Columnar Cell Neoplasia of the Cervix

Adenocarcinoma in Situ Mistaken for Menstrual or Directly Sampled Endometrial Cells

In AIS, as with menstrual endometrium, the cells are extremely crowded but, unlike the latter, the cells in AIS have well-preserved nuclei and at least subtle feathering or small rosettes. The absence of endometrial stromal fragments, cells in honeycombed sheets, or three-dimensional tubular groups rules against direct sampling of the endometrium (discussed later; Figs. 14.11 and 14.12 ).

Adenocarcinoma in Situ or Adenocarcinoma Mistaken for Reactive Endocervical Cells

In AIS or adenocarcinoma, the abnormal cell population is relatively monomorphous, not a spectrum, as is commonly seen with reactive endocervical cells. Neoplastic groups have overlapping or crowded nuclei that have a range of chromatin coarseness and either small or prominent nucleoli ( Figs. 14.13 to 14.16 ). In some cases, this distinction is admittedly difficult. Smears with only slightly atypical endocervical-appearing cells (see Fig. 14.10 ) or bloody smears with only a few atypical cells are two patterns of adenocarcinoma that are often underdiagnosed.

Tubal and Tuboendometrial Metaplasia

In the most common benign mimic of AIS, tubal metaplasia, the cells, although enlarged and crowded, are usually less so than in AIS. Nuclear chromatin is finer than in AIS, and feathering and rosettes are absent ( Figs. 14.17 and 14.18 ). Cytoplasmic cilia are a key finding, confirming tubal metaplasia, but often are difficult to visualize. One needs to see discrete hairlike cytoplasmic processes because adherent background debris can mimic cilia.

Lower Segment Endometrium, Directly Sampled

Cells directly scraped from the lower segment of the endometrium are not uncommon. This is especially true in women who have had a cone biopsy. They appear in fragments that may resemble those of AIS and may include cells with mitotic figures. However, in lower segment endometrium, the cells are arranged in flat sheets or three-dimensional tubules that lack feathering or rosettes ( Fig. 14.19 ). Cells are small, uniform, and evenly dispersed, with fine or dense (India ink) nuclear chromatin ( Fig. 14.20 ). Groups of endometrial stromal cells (tangles of spindled cells with bland oval nuclei and wispy cytoplasm), attached to or independent of the epithelial groups, are an important clue ( Fig. 14.21 ).

Menstrual Endometrial Cells

Perhaps surprisingly, clusters of menstrual endometrial cells may sometimes be difficult to separate from AIS or a high-grade squamous intraepithelial lesion ( Fig. 14.22 ). Also, sometimes, large pseudopapillary endometrial fragments are encountered that are frightening in their similarity to adenocarcinoma. In these situations, as in all difficult cytologic interpretations, evaluation of the worrisome cells in the context of the entire spectrum of cells on the smear is critical. Most groups of menstrual endometrial cells have small molded nuclei with scant or absent cytoplasm and interspersed pyknotic nuclear fragments or are in tight balls of stromal cells surrounded by larger epithelial cells. Hyperchromasia is present but is degenerative, with dense, smudged, or beaded chromatin. Feathering, rosettes, or strips of columnar cells, characteristic of AIS, are absent.

Reactive Endocervical Cells

Reactive endocervical cells, whether from cervicitis, endocervical polyps, microglandular hyperplasia, irradiated endocervix, or the Arias-Stella reaction, may resemble AIS or adenocarcinoma. As opposed to neoplasia, in cervicitis or polyps there are sheets of repair-like cells containing evenly distributed nuclei with fine chromatin, sometimes prominent nucleoli, and delicate nuclear membranes. Nuclear size may vary both within and among groups, but the nuclear-to-cytoplasmic ratio is generally retained ( Fig. 14.23 ).

Microglandular hyperplasia may yield groups with enlarged, sometimes vacuolated cells that raise the possibility of adenocarcinoma. However, nuclei are variably sized, rather than monomorphic, the chromatin is fine, and there may be adherent crowded reserve cells or immature metaplastic cells, some with recognizable intercellular bridges ( Fig. 14.24 ).

