Abstract
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.
Keywords: Endometrial cancer, cervical cytology, POLEmutsubtype, surface epithelial slackening, genome profiling
Comprehensive examination of cancer gene alterations is useful for cancer subtype classification, molecularly targeted drug selection, and prognostic speculation (1-3). The Cancer Genome Atlas (TCGA)-based integrative genomic classification system for endometrial tumors, which is introduced in the latest WHO classification of female genital tract tumors (4), includes four subtypes: mismatch repair (MMR)-deficient (MMRd), p53 mutation (p53mut), DNA polymerase epsilon (POLE) mutation (POLEmut), and no specific molecular profile (NSMP). These categories, which correspond well with prognosis, are helpful in managing patient care in endometrial cancer (5).
MMR and p53 expression provide surrogate markers for diagnosing the MMRd- and p53mut-subtypes, respectively (4,6). In contrast, because there is no specific antibody for detecting mutant POLE, genomic examination is essential for differentiating between the POLEmut- and NSMP-subtypes. We recently found that the POLEmut-subtype often harbors heterozygous ATM nonsense mutations, with subsequent loss of ATM expression; nonetheless, ATM immunohistochemistry (IHC) does not provide a complete surrogate marker for diagnosing the POLEmut-subtype (7). POLEmut-subtype diagnosis therefore requires identification of characteristic histological features such as giant tumor cells (GTCs); however, GTCs are often observed in other subtypes (8,9). Further, we have recently found that POLEmut-subtype endometrial cancers often exhibit a characteristic surface papillary proliferation pattern, which we have named “surface epithelial slackening” (SES). This unique SES pattern, which differs from the hierarchical micropapillary pattern of p53mut-subtype serous carcinoma, was observed in tumor cells facing the uterine lumen (9). Owing to this cell slackening and the ease with which cells detach at the tumor surface facing the uterine lumen, tumor cells may become dissociated from the tumor surface and scatter into cervical cytology specimens. Therefore, we speculate that the histologically detected SES pattern is likely to be reproducible in POLEmut-subtype cervical cytology.
To identify cytological features unique to the POLEmut-subtype, we compared the cytology of the four endometrial cancer subtypes, confirming the molecular profiling via next-generation sequencing (NGS) (10). In the POLEmut-subtype cervical cytology, atypical cells frequently formed smaller clusters and exhibited an SES pattern. These features may be characteristic of the POLEmut-subtype and hence useful for differentiating it cytologically from the NSMP-subtype.
Materials and Methods
Sample collection. In total, 108 patients with endometrial cancer were registered in the Clinical Research of Cancer Gene Panel Analysis of Gynecologic Cancer Study, conducted between January 2019 and April 2023 at Kagoshima University Hospital, Japan.
Preparation of tissue and cytology specimens, immuno-histochemistry, and next-generation sequencing. The resected tissues were fixed in 10% neutral phosphate-buffered formalin for 24-48 h. The tissues were properly trimmed, processed to prepare formalin-fixed paraffin-embedded (FFPE) specimens, and sectioned for hematoxylin and eosin (H&E) staining, IHC, and next-generation sequencing (NGS). MMR deficiency was defined as the complete loss of nuclear expression of either MLH1 and PMS2, MSH2 and MSH6, MSH6, or PMS2 alone. Diffuse and strong nuclear expression or complete loss of p53 expression were defined as the mutation patterns. Scattered nuclear staining with variable p53 expression intensity was categorized as the wildtype pattern. All antibodies used for IHC analysis were purchased from DAKO (Glostrup, Denmark). The cytological specimens were processed using conventional smear or liquid-based cytology (LBC). To prepare LBC specimens, cervical cytology specimens were immediately fixed with CytoRich Red solution (Becton Dickinson, Franklin Lakes, NJ, USA). The cytology slides were then processed using a BD SurePath liquid-based Pap Test System (Becton Dickinson) and stained with Papanicolaou staining solution.
Genome analysis and integrative diagnosis. Genomic profiles were examined using a custom NGS gene panel as previously reported (9-11). The Gynecologic Cancer Panel Ver. 2 (Qiagen, Hilden, Germany), containing 56 cancer-related genes and 17 microsatellite regions, was used for NGS analysis to determine gene alterations, tumor mutation burden, and microsatellite instability, as previously reported (9-11). After DNA was obtained from the FFPE sections (10-μm thickness) representing ≥30% of the cancerous tissue area, NGS was performed using a MiSeq sequencer (Illumina, San Diego, CA, USA). No fresh frozen or normal tissue were used for the study. The sequence data were annotated using the Qiagen web portal (https://www.qiagen.com./us/shop/genes-and-pathways/data-analysis-center-overview-page/) and Mitsubishi Electronic Software (Amagasaki, Hyogo, Japan) (12) using reference data from the COSMIC database (v.90.0; https://cancer.sanger.ac.uk/cosmic) and the reference human genome GRCh37/hg19 (https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.13/). The sequence data obtained from whole blood DNA were used only as a reference, and germline analysis was not performed. Integrated pathological diagnoses were made according to the WHO and TCGA classification systems (4,5) by two board-certified surgical and molecular pathologists (IK and AT). TNM clinical classification was performed according to the Union for International Cancer Control (UICC) system (13).
