells are remarkably abnormal, and yet the upper two-thirds of the epithelium is relatively normal ( Figures 10.40 and 10.41 ). Emphasis on the upper layers leads to a diagnosis of basal cell hyperplasia, whereas emphasis on the basal layers leads to a diagnosis of HSIL (CIN 2-3). Ki-67 (MIB1) and p16 immunohistochemistry can be helpful in making the distinction, with Ki-67 highlighting the increased proliferation and p16 the presence of a high-risk HPV infection in HSIL.




Figure 10.40


‘Excessive differentiation.’ The basal and parabasal epithelium show features suggesting HSIL (CIN 2-3), but the upper epithelium is normal. The quandary exists whether such a case should be categorized as HSIL (CIN 2-3) or as basal cell hyperplasia.



Figure 10.41


High magnification of ‘excessive differentiation.’


Immature Squamous Metaplasia and Atypical Squamous Metaplasia


Squamous metaplasia is a physiologic process characterized by reserve cell hyperplasia, early squamous differentiation, variable polarity, and nuclear enlargement, any component of which may sometimes be quite exaggerated (see Chapter 8 ). Nuclear pleomorphism and hyperchromasia are absent, thus rendering to the epithelium an appearance of somewhat bland nuclei of uniform size and shape (the nuclei are typically round). During the time when metaplastic squamous cells develop, they may undermine and replace endocervical columnar cells. At times the nuclei can be enlarged and yet be relatively uniform, which elicits differences in diagnoses among pathologists as to whether the lesion represents immature squamous metaplasia or SIL, especially when columnar cells are present on the surface ( Figure 10.42 ). HPV-associated features such as koilocytosis are rarely seen in immature metaplastic squamous epithelium, presumably as a result of the lack of differentiation.




Figure 10.42


Immature squamous metaplasia versus HSIL (CIN 2), with surface columnar epithelium. Ki-67 and p16 immunostaining may help to make this distinction.


It can be difficult to distinguish immature squamous metaplasia with minimal nuclear changes from SIL (CIN) ( Table 10.5 ). Typically, immature metaplastic squamous cells have abundant cytoplasm and homogeneous round nuclei with fine speckled chromatin and a small nucleolus. The frequency of Ki-67 (MIB1)-reactive nuclei is low and the staining intensity minimal. Cylindrical cells can occur intermingled with, or at the surface of, the immature cells and then are a strong diagnostic criterion, but are not always present. Whenever substantial nuclear pleomorphism is found, SIL (CIN) should be diagnosed even if columnar cells are present on the surface ( Figure 10.43 ). Ki-67 staining is useful in resolving the differential diagnosis, as it is typically unremarkable in immature squamous metaplasia but appears at high density throughout the epithelium in SIL (CIN). Similarly, p16 is typically diffusely expressed in SIL (CIN) but absent or expressed only focally in immature metaplastic squamous epithelium.



Table 10.5

Differential Diagnostic Features of Immature Squamous Metaplasia and SIL (CIN)
































Feature Immature squamous Metaplasia SIL (CIN)
Nuclei Round Variable
Chromatin Homogeneous Coarse, clumped
Nucleoli Single and prominent Usually inconspicuous.
When present, often large and multiple
Cylindrical cells Useful when present but often absent Absent
Ki-67 (MIB1) Scattered positive cells Often diffusely positive
p16 Negative or focally positive Diffusely positive



Figure 10.43


HSIL (CIN 2) with pleomorphic nuclei beneath the surface columnar mucinous epithelium. Mucin stain.


Repair (Reactive Epithelial Changes)


Repair of the squamous epithelium is a condition that commonly mimics the features of SIL (CIN). Unlike SIL (CIN), the stroma in repair is virtually always chronically and often floridly inflamed. The nuclei are uniform, with no or minimal pleomorphism. The chromatin is bland and evenly distributed. Nucleoli of ‘bull’s eye’ or macronucleolar appearance are often easily found ( Figure 10.44 ). The epithelium in SIL (CIN) may be incidentally associated with an intensely inflamed stroma, but can be recognized by nuclei that are pleomorphic and commonly display coarse chromatin and mitoses.




Figure 10.44


Repair. The nuclei lack pleomorphism and contain prominent nucleoli.


Low Estrogen States and Atrophy


In low estrogen states (i.e., after menopause), and in high progesterone states (e.g. during pregnancy or as a result of progestogen therapy), the cervical squamous epithelium is usually thin and may be composed entirely of parabasal cells. While mild nuclear hyperchromasia is often seen, the nuclei are uniform in size and shape and a constant amount of cytoplasm surrounds most nuclei. Nuclear pleomorphism is absent. In some postmenopausal women, the cervix may exhibit a spectrum of epithelial alterations, including prominent perinuclear halos, nuclear hyperchromasia, some variation in nuclear size, and multinucleation. In one study, all cases showing these changes were negative for HPV by polymerase chain reaction (PCR) analysis. Several features help to distinguish postmenopausal atrophy from the HPV-associated changes of SIL (CIN). They include less variation in nuclear size and staining intensity, more finely and evenly distributed nuclear chromatin, and greater uniformity of perinuclear halos in the former. Ki-67 (MIB1) expression can also be useful, as positive nuclei are typically limited to the lower layers. However, this must be interpreted with care as very thin epithelium may lack superficial cells and can mimic HSIL (CIN 2-3). However, correlation with HPV testing is important as many studies demonstrate a ‘bump’ in HPV prevalence in the postmenopausal age group. p16 immunostaining can be helpful as a surrogate marker of high-risk HPV infection in this situation as it is typically diffusely positive in SIL (CIN) but negative in atrophic epithelium. Hence, a combination of Ki-67 and p16 immunostaining may be particularly useful in this context.


Thin Epithelium


In an epithelium that has only a few layers of cells, it is commonly impossible to confidently diagnose the presence of SIL (CIN). There is often the concern that the epithelium is not naturally thin, and that some artifactual process is responsible for the removal of multiple superficial layers. In the absence of severe inflammation, SIL (CIN) should usually be diagnosed if there is cytologic atypia ( Figure 10.45 ). Sometimes, the number of cell layers present is sufficient to diagnose SIL (CIN), or even probable HSIL (CIN 2-3), but further definition is precluded ( Figure 10.46 ). It may be helpful to designate these lesions as SIL (CIN) of indeterminate grade (or ungradeable SIL (CIN)), using the cytologic features to indicate whether the lesion is likely to be low or high grade. Correlation with the Pap smear may be useful in this situation, as may immunostaining for Ki-67 and p16, as discussed previously for atrophic epithelium.




