Molecular Classification Of Endometrial Carcinoma Research Articles

Predicting Mismatch Repair Deficiency Status in Endometrial Cancer through Multi-Resolution Ensemble Learning in Digital Pathology.

For molecular classification of endometrial carcinoma, testing for mismatch repair (MMR) status is becoming a routine process. Mismatch repair deficiency (MMR-D) is caused by loss of expression in one or more of the 4 major MMR proteins: MLH1, MSH2, MSH6, PHS2. Over 30% of patients with endometrial cancer have MMR-D. Determining the MMR status holds significance as individuals with MMR-D are potential candidates for immunotherapy. Pathological whole slide image (WSI) of endometrial cancer with immunohistochemistry results of MMR proteins were gathered. Color normalization was applied to the tiles using a CycleGAN-based network. The WSI was divided into tiles at three different magnifications (2.5 × , 5 × , and 10 ×). Three distinct networks of the same architecture were employed to include features from all three magnification levels and were stacked for ensemble learning. Three architectures, InceptionResNetV2, EfficientNetB2, and EfficientNetB3 were employed and subjected to comparison. The per-tile results were gathered to classify MMR status in the WSI, and prediction accuracy was evaluated using the following performance metrics: AUC, accuracy, sensitivity, and specificity. The EfficientNetB2 was able to make predictions with an AUC of 0.821, highest among the three architectures, and an overall AUC range of 0.767 - 0.821 was reported across the three architectures. In summary, our study successfully predicted MMR classification from pathological WSIs in endometrial cancer through a multi-resolution ensemble learning approach, which holds the potential to facilitate swift decisions on tailored treatment, such as immunotherapy, in clinical settings.

Integration of the Molecular Classification of Endometrial Carcinoma to Select Patients for Fertility Sparing Strategies.

Fertility-sparing treatment (FST) for endometrial carcinoma (EC) is an option for a subgroup of young women with low-risk disease. The low-risk group comprises patients with endometrioid EC stage IA, grade 1, with or without focal lymphovascular invasion. In the era of molecular subtyping, treatment de-escalation for some EC subtypes is recommended. Recommendations for fertility-preserving treatments were developed regardless of the molecular classification of EC. However, few studies have focused on this topic. In this review, we summarize the actual data available in the literature and discuss the impact of some molecular subtypes of FST.

Molecular classification guides for the postoperative adjuvant therapy of early-stage endometrial carcinoma

Surgical treatment has been widely used for early-stage endometrial carcinoma patients, but 15-20% of patients had a poor prognosis, requiring a postoperative adjuvant therapy. With the development of molecular classification of endometrial carcinoma, the combination of molecular and clinicopathological factors can guide the prognosis risk assessment and make the adjuvant therapy selection more accurate, as a result, the survival outcome of patients can be greatly improved. In this article, the molecular classification of endometrial carcinoma is reviewed, and its guidance to the postoperative adjuvant therapy for early-stage endometrial carcinoma is discussed. It concludes that the molecular classification opens up the opportunity of creating new ideas for adjuvant treatment strategies for early endometrial cancer.

Comprehensive assessment of mismatch repair and microsatellite instability status in molecular classification of endometrial carcinoma