Evaluation of cytology specimens. Cervical cytological features were classified as negative for intraepithelial lesion or malignancy (NILM), or as having atypical glandular cells (AGC), AGC-favor neoplastic cells, or as adenocarcinoma cells, according to the Bethesda System (14). Atypical or adenocarcinoma cells were classified based on their dominant grouping (small or large clusters, or single cells). Clusters were considered large with >20 atypical cells or small with ≤20 atypical cells. The category comprising >50% of the combined clusters and single cells was selected as the dominant category for that sample. We detected the presence or absence in the clusters of tumor infiltrating lymphocytes (TILs) and tumor-infiltrating neutrophils (TINs).
Cytologically, SES was identified in atypical cell clusters based on the loosening or detachment of individual cells from the cell cluster periphery. An SES-positive cluster was defined as one exhibiting cellular loosening or dissociation in ≥50% of the cluster perimeter. Clusters exhibiting cytologically detected SES (cSES) was counted. The presence or absence of GTCs was determined.
Statistical analysis. Statistical comparisons were performed using Kruskal-Wallis, Wilcoxon, and Fisher’s exact tests. Differences were considered statistically significant at p<0.05 and borderline significant at p<0.1. The cSES count was evaluated using a receiver operator characteristic (ROC) curve.
Ethical approval. The use of the clinical samples was approved by the Ethics Committee for Clinical and Epidemiologic Research of Kagoshima University (approval no. 180215) and the 1964 Helsinki Declaration, including its later amendments and comparable ethical standards. Written informed consent was obtained from all the participants prior to the study. Participants younger than 20 years of age were excluded.
Results
Clinical summary. Among the 108 registered endometrial cancer cases, 103 were available for evaluation via cervical cytology and were included here. Table I presents the clinical findings, staging, and pathological diagnoses of these 103 cases, among these, there were eight POLEmut-, 22 MMRd, 28 p53mut-, and 45 NSMP subtypes cases. The POLEmut-subtype was observed in endometrioid carcinomas and mixed carcinomas (those with components of endometrioid and clear cell carcinomas). The MMRd-subtype was observed in endometrioid carcinomas, dedifferentiated carcinomas, serous carcinomas, carcinosarcomas, and clear cell carcinomas. Most of the p53mut-subtpye cases were serous carcinomas or carcinosarcomas. Most of the NSMP-subtype cases were endometrioid carcinomas.
Table I. Clinical, pathological, and cytological summaries.
SES, Surface epithelial slackening; TIN, tumor infiltrating neutrophil; TIL, tumor infiltrating lymphocyte; GTC, giant tumor cell; CCC, clear cell carcinoma; EC, endometrioid carcinoma; SC, serous carcinoma; SEIC, serous intraepithelial carcinoma; POLEmut, DNA polymerase epsilon mutation; p53mut, p53 mutation; MMRd, mismatch repair-deficient; NSMP, no specific molecular profile; conv, conventional; LBC, liquid-based cytology; ca(+), adenocarcinoma; ca(s/o), AGC-favor neoplastic; AGC, atypical glandular cell; NILM, negative for intraepithelial lesion or malignancy; ne, no evaluation; na, not available; *FIGO Stage classification 2008.
The UICC-based International Federation of Gynecology and Obstetrics (FIGO)-stage (2008) distribution varied significantly among the four subtypes (Figure 1A): the p53mut-subtype exhibited more-advanced FIGO stages than the MMRd- and NSMP-subtypes (Table II). UICC pathological T category frequencies showed a significant difference among the four subtypes (Figure 1B): the p53mut-subtype exhibited more-advanced pathological T categories than the NSMP-subtypes (Table II).
Figure 1. Endometrial cancer subtype clinical stages. (A) Based on the International Federation of gynecology and Obstetrics (FIGO) stage distribution, FIGO stage is significantly different among the subtypes (Kruskal-Wallis test): the p53mut-subtype exhibits significantly more-advanced stages on admission (Fisher’s exact test) (Table II). (B) Pathological T (pT) category frequency is significantly different among the subtypes (Kruskal-Wallis test). MMRd, Mismatch repair-deficient; NSMP, no specific molecular profile; p53mut, p53 mutation; POLEmut, DNA polymerase epsilon mutation.