Figure 10.45


Thin epithelium, probably SIL (CIN). The nuclei appear atypical. Immunostaining for Ki-67 and p16 is useful in this situation, as diffuse and widespread expression of these markers, particularly p16, supports a diagnosis of SIL (CIN).



Figure 10.46


Thin epithelium with ungradeable SIL (CIN). The number of cell layers present is sufficient to diagnose SIL (CIN), but further formal definition is precluded. However, the cytologic features including atypical mitoses suggest a high-grade lesion.


Invasive Squamous Cell Carcinoma


Not uncommonly, one of the most difficult entities to distinguish from HSIL (CIN 2-3) is invasive squamous cell carcinoma. This occurs most commonly if the biopsy is superficial and is devoid of an obvious stromal component. In such examples, the lesion appears as sheets of irregularly folded dysplastic epithelium lying on a thin basement membrane. The most appropriate diagnosis is that of ‘at least HSIL (CIN 2-3), cannot exclude invasion. In one study testing for cellular features that distinguish invasive and noninvasive disease, several histologic features found in the cone biopsy specimens were preferentially associated with invasive tumor. These included giant bizarre cells that were irregular, hyperchromatic, and up to five times the size of a basal cell (67% vs 6%), large keratinized cells with distinct cell borders (87% vs 0%), keratin pearls (41% vs 0%), necrosis, often comedo-like (80% vs 8%), and neovascularized tumor cells close to the endothelial lining and lacking intervening connective tissue (57% vs 0%). In 74% of invasive carcinomas, a component of CIN 3 was present, of which 35% showed large keratinized cells or keratin pearls in the in situ components. This suggested that the presence of either feature in biopsy specimens showing CIN 3 might signify the presence of invasive lesions elsewhere in the cervical mucosa. In another study, several histologic features were associated with the presence of invasive disease: these included extensive and expansile involvement of endocervical crypts; and the presence of comedo-type necrosis in the center of involved crypts (see later).


It is not unusual for the diagnosis of HSIL (CIN 2-3) to be correct, but for the results of hysterectomy to show the disease process to be more extensive than expected. Occasionally, the surface and the endocervical crypts are involved only by HSIL (CIN 2-3) in a biopsy, as suggested by a history of only HSIL (CIN 2-3) on smears (even when reviewed retrospectively) and the absence of any abnormality clinically. However, the hysterectomy specimen shows that the cervical wall harbors a small carcinoma or on occasion is permeated by a larger carcinoma (see Chapter 11 ).


Artifact


Fragmentation and thermal artifact in cone, loop electrosurgical excision procedure (LEEP) or loop excision of the transformation zone (LETZ) biopsy specimens are major problems affecting correct diagnosis. When specimens are fragmented into multiple small pieces, it is difficult, if not impossible, to evaluate margins. Thermal artifact, which is often caused by low-voltage techniques, results in an epithelium that appears smudged and uninterpretable. Cellular and nuclear details are lost ( Figure 10.47 ). In a LEEP biopsy correctly done, the thermal artifact produces a very thin rim, usually a fraction of a millimeter wide, at the periphery of the specimen. The outermost layer, the carbonization zone, is usually quite thin. The coagulation zone is deeper and is significantly larger and more readily apparent. Unacceptable thermal artifact occurs when the coagulation zone is wide ( Figure 10.48 ), resulting in extensive loss of cellular detail. A randomized controlled trial that evaluated pure cut versus traditional blending settings in large LETZs found no significant differences in thermal artifact. In the deep stroma, however, the blended setting had a thicker thermal artifact band (0.382 mm) than the pure cut setting (0.325 mm).




Figure 10.47


Thermal artifact with LEEP biopsy. The epithelium has lost all cellular detail.



Figure 10.48


Thermal artifact with LEEP biopsy. The stroma shows extensive coagulative changes.


Stratified Mucin-Producing Intraepithelial Lesion


Some intraepithelial lesions show features intermediate between HSIL (CIN) and cervical adenocarcinoma in situ (AIS)(also termed high-grade cervical glandular intraepithelial neoplasia) (see Chapter 12 ). Where mucin-producing cells are admixed with non-mucin-producing cells in an atypical stratified intraepithelial lesion, the term stratified mucin-producing intraepithelial lesion or SMILE has been proposed. This entity is uncommon and when observed is best diagnosed as a histologic variant of cervical AIS: ‘cervical adenocarcinoma in situ , stratified mucin-producing intraepithelial type.’ In many cases features of ‘pure’ AIS are also present in the histologic slides.


Congenital Transformation Zone (See also Chapter 8 )


Immature squamous epithelium may be present distal (caudal, vaginal) to the last endocervical gland (discussed later) in an anatomic position, peripheral to the transformation zone, which is normally occupied by native, mature squamous epithelium. Recognition of the appearance of this epithelium, which has been termed the ‘congenital transformation zone,’ is therefore important. Surface hyperkeratosis or parakeratosis is often present and this can produce a leukoplakic appearance colposcopically. Histologically, the typical features are a relative lack of epithelial maturation of squamous epithelium that has an irregular, dentate, contour. The lack of maturation raises the possibility of SIL (CIN) but cytologic atypia is absent. Specific recognition of this entity is important for colposcopy–histopathology correlation.


Miscellaneous Conditions


One of the more bizarre situations rarely encountered is when the cervix is treated for SIL (CIN), but the results of hysterectomy show the SIL (CIN) process involves more than the cervix. There are rare reported cases where hysterectomy and bilateral salpingo-oophorectomy performed to treat SIL (CIN) disclosed that the disease had extended to the endometrium and fallopian tubes, and there extensively replaced the normal glandular mucosa.


Biomarkers


p16


The cyclin-dependent kinase 2A inhibitor, p16 INK4 , or simply p16, helps distinguish SIL (CIN) from reactive and atrophic lesions. Since a retinoblastoma protein (pRb)-dependent negative feedback loop regulates p16 expression, continuous inactivation of pRb by high-risk (but not low-risk) HPV E7 results in increased p16 levels. Hence, increased p16 levels may reflect HPV-induced SIL (CIN) with deregulated E7 expression. Marked overexpression of p16 protein, i.e., diffuse and strong immunostaining ( Figure 10.49 ), is present in virtually all cervical squamous cell and adenocarcinomas, and high-grade squamous and glandular preinvasive lesions (HSIL (CIN) and AIS) infected by high-risk HPVs, e.g., types 16, 18, 31, 33, 52, and 58, in contrast to the weak/focal staining in lesions infected with HPV 6/11 or other low-risk HPVs. p16 overexpression is sensitive (84%) and specific (98%) for the detection of high-risk HPV. At low magnification, p16 staining facilitates finding a dysplastic area, especially if the epithelium is heavily infiltrated by leukocytes, as often occurs in SILs (CINs). In addition, overexpressed p16 helps identify individual SIL cells in fluid-based cytologic smears. This biomarker is particularly useful in the distinction between SIL (CIN) and reactive and atrophic epithelia, especially where the epithelium is thin (see earlier).




