Akihide Tanimoto

Endometrial Liquid‐Based Cytology Specimens Preserve High Genome Quality for Molecular Classification After Long‐Term Storage

ABSTRACTBackgroundMolecular classification of endometrial cancer is useful for predicting prognosis. Genomic examinations are performed using formalin‐fixed paraffin‐embedded (FFPE) tissues; however, we previously reported that liquid‐based cytology (LBC) specimens can be used for next‐generation sequencing (NGS). In this study, we evaluated long‐term storage effects of LBC specimens on NGS‐based genomic profiling, including gene mutations, tumor mutation burden (TMB), and microsatellite instability (MSI).MethodsFour LBC fixatives (CellPrep, ThinPrep, CytoRich Red, and SurePath) were used to prepare NGS samples from cultured endometrioid carcinoma HEK‐251 cells. Twelve endometrial LBC specimens from patients with endometrioid carcinoma were fixed with CytoRich Red. The TMB, MSI, and gene mutations were analyzed after 1 week, 6 months, and 12 months of storage in cultured HEK‐251 cells. Paired LBC and FFPE specimens of endometrioid carcinoma stored for 15–45 months were subjected to NGS‐based analysis, and their molecular profiles were compared to those at the initial diagnosis.ResultsThe TMB and MSI did not differ during the storage periods for any of the LBC fixatives in the cultured cells; in addition to gene mutations, they were comparable between the initial and second analyses of the clinical FFPE and LBC specimens. There were no changes in the integrative diagnosis of the endometrioid carcinoma subtype classification.ConclusionLBC specimens, which preserved high‐quality genomes for molecular classification after long‐term storage, may be an alternative source of genomic examination for the integrative diagnosis of endometrial cancer.

“Surface epithelial slackening” pattern in endometrioid carcinoma: A morphological feature for differentiating the POLE mutation-subtype from the no specific molecular profile subtype

Endometrial cancers are classified into mismatch repair (MMR) deficient- (MMRd), p53 mutation- (p53mut), DNA polymerase epsilon (POLE) mutation (POLEmut), and no specific molecular profile (NSMP) subtypes according to The Cancer Genome Atlas (TCGA). The distinction between POLEmut and NSMP subtypes is made on the basis of molecular analysis because the specific histological and immunohistochemical features of these two subtypes are still unknown. In this study, we analyzed histological features by scoring the presence of a mucinous pool, giant cells, clear cells, keratinization, neutrophilic abscess, and surface proliferating pattern in 82 cases of endometrial cancers in which an integrative diagnosis was confirmed by immunohistochemistry and genomic profiles showing POLE mutations, tumor mutation burden, and microsatellite instability. In contrast to the hierarchical branching of micropapillary proliferation observed in serous carcinoma, POLEmut-subtype endometrioid carcinomas often showed a surface epithelial slackening (SES) pattern in the tumor cells facing the uterine surface. The POLEmut subtype exhibited higher scores for clear cells and SES patterns than the other three subtypes. The scores for giant cells, clear cells, and the SES pattern were significantly higher in the POLEmut subtype than in the NSMP subtype, suggesting that these morphometric parameters are useful for differentiating POLEmut- and NSMP-subtype endometrioid carcinomas, although genomic profiling is still necessary for a definite molecular diagnosis.

A potential inflammatory biomarker for advanced endometrial cancer treated with lenvatinib plus pembrolizumab

AbstractIntroductionTo identify prognostic biomarkers that could predict how well patients will respond to lenvatinib/pembrolizumab (LEN/PEM). The utility of certain inflammatory biomarkers in endometrial liquid‐based cytology (LBC) or peripheral blood samples, such as neutrophil counts, lymphocyte counts, and neutrophil‐to‐lymphocyte ratio (NLR) were explored.MethodsThe study included 25 patients with advanced or recurrent endometrial cancer who had received LEN/PEM between August 2018 and March 2024. Predictors for overall response (OR), disease control, and progression‐free survival, based on neutrophil/lymphocyte counts, NLR scores of the endometrial LBC prior to initial treatment, and peripheral blood prior to initial treatment and prior to LEM/PEM treatment were compared using a receiver operating characteristic curve. Significant predictors were evaluated using the log‐rank test, and multivariate analysis.ResultsAlthough neutrophil counts and NLR score in endometrial LBC prior to initial treatment were better effective predictors for OR, the most accurate predictor of a progression‐free status was NLR score in peripheral blood prior to LEM/PEM (0.722, 95% CI: 0.45–0.99, sensitivity: 57.1%, specificity: 94.4%). In peripheral blood prior to LEN/PEM, the lower NLR (NLR <5.39) group had a significantly longer PFS than the higher NLR (5.39 ≤ NLR) group (p = 0.023, median survival: 13.5 vs. 3.0 months), and tended to be independently correlated with PFS (hazard ratio = 2.571; 95% CI = 0.857–7.719; p = 0.092).ConclusionInflammatory biomarkers in endometrial LBC failed to predict the efficacy of LEN/PEM, while peripheral blood NLR score sampled prior to LEN/PEM potentially could be a significant predictor.

Pelvic Carcinosarcoma Showing a Diverse Histology and Hierarchical Gene Mutation with a Common POLE Mutation to Endometrial Endometroid Carcinoma: A Case Report

POLE mutation-type endometrial cancer is characterized by an extremely high tumor mutation burden. Most POLE mutation-type endometrial cancers are histologically endometrioid carcinomas, and POLE mutation-type carcinosarcomas are rare among endometrial cancers. We report a case of endometrial and pelvic cancer in a 53-year-old woman who was analyzed using next-generating sequencing. The endometrial lesion harbored a p.T457del POLE mutation with an elevated tumor mutation burden and low microsatellite instability. The pelvic lesion showed divergent histological features, consisting of high-grade endometrioid carcinoma, neuroendocrine carcinoma, and chondrosarcoma. In addition to the common POLE mutation detected in the endometrial lesion, the pelvic lesion in each element showed additional gene mutations in a hierarchical manner. Therefore, it is indicated that the p.T457del POLE mutation is a pathogenic mutation and may be related to POLE mutation-induced carcinogenesis and divergent morphogenesis in endometrial cancer.