Table II. Distribution of clinical stage, tumor progression, and cytomorphometric features.
POLEmut, DNA polymerase epsilon mutation; p53mut, p53 mutation; MMRd, mismatch repair-deficient; NSMP, no specific molecular profile; GCT, giant tumor cell; TIN, tumor infiltrating neutrophil; TIL, tumor infiltrating lymphocyte; ns, not significant; bs, borderline significance. *In each one case of MMRd- and NSMP-subtypes, no TNM information was obtained due to no operation. The frequencies in italics were compared via the Fisher’s exact test. Bold values represent statistical significance.
Cervical cytology of POLEmut-subtype endometrial cancer. Abnormal cervical cytology, including atypical or adenocarcinoma cells, was observed in 55-75% of specimens. The POLEmut-subtype appeared predominantly as small clusters or as single atypical cells with moderate nuclear atypia (Figure 2A). Most clusters exhibited cell dissociation around the entire circumference of the clusters, forming the cSES pattern (Figure 2B and C). Many of the clusters contained inflammatory cells (TILs, TINs, or both). GTCs were detected (Figure 2D). Although cSES was recognizable in conventional smear cytology specimens, the clusters were distorted, and cell detachment was less obvious than in the LBC specimens.
Figure 2. Cytological features of POLEmut-subtype. (A) In the POLEmut-subtype, atypical cells occur frequently in small clusters or as single cells with moderate nuclear atypia (case 5). (B, C) Larger cell clusters exhibit partial (B) or complete cell dissociation at the perimeter (C), forming the cSES pattern (cases 7 and 5, respectively). Many of the clusters contain inflammatory cells (C). (D) Giant tumor cells are present in the atypical cells (case 6). Papanicolaou staining, ×40 magnification for scanning view (A) and ×400 magnification (B, C, D). POLEmut, DNA polymerase epsilon mutation.
Cervical cytology of the other subtypes. The cytological features already mentioned were not specific to the POLEmut-subtype. Figure 3 presents the representative cytological findings for the other subtypes. In the p53mut-subtype, the tumor cells were arranged in small clusters or single cells (as observed in the POLEmut-subtype), exhibiting higher-grade nuclear atypia with occasional GTCs present (Figure 3A). These p53mut-subtype clusters exhibited cSES (Figure 3B) and included inflammatory cells. The MMRd-subtype exhibited large clusters containing TILs, TINs, or both (Figure 3C). The NSMP-subtype mostly appeared in large clusters, and cSES, TILs and TINs were less frequent than in the other subtypes (Figure 3D).
Figure 3. Cytological features of the other subtypes. (A) In the p53mut-subtype, the atypical cells occur in small clusters or as single cells, with high-grade nuclear atypia; giant tumor cells are occasionally present (case 48). (B) The atypical cell clusters of the p53mut-subtype exhibit cSES (case 47). (C) In the MMRd-subtype, the atypical cells are present in large clusters that contain TILs, TINs, or both (case 16). (D) In the NSMPsubtype, the atypical cells occur in large clusters with low frequencies of cSES, TILs, and TINs (case 83). Papanicolaou staining, ×100 (A) and ×400 magnification (B, C, D). cSES: Cytologically detected surface epithelial slackening; MMRd: MMRd, Mismatch repair-deficient; TILs: tumorinfiltrating lymphocytes; TINs: tumor-infiltrating neutrophils; NSMP, no specific molecular profile; p53mut, p53 mutation.
Quantitative comparison of cervical cytology. We performed quantitative analysis to extract cytomorphological features characteristic of the POLEmut-subtype to distinguish it from the NSMP-subtype. Abnormal cytology (AGC+AGC-favor neoplastic+adenocarcinoma) was most frequent in the POLEmut-subtype (75%), followed by the p53mut- (61%), NSMP- (60%), and MMRd- (55%) subtypes. However, the frequency of abnormal cytology was not considerably different among the 4 subtypes (Figure 4A, Table II). In contrast, cell grouping differed significantly among the four subtypes (Figure 4B). Specimens classified as having predominantly smaller clusters or single cells were more frequent in the p53mut-subtype than in the NSMP-subtype. The proportion of the different cell groupings exhibited borderline significant differences between the MMRd- and p53mut-subtypes (p=0.092) and between the POLEmut- and NSMP-subtypes (p=0.085) (Table II).
Figure 4. Abnormal cytology and cell grouping in the four subtypes. (A) The frequency of abnormal cervical cytology (AGC + AGC-favor neoplastic + adenocarcinoma) is similar among the four subtypes. (B) In contrast, the cell grouping varies among the subtypes, with small clusters being more frequent in the p53mut- and POLEmut-subtypes (Table II). Differences among the four subtypes were evaluated using Fisher’s exact test. MMRd, Mismatch repair-deficient; NSMP, no specific molecular profile; p53mut, p53 mutation; POLEmut, DNA polymerase epsilon mutation; AGC, atypical glandular cell.