Figure 10.49


There is diffuse, strong p16 and Ki-67 expression in this HSIL (CIN 3). Note that expression becomes less intense and widespread, respectively, in parallel with superficial maturation at the surface of the lesion. (A) H&E. (B) p16. (C) Ki-67.


p16 positivity should not be used directly to grade SIL (CIN), a distinction that must be based upon the diagnostic appearance on H&E as outlined previously. This is because high-risk viruses infect some LSIL epithelia, and these will be p16 positive and should not be overdiagnosed as HSIL. A negative p16 result, however, can be used to rule out an HSIL where the differential diagnosis includes mimics such as atrophy or reactive change. Detailed discussion of the use of p16 is included in reference 6.


Ki-67


The expression of Ki-67 can also be used to support a diagnosis of SIL (CIN) as its expression signifies continued cellular proliferation and, if present beyond the parabasal cells, may indicate SIL (CIN). Used in conjunction with p16 immunostaining, it can help to distinguish between reactive and atrophic states and SIL (CIN).


ProExC


More recently, immunostaining using ProExC, which detects the combination of minichromosome maintenance (MCM) protein 2 (MCM2) and topoisomerase 2α (TOPO2α), has been suggested as an alternative biomarker for the diagnosis of SIL (CIN). This performs well in some studies and there is some evidence that the combination of ProExC and p16 may be diagnostically useful.


HPV Typing


There is a large amount of literature addressing the role of HPV testing in cervical screening, particularly the triage of women with low-grade cervical cytologic abnormalities, or during follow-up of patients with SIL (CIN), but the role of HPV typing in the assessment of biopsy material is limited. The detection of HPV DNA by in situ hybridization can aid the identification of HPV-associated carcinomas, particularly adenocarcinomas, and in the distinction between cervical and endometrial adenocarcinomas. However, its use in the assessment of SIL (CIN) is limited, particularly in view of the excellent performance of immunohistochemical biomarkers such as p16, which are more easily implemented in a routine laboratory context.


Histologic Features of SIL (CIN) Affecting Management and Prognosis


The specification of a biopsy-proven SIL (CIN) as low or high grade greatly influences management, with the former usually eliciting prospective surveillance and the latter ablative therapy. This was reaffirmed in the 2012 consensus recommendations by the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology. There is some controversy, however, regarding whether HSIL (CIN 2) should be managed differently from HSIL (CIN 3), especially in the younger patient. This is fueled by the observation that HSIL (CIN 2) is a less reproducible diagnosis than HSIL (CIN 3), with the implication that some proportion of HSIL (CIN 2) cases are low clinical risk lesions overdiagnosed by the pathologist.


Involvement of Endocervical Gland Crypts


Consideration of the involvement of endocervical gland crypts in addition to surface epithelium is important for patient management ( Figures 10.50–10.52 ). The number and depth of involved crypts increases with the grade of SIL (CIN). In CIN 3 cone biopsy specimens, nearly 90% had involved crypts. The mean depth of involvement was 1.2 mm, but 5% extended deeper than 3 mm and some reached a depth of over 5.2 mm or even over 6 mm.




Figure 10.50


HSIL (CIN 3) with endocervical crypt involvement. Several crypts are only partially replaced; the endocervical mucosa remains intact. Where the HSIL (CIN 3) has entirely replaced the crypt, the smooth perimeter indicates that the lesion is in situ and has not invaded into the cervical stroma.



Figure 10.51


HSIL (CIN 3) with endocervical crypt involvement. The perimeter is smooth, indicating that the HSIL (CIN 3) is replacing the gland crypt rather than invading into the underlying stroma.



Figure 10.52


HSIL (CIN 3) with endocervical crypt involvement. Note the abrupt transition between the lesion and the adjacent normal endocervical epithelium.


Extreme care must be taken not to mistake crypt involvement for invasive carcinoma. When HSIL (CIN 2-3) involves only a portion of the crypt, thus exposing some of the lining composed of mucinous columnar cells, the correct diagnosis is usually obvious. Difficulties arise when SIL (CIN) replaces the crypts in their entirety, especially in a biopsy specimen. In this instance the clue that the disease process is not invasive carcinoma, but HSIL (CIN 2-3) that involves a crypt, lies with its shape. Crypts that are involved have perimeters that are round to oblong and smooth, reflecting the fact that the normal crypt lining has been replaced. Sometimes the crypt diameter may be expanded. The stroma around the crypt is often inflamed, and may be edematous, complicating assessment of the epithelial–stromal interface. Tumor that is invasive usually has irregular and sharply angulated borders, and frequently exhibits features of increased squamous differentiation (e.g., increased cytoplasmic eosinophilia, reduced nuclear to cytoplasmic ratio, dyskeratosis) by comparison with the overlying or adjacent SIL (CIN).


Resection Margins


Not infrequently, LEEP, LETZ, or cone biopsy specimens disclose HSIL (CIN 2-3) that reaches the resection margins either on the surface or in involved endocervical crypts ( Figure 10.53 ), both of which may confer an increased risk of residual disease. Residual CIN has been detected in 8–85% of women with positive margins on cone biopsy and in 0–55% of women with negative margins. In a study of 782 women treated for CIN with large loop excision, 9% of margins were involved but, within 2 years, the treatment failure rate was 30%. With uninvolved margins, 5% of these patients proved still to have residual disease. Endocervical glandular involvement, age exceeding 40 years, and the presence of satellite lesions were all identified as independent risk factors for the appearance of a subsequent lesion. Among a cohort of 390 patients, 22% had recurrent CIN or developed invasive carcinoma after cold knife conization with positive margins. Persistent or recurrent disease was more common in women with both ectocervical and endocervical margin involvement as opposed to singular ectocervical or endocervical positive margins.




Figure 10.53


HSIL (CIN 3) involving resection margins both on the surface and within endocervical crypts.


