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.

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.

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 change¹⁹ (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.20,21 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.^22,23 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.

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.24,25

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.

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.

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.²⁶ 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.

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.

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.