The POLEmut-subtype exhibited the highest frequency of atypical cell clusters with cSES (Figure 5A and B). ROC curve analysis revealed optimal cut-off value of 0.00 for the use of cSES frequency to distinguish the POLEmut-subtype from the other subtypes (Figure 5C), suggesting that the detection of even one cSES-positive cluster indicates a possible POLEmut-subtype diagnosis.
Figure 5. SES frequency in atypical cell clusters. (A) Frequency of cSES in the four subtypes (Kruskal-Wallis test). (B) cSES frequency is significantly higher in the POLEmut-subtype than in the others (Wilcoxon test). (C) The receiver operator characteristic (ROC) curve reveals that the cut-off value of cSES frequency for distinguishing the POLEmut-subtype is 0.000, indicating that the presence of cSES is highly suggestive for the cytological diagnosis of POLEmut-subtype. SES, Surface epithelial slackening; cSES, cytologically detected SES; MMRd, mismatch repair-deficient; NSMP, no specific molecular profile; p53mut, p53 mutation; POLEmut, DNA polymerase epsilon mutation.
The frequency of GTC varied among the four subtypes (Figure 6A), being more frequent in the p53mut-subtype than in the NSMP-subtype. GTC frequency was significantly higher in the p53mut- and POLEmut-subtypes than in the NSMP-subtypes (Table II). TIN frequency was similar among the four subtypes (Figure 6B), whereas TIL frequency varied (Figure 6C). TIL frequency was significantly higher in the POLEmut- and MMRd-subtypes than in the NSNP-subtype (Table II). In summary, for the POLEmut-subtype, cervical cytology revealed frequent atypical cells with moderate nuclear atypia, in small clusters or as single cells, and with the presence of cSES and TILs. In contrast, the NSMP-subtype exhibited large clusters with less frequent cSES, TIL, and GTCs. Table III summarizes the representative cervical cytology features estimated via cytomorphological analyses of each subtype.
Figure 6. GTC and tumor-infiltrating inflammatory cell frequency. (A) GTC frequency varies among the four subtypes, being higher in the p53mutand POLEmut-subtypes. (B) Tumor-infiltrating neutrophils (TIN) frequency is similar among the four subtypes. (C) Tumor-infiltrating lymphocytes (TIL) frequency varies among the subtypes, being higher in the MMRd- and POLEmut-subtypes (Table II). The frequencies were compared via the Fisher’s exact test. GTC: Giant tumor cells; MMRd, mismatch repair-deficient; NSMP, no specific molecular profile; p53mut, p53 mutation; POLEmut, DNA polymerase epsilon mutation.
Table III. Representative cytomorphology in each subtype of endometrial cancer.
SES, Surface epithelial slackening; POLEmut, DNA polymerase epsilon mutation; p53mut, p53 mutation; MMRd, mismatch repair-deficient; NSMP, no specific molecular profile; GCT, giant tumor cell; TIN, tumor infiltrating neutrophil; TIL, tumor infiltrating lymphocyte.
Discussion
Among the four subtypes, the POLEmut-subtype exhibited a higher incidence of abnormal cervical cytology in the form of cSES. Together with the presence of GTCs, cSES is suggestive of POLEmut subtype diagnosis (8,9). Although an integrative diagnosis should be made using a combination of histology, IHC, and molecular examinations (9,10), these findings reflect the usefulness of cervical cytology specimens in distinguishing the POLEmut-subtype from the NSMP-subtype.
In addition to glandular proliferation, endometrial cancers exhibit cellular features, such as mucin production, squamous metaplasia (morula) or obvious keratinization, clear cell changes, bizarre GTCs, and inflammatory cell infiltration into the cancer stroma and tumor nests (8,15,16). Endometrial cancers exhibit serous morphology, in which papillary growth generates a micropapillary pattern with hierarchical branching and condensation, leading to the formation of solid nests; this is diagnostic of serous carcinoma (4,17,18).
In contrast to serous morphology, SES occurs only in tumor cells facing the uterine lumen, and causes cell clusters to have more irregular contour, without hierarchical branching (9). In endometrioid carcinomas, SES-like papillary morphology has previously been referred to as “surface epithelial changes” (thin micropapillae without hierarchal branching) (19), or as small nonvillous papillae (20). These histological features may be reserved in cytological specimens, especially in LBC, which preserves cytomorphological features well (21,22). Our findings reveal that the histologically detected SES morphology was well preserved in the cervical cytology specimens. Similarly, in our study, the histologically detected serous morphology of the p53mut-subtype was reflected in its cytomorphology, which revealed cSES-like cell dissociation from cell clusters. Consequently, we were unable to distinguish cytologically between SES and serous morphology. The cytomorphological detection of cSES involves cytological detection of serous morphology. Therefore, presumptive or differential cytological diagnosis of the POLEmut-subtype should be made via comprehensive cytological observation, and not only based on the detection of cSES.