In another study, if an ECC was positive for CIN, 65% had CIN 2–3 in a cone biopsy. A retrospective study of 152 women who underwent ECC at the time of conization concluded that only the endocervical margin status predicted residual disease (and not ECC).


In one study where hysterectomy was performed soon after the cone biopsy, 61% of patients who had both involved margins and involved crypts had residual CIN: 29% of women with uninvolved margins and crypts also had residual CIN in the hysterectomy specimen. In a second phase of the same investigation, women treated with cone biopsy alone were followed over a long period. Of women with both involved margins and involved crypts, 23% developed a subsequent recurrence, which compared to only 8% of women in whom both the margins and crypts were initially normal. The average time to the first recurrence was 2.2 years. In predicting recurrence, positive margins and involved crypts were each found to be independent prognostic factors with equal predictive value.


It is of note that margin status has been shown to be less sensitive than cytology or high-risk HPV testing in predicting post-treatment disease. In addition, a recent systematic review concluded that high-risk HPV testing had higher sensitivity than, but similar specificity to, cytology for the detection of post-treatment high-grade disease.


With the knowledge that the status of the endocervical margin can have a substantial effect on immediate therapy and later disease progression, a lively debate has ensued over the years about the wisdom of using frozen sections to examine cone biopsy specimens. The consensus has been that complete examination of specimens by frozen section is less thorough than if the specimen is first fixed in formalin and then carefully blocked. It is the common experience that cone specimens cut into 12 or more pieces not infrequently show only a small focus of high-grade disease, which would easily have been missed by frozen section examination. In the example shown, the entire focus of HSIL (CIN 3) was under 1 mm in total size ( Figure 10.54 ). Frozen-section examination of large specimens is also time-consuming and expensive. However, in those institutions where frozen-section evaluation of the endocervical margins is performed routinely, the overall experience has been highly satisfactory. A number of institution also report excellent correlation between the frozen sections and permanent sections in cases of CIN 3 and superficial invasive carcinoma. Yet the artifacts and tissue loss at frozen section can compromise the evaluation of early invasion. Furthermore, rarely will the astute gynecologist need to alter the approach based on the finding of superficial invasion. Hence, frozen-section evaluation of conization is generally discouraged.




Figure 10.54


A focus of HSIL (CIN 3) under 1 mm in total size.


Invasive Disease


An obvious concern in the management of SIL (CIN) is to ensure that the therapy is not excessive for the degree of abnormality present. Conversely, it is important not to treat lesions inadequately and miss an occult carcinoma as a result. In a meta-analysis examining the use of various diagnostic techniques, both punch biopsy and cone biopsy with or without colposcopy missed significant numbers of invasive carcinomas ( Table 10.6 ).



Table 10.6

Occult Invasive Carcinoma as an Unexpected Finding after Biopsy or Conization
























Procedure Total Patients ( n ) Patients with More Severe Lesion on Subsequent Surgical Specimen (%) Invasive Lesions Missed (%)
Punch biopsy 4334 16.7 6.0
Colposcopically directed biopsy 1930 4.0 0.8
Cone biopsy without invasive carcinoma 1734 7 2.1


In one of the largest case series, 2.7% of 600 patients with CIN 3 had invasive carcinomas detected in a subsequent cone biopsy specimen. Another 1% had microinvasive carcinomas. The major features identified in CIN 3 associated with carcinoma in another study were as follows:




  • Extensive involvement of surface epithelium, often with multiquadrant disease



  • Deep involvement of endocervical crypts by expansile CIN 3



  • Luminal necrosis.

One study has shown that the mean size of CIN 3 lesions exhibiting microinvasion is seven times greater than CIN 3 lesions without invasion and 100-fold greater than CIN 1 lesions. Several studies have shown that care must also be exercised to remove the entire lesion when locally destructive methods are used in the treatment of HSIL (CIN 2-3). In a multicenter retrospective study, the British Society for Colposcopy and Cervical Pathology identified 49 women who subsequently developed invasive carcinoma following therapy with laser vaporization, cold coagulation, diathermy, or cryosurgery. Most of these tumors were ascribed to failure to recognize the early invasive disease at the time the patients were initially assessed. Fortunately, radical reoperation has been performed in such patients with low morbidity and excellent cure rates.


Regression and Progression to Invasive Carcinoma


Regression


Practical data are difficult to find regarding the transit times from SIL (CIN) to invasive carcinoma. In one of the better older studies, the median transit times for progression from mild, moderate, and severe dysplasia to CIS were 5 years (58 months), 3 years (38 months), and 1 year (12 months), respectively. The regression rate for the high-grade abnormality reverting to normal was 6%, a lower number than anticipated, attributed to the fact that biopsies were not taken. It was believed that removing even a small piece of tissue from a field of dysplasia materially altered the disease’s natural history. In contrast, others found higher regression rates: 50% of patients followed by cytology and biopsy experienced regression during the next 10 years. In the same interval, only 1.4% of all patients with dysplasia showed progression to CIS. In part the rates of progression and regression are related to the initial grade of the CIN, as shown by a meta-analysis of papers published since 1950 ( Table 10.7 ).



Table 10.7

Natural History of CIN


































Regress (%) Persist (%) Progress to CIS (%) Progress to Invasion (%)
HPV without CIN 80 15 5 0
CIN 1 57 32 11 1
CIN 2 43 35 22 5
CIN 3 32 <56 >12


In a more recent study based on a historical cohort of women whose Pap smear histories were recorded continuously between 1962 and 1980, and during which time CIN was managed conservatively, both CIN 1 and CIN 2 were more likely to regress (usually within 2 years) than to progress. The risk of progression from CIN 1 to CIN 3 or worse was only 1% per year, but the risk of progression from CIN 2 was 16% within 2 years and 25% within 5 years, in agreement with meta-analyses. Most of the excess risk for carcinomas developing from CIN 2 or 3 occurred within the first 2 years after the initial cervical abnormality was identified.


Two decades ago, it was generally assumed that progression through SIL (CIN) grades occurred incrementally, starting as LSIL (CIN 1) and over many years progressing through HSIL (CIN 2) to HSIL (CIN 3) before becoming invasive. However, this is not based on actual observations. The biopsies necessary to confirm the diagnoses on each occasion would destroy the tissue under study and colposcopic observations are insufficiently reliable to diagnostically render such a claim. Moreover, there are very rare reports of cases in which HSIL (CIN 2)and even LSIL (CIN 1) have apparently developed directly into invasive carcinoma, without reaching the stage of HSIL (CIN 3). It is possible that a small, very aggressive, newly formed clone of abnormal cells appearing in the deeper epithelium acquires sufficient molecular abnormality to drive the daughter cells to acquire invasive capacity so that these lesions immediately invade, bypassing HSIL (CIN 3). On the basis of such observations, some have argued for treating all patients who have SIL (CIN), irrespective of its grade, but this would lead to enormous overtreatment and has not been widely adopted. Also, it has never been fully established whether the lower grade SIL (CIN) lesions that appeared to progress rapidly to invasive disease had been adequately sampled.


