Cervical Pre-cancers: Biopsy and Immunohistochemistry

Abstract

The existence of precursor lesions for invasive cervical cancer has been recognized for more than 50 years. Our understanding of the pathobiology and behavior of cervical cancer precursors has evolved considerably over the past five decades. Furthermore, the terminology used to classify pre-invasive lesions of the cervix has frequently changed. The realization that human papillomavirus (HPV) infections constitute a morphologic continuum has prompted efforts to include them within a single classification system, specifically the squamous intraepithelial lesions (SILs) which have now been embraced by the surgical pathologists. The reduced number of specific pathological categories has made clinical decision-making more straightforward. The generic criteria for SIL have two important histological parameters: Alterations in the density of superficial epithelial cells and superficial squamous atypia. The flat condyloma or cervical intraepithelial neoplasia (CIN) I is generally associated with intermediate and high-risk HPV types as against the low-risk viruses that cause exophytic/papillary growth patterns of condylomas. The diagnosis of low-grade SIL (LSIL) (flat and exophytic condylomas) requires first excluding benign mimics of LSIL and second to confirm the characteristic cytologic atypia. For high-grade SILs (HSILs), the extent and degree of atypia generally exceed the limits of that described in flat or exophytic condylomas (LSILs). Less maturation, abnormal cell differentiation, loss of cell polarity, and increased mitotic index with abnormal mitotic figures occupying increasing thickness of the epithelium define a lesion as CIN II or CIN III. Atypical immature metaplasia associated with inflammation and atrophy is a challenge in cervical biopsy interpretation. Careful attention to the growth pattern of the epithelium, the distribution of the atypia, nuclear spacing, and the degree of anisokaryosis and the presence of enlarged hyperchromatic nuclei help in differentiating a non-neoplastic from a neoplastic process. This chapter describes in depth the diagnostic difficulties in the interpretation of cervical biopsies. It also provides useful criteria in distinguishing benign mimics from true precancerous lesions and the role of biomarkers such as the p16ink4 and Ki-67 in the differential diagnosis of precursor lesions and the reactive and metaplastic epithelium.

Keywords: Atypical Immature metaplasia; Mitosis in cervical biopsy; SILs-diagnostic difficulties in cervical biopsy; biomarkers in cervical precancers; pseudokoilocytes in cervical biopsies.

Figure 1:

Cervical biopsy – normal basket weave appearance with equidistant regular proliferating capillaries at the SCJ. Glands are seen beneath the squamous epithelium.

Figure 2:

Exophytic condyloma (×5) showing papillomatosis and acanthosis in a warty lesion.

Figure 3:

(a and b) Condyloma showing hyperplasia of basal layers in (a) and absence of distinct basal cell layer but focally present as seen in the lower left corner (b). Bizarre nuclei are seen within the proliferating epithelium along with orderly maturation.

Figure 4:

Cervical biopsy showing a normal basal layer with maturation and koilocytotic atypia.

Figure 5:

Cervical tissue is showing indistinct basal layer with hyperplasia of parabasal cells with minimal nuclear atypia. Koilocytosis and loss of polarity of intermediate and superficial cells are present along with significant nuclear atypia in the upper third of the epithelium.

Figure 6:

Multinucleation and single-cell dyskeratosis are seen in HPV infection.

Figure 7:

Clones of koilocytes are seen concentrated randomly throughout the epithelium. Perinuclear halos and nuclear atypia are characteristic features of koilocytes.

Figure 8:

(a and b) Spiky (a) condyloma. The surface of the spikes shows hyperkeratosis, parakeratosis, and koilocytes beneath (×10). (b) Flat condyloma lacks the papillary architecture of an exophytic lesion (×10). Both these generally present as subclinical condylomas and are detected only when they turn acetowhite after application of 5% acetic acid or during colposcopy.

Figure 9:

(a-c) For the diagnosis of pure CIN1, it is necessary to identify alterations in the lower third of the epithelium with mitosis (a and b blue circles) ×40, loss of picket fence arrangement of the basal cells, and focal loss of polarity of atypical hyperchromatic cells, acanthosis, and mitosis ×5.

Figure 10:

CIN1 showing high mitotic index. The mitotic figures are seen in the blue circles.

Figure 11:

Cervical biopsy showing features of both CIN1 and HPV in the form of koilocytotic atypia, bizarre nuclei, and multinucleation. This patient was HPV DNA positive. Colposcopic impression was HSIL.

Figure 12:

(a and b) Cervical biopsies showing hyperkeratosis (a), atypical or pleomorphic parakeratosis, individual cell keratinization, and dyskeratosis (b).

Figure 13:

Section from tissue obtained by LEEP is showing less maturation, higher nuclear density, and a less orderly transition from immature to mature epithelial layers ×40.

Figure 14:

Section from cervical biopsy from a HPV DNA-positive woman shows histological features of LSIL (CIN2) with surface showing koilocytotic atypia. The distinction of LSIL and HSIL is based primarily on the presence of atypia in the latter that does not fit within the spectrum of a flat or exophytic condyloma. Assessment of lower epithelial layers for hypercellularity, nuclear pleomorphism, and abnormal mitosis can help in distinguishing between these two entities with some consistency. Such lesions are referred to as mature or koilocytotic HSIL (CIN2).

Figure 15:

(a-c) Section from tissue obtained by LEEP is showing full-thickness nuclear atypia in basaloid cells, loss of cell polarity, and a high mitotic index ×40. Thorough sampling of sufficiently excised tissue (b) is required to rule in/out invasive malignancy. (c) HSIL with a high mitotic index. Swirling of nuclei, known as the wind swept appearance, a feature of SILs is seen.