In addition to the SES pattern, our detection of GTCs in cervical cytology corresponds to that previously reported using histological specimens (8,9). Among the subtypes, TILs are observed predominantly in MMRd- and POLEmut-subtype histological and endometrial cytology specimens (8,23), and TINs occur less frequently (9). Here, we found that the presence of TILs and TINs was preserved in cervical cytology. Therefore, these findings provide evidence that cervical cytology specimens can preserve not only the tumor cell structure or arrangement but also the tumor constituents detected in histological sections.
Endometrial cytology is not a popular procedure worldwide, whereas cervical cytology is a practical and less invasive screening tool for the detection of both cervical and endometrial cancers (24,25). The reported frequencies of cervical cytology for the detection of endometrial cancers varies from 25.5% to 45.5% (26-29), while the non-endometrioid subtype and serous carcinoma exhibit higher frequencies (77% and 65.7%, respectively) (26,28). Consistent with this, in the present study, the frequency of abnormal cervical cytology was 60% (62/103 cases).
We have previously reported that a custom NGS cancer gene panel is useful for genomic classification of endometrial cancer according to the WHO system (10,11). The approach of the WHO integrative diagnostic system, which begins with genomic detection of the POLE mutation to differentiate the POLEmut-subtype (4,6), might lead to incorrect molecular classification in cases with multiple-classifier phenotypes (30). The ProMisE system, which meets the minimal requirements of the WHO classification system (31), provides an alternative approach. This system begins with IHC to detect MMR, followed by hotspot Sanger sequencing of the POLE exonuclease domain, and loss of MMR expression is diagnostic of the MMRd-subtype (32). However, as with the WHO integrative diagnostic system, the ProMisE strategy can lead to misclassification in multiple-classifier phenotype cases, such as those with both MMR deficiency and the POLE mutation (30). Based on our findings, cytological detection of features such as cSES are suggestive of a possible POLEmut-subtype diagnosis and can help to prevent misclassification. Nonetheless, we recommend additional genomic examination via an NGS panel. These findings reveal that cervical cytological specimens provide an essential resource for integrative diagnosis in endometrial cancer. This approach is made easier by the fact that residual LBC specimens are widely available for use in molecular analyses including DNA-, RNA-, and methylation-based NGS analyses (33-37).
Conclusion
Our findings show that, in cervical cytology, the cSES pattern is unique to POLEmut-subtype endometrial cancer and occurs frequently in this subtype. Detection of cSES in cervical cytology specimens indicates a possible diagnosis of the POLEmut-subtype, although an integrative diagnosis including genomic profiling remains critical for diagnostic confirmation.
Availability of Data
The data supporting the findings of the study are available upon reasonable request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
Conflicts of Interest
The Authors have no conflicts of interest to declare.
Authors’ Contributions
Toshiaki Akahane, Emi Kubota, and Yukari Nishida-Kirita contributed as cytotechnologists; Toshiaki Akahane and Ikumi Kitazono analyzed and interpreted the sequencing data; Seiya Yokoyama performed statistical analyses; Ikumi Kitazono and Hirotsugu Noguchi contributed to cytopathological and histopathological diagnosis; Shintaro Yanazume and Miki Murakami summarized the clinical data; Hiroaki Kobayashi and Akihide Tanimoto organized the study design, and wrote the article; and all authors have read and approved the final manuscript.
Acknowledgements
We would like to thank Editage (www.editage.com) for English language editing.