Progression


The goal of early treatment is to prevent squamous cell carcinoma by eradicating preinvasive lesions. Cervical screening programs to detect SIL (CIN) by cytology and treat during the preinvasive phase are based on two assumptions: (1) a significant proportion of women with SIL (CIN) would eventually develop invasive carcinoma if not treated and (2) most invasive squamous cell carcinomas are preceded by a demonstrable intraepithelial phase. Despite the fact that cervical screening is the most effective cancer preventive program currently available, total eradication has not been obtained. There are numerous reasons why this hope has not been fully realized, including poor coverage by the screening programs of the population most at risk, poor quality of screening, and the possibility that some invasive carcinomas may not be preceded by a demonstrable preinvasive phase. Even so, a woman who has been screened even once during her lifetime will have a nearly sixfold lower incidence rate of cervical cancer (decreasing from about 34 to 4.2/100,000 women-years).


Several early studies provided insights into the relationship between SIL (CIN) and invasive carcinoma. One highly quoted study showed that 14% of 59 women whose CIS (now HSIL or CIN 3) remained untreated developed invasive carcinoma. It was later admitted that many of the original diagnoses were incorrect and that only 14 of the patients had acceptable CIS. Thus the percentage rose to 57%, i.e., 8 of 14 women developed invasive carcinoma over a period of 10 years. Later still, it was reported that 31 of these women had been followed for at least 12 years and that 22 (71%) of them had developed invasive disease. Other reports from that time were similar. Of 127 women with ‘epithelial atypia,’ 26% ultimately developed invasive carcinoma. In a fuller account of the same patients, it was admitted that only 67 had a recognizable abnormality still present at the end of the first year of follow-up. Of the 67 who remained in the series, two-thirds eventually developed invasive carcinoma. While the more recent amended rates are rarely quoted, they may well provide a more reliable indication of the malignant potential of SIL (CIN) than their earlier and more widely quoted figures. Most investigations have found that the time for progression from CIN 3 to occult invasive cancer takes between 5 and 25 years, with most series reporting times of over 10 years. Estimates of transit time from CIN to microinvasive/subclinical carcinoma are about 10 years with another 4–5 years elapsing until the tumor causes symptoms.


These estimates are in accord with the average ages when women develop SIL (CIN) (25–40 years), microinvasive carcinoma (43 years), stage 1 squamous cell carcinoma (48 years), and stage 4 squamous cell carcinoma (58 years) ( Figure 10.55 ).




Figure 10.55


Ages of occurrence of squamous cell carcinoma of the cervix and its precursor states. Mean ages are presented for each category.


A common issue in evaluating the natural progression of SIL (CIN) is the effect introduced by both biopsy and conization. The former can theoretically affect the diseased tissues that remain in place, while conization certainly can ablate all tissues, removing the entire lesion or even the entire transformation zone. To overcome the difficulty that the method of diagnosis may interfere with the natural history of the disease, 52 women were traced who had positive smears diagnosed at least 2 years previously but had had neither biopsies nor any treatment. Ten of these women (19%) developed invasive carcinoma, including some preclinical invasive carcinomas.


A notable study involved 948 women who had CIS diagnosed by histology, most of whom had cone biopsies. Of this group, 131 continued to have abnormal cytology, indicative of residual disease. No further treatment was given to these patients but they were followed closely. After 10 years 18% had developed invasive carcinoma, and after 20 years the number had risen to 36%. Of those whose cytology was normal after the initial treatment, 1.5% developed invasive carcinoma and 0.8% developed recurrent CIS. One explanation of the latter figures is that the propensity to develop new disease may remain where there is residual or new infection or other promoting factors (relative risk of 3.2). A retrospective study of 33 women revealed that the invasive carcinoma occurred within 5 and 10 years in 67% and 94% of women, respectively, after their CIN was treated.


The figures quoted for the progression from SIL (CIN) to invasive carcinoma thus vary widely, from 0.17% to 71%. Several difficulties exist in characterizing this relationship. First, all reliable methods for diagnosing SIL (CIN) involve the removal of tissue with the associated risk of interfering with the disease’s natural progression. Second and more importantly is an ethical consideration. Once it becomes apparent that there is a substantial risk of a woman developing invasive carcinoma from unattended SIL (CIN) it is unthinkable not to provide treatment. Because currently available methods to assess follow-up are destructive (biopsy), progression to cancer cannot be observed and therefore precise rates of progression from SIL (CIN) to invasive carcinoma are now virtually impossible to determine. Another factor to consider when evaluating follow-up studies, making certain conclusions difficult, is that the invasive or higher grade lesions may be unrelated to the original lesion, but caused by reinfection.


In examining the natural history of SIL (CIN) following conization, caution must be exercised in attributing the reappearance of an abnormality to the development of new disease. In one large study, 672 women were treated with conization for CIS and had resection margins free of tumor. Of this group, 4% had abnormal smears detected within 3 months and 5% within 1 year, suggesting that the original tumor had never been fully resected. Another 2.5% developed abnormal smears during the ensuing 2–6 years. In general, the recurrence rate and time of recurrence correlated strongly with the presence or absence of initial disease at the resection margins ( Table 10.8 ).



Table 10.8

Reappearance of Disease after Conization





























Results of Follow-Up Free Margins Ambiguous Margins Involved Margins
Women treated by conization for CIS 672 40 6
Abnormal <3 months 3.6% 20% 100%
Abnormal 4 months to 1 year 1.3% 12.5%
Abnormal >1.1 year 2.5% 0%


During the past several decades disquieting new trends have developed. Since the 1960s and the era of the ‘sexual revolution,’ the average age of SIL (CIN) development has fallen progressively to between 25 and 30 years of age and more recently even further. There is concern that the age when invasive carcinoma will develop is also dropping.