Figure 16:

HSIL with an immature metaplastic phenotype. Observe the rounded stromal-epithelial interface. Characteristic is increased nuclear density in the upper layers, the appearance of syncytium of nuclei in the superficial epithelium, nuclear hyperchromasia, and increased mitosis.

Figure 17:

(a and b) HSIL involving glandular crypts. Colposcopic diagnosis was HSIL (a) ×5 and (b) ×40.

Figure 18:

hrHPV DNA-positive patient with colposcopic findings that favored LSIL. Section showed mild atypia in the basal layer with mitosis figures in the blue circle.

Figure 19:

Section is showing a small focus favoring LSIL adjacent to normal epithelium. The abnormalities in a true neoplastic lesion are generally strikingly focal. The presence of a single mitosis in the light of positive hrHPV DNA and colposcopic findings favoring LSIL helped in signing it out as LSIL.

Figure 20:

Increasing CIN grade is associated with increasing mitotic index (MI). A lesion difficult to label as CIN1 as there are atypia and mitosis seen in upper two-thirds of the epithelium also. This patient was hrHPV DNA positive with colposcopic impression as HSIL.

Figure 21:

(a-c) Biopsies from both these patients (a and b) who were hrHPV DNA positive had colposcopic findings that favored LSIL. However, there is the absence of variance in size of cells and nuclei as compared to figure (c) which is an out and out LSIL. Multiple biopsies during colposcopy improve significantly the detection rate of SILs.

Figure 22:

hrHPV DNA positive. Colposcopic findings favored LSIL. Section shows frequent binucleation of large hyperchromatic nuclei in the mature squamous cells.

Figure 23:

(a and b) If slight nuclear enlargement affects a large proportion of squamous epithelium in a biopsy, it is unlikely to be neoplastic. Observe the narrowness and regular appearance of the perinuclear clear zones along with fairly well-preserved horizontal stratification of nuclei.

Figure 24:

(a) Cytoplasmic vacuolation alone should not be diagnosed as koilocytosis. A common source of pseudo-koilocytes is the intermediate squamous cells, filled with glycogen and/or reactive atypia, and is not accompanied by nuclear atypia. Under higher magnification, the nuclei are vesicular with nuclear grooves and are not hyperchromatic. (b and c) Mild degree of loss of polarity in the basal layers with mild-to-moderate atypia in upper three layers of the epithelium along with perinuclear clearing. One may be tempted to call this as a LSIL encompassing HPV infection despite the presence of inflammation in the stroma and also encroaching the squamous epithelium.

Figure 25:

Vesicular nuclei with prominent nucleoli are most unusual of dysplastic epithelium. Even if the nuclear atypia is not marked, the presence of many mitotic figures favors a neoplastic process. Nuclear appearance hyperchromasia or vesicular – although important – is not statistically significant in differentiating neoplastic from non-neoplastic lesions.

Figure 26:

Vertical orientation of nuclei favors a neoplastic process. hrHPV DNA was positive in this patient.

Figure 27:

Atrophic epithelium may show thinning or very few layers and also focal hyperplasia making it difficult to distinguish between a LSIL and HSIL.

Figure 28:

(a-c) Quite often normal slides are miscategorized as LSIL and vice versa as a result of focal loss of a regular basal layer. Basal cell hyper hyperplasia may be associated with the use of oral contraceptives or use of IUCD, during pregnancy, inflammation and repair and infections.

Figure 29:

(a and b) Possibility of LSIL in immature or atypical metaplasia cannot be ruled out when the basal layers start showing all features of CIN I and there is marked atypia with mitosis (a). This metaplastic epithelium in (b) is showing koilocytosis with nuclear atypia in addition to immature metaplasia.

Figure 30:

(a and b) Atypical immature metaplasia showing mild crowding of nuclei, mitosis, and rounded or undulating epithelial stromal interface. Desmosomes are not visible in the lower third of the epithelium. Maturation is seen in the upper layers. The area circled shows features of HSIL. (b) section shows variably hyperchromatic and vertically orientated nuclei with mitotic activity in upper layers clearly favor a neoplastic and a HSIL lesion. The very thin epithelium may reflect loss of upper epithelial layers because of fragility of these lesions. These cases are reminiscent of the “clinging” form of carcinoma in situ of the bladder.

Figure 31:

Biopsy from a benign atrophic epithelium showing presence of hyperchromatic but generally uniform nuclei. The epithelium is thin with maintained polarity. The nuclear appearance is predominantly smudgy, hyperchromatic without recognizable nucleoli, and internal structure is no longer discernible. Colposcopic impression was HSIL.

Figure 32:

(a and b) A negative p16 immunostain is of significance to differentiate between metaplasia and HSIL small cell type found high up in the canal.

Figure 33:

(a and b) Stripping away of fragile cells from the surface gives a false impression of HSIL. Ki 67 is negative because overall proliferative activity is very low in atrophy.

Figure 34:

(a-d) Metaplastic/reactive changes show the presence of Mib1 positive nuclei in intermediate layers along with focal positivity in AIM (a and b) as against continuous and quantitatively more positivity is seen in HSIL (c and d).

Figure 35:

(a-c) p16^INK4a is a surrogate marker for HPV infection. In an uncertain or borderline condylomatous lesion, assessment of p16^INK4a overexpression might be more specific for discriminating neoplastic groups of ASLs than for the detection of high-risk HPV types. p16 expression is linearly and significantly associated with the degree of dysplasia.

Figure 36:

(a and b) Ki-67 positivity may be discordant with morphology. (a) The spacing of the abnormal nuclei is more and there are no nuclear overlap creating doubts about LSIL or HSIL whereas Ki-67 is positive.