References
- 1.Lappalainen T, Scott AJ, Brandt M, Hall IM. Genomic analysis in the age of human genome sequencing. Cell. 2019;177(1):70–84. doi: 10.1016/j.cell.2019.02.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Holch JW, Metzeler KH, Jung A, Riedmann K, Jost PJ, Weichert W, Kirchner T, Heinemann V, Westphalen CB. Universal Genomic Testing: The next step in oncological decision-making or a dead end street. Eur J Cancer. 2017;82:72–79. doi: 10.1016/j.ejca.2017.05.034. [DOI] [PubMed] [Google Scholar]
- 3.Horak P, Fröhling S, Glimm H. Integrating next-generation sequencing into clinical oncology: strategies, promises and pitfalls. ESMO Open. 2016;1(5):e000094. doi: 10.1136/esmoopen-2016-000094. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Kim KR, Lax SF, Lazar AJ, Longacre TA, Malpica A, Matias-Guiu X, Nucci MR, Oliva E. Tumours of the uterine corpus. In: WHO classification of tumours female genital tumours, 5th edition. Lyon, France, IARC. 2020:pp. 245–308. [Google Scholar]
- 5.Cancer Genome Atlas Research Network, Kandoth C, Schultz N, Cherniack AD, Akbani R, Liu Y, Shen H, Robertson AG, Pashtan I, Shen R, Benz CC, Yau C, Laird PW, Ding L, Zhang W, Mills GB, Kucherlapati R, Mardis ER, Levine DA. Integrated genomic characterization of endometrial carcinoma. Nature. 2013;497(7447):67–73. doi: 10.1038/nature12113. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Vermij L, Smit V, Nout R, Bosse T. Incorporation of molecular characteristics into endometrial cancer management. Histopathology. 2020;76(1):52–63. doi: 10.1111/his.14015. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Kitazono I, Kobayashi Y, Akahane T, Yamaguchi T, Yanazume S, Nohara S, Sakamoto I, Tabata K, Tasaki T, Kobayashi H, Tanimoto A. ATM immunohistochemistry as a potential marker for the differential diagnosis of no specific molecular profile subtype and POLE-mutation subtype endometrioid carcinoma. Pathol Res Pract. 2022;230:153743. doi: 10.1016/j.prp.2021.153743. [DOI] [PubMed] [Google Scholar]
- 8.Van Gool IC, Ubachs JEH, Stelloo E, de Kroon CD, Goeman JJ, Smit VTHBM, Creutzberg CL, Bosse T. Blinded histopathological characterisation of POLE exonuclease domain-mutant endometrial cancers: sheep in wolf’s clothing. Histopathology. 2018;72(2):248–258. doi: 10.1111/his.13338. [DOI] [PubMed] [Google Scholar]
- 9.Kitazono I, Akahane T, Yokoyama S, Kobayashi Y, Togami S, Yanazume S, Tasaki T, Noguchi H, Tabata K, Kobayashi H, Tanimoto A. “Surface epithelial slackening” pattern in endometrioid carcinoma: A morphological feature for differentiating the POLE mutation-subtype from the no specific molecular profile subtype. Pathol Res Pract. 2023;247:154563. doi: 10.1016/j.prp.2023.154563. [DOI] [PubMed] [Google Scholar]
- 10.Kobayashi Y, Kitazono I, Akahane T, Yanazume S, Kamio M, Togami S, Nohara S, Sakamoto I, Yokoyama S, Tabata K, Kobayashi H, Tanimoto A. Molecular evaluation of endometrial dedifferentiated carcinoma, endometrioid carcinoma, carcinosarcoma, and serous carcinoma using a custom-made small cancer panel. Pathol Oncol Res. 2021;27:1610013. doi: 10.3389/pore.2021.1610013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Akahane T, Kitazono I, Yanazume S, Kamio M, Togami S, Sakamoto I, Nohara S, Yokoyama S, Kobayashi H, Hiraki T, Suzuki S, Ueno S, Tanimoto A. Next-generation sequencing analysis of endometrial screening liquid-based cytology specimens: a comparative study to tissue specimens. BMC Med Genomics. 2020;13(1):101. doi: 10.1186/s12920-020-00753-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Xu C, Gu X, Padmanabhan R, Wu Z, Peng Q, DiCarlo J, Wang Y. smCounter2: an accurate low-frequency variant caller for targeted sequencing data with unique molecular identifiers. Bioinformatics. 2019;35(8):1299–1309. doi: 10.1093/bioinformatics/bty790. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Brierley JD, Gospodarowicz MK, Wittekind C. Gynaecological tumours: TNM classification of malignant tumours, eighth edition. Oxford, UK, Wiley Blackwell. 2017:pp. 171–174. [Google Scholar]