The second issue involves reports of young women developing invasive carcinoma of the cervix after recent prior negative cytology. Some of these lesions have been called rapidly progressive cancer, a condition discussed elsewhere (see Chapter 9 ). There are several explanations for these findings. Some of the smears were probably false negatives that would have been found to contain malignant cells or at least dyskaryotic/dysplastic cells on review. Others may have been genuine false negatives, in which either the cytologic sample was inadequate, the tumor was located high in the endocervical canal, or the tumor did not exfoliate cells and none were present in the smears. However, it is impossible to escape the conclusion that at least some of these women had true progression from normal histology to invasive carcinoma. This has occurred in a short period between the collection of the smear and the diagnosis of the carcinoma, sometimes in less than a year. It seems reasonable to speculate that, although the mean time interval for the progression through SIL (CIN) to invasive carcinoma may be 10–15 years, a few women will fall at the two extremes of the distribution curve of the length of natural history. Some may have such a long natural history that progression to invasion will never occur in the course of their lifetime; these women would be categorized as nonprogressive. On the other hand, there may be those women at the other extreme, in whom the natural history of the disease runs a very rapid course, measured in months rather than years. These may be the patients described in the previous reports. Indeed, very rarely do women under age 20 develop rapidly lethal invasive cancer. This hypothesis presumes that one disease process is occurring and that the difference in the behavior in different women is the result of extreme variations in the length of the natural history of the same disease. Another suggestion for which there are only scant data is that certain HPV types associated with cervical cancer are intrinsically associated with more rapid progression. This may correlate with the finding that HPV type 18 is rarely detected in cases of HSIL (CIN 2-3), but commonly in cervical cancers that have metastasized.




Nomenclature


The nomenclature used to describe the precursor conditions of invasive squamous cell carcinoma continues to be a subject of some debate. In the 1950s and into the 1960s, the term ‘dysplasia’ was equated to a graded lesion something less than CIS, which was diagnosed and managed separately. Both were composed of basal-type cells extending upward in the epithelium, to almost full thickness in the case of severe dysplasia. Flattening of the topmost surface cells, construed as a sign of differentiation, was said to occur in severe dysplasia, but not CIS. These diagnoses led to widely divergent interventions: the former passively followed, and the latter often to hysterectomy. Critical examination of the data subsequently showed that distinction between severe dysplasia and CIS was not only poorly reproducible by pathologists, but, when applied, failed to stratify patients into differing risk groups for invasive carcinoma. Lack of clinical significance of the dysplasia/CIS distinction led to the simplified classification of CIN in which three grades of precancerous lesions were acknowledged, but the term ‘carcinoma’ was reserved for invasive processes. New treatment options for intraepithelial precursor lesions, intended to ablate only the cervical lining, were introduced. These included topically applied liquid nitrogen (cryosurgery), laser ablation, and local excision by electrocautery loop.


Knowledge of the role of HPV infection (see Chapter 9 ) and more specifically the roles of individual HPV types, suggested division according to the presence of the low-risk HPV types such as HPV 6 and 11 and the high-risk HPV types, most commonly HPV 16 and 18. Since the late 1980s, the Bethesda classification scheme, introduced for use in reporting cervical cytology smears, has incorporated these concepts into a bimodal classification system of LSIL/HSIL, which differ in their risk for subsequent carcinoma. HPV lesions previously considered condylomata or CIN 1 (mild dysplasia) are grouped into the single category of LSIL. These low-grade lesions have a low growth fraction, koilocytotic atypia (also called ‘HPV cytopathic effect’), and are the morphologic manifestation of productive HPV infection. They might perhaps be better called ‘low risk for the development of high-grade intraepithelial neoplasia or invasive carcinoma.’ All HPV types functionally produce LSIL at some point in their life cycle (or they would not survive). In contrast, HSILs are the approximate equivalent of CIN 2 and CIN 3 (moderate dysplasia, severe dysplasia, and CIS). HSIL is sometimes subdivided into HSIL-2 and HSIL-3 to reflect this comparison with CIN 2 and CIN 3. The relationship between these various terms is shown in Figure 10.1 . A strength of the Bethesda System is that it may be applied to both cytologic and histologic specimens. The cells from an HPV-associated lesion are all included under the umbrella of SIL, as there is no underlying biologic or diagnostic basis to maintain a separate category of non-SIL condyloma. As suggested earlier, the concept of progression is best considered probabilistically, incorporating independent assessment of the results of cytology and biopsy and, when appropriate, HPV testing.


The cytopathologist, in contrast to the histopathologist, can only examine cells that have been exfoliated, which tend to be the most superficial cells in the mucosa. Yet, in some ways, the cytologic sample may be more representative of the spectrum of pathology in the cervix because of its ability to sample a much larger surface area compared to the punch biopsy. The fact that smear and biopsy findings from samples obtained during the same patient examination show high degrees of correlation supports the concept that SIL, even when low-grade, affects all layers of the epithelium. The sections that follow, therefore, emphasize the quantitative and qualitative changes that occur throughout the epithelium in the various degrees of SIL.




Features


The histopathologic assessment of a cervical biopsy must determine whether SIL is present in a sample of epithelium and, if so, the grade of SIL ( Figure 10.2 ). Both of these decisions may be difficult to make: the former because benign and reactive changes may be mistaken for SIL; the latter because interpretation of features used for grading is complex ( Table 10.3 ).




Figure 10.2


Abrupt transition between normal squamous epithelium (right) and an adjacent HSIL (left).


Table 10.3

Histopathologic Features of SIL/CIN







  • 1.

    Nuclear abnormalities



    • a.

      Nuclear to cytoplasmic ratio (↑)


    • b.

      Hyperchromasia (↑)


    • c.

      Nuclear pleomorphism and anisokaryosis (↑)


    • d.

      Nuclear polarity (↑ irregular)


    • e.

      Wrinkling of nuclear membrane (↑)



  • 2.

    Mitotic activity



    • a.

      Number of mitotic figures (↑)


    • b.

      Height in epithelium (↑)


    • c.

      Abnormal mitotic figures (↑)



  • 3.

    Differentiation (maturation, stratification)



    • a.

      Proportion of epithelium showing differentiation (↓)


    • b.

      Proportion of unit area occupied by nuclei (↑)



These features become progressively more prominent with increasing grade.


SIL is divided into grades as a prognostic aid, implying it can be used as a guide in managing the patient. As a result, criteria that are purely morphologic are used to predict the clinical behavior of the abnormal epithelium. The correlation between histologic appearance and behavior is, however, imperfect. This may be in part due to diagnostic imprecision, in addition to modifying host factors not reflected in the classification system itself. An additional complication in the cervix is that the current aim of treatment is to eradicate the disease before it becomes invasive. Thus the clinical outcomes observed are rarely unaltered by therapy, especially for HSILs. True invasion is found only in a minority of SIL cases, even in untreated HSILs, not exceeding 30% after many years.