Objective: To explore the concordance and causes of different mismatch repair (MMR) and microsatellite instability (MSI) detection results in endometrial carcinoma (EC) molecular typing. Methods: A total of 214 EC patients diagnosed from January 2021 to April 2023 were selected at the Department of Pathology, Peking University Third Hospital. The immunohistochemistry (IHC) results of MMR protein were reviewed. Tumor specific somatic mutations, MMR germline mutations, microsatellite scores and tumor mutation burden (TMB) were detected by next-generation sequencing (NGS) with multi-gene panel. Methylation-specific PCR was used to detect the methylation status of MLH1 gene promoter in cases with deficient MLH1 protein expression. In cases with discrepant results between MMR-IHC and MSI-NGS, the MSI status was detected again by PCR (MSI-PCR), and the molecular typing was determined by combining the results of TMB and MLH1 gene promoter methylation. Results: (1) In this study, there were 22 cases of POLE gene mutation subtype, 55 cases of mismatch repair deficient (MMR-d) subtype, 29 cases of p53 abnormal subtype, and 108 cases of no specific molecular profile (NSMP). The median age at diagnosis of MMR-d subtype (54 years old) and the proportion of aggressive histological types (40.0%, 22/55) were higher than those of NSMP subtype [50 years old and 12.0% (13/108) respectively; all P<0.05]. (2) Among 214 patients, MMR-IHC test showed that 153 patients were mismatch repair proficient (MMR-p), 49 patients were MMR-d, and 12 patients were difficult to evaluate directly. MSI-NGS showed that 164 patients were microsatellite stable (MSS; equal to MMR-p), 48 patients were high microsatellite instability (MSI-H; equal to MMR-d), and 2 patients had no MSI-NGS results because the effective sequencing depth did not meet the quality control. The overall concordance between MMR-IHC and MSI-NGS was 94.3% (200/212). All the 12 discrepant cases were MMR-d or subclonal loss of MMR protein by IHC, but MSS by NGS. Among them, 10 cases were loss or subclonal loss of MLH1 and (or) PMS2 protein. Three discrepant cases were classified as POLE gene mutation subtype. In the remaining 9 cases, 5 cases and 3 cases were confirmed as MSI-H and low microsatellite instability (MSI-L) respectively by MSI-PCR, 6 cases were detected as MLH1 gene promoter methylation and 7 cases demonstrated high TMB (>10 mutations/Mb). These 9 cases were classified as MMR-d EC. (3) Lynch syndrome was diagnosed in 27.3% (15/55) of all 55 MMR-d EC cases, and the TMB of EC with MSH2 and (or) MSH6 protein loss or associated with Lynch syndrome [(71.0±26.2) and (71.5±20.1) mutations/Mb respectively] were significantly higher than those of EC with MLH1 and (or) PMS2 loss or sporadic MMR-d EC [(38.2±19.1) and (41.9±24.3) mutations/Mb respectively, all P<0.01]. The top 10 most frequently mutated genes in MMR-d EC were PTEN (85.5%, 47/55), ARID1A (80.0%, 44/55), PIK3CA (69.1%, 38/55), KMT2B (60.0%, 33/55), CTCF (45.5%, 25/55), RNF43 (40.0%, 22/55), KRAS (36.4%, 20/55), CREBBP (34.5%, 19/55), LRP1B (32.7%, 18/55) and BRCA2 (32.7%, 18/55). Concurrent PTEN, ARID1A and PIK3CA gene mutations were found in 50.9% (28/55) of MMR-d EC patients. Conclusions: The concordance of MMR-IHC and MSI-NGS in EC is relatively high.The discordance in a few MMR-d EC are mostly found in cases with MLH1 and (or) PMS2 protein loss or MMR protein subclonal staining caused by MLH1 gene promoter hypermethylation. In order to provide accurate molecular typing for EC patients, MLH1 gene methylation, MSI-PCR, MMR gene germline mutation and TMB should be combined to comprehensively evaluate MMR and MSI status.

787P A NGS panel for molecular classification of endometrial carcinoma

787P A NGS panel for molecular classification of endometrial carcinoma

Practical lessons learned from real-world implementation of the molecular classification for endometrial carcinoma

ObjectivesThis study aimed to explore the practical organisational aspects and difficulties in the implementation of the molecular classification of endometrial carcinoma (EC), and to demonstrate its potential impact in prognostic risk group classification. MethodsWe conducted a multicentre, retrospective cohort study of 230 patients with EC diagnosed between 2019 and 2022. Sample processing, clinicopathological, treatment and follow-up data were collected. Molecular classification was obtained by p53 and mismatch repair proteins immunohistochemistry, and POLE next-generation sequencing. ResultsImplementation was achieved through centralization of molecular analysis. In practice, it was possible to optimise turnaround times of complete integrative reports for hysterectomy specimens to a median time of 18 workdays. If genetic study was started in endometrial biopsies before surgery, 82.0% were available at the time of multidisciplinary tumour board, compared to 8.4% if performed in hysterectomy. ECs were classified as follows: 37.8% no specific molecular profile, 31.7% p53 abnormal, 24.3% mismatch repair deficient, and 6.1% POLE mutant. Integration of these results with traditional clinicopathologic factors led to a change in prognostic risk group in 15 (6.5%) patients, most being initially allocated to high-intermediate (n = 8) and low (n = 5) risk groups. Eight patients changed to a higher risk, and 7 to a lower risk group, whereas 2 remained in the same group. ConclusionsCentralization of EC molecular classification is a feasible option for countries with limited resources. Optimization of workflows may be achieved by earlier analysis in biopsies and prioritisation of patients whose results imply changes in risk group classification.