With irradiation and the Arias-Stella reaction, there are gigantic cells with voluminous, sometimes vacuolated cytoplasm. Nuclei vary from normal-sized to very large, often within the same group, and have smudged chromatin and intranuclear vacuoles ( Fig. 14.25 ).

Atrophy

Aside from the well-known problem of benign nuclear atypia in atrophy mimicking squamous dysplasia, atrophic cells may sometimes shed some or all of their cytoplasm and aggregate in pseudoglandular groups, which may be mistaken for endometrial cells, endometrial carcinoma, or even metastatic carcinoma. The atrophic nuclei are small, bland, and uniform. Importantly, they are identical to those of the intact cells and cells that have lost only part of their cytoplasm that are present in the background ( Fig. 14.26 ).

Misdiagnosis: High-Grade Squamous Intraepithelial Lesion Mistaken for Adenocarcinoma in Situ

A high-grade squamous intraepithelial lesion (HSIL) is the most frequent neoplastic mimic of AIS. Most biopsies that contain a neoplastic lesion following a report of glandular atypia in Pap smears have HSIL without AIS. In AIS-like HSIL, the abnormal cells are in very crowded groups, with coarsely hyperchromatic nuclei and scant cytoplasm ( Fig. 14.27A ), or are loosely arranged, with a pale, finely vacuolated cytoplasm (see Fig. 14.27B ); both appearances mimic glandular differentiation. However, the absence of distinctly columnar shapes, pronounced feathering, or rosettes argues against this. As a general rule, if there is uncertainty as to whether the cells are HSIL or AIS, they are likely to be the former. As a corollary, glandlike HSIL may be mistaken for reactive rather than neoplastic glandular cells (similar to the problem of false- negative interpretations with AIS, discussed later).

Classification of Columnar Cell Abnormalities

The Bethesda 2014 terminology for reporting glandular lesions remains similar to the Bethesda 2001 terminology ( Box 14.2 ). Of note, the reporting of a finding of otherwise benign-appearing endometrial cells has been moved from an age of 40 years at initiation (in the 2001 Bethesda classification) to an age of 45 years to improve the predictive value of the result.

In addition, endocervical adenocarcinoma in situ is a separate diagnostic category now that enough experience with its cytologic appearance has accrued. Despite recognition of AIS as a distinct category, in practice this diagnosis is more difficult to recognize than other serious diagnoses. Nevertheless, the 2001 Bethesda classification scheme overall is more likely to correlate with a significant lesion than the 1991 terminology.

Interpretation of Cervical Glandular Abnormalities Using Liquid-Based Preparations

Most cytologic preparations are now liquid-based. A comparison of conventional smears, ThinPrep and SurePath (BD Diagnostics, TriPath, Burlington, NC), and liquid-based preparations found similar criteria applied for detecting endocervical glandular neoplasia in all three specimen preparations. ThinPrep was less likely to exhibit large dark sheets of cells and individual cells than the other two methods, whereas feathering was more pronounced in conventional smears.

Human Papillomavirus Testing Following Atypical Glandular Cells or Atypical Endocervical Cells

The 2012 consensus guidelines from the American Society for Colposcopy and Cervical Pathology (ASCCP) for the initial management of all categories of atypical glandular cells, except atypical endometrial cells, did not recommend high-risk HPV triage for initial presentations of atypical glandular cells. Based on two large US studies, 25% of cases in the AGC category test positive for high-risk HPV, of which the most prevalent genotypes are 18 and 45, followed by 16. Although several studies have indicated that the presence of high-risk HPV is highly predictive of high-grade lesions (glandular and squamous) following AGC, and that false-negative interpretations are infrequent, caveats are that in some cases, the atypical glandular cells are from endometrial, tubal, or even ovarian carcinomas that are not HPV-related. Also, a significant minority of cervical adenocarcinomas in situ and invasive cervical adenocarcinomas are HPV negative. Thus, reliance on a negative HPV test result to avoid colposcopy or endometrial biopsy following the reporting of atypical endocervical cells or atypical glandular cells on a smear may lead to underdiagnosis of a serious lesion. In fact, the heterogeneity of the lesions associated with AGC means that no single approach is adequately sensitive for detecting disease in this high-risk category. Consequently, all patients with AGC are recommended to undergo initial colposcopy with endocervical sampling, and endometrial sampling if older than 35 years, with or without initial colposcopy. In patients for whom a high-grade lesion (cervical intraepithelial neoplasia [CIN]2+) is not identified, two HPV co-test results are then used to determine the need for colposcopic evaluation and the interval for subsequent cytology screening.

