Polymerase Epsilon Mutations Research Articles

Excellent concordance of the molecular classification between preoperative biopsy and final hysterectomy in endometrial carcinoma

ObjectiveThe 2023 International Federation of Gynecology and Obstetrics classification with molecular classification shows superior discriminatory ability compared to staging systems lacking molecular data. However, the accuracy of endometrial biopsy data in molecular classification remains uncertain. This study aimed to assess the concordance of molecular classifications between preoperative biopsy and hysterectomy to predict prognosis before surgical staging. MethodsEndometrial biopsies and corresponding hysterectomy specimens were collected at the National Cancer Center Hospital between 2012 and 2023. Immunohistochemistry for p53 and mismatch repair (MMR) proteins and next-generation sequencing of all exons of polymerase epsilon (POLE) were performed. Given the limited number of POLE mut cases in prior studies, we prepared a POLE mut-enriched cohort. Cohen's kappa estimates were used to determine concordance for molecular and clinicopathological subgroup assignments. ResultsAmong 70 patients classified into four molecular subtype groups, 33 exhibited POLE mutations, 15 showed loss of MMR protein expression, 13 had p53-abnormality, and 9 had no specific molecular profile. Concordance between biopsy and hysterectomy specimens was 100% (κ = 1.000). In contrast, histological types and grades between biopsy and surgical specimens showed moderate and substantial agreement (κ = 0.420 and κ = 0.780, respectively). ConclusionsMolecular subtypes were completely consistent with those derived from surgical specimens, demonstrating high concordance between preoperative and postoperative molecular classifications. This suggests that endometrial biopsies could reliably predict prognosis. Future studies should investigate how biopsy-based molecular profiling influences treatment planning and patient outcomes.

Morphomolecular Correlation and Clinicopathologic Analysis in Endometrial Carcinoma.

Research groups have identified 4 groups [polymerase epsilon (POLE) mutant, mismatch repair-deficient, p53-abnormal, and no specific molecular profile)] reflecting the Tumor Cancer Genomic Atlas Research Network subgroups in endometrial carcinomas, improving the clinical applicability of molecular classification. We have analyzed the histopathologic and prognostic characteristics of our cases based on the ProMisE classification, supported by growing data on recommended treatment regimens. The study included 118 cases of endometrial carcinoma diagnosed between 2016 and 2020, which underwent mismatch repair and p53 immunohistochemistry. Next-generation sequencing was performed for POLE mutation analysis, dividing the cases into 4 subgroups. The histopathologic and clinical characteristics of these groups were then analyzed statistically. Four cases(3.4%) were classified as POLE mutant, 31 (26.3%) as mismatch repair-deficient, 22 (18.6%) as p53 mutant, and 61 (51.7%) as no specific molecular profile. We categorized 118 patients with endometrial carcinoma into low (n=43), intermediate (n=28), high-intermediate (n=21), high (n=22), and advanced metastatic (n=4) risk groups regardless of the molecular subtypes of their disease. When we reclassified all cases according to the molecular subtypes of endometrial carcinoma only the risk group of 3 (2.5%) cases changed. Using the new algorithm we designed, after narrowing down the number of patients, the microcystic, elongated, and fragmented pattern of invasion was revealed as an independent prognostic factor that reduces overall survival time (hazard ratio: 16.395, 95% CI: 2.140-125.606, P =0.007). In conclusion, using the new algorithm we have designed, and by identifying patients for whom molecular classification could alter risk groups, we observed that molecular tests can be utilized more efficiently in populations with limited economic resources and, in doing so, we discovered a new morphologic marker with prognostic significance.

Morules and β-catenin predict POLE mutation status in endometrial cancer: A pathway to more cost-effective diagnostic procedures.