Comparative study of next generation sequencing and immunohistochemistry on molecular classification of endometrial carcinoma

Objective: To investigate the differences in molecular classification of endometrial carcinoma (EC) between various technical methods and to explore molecular classification schemes suitable for Chinese population. Methods: The study used a comprehensive scheme of next generation sequencing (NGS) and immunohistochemistry for molecular classification of 254 EC cases that were obtained at Department of Pathology, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China from April 2021 to March 2022. According to the recommended threshold of Sanger sequencing which was approximate-20% variant allele fraction (VAF), NGS data were extracted to simulate the results of Sanger sequencing. Results: The 254 EC patients had a mean age of 51 years (range, 24 to 89 years). Combination of POLE (9-14 exons), TP53 total exons and microsatellite instability (MSI) detection was a better single scheme than NGS alone, while combination of MSI fragment analysis and conventional immunohistochemistry was the best solution and seemed best aligned with TCGA data and recent studies. POLE ultramuted type, mismatch repair defect type, TP53 mutant type and non-specific molecular characteristic type accounted for 11.4% (29/254), 31.5% (80/254), 22.4% (57/254) and 34.6% (88/254) of the cases, respectively. If Sanger sequencing was adopted for POLE and TP53 detection, the frequencies of these EC types were 9.1% (23/254), 31.5% (80/254), 12.9% (33/254) and 46.6% (118/254), respectively, with greatly increasing non-specific molecular characteristics cases. If POLE was detected by Sanger sequencing and others by immunohistochemistry, they were 9.1% (23/254), 42.2% (92/218), 13.8% (35/254) and 40.9% (105/254), respectively, with increasing the false positive rates of the mismatch repair defect group. Conclusions: Small and medium-sized NGS panels with MSI detection is a better solution than NGS alone. Sanger sequencing is currently available for POLE mutation detection, which is not sensitive enough for TP53 mutation detection, and seems equivalent to the efficiency of TP53 by immunohistochemistry. Further optimization of small and medium-sized NGS panels covering MSI detection and POLE and TP53 full exons may be the best choice for the future to meet national conditions.

Tumor-Infiltrating CD8-Positive T-Cells Associated with MMR and p53 Protein Expression Can Stratify Endometrial Carcinoma for Prognosis.

Inspired by the molecular classification of endometrial carcinoma (EC) proposed by The Cancer Genome Atlas Research Network (TCGA), we investigated tumor-infiltrating CD8-positive T-cell as well as DNA mismatch repair (MMR) protein and p53 protein expression, and we developed a new classification system for ECs to predict patients' prognosis using immunohistochemical methods. The study included 128 patients with ECs who underwent surgery. Paraffin-embedded tissue sections of the tumor were stained using antibodies against MMR protein, p53, and CD8. Cases were stratified into four classes by a sequential algorithm. An immunohistochemical classification system for ECs (ICEC) was created, including HCD8, MMR-D, LCD8, and p53 LCD8. In ICEC, 16 cases (12.5%), 27 cases (21.09%), 67 cases (52.34%), and 18 cases (14.06%) belonged to HCD8, MMR-D, LCD8, and p53 LCD8, respectively. ICEC did not show any correlation with clinical stage, lymphovascular space invasion, or lymph node metastasis. However, the p53 LCD8 class contained a significantly higher proportion of G3 ECs and serous carcinoma (p < 0.0001). ICEC showed prognostic significance in overall survival (OS) (p < 0.0001) and disease-free survival (DFS) (p < 0.0001). The class of p53 LCD8 showed the worst prognosis among the classes. ICEC classification is useful in predicting the prognosis of ECs.

Molecular classification of endometrial carcinoma: Analysis of a multicentre Portuguese cohort.