Distinguishing Features

Subtypes

Intestinal Subtype

This is distinguished by a variable proportion of goblet cells ( Fig. 14.29A ). Immunohistochemical staining for CDX2 in intestinal-type AIS typically demonstrates positive staining in AIS epithelium, both in areas of obvious intestinal differentiation and in areas without prominent goblet cells (conventional-type AIS adjacent to intestinal-type AIS). This suggests that morphologic changes of intestinal differentiation may not be evident early in the process. There is less nuclear crowding and hyperchromasia in this variant, making confirmation more difficult. However, intestinal differentiation alone is an important clue to AIS, because benign intestinal metaplasia of the cervix is rare. Occasionally, gastric (pyloric) or pancre-aticobiliary differentiation will be seen (see Fig. 14.29B ). The intestinal-type AIS often stains less intensely with p16. Interestingly, patients with intestinal-type endocervical AIS are on average older (44.5 vs. 32.6 years), and multiple studies have shown that the frequency of HPV detection in intestinal-type AIS is less than that for other forms of AIS, with a corresponding lower rate of p16 diffuse positivity. Together, these data suggest more than one pathway of pathogenesis for this AIS subtype.

Subtle Variants of Adenocarcinoma in Situ: Superficial (Early) Type

It is reasonable to assume that in its earliest form, AIS may not manifest with the full range of atypia that characterizes well-established lesions. We previously reported a variant of AIS termed superficial or early AIS, which had the following characteristics: (1) occurrence in younger women, with a mean age in the low 20s; (2) a more restricted distribution, which was principally superficial; and (3) less conspicuous mitotic activity, nuclear atypia, and apoptosis. Like other forms of AIS, these lesions were HPV positive and contained a discrete punctate nuclear signal, with high-risk HPV probes characteristic of integrated DNA. At its most recognizable form, early AIS manifests as discrete hyperchromatic and nonciliated foci on the surface, with apoptosis or mitotic activity ( Fig. 14.30A and B ). Less conspicuous are lesions that are not hyperchromatic but demonstrate small but discrete differences in nuclear morphology, with mitotic activity (see Fig. 14.30C and D ). Immunostains for MIB-1 and p16 are typically strongly positive and can be helpful in confirming the diagnosis (see Fig. 14.30B ).

p16ink4

Because they are strongly associated with HPV, most AIS will stain strongly for p16. This biomarker is occasionally used to distinguish AIS from tubal or tubal-endometrial metaplasia from AIS. The latter will stain but typically in a heterogeneous fashion.

Ki-67 (MIB-1)

One series has concluded that although benign proliferations (e.g., reparative or inflamed endocervical epithelium, microglandular hyperplasia, tubal metaplastic epithelium) typically have a proliferative index less than 10%, the proliferative index in AIS often exceeds 50%. There are exceptions, however, particularly with inflamed endocervical mucosa, and we rely principally on p16 to aid in making the distinction between reactive and neoplastic columnar epithelium.

Stathmin-1

Stathmin-1 is a microtubule destabilizing protein shown to be overexpressed in a number of cancer types. A single series has shown that all cases of benign endocervix are negative for the biomarker, whereas all cases of AIS show diffuse positivity and all cases of adenocarcinoma show patchy or diffuse positivity. The initial results are promising; further studies with larger sample sizes will be needed to corroborate and generalize these findings.