Keratinization in atypical glandular cell clusters as a cytological clue to endometrioid carcinoma on cervical cytology

Specific diagnosis of endometrial carcinomas on cervical cytology is difficult with few useful cytomorphological clues reported. This study reviews a cohort of cervical cytology to investigate the presence of keratinization in atypical glandular cells (AGC), an undescribed cytomorphological clue for identifying endometrial endometrioid carcinomas on cervical cytology.

morphologically variable, with many architectural patterns (microglandular, papillary and villoglandular), variant differentiation (osteoid, sex cord-like and de-differentiation). 5 Specific cytomorphologic features indicative of upper tract origin, or features that discriminate histotypes of endometrial carcinomas are lacking, 6 and cytomorphologic descriptions of endometrial carcinomas are mostly based on comparison of high-grade endometrial carcinomas against human papillomavirus (HPV)-dependent adenocarcinomas. 6,7Discriminating and suggesting the possibility of an upper tract lesion can alert the clinician for prompt investigation.
In this study, a cohort of cervical cytology specimens was reviewed for the presence of keratinization in AGCs, a cytomorphologic clue proposed to identify endometrial endometrioid carcinomas, from patients with histologically proven endometrial carcinoma.

| ME THODS
A computerized search of the department archives for cervical cytology, from the year 2001 to 2020, was performed within up to 180 days prior to a histologic diagnosis of endometrial endometrioid carcinoma.All slides with an abnormal cytologic diagnosis were included (i.e.all negative or inadequate cases were excluded).
The cervical cytology slides, including smear and liquid-based preparations, were retrieved and reviewed for the presence of keratinization associated with AGCs.Keratinization was defined as orangeophilic, rigid and acellular fragments and must be either present within clusters of AGCs or associated with the AGCs by connecting extracellular materials (Figure 1A-D).Corresponding clinical and staging data were retrieved from the pathology reports and case records.Histology slides, when retrievable, were reviewed for the percentage of squamous differentiation, defined by the area of squamoid morules and frank squamous differentiation over total tumour area, and for the presence of keratinization.
Assessment was performed by scanning at least two representative tumour slides if available and viewing all slides of each case in parallel, at high-power magnification to very low-power magnifications displaying the entire tumour area of each slide.A summation of total tumour area and squamous differentiation for calculation was made when multiple slides were available for a case.Slides were assessed jointly by two board-certified pathologists, and discrepancies were resolved by viewing the slides together until a consensus was reached.
Statistical analysis was performed using SPSS (version 26.0).

F I G U R E 1 Cervical cytology shows keratinization in atypical glandular cells (AGC). (A) A large cluster of AGCs surrounding a fragment of orangeophilic acellular keratin, 400× magnification. (B) Atypical cells and keratinization among tumour diathesis, 400× magnification. (C)
A large fragment of keratinous material connected to AGCs by extracellular material, 400× magnification.(D) A cluster of atypical cells associated with a squamous morule and keratin fragment, 400× magnification.

| RE SULTS
In total, 42 cases of cervical cytology specimens from 41 patients were retrieved, including 7 (16.7%)with keratinization associated with AGCs seen and 35 (83.3%) without.Hysterectomy was performed for 39 patients (40 cases) with staging data available, corresponding to FIGO (The International Federation of Gynaecology and Obstetrics) stage I, II, III and IV disease in 25, 9, 3 and 2 cases respectively (Table S1).Tumour histologic grading was grade I, II and III for 26, 11 and 5 cases respectively (Table 1).
Histology slides were available for 37 cases for review.Cases with keratinization seen on cervical cytology had an average of 7.43% are squamous differentiation, compared with cases without keratinization on cytology at 3.87%, but the difference did not reach statistical significance.Cytologic and histologic keratinization were associated statistically (p = 0.002).Frank keratinization was seen on histologic slides of five cases, with four also showing cytologic keratinization (Table 2, Figure 2A-C).