e17615 Background: Management of endometrial carcinoma (EC) relies on prognostic risk group classification to help to determine the individual risk of recurrence and the need for adjuvant treatment after surgery. A molecular classification with four distinct prognostic EC subtypes based on genomic abnormalities - DNA polymerase epsilon ( POLE) mutated ( POLEmut), mismatch repair deficient (MMRd), p53 abnormal (p53abn) and no specific molecular profile (NSMP) - has emerged, that raises the possibility of a more precise tailoring of adjuvant therapy. This study aimed to describe the clinicopathological and molecular characteristics of an EC Portuguese cohort, assess its potential impact in patient management and evaluate its prognostic value. Methods: Multicentre, retrospective cohort study of 230 patients with EC diagnosed between 2019 and 2022. Sample processing, clinicopathological, treatment and follow-up data was collected. Molecular classification was obtained by p53 and mismatch repair proteins immunohistochemistry, and POLE next-generation sequencing. Results: Overall, 230 patients from two institutions were included. Median age at diagnosis was 68 years. The most frequent histology was endometrioid (n=163; 70.9%) and most were low-grade (n=129, 56.1%).At diagnosis, disease was confined to the uterus in the majority of patients (FIGO stage I/II, n=153, 66.5%). Most had surgery upfront (n=196, 85.2%) and, of those, 120 (52.2%) received adjuvant treatment. 26 (11.3%) had metastatic disease. Median follow-up time was 15.4 months. Regarding molecular subgroups, the majority were classified as NSMP (n=87, 37.8%), followed by p53abn (n=73, 31.7%), MMRd (n=56, 24.3%), and POLEmut (n=14, 6.1%); 11 (4.8%) were multiple-classifier. Integration of these results led to a change in adjuvant treatment in 3 patients. Median disease-specific survival (DSS) was 108 months. Factors that significantly influenced survival included age (p=0.030), histological type (p&lt;0.001), grade (p&lt;0.001) and FIGO stage (p=0.006). Overall, 35 (15.2%) patients developed recurrences. Median recurrence-free survival (RFS) was 46.7 months. Kaplan-Meier survival analysis showed that molecular alterations were significantly associated both with DSS and RFS (p=0.0092 and p=0.0139, respectively). Tumours with p53abn had the worst prognosis, and patients with POLEmut tumours experienced an excellent prognosis. Comparative measures (BIC, C-index, log-likelihood) showed that considering the molecular classification in establishing risk groups for adjuvant treatment had prognostic significance. Conclusions: The molecular classification has prognostic value and should be considered in adjuvant treatment decisions. It allows for a more personalised approach, helps to reduce under and overtreatment, and therefore reduce patient morbidity associated with treatment toxicities, and healthcare related costs.

Integration of clinical sequencing and immunohistochemistry for the molecular classification of endometrial carcinoma

PurposeUsing next generation sequencing (NGS), The Cancer Genome Atlas (TCGA) found that endometrial carcinomas (ECs) fall under one of four molecular subtypes, and a POLE mutation status, mismatch repair (MMR) and p53 immunohistochemistry (IHC)-based surrogate has been developed. We sought to retrospectively classify and characterize a large series of unselected ECs that were prospectively subjected to clinical sequencing by utilizing clinical molecular and IHC data. Experimental designAll patients with EC with clinical tumor-normal MSK-IMPACT NGS from 2014 to 2020 (n = 2115) were classified by integrating molecular data (i.e., POLE mutation, TP53 mutation, MSIsensor score) and MMR and p53 IHC results. Survival analysis was performed for primary EC patients with upfront surgery at our institution. ResultsUtilizing our integrated approach, significantly more ECs were molecularly classified (1834/2115, 87%) as compared to the surrogate (1387/2115, 66%, p < 0.001), with an almost perfect agreement for classifiable cases (Kappa 0.962, 95% CI 0.949–0.975). Discrepancies were primarily due to TP53 mutations in p53-IHC-normal ECs. Of the 1834 ECs, most were of copy number (CN)-high molecular subtype (40%), followed by CN-low (32%), MSI-high (23%) and POLE (5%). Histologic and genomic variability was present amongst all molecular subtypes. Molecular classification was prognostic in early- and advanced-stage disease, including early-stage endometrioid EC. ConclusionsThe integration of clinical NGS and IHC data allows for an algorithmic approach to molecularly classifying newly diagnosed EC, while overcoming issues of IHC-based genetic alteration detection. Such integrated approach will be important moving forward given the prognostic and potentially predictive information afforded by this classification.

Abstract 4542: Development of a one-step molecular classifier for endometrial carcinoma using an amplicon-based gene panel and next generation sequencing technology