Changes of HPV Infection


In cervical epithelia, all productive HPV infections commonly manifest themselves both cytologically and histologically through a distinctive cytopathic effect. The cell in which this is found has been termed the ‘koilocyte’ ( Figure 10.3 ). This feature, first recognized more than 50 years ago, was given the term ‘koilocytotic atypia’ (some use koilocytic) as these cells histologically resembled those in skin warts. Some 20 years later it was recognized that this same cell type occurred in genital warts and was associated with both HPV infection and preinvasive squamous intraepithelial lesions.




Figure 10.3


Koilocytes (arrows) in a cervical smear.


The koilocyte is an intermediate cell that has a prominent cytoplasmic space around an atypical nucleus ( Table 10.4 ; Figures 10.3–10.8 ). Due to an extensively marginated cytoplasm, the halo has a sharp edge. The cytoplasmic change is thought to be due to abundant expression of the HPV E1∧E4 fusion protein, which binds with cytoplasmic keratin. Even in the state where the nuclei are not overtly dysplastic by size criteria, they are still, nonetheless, irregular and hyperchromatic and, if near the surface, may show a wrinkled nuclear membrane. The nuclei, which lack nucleoli, are usually two to four times larger in nuclear area than those of the adjacent, nonballooned cells. Koilocytotic atypia, which under the Bethesda classification is considered a low-grade SIL, is the most common definite abnormality in cytologically screened women and is found in up to 4% of all cervical smears.



Table 10.4

Koilocytotic Change: Diagnostic Criteria



















Well-defined and exaggerated perinuclear halo
Cytoplasmic condensation around the halo
Nuclear area at least 2–3 times that of a normal intermediate cell nucleus
Increased nuclear to cytoplasmic ratio
Mild nuclear hyperchromasia (usually)
Wrinkled nuclear membrane
Nuclear membrane chromatin condensation
Degenerative nuclear changes (variable)



Figure 10.4


HPV-associated koilocytes in an LSIL. The cells in the intermediate layers are ballooned with copious clear cytoplasm. One cell is binucleate (arrow). The lowermost layer of cells against the basement membrane is orderly.



Figure 10.5


LSIL. The basal layer is slightly thickened. Koilocytes are prominent.



Figure 10.6


LSIL (CIN 1). Several of the koilocytes show nuclear atypia, which persists in the parakeratotic scale. Figures 10.4–10.6 are all low-risk viral lesions.



Figure 10.7


The nuclei of koilocytes are wrinkled and enlarged but show neither mitotic activity nor nucleoli.



Figure 10.8


High-power magnification of koilocytes. The nuclei are wrinkled and enlarged with coarse chromatin.


While the koilocyte is often described to be pathognomonic for HPV infection, perinuclear halos that mimic koilocytotic atypia may be caused by other infectious diseases and at times may be due to artifact. Epstein–Barr virus, when present in the cervix, may be associated with koilocytic change (although this may also be due to coinfection with HPV). Likewise, when the cytoplasmic halo is less than morphologically perfect, i.e., when the borders are smooth or when the nuclei have smooth borders, trichomoniasis may be a consideration. It is therefore important that the presence of a cytoplasmic halo is not overinterpreted as due to HPV infection. Rather, it is the combination of a well-defined halo plus definite nuclear atypia as defined previously that confers specificity to the morphologic findings. Other features associated with HPV infection include binucleation ( Figure 10.4 ) and meganuclei. Both are found in the mid to superficial levels. Some investigators have suggested that the latter feature, or more severely pleomorphic koilocytes, is more associated with high-risk HPV types. However, since over 85% of cervical HPV infections are due to viruses from the high-risk group, this is not a practically useful concept and LSILs of the cervix cannot be reliably genotyped by morphology.


Nuclear Abnormalities


The defining hallmarks of SIL are its nuclear abnormalities. These include nuclei that are enlarged, pleomorphic (irregular in size and shape), and often have a wrinkled nuclear membrane. The chromatin is increased in amount (hyperchromasia) and irregularly clumped, often condensing along the inside of the nuclear membrane. Collectively, this constellation of features is described as ‘nuclear atypia.’ These changes may reflect the polyploid and/or aneuploid DNA content of the cells induced by the action of HPV E6 and E7 on host DNA synthesis and cell cycle checkpoints (see Chapter 9 ) and are important for the diagnosis of SIL. Neoplastic atypia is distinguished from reactive changes by the heterogeneity of the nuclear changes in SIL, contrasting with the relatively homogeneous changes in reactive atypia. It is of note that nucleoli are rare in preinvasive lesions, especially in smears, but are commonly seen in reactive atypia.


Cytologic atypia must always be interpreted within the local context of level in the epithelium, and by comparison with adjacent cells. It is common for the koilocytes of LSILs, which reflect HPV DNA amplification and viral propagation, to demonstrate bizarre nuclear pleomorphism in an irregular distribution. These are generally confined to the superficial layers, have classic perinuclear halos, and may be polynucleated. Clonal HSILs may contain koilocytes, but their more characteristic feature is an increased nuclear to cytoplasmic ratio which creates a basal-type appearance to cells high in the epithelium. This is in addition to the previously mentioned pleomorphism and chromatin changes, which are also present.


If a particular epithelium is extremely thin, it may be difficult to relate changes in cytology to position within the vertical height of the epithelium, thus confounding evaluation of maturation from base to surface. In this situation, the cytology alone may provide some clues as to grade, or the specimen may be regarded as ungradeable (or the grade undetermined/indeterminate).


Mitotic Activity


Mitotic activity is the histologic hallmark of cell proliferation. The conceptual distinction between a normal epithelium and SIL has much to do with the frequency, distribution, and type of mitotic activity. HSIL has a proliferative phenotype. Though LSIL is slightly more proliferative than normal epithelium, it does not display the same viral oncogene-driven proliferation as HSIL: this is related at least in part to the switch to productive HPV infection, which occurs in the suprabasal keratinocytes of low-grade lesions.


In normal epithelium, mitotic figures are rare and are restricted to the parabasal cells. In contrast, SIL shows an increased number of mitoses, and they may be present at any level in the epithelium. The frequency of mitoses in the epithelium increases from normal epithelium to LSIL to HSIL. Moreover, the density of mitotic figures in the superficial third of the epithelium increases with the degree of abnormality. Thus, vertical position in the epithelium at which mitotic figures are found is a useful diagnostic indicator when contemplating the grade of SIL. However, not all SILs show mitoses above the parabasal layer.