- 14.Wilbur DC, Chhieng DC, Guidos B, Mody DR. Epithelial abnormalities: Glandular. The Bethesda System for Reporting Cervical. 2015;Cytology:193–240. doi: 10.1007/978-3-319-11074-5_6. [DOI] [Google Scholar]
- 15.Murali R, Davidson B, Fadare O, Carlson JA, Crum CP, Gilks CB, Irving JA, Malpica A, Matias-Guiu X, McCluggage WG, Mittal K, Oliva E, Parkash V, Rutgers JKL, Staats PN, Stewart CJR, Tornos C, Soslow RA. High-grade endometrial carcinomas: Morphologic and immunohistochemical features, diagnostic challenges and recommendations. Int J Gynecol Pathol . 2019;38 Suppl 1(Iss 1 Suppl 1):S40–S63. doi: 10.1097/PGP.0000000000000491. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Lucas E, Carrick KS. Low grade endometrial endometrioid adenocarcinoma: A review and update with emphasis on morphologic variants, mimics, immunohistochemical and molecular features. Semin Diagn Pathol. 2022;39(3):159–175. doi: 10.1053/j.semdp.2022.02.002. [DOI] [PubMed] [Google Scholar]
- 17.Wei JJ, Paintal A, Keh P. Histologic and immunohistochemical analyses of endometrial carcinomas: experiences from endometrial biopsies in 358 consultation cases. Arch Pathol Lab Med. 2013;137(11):1574–1583. doi: 10.5858/arpa.2012-0445-OA. [DOI] [PubMed] [Google Scholar]
- 18.Hoang LN, McConechy MK, Köbel M, Han G, Rouzbahman M, Davidson B, Irving J, Ali RH, Leung S, Mcalpine JN, Oliva E, Nucci MR, Soslow RA, Huntsman DG, Gilks CB, Lee C. Histotype-genotype correlation in 36 high-grade endometrial carcinomas. Am J Surg Pathol. 2013;37(9):1421–1432. doi: 10.1097/PAS.0b013e31828c63ed. [DOI] [PubMed] [Google Scholar]
- 19.Singh K, Simon RA, Zhang C, Quddus MR. “Surface epithelial changes” in uterine endometrioid carcinoma mimicking micropapillary serous borderline tumor of ovary: report of two cases and review of the literature. Diagn Pathol. 2011;6:13. doi: 10.1186/1746-1596-6-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Murray SK, Young RH, Scully RE. Uterine endometrioid carcinoma with small nonvillous papillae: an analysis of 26 cases of a favorable-prognosis tumor to be distinguished from serous carcinoma. Int J Surg Pathol. 2000;8(4):279–289. doi: 10.1177/106689690000800407. [DOI] [PubMed] [Google Scholar]
- 21.Tanaka R, Fujiwara M, Sakamoto N, Kanno H, Arai N, Tachibana K, Kishimoto K, Anraku M, Shibahara J, Kondo H. Cytological characteristics of histological types of lung cancer by cytomorphometric and flow cytometric analyses using liquid-based cytology materials. Diagn Cytopathol. 2023;51(6):356–364. doi: 10.1002/dc.25118. [DOI] [PubMed] [Google Scholar]
- 22.Tanaka R, Fujiwara M, Sakamoto N, Suzuki H, Tachibana K, Ohtsuka K, Kishimoto K, Kamma H, Shibahara J, Kondo H. Cytomorphometric and flow cytometric analyses using liquid-based cytology materials in subtypes of lung adenocarcinoma. Diagn Cytopathol. 2022;50(8):394–403. doi: 10.1002/dc.24978. [DOI] [PubMed] [Google Scholar]
- 23.Yanazume S, Iwakiri K, Kobayashi Y, Kitazono I, Akahane T, Mizuno M, Togami S, Tanimoto A, Kobayashi H. Cytopathological features associated with POLE mutation in endometrial cancer. Cytopathology. 2023;34(3):211–218. doi: 10.1111/cyt.13215. [DOI] [PubMed] [Google Scholar]
- 24.Munakata S. Diagnostic value of endometrial cytology and related technology. Diagn Cytopathol. 2022;50(7):363–366. doi: 10.1002/dc.24956. [DOI] [PubMed] [Google Scholar]
- 25.Toyoda S, Kawaguchi R, Kobayashi H. Clinicopathological characteristics of atypical glandular cells determined by cervical cytology in Japan: Survey of gynecologic oncology data from the Obstetrical Gynecological Society of Kinki District, Japan. Acta Cytol. 2019;63(5):361–370. doi: 10.1159/000498977. [DOI] [PubMed] [Google Scholar]
- 26.Skaznik-wikiel ME, Ueda SM, Frasure HE, Rose PG, Fleury A, Grumbine FC, Fader AN. Abnormal cervical cytology in the diagnosis of uterine papillary serous carcinoma: earlier detection of a poor prognostic cancer subtype. Acta Cytologica. 2011;55(3):255–260. doi: 10.1159/000324052. [DOI] [PubMed] [Google Scholar]