Abstract Objectives: Molecular classification of endometrial carcinoma (EC) is now recommended by the WHO, ESGO/ESTRO/ESP and NCCN guidelines. The pragmatic molecular classification tool, ProMisE, identifies four molecular subtypes based on next generation sequencing (NGS) for the detection of somatic pathogenic POLE mutations, and immunohistochemistry for mismatch repair and p53 proteins. ProMisE provides valuable prognostic and predictive information to direct care, however, multiple molecular results are often received from different time periods and/or from different centers which can cause delays. Therefore, we developed a classifier that relies on a single DNA-based NGS test with the goal of recapitulating the prognostic value of ProMisE by producing concordant results. Methods: Formalin fixed paraffin embedded (FFPE) EC tumor DNA was sequenced using the clinically validated Imagia Canexia Health Find ItTM amplicon-based next generation sequencing gene panel assay. The 38 gene panel assessed single nucleotide variants (SNVs), indels (insertions and deletions), gene amplification and microsatellite instability (MSI) using 21 MSI loci. To compare to the original ProMisE classifier, we identified somatic mutations in POLE, TP53 and MSI-High or MS-Stable for molecular classification. Molecular subtypes assigned by both classifiers were assessed for concordance metrics and Kaplan-Meier survival curves. Results: The one-step NGS molecular classifier assessed 165 unique patient FFPE EC samples that had previously been assessed by ProMisE. There were 152/165 cases that were concordant between molecular subtype assignment from the original ProMisE and the one-step NGS assay with a kappa statistic of 0.88 and accuracy of 0.92. There were 13 samples that were discordant (original classifications: 3 POLE, 3 MMRd, 2 p53abn, 5 NSMP/p53wt), which will be reviewed in detail. Results were concordant in 14 of 15 cases (93%) when both diagnostic biopsy and hysterectomy specimens were tested. Molecular subtypes maintained their associations with clinical outcomes (progression free survival, disease specific survival, overall survival). Conclusion: The one-step NGS molecular classifier demonstrates high concordance with the original ProMisE classifier including between biopsy and hysterectomy samples. This shows that reliable molecular testing could be obtained from time of first diagnosis. Prognostic value of this new one-step classification tool is maintained. Appropriate interpretation of results is critical, including limiting POLE mutation assignment to a confirmed list of pathogenic mutations and correct order of segregation for ECs with more than one molecular feature. Further validation is needed in a larger cohort to implement into standard of care testing. Citation Format: Melissa K. McConechy, Amy Jamieson, Amy Lum, Samuel Leung, Adrian Kense, Sonal Brahmbhatt, Ka Mun Nip, Ebru Baran, Dilmi Perera, Kurt Yakimovich, Ruth Miller, C. Blake Gilks, David Huntsman, Jessica N. McAlpine. Development of a one-step molecular classifier for endometrial carcinoma using an amplicon-based gene panel and next generation sequencing technology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4542.

Spatial distribution of CD3- and CD8-positive lymphocytes as pretest for POLE wild-type in molecular subgroups of endometrial carcinoma.

Over the years, the molecular classification of endometrial carcinoma has evolved significantly. Both POLEmut and MMRdef cases share tumor biological similarities like high tumor mutational burden and induce strong lymphatic reactions. While therefore use case scenarios for pretesting with tumor-infiltrating lymphocytes to replace molecular analysis did not show promising results, such testing may be warranted in cases where an inverse prediction, such as that of POLEwt, is being considered. For that reason we used a spatial digital pathology method to quantitatively examine CD3+ and CD8+ immune infiltrates in comparison to conventional histopathological parameters, prognostics and as potential pretest before molecular analysis. We applied a four-color multiplex immunofluorescence assay for pan-cytokeratin, CD3, CD8, and DAPI on 252 endometrial carcinomas as testing and compared it to further 213 cases as validation cohort from a similar multiplexing assay. We quantitatively assessed immune infiltrates in microscopic distances within the carcinoma, in a close distance of 50 microns, and in more distant areas. Regarding prognostics, high CD3+ and CD8+ densities in intra-tumoral and close subregions pointed toward a favorable outcome. However, TCGA subtyping outperforms prognostication of CD3 and CD8 based parameters. Different CD3+ and CD8+ densities were significantly associated with the TCGA subgroups, but not consistently for histopathological parameter. In the testing cohort, intra-tumoral densities of less than 50 intra-tumoral CD8+ cells/mm2 were the most suitable parameter to assume a POLEwt, irrespective of an MMRdef, NSMP or p53abn background. An application to the validation cohort corroborates these findings with an overall sensitivity of 95.5%. Molecular confirmation of POLEmut cases remains the gold standard. Even if CD3+ and CD8+ cell densities appeared less prognostic than TCGA, low intra-tumoral CD8+ values predict a POLE wild-type at substantial percentage rates, but not vice versa. This inverse correlation might be useful to increase pretest probabilities in consecutive POLE testing. Molecular subtyping is currently not conducted in one-third of cases deemed low-risk based on conventional clinical and histopathological parameters. However, this percentage could potentially be increased to two-thirds by excluding sequencing of predicted POLE wild-type cases, which could be determined through precise quantification of intra-tumoral CD8+ cells.