The characterization of DNA polymerase epsilon (POLE) mutations has transformed the classification of endometrial endometrioid carcinomas (EECs), highlighting the need for efficient identification methods. This study aims to examine the relationship between distinct morphologic features-namely, squamous morules and squamous differentiation (SD), as well as β-catenin expression-and the POLE mutation status in endometrial cancer (EC). Our study included 35 POLE-mutated (POLEmut) EC cases and 395 non-POLEmut EEC cases. Notably, we observed no presence of morules in POLEmut cases, while SD was identified in 20% of instances. Conversely, morules and SD were identified in 12.7% and 26.1% of non-POLEmut EC cases, respectively, with morules consistently linked to a POLE wild-type status. The nuclear β-catenin expression is typically absent in tumors with wild-type POLE (wt-POLE) status. Our findings suggest that the presence of either morules or nuclear β-catenin expression in EEC could practically rule out the presence of POLE mutations. These morphologic and immunohistochemical features can be used as preliminary screening tools for POLE mutations, offering significant savings in time and resources and potentially enhancing clinical decision-making and patient management strategies. However, further validation in larger, multi-institutional studies is required to fully understand the implications of these findings on clinical practice.

Clinical characterization and therapeutic outcomes of patients (pts) with colorectal cancer (CRC) harboring somatic BRCA1/2 mutations.

134 Background: Somatic BRCA1/2 mutations in BRCA-associated cancers increase therapeutic sensitivity to platinum-based chemotherapies and PARP inhibitors, but the implications of BRCA mutations in non- BRCA-associated cancers such as CRC is unknown and hypothesized to be shaped by tumor lineage. We thus conducted a retrospective analysis of outcomes with platinum-based chemotherapy and PARP inhibition in pts with CRC, where the estimated prevalence of BRCA1/2 alterations is 3-4%. Methods: Pts with blood or tissue next generation sequencing (NGS) positive for a somatic BRCA1/2 variant and a diagnosis of CRC were identified from a single institution database. The pathogenicity of each mutation was annotated using public genomic databases (e.g. ClinVar). Pt characteristics were compared using Fisher’s exact test. Log-rank test was used for comparison of survival distribution among groups. Results: 8,258 NGS reports with a BRCA1/2 alteration from 1760 cancer pts were identified of which 189 (11%) had a diagnosis of CRC. 54/189 CRC pts (29%) had ≥1 pathogenic BRCA1/2 variant, 19 pts (10%) had ≥1 benign variant, and 134 pts (69%) had 1 ≥ variant of unknown significance (VUS); 26 pts (14%) had more than one BRCA1/2 variant. Pts with ≥1 pathogenic mutation (n=54) were more likely to have an inactivating DNA polymerase epsilon (POLE) mutation versus pts with only benign/VUS BRCA1/2 variants (n=135) (20% vs 6%, p=0.004) while prevalence of microsatellite instability (MSI-H) (19% vs 13%, p=0.37) and Ras/Raf (53% vs 53%, p=1.00) were similar between groups. Median progression free survival (PFS) among 115 CRC pts (unselected for BRCA1/2 pathogenicity) who received an oxaliplatin-based regimen for advanced disease in the 1st line (n=52) was similar to the median PFS among pts who received an irinotecan-based regimen (n=63) (7.0 mths vs 7.0 mths, p=0.48). Median PFS with an oxaliplatin-based regimen in the 1st line among 52 pts with pathogenic BRCA1/2 variants (n=13) was 5.1 mths vs 7.4 mths among pts with benign/VUS BRCA1/2 variants (n=39) (p=0.398). There were no significant differences in median PFS among pts with a pathogenic vs benign/VUS BRCA1/2 variants when the analysis was restricted to pts with BRCA1/2 variants identified in tissue (p=0.76) and to pts without co-occurring driver mutations (Ras/Raf, MSI-H, and/or POLE) (p=0.1). Overall survival in patients with advanced disease was similar between pts with a pathogenic (n=52) vs benign/VUS (n=122) BRCA variants (37.1 mths vs 45.1 mths, p=0.14). 6 pts (4 with pathogenic BRCA1/2 and 3 with VUS) received a PARP inhibitor after a median of 3 lines of treatment and the best response was disease progression in 6 pts (100%). Conclusions: In the context of CRC, somatic BRCA1/2 mutations frequently co-occur with pathogenic POLE mutations, but do not appear to confer therapeutic benefit to platinum-based chemotherapy and PARP inhibitors.

