Final Diagnosis & Discussions: Liquid-Based Pap Test on 36-Year-Old Non-Gravid Woman

FINAL DIAGNOSIS: Endocervical Adenocarcinoma in Situ (AIS); features suspicious for invasion


The Papanicolaou test was designed and is used to primarily detect abnormalities of the ectocervix, which mainly covers squamous intraepithelial lesions and squamous cell carcinoma. Due to technical (sampling) aspects of the test, glandular lesions are more difficult to detect. The 2001 Bethesda reporting system has several categories of glandular epithelial abnormalities which reflect current understanding of glandular neoplasia, including endocervical adenocarcinoma in situ (AIS) (1).

AIS is a recognized high grade glandular precursor lesion to invasive adenocarcinoma. High risk subtypes 16 and 18 of human papillomavirus (HPV) DNA have been identified in both lesions. It appears that subtype 18 is preferentially associated with adenocarcinoma versus subtype 16 which is more commonly linked with squamous cell carcinoma (2). HPV DNA testing is not only part of the management of patients with squamous lesions but also with glandular lesions such as atypical glandular cells (AGC) and AIS. An important finding by Fetterman and colleagues is that women who had a negative initial evaluation and were HPV DNA positive had a 24% risk of having high-grade cervical intraepithelial neoplasia (CIN) or AIS in the follow-up (3).

Cytologically, AIS is characterized by nuclear enlargement with resulting increased nuclear to cytoplasmic ratio, hyperchromasia, stratification and mitotic activity. Major cytologic features include:

  • Columnar cells arranged in sheets, clusters, strips and rosettes with nuclear crowding and overlap. There is a loss of the honeycomb pattern.
  • Cell clusters may have a palisading, pseudostratified appearance with some nuclei or cytoplasm protruding at the edge. This is frequently referred to as "feathering" or "bird-tail" appearance.
  • The chromatin is evenly dispersed, coarsely granular without prominent nucleoli.
  • Mitoses and apoptotic bodies are commonly seen.
  • No tumor diathesis.

Architecture is one of the most important defining features of AIS. Rabelo-Santos et al. evaluated 35 different cytomorphological criteria in smears with atypical glandular cells and AIS in 155 patients and found "feathering" to have the strongest predictive value (80%) for glandular neoplasia (4).

The differential diagnosis for endocervical adenocarcinoma in situ includes several benign entities, pre-malignant lesions and actual malignancy.

Endometrial hyperplasia is a recognized precursor to endometrial adenocarcinoma (EMAC). Cytologically, cells of atypical hyperplasia cannot be differentiated from well-differentiated EMAC. As with diagnosis of atypical glandular cells (AGC), confirmation by histology is needed.

Of the benign entities, some common considerations should include tubal metaplasia, lower uterine segment sampling, endocervical polyp, reactive/ reparative changes, and Arias-Stella phenomenon. Tubal metaplasia cells maybe enlarged and slightly hyperchromatic but bear characteristic cilia. Endometrial cells tend to be smaller than endocervical cells with very little cytoplasm. Although endometrial cell groups tend to be crowded, they retain an orderly pattern unlike the haphazard AIS arrangement. Endocervical polyps will have a papillary type architecture and smoother outline than the feathery edge of AIS. Reactive endocervical cells tend to have greater nuclear pleomorphism but less hyperchromasia than AIS. They also feature more prominent but small nucleoli which are less frequent in AIS (5). Our case showed very large and eosinophilic nucleoli which suggested a more malignant diagnosis (Figures 4 and 5). Arias-Stella cells are large and hyperchromatic with finely vacuolated cytoplasm and are seen in pregnancy. They also tend to be very few in a Pap smear sample, if seen at all. Clinical history is important in making the distinction between AIS and Arias-Stella phenomenon.

Additional lesion that should be considered and may coexist with AIS is high grade squamous intraepithelial lesion (HSIL). HSIL may perfectly resemble AIS, therefore diagnosis of AIS should be made only when there is clear evidence of glandular differentiation: strips or rosettes of columnar cells and feathering. Our case demonstrated feathering and pseudostratified cells arranged in strips (Figures 2 and 3). Another potential diagnostic pitfall is the extension of HSIL into endocervical glands. HSIL involving gland spaces lack AIS architecture such as feathering, rosettes, and strips. The edges of the groups tend to be flattened and scattered single dysplastic cells may be seen. AIS and HSIL coexist in approximately 50% of cases.

Separation between AIS and invasive endocervical adenocarcinoma can be challenging on a Pap smear. In general, adenocarcinoma cells are larger with uneven chromatin distribution and macro-nucleoli. Tumor diathesis when seen can be a helpful clue to presence of invasion. According to the 2006 American Society for Colposcopy and Cervical Pathology (ASCCP) guidelines a patient with cytologic diagnosis of AIS should undergo colposcopy with excisional procedure and histological evaluation for invasion (6).

Other malignancies that should be considered in the differential diagnosis are endometrial adenocarcinoma, adenocarcinoma arising from other gynecological sites such as vagina, ovary and fallopian tube and, metastasis from non-gynecological sites, primarily breast and colon. Endometrial adenocarcinoma cell groups tend to be smaller and more three dimensional. Intracytoplasmic neutrophils may also be seen. The background is also helpful in making the diagnosis. Tumor diathesis that is either watery or granular in case of endometrial adenocarcinoma or the "dirty necrosis" of colon adenocarcinoma should clue in the slide evaluator. Breast cancer metastases do not usually produce a tumor diathesis. Breast cancer cells tend to have a more typical signet ring appearance and may show cannibalism. Usually the patient’s clinical history is known and therefore the correct diagnosis can be made without too much difficulty.

Endocervical adenocarcinoma in situ (AIS) is now a recognized precursor glandular lesion to invasive carcinoma with reliable cytological criteria; "feathering" being one of the most important diagnostic clues. High risk HPV DNA, particularly subtype 18 is associated with AIS and endocervical carcinoma. The lesion frequently affects younger women (average age 38 versus 45 for patients with adenocarcinoma), therefore appropriate early diagnosis is vital to appropriate treatment.


  1. Thrall, MM et al. Rate of endometrial adenocarcinoma in women screened before and after implementation of the Bethesda 2001 reporting system. Acta Cytol 2008;52:1–7.

  2. Giatromanolaki A. et al. Human papillomavirus in endometrial adenocarcinomas: infectious agent or a mere "passenger"? Infect Dis Obstet Gynecol 2007.

  3. Cibas ES, Ducatman BS. 2003. Cytology: diagnostic principles and clinical correlates. Saunders Ltd., 2nd ed.

  4. Koss L, Melamed M. 2006. Koss’ diagnostic cytopathology and its histopathologic bases. Philadelphia: Lippencott Williams & Wilkins.