Significance of molecular classification in fertility-sparing treatment of endometrial carcinoma and atypical endometrial hyperplasia

Objective: To investigate the molecular classification of endometrial carcinoma (EC) and atypical endometrial hyperplasia (AEH) treated with fertility-sparing therapy, and to analyze its relationship with clinicopathological factors and treatment efficacy. Methods: A total of 46 EC and AEH patients who received fertility-sparing therapy and molecular classification tested by next generation sequencing in Peking University People's Hospital from June 2020 to December 2021, were retrospectively collected. The relationships between molecular classification and clinicopathological factors and treatment outcomes were analyzed. Results: (1) Of the 46 patients, including 40 EC and 6 AEH patients, 32 cases (71%, 32/45) had complete response (CR) after treatment, with median CR time of 8 months, 6 cases (13%, 6/45) had partial response, and 8 cases (25%, 8/32) had recurrence. (2) The cases were distributed as no specific molecular profile (NSMP) 34 cases (74%, 34/46) subtype mainly, high microsatellite instability (MSI-H) 7 cases (15%, 7/46), POLE ultra-mutated 3 cases (7%, 3/46), and copy number high (CNH) 2 cases (4%, 2/46). Patients with CNH had the hightest serum cancer antigen 125 (CA125) level [(34.3±35.2) kU/L]. MSI-H subtype had more family history of tumors (6/7), more with loss of mismatch repair (MMR) protein expression by immunohistochemical (7/7), and higher nuclear antigen associated with cell proliferation (Ki-67) expression level (3/3). (3) Patients in MSI-H subgroup had the lowest CR rate at 6 months (0/6; P=0.019), and survival analysis showed that they were less likely to achieve CR than those with NSMP subtype (P=0.022). Subgroup analysis of patients with NSMP showed that age ≥30 years related with longer treatment time to CR (P=0.010). In addition, CR was obtained after treatment in 2/3 POLE ultra-mutated cases and 2/2 CNH, respectively. Conclusions: Molecular classification relates with the treatment response in patients with EC and AEH treated with fertility-sparing therapy. Patients with MSI-H subtype have poor treatment efficacy, and patients with NSMP need to be further studied and predict treatment benefit. However, there are few cases in POLE ultra-mutated and CNH subtypes, which need further clinical research.

576P Genome instability index used for the optimization of molecular classification of endometrial carcinoma

The molecular classification of endometrial carcinoma (EC) has divided patients into four distinct prognostic subtypes based on the genomic abnormalities. However, in clinical application, the genomic copy number index lacks a simple and accurate detection method, and the method of using p53 immunohistochemistry or TP53 mutation as a surrogate for copy number status needs to be optimized. Here, we explored the application of genome instability (GI) index in molecular classification of EC. We assessed a retrospective cohort of EC from Beijing Cancer Hospital between 2011 and 2020. Genomic profiling of tumor tissues from EC patients was sequenced by OncoScreen Plus Panel of 520 cancer-related genes, and tumor immune microenvironment was evaluated using multiplex immunofluorescence staining. GI index was calculated as the sum of loss of heterozygosity, telomeric allelic imbalance, and large-scale state transitions scores. 143 patients were enrolled in this study, with the median follow-up time of 38.77 months. Molecular analysis firstly identified four different prognostic subgroups: POLE mutations (mut), MSI-H, TP53 wildtype, and TP53 mut. We also divided patients into two groups based on GI index: GI-high and GI-low, and patients with GI-high were found in three subgroups except for POLE mut. Further analysis showed that GI status had a more important effect on prognosis than TP53 mutation. Therefore, we proposed a three-group molecular classification based on POLE mutation and GI index: POLE mut (n=9, 6.5%), GI-low (n=116, 82.9%), and GI-high (n=15, 10.7%). Patients with GI-high had much poorer survival than those with GI-low [disease-free survival: hazard ratio (HR), 17.87, 95% CI, 6.67-50.00, P < 0.001; overall survival: HR, 16.75, 95% CI, 4.76-50.00, P < 0.001]. The application in TCGA-UCEC cohort containing 481 EC patients confirmed that this new method worked well for risk stratification of EC. Further analysis showed that patients in GI-high group had higher tumor grade, aneuploidy and lower T cell infiltrations. The one-stop method of GI index combined with POLE mutation offers more accurate and stable disease risk stratification and demonstrates the potential for guiding therapy for EC patients.

One-stop molecular classification of endometrial carcinoma using comprehensive next-generation sequencing.

