Can Endometrial Cytology Identify Patients Who Would Benefit from Immunotherapy?

INTRODUCTION: Patients with polymerase epsilon (POLE) mutation (POLEmut) subtype, MMR-deficient (MMR-d) subtype as classified by The Cancer Genome Atlas (TCGA), and a high tumor mutation burden (TMB-high) potentially benefit from immunotherapy. However, characteristics of the cytological morphology within these populations remain unknown. METHODS: DNA extracted from formalin-fixed paraffin-embedded tissues was subjected to next-generation sequencing analysis. Genomic mutations related to gynecological cancers, TMB, and microsatellite instability were analyzed and were placed in four TCGA classification types. The following morphological cytological investigations were conducted on endometrial cancer using a liquid-based preparation method, prior to the commencement of initial treatment: (i) cytological backgrounds; (ii) differences between each count of neutrophils and lymphocytes as described below. RESULTS: Insignificant differences in the cytological background patterns of TCGA groups and TMB status were found. Although there was no significant difference in neutrophil count (p = 0.955) in the TCGA groups, POLEmut and MMR-d had significantly higher lymphocyte counts than no specific molecular profile (NSMP) (p = 0.019 and 0.037, respectively); furthermore, p53mut also tended to be significant (p = 0.064). Lymphocyte counts in TMB-high were also significantly greater than TMB-low (p = 0.002). POLEmut showed a positive correlation between TMB levels and lymphocyte counts. For predicting patients with POLEmut plus MMR-d, lymphocyte counts demonstrated a superior diagnostic accuracy of area under the curve (AUC) (0.70, 95% CI: 0.57-0.84), with a cutoff value of 26 high-power field. CONCLUSION: Lymphocyte count using liquid-based cytology for patients with endometrial cancer may predict POLEmut plus MMR-d of TCGA groups and TMB-high in those who can benefit from immunotherapy.

Cervical Cytology Preserves Histologically Detected Surface Epithelial Slackening, Unique to the POLE Mutation-subtype in Endometrial Cancer.

BACKGROUND/AIM: Among the four genomic subtypes of endometrial cancer, distinguishing between the DNA polymerase epsilon mutation (POLEmut) and no specific molecular profile (NSMP) subtypes requires genomic profiling owing to the lack of surrogate immunohistochemical markers. We have previously found that, histologically, the POLEmut-subtype exhibits surface epithelial slackening (SES). Therefore, to improve subtype identification, we aimed to extract cytological features corresponding to SES in POLEmut-subtype cervical cytology specimens. MATERIALS AND METHODS: We analyzed 104 endometrial cancer cervical cytology specimens, with integrative diagnosis confirmation via histology, immunohistochemistry, and genomic profiling. Cytological features were evaluated for the presence of atypical glandular cells, atypical cell appearance in single cells and clusters, and cytological SES and the presence of tumor-infiltrating inflammatory cells in clusters. RESULTS: Based on cervical cytology, the POLEmut- and p53mut-subtypes exhibited more frequent atypical cells in smaller clusters, giant tumor cells, and cytological SES patterns than the NSMP-subtype. Tumor-infiltrating lymphocytes were frequent in the POLEmut- and mismatch repair-deficient subtypes. CONCLUSION: Histologically-detected SES as well as other endometrial cancer features may be preserved in the atypical cell clusters observed in cervical cytology specimens. Cytological detection of SES and of smaller clusters of atypical cells and inflammatory cells with moderate atypia are suggestive of POLEmut-subtype. Integrative diagnosis including genomic profiling remains critical for diagnostic confirmation.

Direct next-generation sequencing analysis using endometrial liquid-based cytology specimens for rapid cancer genomic profiling.

BACKGROUND: Genomic examination of cytology specimens is often performed on cell blocks or conventional smears rather than on liquid-based cytology (LBC) specimens. Since LBC specimens preserve high-quality DNA, cancer genome profiling using next-generation sequencing (NGS) is also attainable from residual LBC specimens. One of the advantages of using LBC specimens for NGS is that it allows direct extraction of DNA from residual specimens, avoiding a sacrifice of smear slides and minimizing genomic profiling processing time. METHODS: Endometrial LBC specimens were subjected to NGS analysis to validate the practicality of rapid cancer genomic profiling in a pathology laboratory. The extracted DNA was subjected to NGS using a customized cancer gene panel comprising 56 genes and 17 microsatellite regions. The workflow strategy was defined, and the processing time estimated for specimen sampling, cell counting, NGS run, and genome profiling. RESULTS: NGS analysis of most LBC specimens revealed somatic mutations, tumor mutation burden, and microsatellite instability, which were almost identical to those obtained from formalin-fixed paraffin-embedded tissues. The processing time for direct NGS analysis and cancer genomic profiling of the residual LBC specimens was approximately 5 days. CONCLUSION: The residual LBC specimens collected using endometrial cytology were verified to carry a high tumor fraction for NGS analysis and could serve as an alternate source for rapid molecular classification and diagnosis of endometrial cancers, as a routine process in a pathology laboratory.