Endocervical AIS is the prototypical endocervical neoplastic lesion which forms the comparator for all discussions of cervical glandular atypia. The cytologic features of endocervical AIS were first described in studies from the mid-1970s [5]. Additional and more detailed descriptions appeared in the 1980s. Prior to TBS 2001, AIS was included only under “Atypical Glandular Cells of Undetermined Significance” (AGUS) because it was not felt to be reliably recognized and reproducible as a discrete category of interpretation. In the intervening time before the publication of the second edition of the Bethesda System (TBS) atlas, the cytologic characterization of AIS had become sufficiently established to be included as a discrete entity. Because cytologists around the world use the TBS atlas as a reference, acceptance of these published criteria for the cytologic diagnosis of AIS has been high and successful application is now more widespread.

The cytologic features of AIS were first described in conventional smears (CS). With the introduction of liquid-based cytology (LBC) preparations in the mid-1990s, subsequent reports described the cytologic features of AIS on LBC along with the differences that had been noted when compared to CS. Although some cytologists, most notably the authors of the second edition of the TBS atlas, felt that the cytologic features of AIS were less readily recognized on LBC because of the three-dimensionality of this specimen type, many studies have concluded that the identification of AIS on LBC samples is also reliable.

The cytologic features of AIS can be subdivided into those that are detectable at low magnification (×10) and those more readily discernible at higher magnifications. Low magnification features are those that are most commonly found on initial screening and include the presence of hyperchromatic crowded groups (HCG) of cells. The presence of such groups should always alert the screener to the possibility of a glandular lesion (Fig. 4.2a), and should be carefully examined. At higher magnifications, characteristic architectural features that may suggest an endocervical neoplasia include marked crowding of very dark cells which are irregularly arranged and show nuclear overlap (Fig. 4.2b). With adjustment of the focal plane, the disorganization of the crowded cell groups will be appreciated. As the objective changes plane, the presence of rosettes, abnormal honeycomb formations, and pseudostratification of nuclei can be appreciated. At the margins of the groups, cells and nuclei protrude from the periphery of the crowded cell groups (Fig. 4.3). This is a characteristic feature of AIS which is termed feathering, and may also be a “screening” feature of glandular neoplasia identified at low magnification. In feathering, portions of cytoplasm or protruding nuclei may appear to be falling off the edges of the crowded cell groupings. Feathering is more prominent in conventionally prepared specimens due to the flattening of the groups during the smearing process. The group edges tend to be more blunted in LBC preparations due to the rounding effects of liquid suspension and fixation. In contrast to the crowded glandular groups, single cells or clusters of only 2–3 cells of endocervical AIS are less commonly encountered, but still should be present in most cases and can aid in diagnosis when cells in the hyperchromatic groups cannot be well visualized (Fig. 4.4a, b, c). Glandular lumen or rosette formation, which is indicative of glandular differentiation, may be identified (Fig. 4.5a, b). Pseudostratified strips of columnar cells are commonly present and recapitulate this feature well described in the histology of AIS (Fig. 4.4c).

The nuclei of AIS are enlarged, generally at least twice the area of intermediate squamous cell nuclei (Fig. 4.6a, b). Increased optical density, the so-called hyperchromasia, of the coarsened but evenly distributed chromatin is present. Nuclei may be rounded but often are more elongate than is seen in normal endocervical cells. The nuclear to cytoplasmic ratio is increased. Mitotic figures will often be identified, as well as apoptotic debris, both indicative of high cell turnover (Fig. 4.7a, b). As noted in the histology descriptions of the usual type of AIS, most lesions are well differentiated; however, occasional examples will show a more poorly differentiated and therefore more highly atypical nuclear morphology. In such cases, the nuclei can be very large and pleomorphic. In such cases it can be very difficult to distinguish an in situ from an invasive lesion.

In a significant number of cases endocervical AIS is associated with a concurrent squamous lesion, and therefore low- or high grade dysplastic squamous cells may be present in the sample.

At times the crowded cell groups are so densely packed that a distinction between glandular and squamous origin may be a challenge. As noted in Chap. 1, high grade squamous intraepithelial (HSIL) lesions are the most common neoplastic diagnosis in follow-up of an interpretation of atypical glandular cells (AGS), and is the outcome in about 50 % of cases. The cause for this cytologic mimic is the manner of growth of HSIL not only along the surface of the transformation zone, but also the manner of extension into the endocervical crypts. When plucked from the glandular crypts by the sampling device, the HSIL epithelial group maintains a cohesive, bulbous profile, mimicking a HCG of endocervical glandular cells. Clues to the cell of origin are gleaned by examination of the periphery of the HCG where cellular detail is better observed. Columnar shape and delicate mucinous cytoplasm suggest endocervical cells (Fig. 4.8). Flattened, stacked cells suggest a squamous origin (Fig. 4.9). A swirling or spindled cell arrangement deep in the HCG may also suggest squamous differentiation. The cells of HSIL are notorious for growing into and filling native endocervical glands. This HSIL growth pattern often will produce crowded groups on cytology that can mimic the groups associated with true glandular lesions (Fig. 4.10a). An important clue to the squamous nature of the lesion will be the presence of single HSIL cells in the background of the slide, which is almost universally noted in specimens found to be derived from squamous mimics of AIS (Fig. 4.10b).