This study aims to investigate the feasibility of molecular classification using only comprehensive next-generation sequencing-based techniques and its relationship with survival outcomes in patients with endometrial cancer. Paired tumor-normal sequencing data of 1021 cancer-related genes using tumor tissues or peripheral blood samples and clinical data were retrospectively collected from a cohort of endometrial cancers. The microsatellite instability status was inferred using the MSIsensor (v0.5) with a cut-off of 8%. Sixty patients were classified into four groups: POLEMUT group (13.3%), MSI-H group (20%), TP53WT group (45%) and TP53MUT group (21.7%). Patients within TP53MUT group were more common in serous carcinoma compared to endometrioid carcinoma (P=.0098). TP53WT was significantly correlated with early stage and low grade. TP53MUT group was associated with significantly worse DFS compared to MSI-H group and TP53WT group (P=.014 and .004, respectively). Comprehensive next-generation sequencing is a reliable and simple method to stratify the prognosis of endometrial carcinoma. It can be potentially used to guide treatment of patients with endometrial cancer in routine practice.

Molecular classification of endometrial carcinoma applied to endometrial atypical hyperplasia biopsy specimens.

e17622 Background: 50% of endometrial atypical hyperplasia (AH) cases on endometrial sampling have concurrent endometrial cancer (EC) on hysterectomy. Little data exists regarding molecular signatures that can predict the presence of concurrent endometrial cancer in patients with AH on biopsy. Therefore, we set to characterize molecular landscapes of endometrial samples diagnosed with AH with EC on the final hysterectomy specimen, using matched controls. Methods: Karmanos Cancer Institute pathology database was searched for patients with AH on endometrial curettage or biopsy who had a hysterectomy within 6 months. Only samples with adequate tissue for next generation sequencing were included. In total, 59 tissue samples from 34 patients were included: there were 15 AH endometrial biopsy samples, 18 AH final hysterectomy samples (13 matched pairs), 13 EC endometrial biopsies and 13 samples that were EC on final hysterectomy samples (12 matched pairs) that were analyzed using next-generation sequencing (WES) and/or whole transcriptome sequencing (WTS) (NovaSeq). TMB was measured by counting all somatic mutations found per tumor (TMB-high: &gt;10 mutations per MB). Immune cell infiltrates were calculated by Quantiseq. Significance was determined using Fisher exact, Chi-square and Mann-Whitney U test and adjusted for multiple comparisons: p &lt; 0.05 but q &gt; 0.05 was considered a trend. Results: Clinical analysis demonstrated 15/34 patients with endometrial AH on initial sampling and EC on the final hysterectomy. 2 out of 15 patients were &gt; stage II at time of surgery. Molecularly, fewer PTEN mutations were found between AH (2/8) and EC (14/17) on final hysterectomy (25% vs 82.4%, p = 0.01). Samples of EC origin were MSI-H by NGS-MSI (3/18) and had no mutations in PPP2R1A (0/16). Comparing hysterectomy samples, EC trended toward increased CTLA4 (FC: 6.97-fold) expression and immune cell infiltration of Macrophage M1 (+1.19%), NK cells (+2.13%), CD8+ T Cells (+1.27%), regulatory T cells (+2.17%) and Dendritic cells (+2.87%) compared to AH samples (all p &lt; 0.05). Similarly, when comparing endometrial biopsies, EC samples trended toward increased expression of CTLA4 (12.6-fold), HAVCR2/TIM3 (FC: 2.59-fold) and IFNG (FC: 17.8-fold) immune checkpoint genes, as well as increased immune cell infiltration of Neutrophils (+11.7%), CD8+ T cells (+2.39%) and regulatory T cells (+2.43%) compared to AH samples (all p &lt; 0.05). Conclusions: There are molecular and tumor microenvironment differences seen between AH endometrial biopsies that have concurrent EC compared to those that do not on the final hysterectomy specimens. These differences may lead to advances in identifying appropriate patients for fertility sparing treatments, versus those that can be managed surgically by a benign gynecologist or a gynecologic oncologist. Analyses with larger sample sizes are needed and are ongoing.