| DISCUSS ION
Cervical cytology in patients with endometrial carcinomas, in the absence of concurrent lower tract pathologies, is frequently negative. 8,91][12] Differentiating endometrial carcinomas from other neoplastic or reactive glandular lesions in cervical cytology is known to be challenging.The limited quantity of lesional cells sampled from the endometrium is detrimental to architectural and cytomorphologic analysis.
There are cytomorphological features described that are useful in distinguishing endometrial carcinoma from endocervical adenocarcinoma-in situ and adenocarcinoma. 7Columnar configuration, feathering and necrotic tumour diathesis favours endocervical lesions, 13,14 whereas loss of nuclear polarity and tumour diathesis indicates an endometrial origin. 7Papillary fragments may be seen in serous, clear cell and endometrioid carcinomas, 6,15 and intracytoplasmic neutrophils are suggestive of endometrioid histology. 7tomorphologic overlaps between endometrial carcinomas and endocervical adenocarcinomas are significant, as nuclear features such as chromatin pattern, nucleolar size and nuclear size are not sufficiently distinct for reliable cytologic diagnosis.Variation in cytomorphology among the major histotypes of endometrial carcinoma is high, 2 complicated by intratumoral heterogeneity in clear cell and serous endometrial carcinomas. 16,17When reactive and metaplastic endometrial lesions, poorly or undifferentiated carcinomas and metastatic adenocarcinomas are included as differentials, cytologic diagnosis of endometrial carcinomas on cervical cytology is often difficult in practice. 18,19is study reports an undescribed cytomorphological feature for identifying endometrioid carcinomas in cervical cytology.Over 70% of endometrial carcinomas are of endometrioid histology, 20,21 of which many show evidence of squamous differentiation with the formation of squamous morules and even frank squamous differentiation or keratinization. 22,23In a case series described by Gupta et al., the authors have attributed the presence of background keratinized squamoid cells, in two cases of endometrioid carcinoma, to squamoid differentiation. 24From the current cohort of cervical cytology in patients with histologically proven endometrioid endometrial carcinomas, seven cytology specimens with keratinization in AGC clusters were found.The keratinous material appears as orangeophilic, rigid and acellular fragments within clusters of AGCs or is associated with the AGCs among extracellular material (Figure 1D).
Cytologic keratinization is strongly associated with keratinization on histologic follow-up but is not correlated with other histological or grading parameters.
The major cytologic differential for endometrial carcinoma on cervical cytology, endocervical adenocarcinoma, is not described to display keratinization. 25Abnormal squamous cells can be concurrently present along with endocervical adenocarcinoma, both resulting from HPV infection. 7Morphologically, keratin fragments are anucleate, squamoid morules show bland nuclear features, while HPV-infected squamous cells display koilocytic atypia or even overt nuclear dysplasia. 26The normal cervical epithelium is non-keratinizing, 27 but keratinization is not uncommonly observed in association with inflammation and/or irritation, or in the case of paradoxical keratinization present in invasive squamous cell carcinoma. 28,29Tight association between the keratin fragments and AGCs also supports genuine tumour squamous differentiation over background contamination.One pitfall of note is cervical adenosquamous carcinoma, 30  This study is limited by a relatively small number of cases.A similar search was performed for non-endometrial carcinomas for comparison but only yielded five cervical cytology specimens.
None of the five specimens displayed evidence of keratinization.
The final histologic diagnosis was indeterminate for four of the cases, which were reported as adenocarcinoma, not specified.
The remaining case was reported as serous carcinoma.In view of the ambiguity in histologic diagnosis, these five cases were not included in the analysis.

| CON CLUS ION
The t-test and the Fisher exact test were used for comparing cytologic parameters as continuous and categorical variables.A p value of <0.05 was considered significant.The study was approved by the Joint Chinese University of Hong Kong -New Territories East Cluster Clinical Research Ethics Committee and was granted the exemption of requiring written informed consent.

F I G U R E 2
Squamous differentiation is commonly observed in endometrioid endometrial carcinomas, the most prevalent histotype of endometrial carcinoma.Such is reflected in cervical cytology by the presence of orangeophilic, rigid and acellular fragments within or associated with AGC clusters.Keratinization, when identified with AGCs, should be regarded as a cytologic clue suggestive of an endometrial origin.Histology of endometrioid endometrial carcinoma with squamous differentiation.(A) Abundant acellular keratinous material adjacent to tumour cells, 100× magnification.(B) Clusters of tumour cells showing gradual squamous maturation with aggregates of polymorphs, 100× magnification.(C) Squamous differentiation as evidenced by intracellular bridging and keratinization, a squamous whorl is also seen, 200× magnification.
but even if keratinization is seen, nucleated atypical squamous cells should also be present.With the Comparison of degree of squamous differentiation with cytologic evidence.
prevalence of squamous lesions and abnormalities in the uterine cervix, only strictly anucleate keratin fragments touching AGCs should be considered a positive sign.In addition, keratinization is not seen in serous, clear cells or other histotypes of endometrial carcinoma.Those endometrial carcinomas are rare and of high histologic grade, whether specific cytomorphologic features exist for these entities remains to be uncovered.