- 27.Amkreutz LCM, Pijnenborg JMA, Joosten DWL, Mertens HJMM, Van Kuijk SMJ, Engelen MJA, Bergmans M, Nolting WE, Kruitwagen RFPM. Contribution of cervical cytology in the diagnostic work-up of patients with endometrial cancer. Cytopathology. 2018;29(1):63–70. doi: 10.1111/cyt.12511. [DOI] [PubMed] [Google Scholar]
- 28.Frias-Gomez J, Benavente Y, Ponce J, Brunet J, Ibáñez R, Peremiquel-Trillas P, Baixeras N, Zanca A, Piulats JM, Aytés Á, Matias-Guiu X, Bosch FX, de Sanjosé S, Alemany L, Costas L, Screenwide Team Sensitivity of cervico-vaginal cytology in endometrial carcinoma: a systematic review and meta-analysis. Cancer Cytopathol. 2020;128(11):792–802. doi: 10.1002/cncy.22266. [DOI] [PubMed] [Google Scholar]
- 29.Frias-Gomez J, Tovar E, Vidal A, Murgui L, Ibáñez R, Peremiquel-Trillas P, Paytubi S, Baixeras N, Zanca A, Ponce J, Pineda M, Brunet J, de Sanjosé S, Bosch FX, Matias-Guiu X, Alemany L, Costas L, Screenwide Team. Sensitivity of cervical cytology in endometrial cancer detection in a tertiary hospital in Spain. Cancer Med. 2021;10(19):6762–6766. doi: 10.1002/cam4.4217. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.León-Castillo A, Gilvazquez E, Nout R, Smit VT, McAlpine JN, McConechy M, Kommoss S, Brucker SY, Carlson JW, Epstein E, Rau TT, Soslow RA, Ganesan R, Matias-Guiu X, Oliva E, Harrison BT, Church DN, Gilks CB, Bosse T. Clinicopathological and molecular characterisation of ‘multiple-classifier’ endometrial carcinomas. J Pathol. 2020;250(3):312–322. doi: 10.1002/path.5373. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Kommoss S, McConechy MK, Kommoss F, Leung S, Bunz A, Magrill J, Britton H, Kommoss F, Grevenkamp F, Karnezis A, Yang W, Lum A, Krämer B, Taran F, Staebler A, Lax S, Brucker SY, Huntsman DG, Gilks CB, McAlpine JN, Talhouk A. Final validation of the ProMisE molecular classifier for endometrial carcinoma in a large population-based case series. Ann Oncol. 2018;29(5):1180–1188. doi: 10.1093/annonc/mdy058. [DOI] [PubMed] [Google Scholar]
- 32.Talhouk A, McConechy MK, Leung S, Yang W, Lum A, Senz J, Boyd N, Pike J, Anglesio M, Kwon JS, Karnezis AN, Huntsman DG, Gilks CB, McAlpine JN. Confirmation of ProMisE: A simple, genomics-based clinical classifier for endometrial cancer. Cancer. 2017;123(5):802–813. doi: 10.1002/cncr.30496. [DOI] [PubMed] [Google Scholar]
- 33.Akahane T, Yamaguchi T, Kato Y, Yokoyama S, Hamada T, Nishida Y, Higashi M, Nishihara H, Suzuki S, Ueno S, Tanimoto A. Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis. PLoS One. 2019;14(6):e0217724. doi: 10.1371/journal.pone.0217724. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Yamaguchi T, Akahane T, Harada O, Kato Y, Aimono E, Takei H, Tasaki T, Noguchi H, Nishihara H, Kamata H, Tanimoto A. Next-generation sequencing in residual liquid-based cytology specimens for cancer genome analysis. Diagn Cytopathol. 2020;48(11):965–971. doi: 10.1002/dc.24511. [DOI] [PubMed] [Google Scholar]
- 35.Akahane T, Kitazono I, Kobayashi Y, Nishida-Kirita Y, Yamaguchi T, Yanazume S, Tabata K, Kobayashi H, Tanimoto A. Direct next-generation sequencing analysis using endometrial liquid-based cytology specimens for rapid cancer genomic profiling. Diagn Cytopathol. 2021;49(9):1078–1085. doi: 10.1002/dc.24841. [DOI] [PubMed] [Google Scholar]
- 36.Yokoyama S, Iwaya H, Akahane T, Hamada T, Higashi M, Hashimoto S, Tanoue S, Ohtsuka T, Ido A, Tanimoto A. Sequential evaluation of MUC promoter methylation using next-generation sequencing-based custom-made panels in liquid-based cytology specimens of pancreatic cancer. Diagn Cytopathol. 2022;50(11):499–507. doi: 10.1002/dc.25022. [DOI] [PubMed] [Google Scholar]
- 37.Akahane T, Isochi-Yamaguchi T, Hashiba-Ohnuki N, Bandoh N, Aimono E, Kato Y, Nishihara H, Kamada H, Tanimoto A. Cancer gene analysis of liquid-based cytology specimens using next-generation sequencing: A technical report of bimodal DNA- and RNA-based panel application. Diagn Cytopathol. 2023;51(8):493–500. doi: 10.1002/dc.25149. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data supporting the findings of the study are available upon reasonable request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.