Optimized molecular classification of endometrial carcinoma-based on the combination of POLE mutations with homologous recombination deficiency (HRD).

e17628 Background: Molecular analysis of endometrial carcinoma (EC) has identified four clinically significant molecular subgroups with different prognoses: POLE mutations, microsatellite instability-high (MSI-H/dMMR), copy number low (CN-L/p53-normal), and copy number high (CN-H/p53-aberrant). However, the convenience and repeatability of detection methods for molecular classification, as well as the evaluation of their prognostic significance, need to be further explored. Recently, homologous recombination deficiency (HRD) has been studied to show the prognostic value in EC. Here, we aimed to establish a next-generation sequencing (NGS)-based one-stop method with the combination of HRD and POLE mutations for molecular classification of EC. Methods: Paraffin-embedded tissues of 143 eligible EC patients were collected from Beijing Cancer Hospital between 2011 and 2020 and sequenced using a panel of 520 cancer-related genes which could also detect MSI status (OncoScreen Plus, Burning Rock Biotech). HRD score was calculated as the sum of loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST) scores based on the sequencing data. Results: The median follow-up time in this cohort was 38.77 months. We firstly classified EC patients into four subgroups: POLE mutations (mut) (n = 9, 6.3%), MSI-H (n = 45, 31.5%), TP53 wildtype (wt) (n = 67, 46.9%), and TP53 mut (n = 22, 15.4%). POLE mut subgroup had the excellent prognosis as no patient in this subgroup relapsed after surgical resection. Compared with TP53 mut subgroup, the other two subgroups had better prognosis [disease free survival (DFS): TP53 wt: hazard ratio (HR), 0.2, 95% CI, 0.07-0.57, P = 0.003; MSI-H: HR, 0.2, 95% CI, 0.06-0.68, P = 0.01; overall survival (OS): TP53 wt: HR; 0.11, 95% CI, 0.02-0.56, P = 0.008; MSI-H: HR, 0.25, 95% CI, 0.06-1.03, P = 0.055]. Except for the patients with POLE mut, the others were then further optimized and divided into two groups based on HRD score with the cutoff of 13: HRD-high (n = 15, 11.5%) and HRD-low (n = 116, 88.5%). Patients in HRD-low subgroup showed much improved survival than those in HRD-high subgroup (DFS: HR, 0.06, 95% CI, 0.02-0.15, P &lt; 0.001; OS: HR, 0.06, 95% CI, 0.02-0.21, P &lt; 0.001). Most notably, the postoperative recurrence rate was 36.4% in the TP53 mut subgroup with the worst prognosis in the traditional classification, and 66.7% in the HRD-high subgroup with the worst prognosis in the optimized classification. Conclusions: The method of classifying EC patients into four subgroups by NGS large panel provides similar clinical features to previous reports classified by other methods but is more convenient and stable. Furthermore, the method of combination of POLE mutations with HRD offers more accurate disease risk stratification and demonstrates the potential for directing therapy for EC patients.

Molecular classification of endometrial carcinoma, is it the new era of precision medicine?

For many years endometrial cancer has been subdivided into oestrogen - dependent (type I) and oestrogen - independent (type II), according to classical Bokhman classification. Histopathological evaluation including type and grade of tumour, along with clinical factors have been considered as very important prognostic factors that impact treatment decision. However, histologically similar tumours may have different outcomes. Recent molecular findings and new histopathological parameters have given new concept on risk stratification. The Cancer Genome Atlas Research Network (TCGA) of tumours have brought new insights into endometrial cancer management. Four molecular subgroups have been described: POLE ultramutated (POLE mut), p53 mutant (p53abn), mismatch repair deficient (MMRd) and non-specific molecular profile (NSMP). This new subdivision has been recently introduced in the European risk stratification system.

Updates and Diagnostic Challenges in Surgical Pathology and Cytopathology.

Updates and Diagnostic Challenges in Surgical Pathology and Cytopathology.

Molecular testing in endometrial carcinoma - joint recommendation of Czech Oncological Society, Oncogynecological Section of the Czech Gynecological and Obstetrical Society, Society of Radiation Oncology, Biology and Physics, and the Society of Czech Pathologists.

Molecular classification of endometrial carcinoma is becoming an important part of the dia-gnostic process with direct therapeutic implications. Recent international guidelines, including the joint recommendation of the European Society of Gynaecological Oncology, the European Society for Radiotherapy and Oncology and the European Society of Pathology include the molecular classification into standard dia-gnostic algorithms. Molecular testing of endometrial carcinomas is also recommended in the latest (5th edition) of the World Health Organization classification of female genital tumors. Due to the need to implement these recommendations in practice, representatives of four professional societies of the Czech Medical Association of J. E. Purkyn&#283; (the Czech Oncological Society, the Oncogynecological Section of the Czech Gynecological and Obstetrical Society, the Society of Radiation Oncology, Biology and Physics, and the Society of Czech Pathologists) organized a meeting focused on this topic. Recommendation for molecular testing of endometrial carcinoma in routine dia-gnostic practice in the Czech Republic.