Mitotic activity above the basal layer, however, is not always by itself pathognomonic for SIL. A reactive or inflamed but otherwise normal differentiated epithelium may also have increased mitotic activity, but the mitotic figures are concentrated in the basal areas. More problematic are reactive changes in metaplastic squamous areas, such as an inflamed transformation zone, as these may have full thickness mitotic activity and fail to demonstrate the polarization of differentiation. The presence of reactive cytologic features lacking the particular chromatin condensation of SIL may assist in the distinction. Special stains for p16, which is positive in epithelia infected with high-risk HPV, may also be helpful in differentiating between reactive metaplasia and SIL.


Abnormal mitotic configurations, which reflect aneuploidy, are common in HSIL, where they account for between 15% and 30% of total mitoses, but rare in LSIL. HSIL associated with HPV type 16 infection has the highest number of mitoses and the most abnormal forms. The most common of these abnormal configurations is the lag-type mitosis, which is defined as a metaphase with non­attached chromatin in the area of the mitotic figure. The ‘three-group metaphase’ ( Figure 10.9 ), which is where the main mass of the chromatin aligns along the equatorial plate and the nonattached condensed chromatin remains laterally at the two polar sites, has been found in 6% of CIN 1–2, 56% of CIN 3, and 93% of high-grade CIN lesions just adjacent to microinvasive carcinoma. Two-group metaphases (displaced chromatin at only one polar site) may also be seen. Less common than either of these forms is the multipolar mitotic figure, either as a triaster (tripolar) ( Figure 10.10 ) or as a more bizarre multipolar figure ( Figure 10.11 ). Tripolar mitoses may reflect polyploidy, rather than aneuploidy, and thus may be seen in both HSIL and LSIL.




Figure 10.9


Three-group metaphase mitosis (arrow) in HSIL.



Figure 10.10


Tripolar mitosis (arrow) in HSIL.



Figure 10.11


Multipolar mitosis in HSIL.


Differentiation, Maturation, and Stratification


‘Differentiation’ and ‘maturation’ are terms that, while having slightly different meanings, are often used synonymously and interchangeably in the cervix.


The proportion of epithelial cells showing differentiation is a useful indicator of the grade of SIL, although it must not be taken as the only criterion. For example, while SIL develops by a dysplastic process and may show little if any differentiation, it may be difficult to distinguish from an immature, but nondysplastic metaplastic squamous epithelium that also lacks any substantial degree of cytoplasmic differentiation. In this case, the distinguishing feature is the lack of nuclear atypia in the metaplastic process as well as much less evidence of mitotic activity. Nonetheless, distinction between the two conditions can be difficult and the presence of atypical changes in immature metaplastic squamous epithelium may indicate SIL, as shown by the presence of high-risk HPV types, high Ki-67 proliferation indices, p16 expression, and follow-up studies.


As normal cells differentiate and mature and migrate toward the surface, stratification is observed. One means of assessing maturation is to look for a decreasing percentage of nuclear area to overall epithelial area, reflecting a decreasing nuclear to cytoplasmic ratio at increasingly more superficial levels of the epithelium. In smears, which sample superficial cell layers preferentially, this change in ratio is quite dramatic. In normal smears, the nucleus has undergone pyknosis by the time the cell reaches the surface, so that in the smear the nuclear to cytoplasmic ratio is quite low. With increasing grade of SIL, the individual cells have matured progressively less so that the amount of cytoplasm present, as well as its differentiation, is less. Thus, mildly dyskaryotic (a synonym of dysplasia used in cytologic terminology)/LSIL cells present in cervical smears/scrapes have ample cytoplasm with a well-defined polygonal squamous shape, and moderately dyskaryotic/HSIL-2 cells possess less cytoplasm and a less well-defined oval or elliptical squamous shape, whereas in severely dyskaryotic/HSIL-3 cells the rim of cytoplasm that encircles the nucleus is small. These features also occur in histopathologically defined lesions and, in SIL, the proportionally decreasing quantity of cytoplasm contributes far more to the increase in the nuclear to cytoplasmic ratio with increasing lesion grade than does the change in nuclear size. This is consistent with the persistence of nuclear abnormality throughout the epithelial thickness in all grades of SIL.


The presence of surface differentiation, which defined the difference between CIS and severe dysplasia, is poorly reproducible and of no biologic import, as mentioned previously. Thus, with the adoption of the CIN terminology and now with SIL terminology, the former class of squamous CIS is encompassed in CIN 3 and HSIL, respectively.


In smears, the Pap stain accentuates states of maturation. The color of staining, which results in either acidophilic (pink) or basophilic (blue-green) cytoplasm, usually corresponds to normal superficial and intermediate cell types, respectively. Although this helps to distinguish the various grades of SIL, it is not a reliable criterion, being influenced, for example, by stain quality control factors.




Changes of HPV Infection


In cervical epithelia, all productive HPV infections commonly manifest themselves both cytologically and histologically through a distinctive cytopathic effect. The cell in which this is found has been termed the ‘koilocyte’ ( Figure 10.3 ). This feature, first recognized more than 50 years ago, was given the term ‘koilocytotic atypia’ (some use koilocytic) as these cells histologically resembled those in skin warts. Some 20 years later it was recognized that this same cell type occurred in genital warts and was associated with both HPV infection and preinvasive squamous intraepithelial lesions.




Figure 10.3


Koilocytes (arrows) in a cervical smear.


The koilocyte is an intermediate cell that has a prominent cytoplasmic space around an atypical nucleus ( Table 10.4 ; Figures 10.3–10.8 ). Due to an extensively marginated cytoplasm, the halo has a sharp edge. The cytoplasmic change is thought to be due to abundant expression of the HPV E1∧E4 fusion protein, which binds with cytoplasmic keratin. Even in the state where the nuclei are not overtly dysplastic by size criteria, they are still, nonetheless, irregular and hyperchromatic and, if near the surface, may show a wrinkled nuclear membrane. The nuclei, which lack nucleoli, are usually two to four times larger in nuclear area than those of the adjacent, nonballooned cells. Koilocytotic atypia, which under the Bethesda classification is considered a low-grade SIL, is the most common definite abnormality in cytologically screened women and is found in up to 4% of all cervical smears.


Oct 5, 2019 | Posted by in GYNECOLOGY | Comments Off on Cervical Squamous Intraepithelial Lesions
Premium Wordpress Themes by UFO Themes