One-step nucleic acid amplification (OSNA) assay for detecting lymph node metastasis in cervical and endometrial cancer: a preliminary study

To evaluate the accuracy of the one-step nucleic acid amplification (OSNA) assay for the diagnosis of lymph node (LN) metastasis in uterine cancer. A total of 116 LNs from 30 patients with cervical and endometrial cancer, enrolled in this prospective study, were used. Excised LNs were cut into 4 to 6 blocks at 2 mm intervals, and nonadjacent blocks were alternately subjected to either histological examination or the OSNA assay. The concordance rate between histological examination and the OSNA assay in cervical cancer and in endometrial cancer was 95.9% and 95.2%, respectively. The sensitivity, specificity, and negative predictive value of the OSNA assay were 80%, 97.7%, and 97.7% in cervical cancer, and 85.7%, 93.3%, and 98.2% in endometrial cancer, respectively. In cervical cancer, discordant results were observed in 2 out of 49 LNs (4.1%); 1 was OSNA assay-positive and histological examination-negative, and 1 was OSNA assay-negative and histological examination-positive. In endometrial cancer, discordant results were observed in 5 out of 67 LNs (7.5%); 4 were OSNA assay-positive and histological examination-negative, and 1 was OSNA assay-negative and histological examination-positive. The OSNA assay showed high concordance rate with histological examination, sensitivity, and specificity in uterine cancer, suggesting that it could enhance the accuracy of conventional pathological examination for the detection of LN metastasis by reducing false negative rate.

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

AbstractBackgroundGenomic 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.MethodsEndometrial 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.ResultsNGS 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.ConclusionThe 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.

Next-generation sequencing analysis of endometrial screening liquid-based cytology specimens: a comparative study to tissue specimens

Abstract Background Liquid-based cytology (LBC) is now a widely used method for cytologic screening and cancer diagnosis. Since the cells are fixed with alcohol-based fixatives, and the specimens are stored in a liquid condition, LBC specimens are suitable for genetic analyses. Methods Here, we established a small cancer gene panel, including 60 genes and 17 microsatellite markers for next-generation sequencing, and applied to residual LBC specimens obtained by endometrial cancer screening to compare with corresponding formalin-fixed paraffin-embedded (FFPE) tissues. Results A total of 49 FFPE and LBC specimens (n = 24) were analyzed, revealing characteristic mutations for endometrial cancer, including PTEN, CTNNB1, PIK3CA, and PIK3R1 mutations. Eight cases had higher scores for both tumor mutation burden (TMB) and microsatellite instability (MSI), which agree with defective mismatch repair (MMR) protein expression. Paired endometrial LBC, and biopsied and/or resected FFPE tissues from 7 cases, presented almost identical mutations, TMB, and MSI profiles in all cases. Conclusion These findings demonstrate that our ad hoc cancer gene panel enabled the detection of therapeutically actionable gene mutations in endometrial LBC and FFPE specimens. Endometrial cancer LBC specimens offer an alternative and affordable source of molecular testing materials.

Two Components of Variant Profiles in Primary Vaginal Carcinosarcoma via Next-Generation Sequencing and a Literature Review

Primary vaginal carcinosarcoma (VCS) is an extremely rare and aggressive tumor consisting of admixed malignant epithelial and mesenchymal elements. We report a case of VCS that was subjected to analysis by immunohistochemistry and next-generation sequencing (NGS). A 53-year-old woman with post-menopausal vaginal bleeding underwent surgical excision followed by concurrent chemoradiation. A well demarcated tumor was growing in a discontinuous fashion at a location some distance from both the cervix and vulva. Microscopically, the tumor consisted of adenocarcinoma components and sarcoma components consisting of a sheet-like growth of spindle-shaped cells, and we diagnosed this tumor as primary vaginal carcinosarcoma. NGS analysis of each component identified the following variants, TP53, PIK3CA, KRAS and FBXW7. A comparison of microsatellite instability (MSI) and tumor mutation burden (TMB) showed that within both tissues the sarcomatous components had a higher MSI and TMB than the carcinomatous components. This case supports “a monoclonal theory” with the genome profile being similar to other malignant mixed Müllerian tumors.

Human Papilloma Virus 18-Positive Submucosal Small Cell Neuroendocrine Carcinoma of the Vagina: An Immunohistochemical and Genomic Study

Primary vaginal neuroendocrine carcinoma (NEC) is extremely rare among female genital tract tumors. Here, we report 2 cases of vaginal small cell NEC (SCNEC) using immunohistochemistry and next-generation sequencing (NGS) analysis. The 2 patients were in their mid-to-late 70s, presented with abnormal vaginal bleeding and had a vaginal submucosal mass. The biopsied or resected tumors showed a typical neuroendocrine morphology consisting of solid nests of atypical tumor cells, with no specific organoid patterns, and proliferating in the vaginal submucosa. Immunohistochemical analysis showed strong and diffuse expression of chromogranin A, synaptophysin, and p16, but no thyroid transcription factor 1 expression. Additionally, both cases were positive for human papillomavirus (HPV) 18. An NGS-based cancer panel analysis revealed that the tumors carried NF1 and AR mutations, but no major driver mutations were detected. The results of this study suggested that HPV18 infection is linked to vaginal SCNEC.