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

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. 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. 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. 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.

CHEK2 deficiency increase the response to PD-1 inhibitors by affecting the tumor immune microenvironment

Immune checkpoint blockade (ICB) therapy has improved treatment effects in multiple cancers. Gene mutations in the DNA damage repair pathway (DDR) may cause genomic instability and may relate to the efficacy of ICB. Checkpoint kinase 2 (CHEK2) and polymerase epsilon (POLE) are important genes in the DDR. In this study, we aimed to study the impact of CHEK2 deficiency mutations on the response to ICB. We found that tumors with CHEK2 mutations had a significantly higher tumor mutational burden (TMB) compared to those with CHEK2-WT in a pancancer database. We noted that CHEK2 deficiency mutations potentiated the anti-tumor effect of anti-PD-1 therapy in MC38 and B16 tumor-bearing mice with the decrease of tumor volume and tumor weight after anti-PD-1 treatment. Mechanistically, CHEK2 deficiency tumors were with the increased cytotoxic CD8+ T-cell infiltration, especially cytotoxic CD8+ T cells, and modulated the tumor-immune microenvironment with an upregulated immune inflammatory pathway and antigen presentation pathway after anti-PD-1 treatment. Furthermore, murine models with POLE mutations confirmed that CHEK2 deficiency shaped similar mutational and immune landscapes as POLE mutations after anti-PD-1 treatment. Taken together, our results demonstrated that CHEK2 deficiency mutations may increase the response to ICB (eg. anti-PD-1) by influencing the tumor immune microenvironment. This indicated that CHEK2 deficiency mutations were a potentially predictive biomarker and CHEK2 deficiency may potentiate response to immunotherapy.

Integrating POLE/POLD1 mutated for immunotherapy treatment planning of advanced stage non-small cell lung cancer.

In this study, we evaluated the potential of DNA polymerase epsilon (POLE) and DNA polymerase delta 1 (POLD1) as prognostic biomarkers for immune checkpoint inhibitor (ICI) treatment in patients with advanced stage non-small cell lung cancer (NSCLC). Disease stage, PD-L1 positivity, histological subtypes, POLE/POLD1 mutation status, tumor mutation burden (TMB), and response to ICIs in NSCLC cases were derived from AACR GENIE dataset (n = 24 120), TCGA-Pan Lung Cancer dataset (n = 1144), AACR GENIE BPC NSCLC v2.0-public (n = 2004), and Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets dataset (n = 350). The smoking history from TCGA and AACR GENIE datasets was grouped into current, former or never-smokers. POLE and POLD1 genetic alterations were identified in 5% and 2.6% of NSCLC patients, respectively. Current smokers had 9% and 4% of POLE/POLD1 mutations, respectively, versus 1.7% for both POLE and POLD1 mutations prevalence in never-smokers. POLE/POLD1 mutations were associated with elevated mutation counts than those with wild-type (median mutation counts 16 vs. 7, p < 0.0001), more advanced disease stages (stage I disease 15.19% vs. 29.42%), more prevalent squamous histology subtype (21.69% vs. 9.05%, p = 0.0427), and a higher percentage of PD-L1 positivity (66.67% vs. 43.87%, p < 0.001). Treatment with ICIs improved survival in patients with both POLE/POLD1 mutated and those with TMB > 18 (p < 0.001). Current smokers have a five-fold increased risk of having POLE mutations than never-smokers. POLE/POLD1 mutation status and TMB > 18 can be a composite biomarker for selecting NSCLC patients with survival benefits to ICI treatment.

“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.

Cytopathological features associated with POLE mutation in endometrial cancer.

For patients with endometrial cancer, the POLE (polymerase epsilon) mutation (POLEmut)-subtype, one of four molecular-analysis-based categories in the Cancer Genome Atlas (TCGA), has the best prognosis. The following histological characteristics are typically observed in endometroid carcinoma cases with the POLEmut-subtype: (1) the presence of tumour giant cells, (2) numerous tumour-infiltrating lymphocytes (TILs) and/or peri-tumoral lymphocytes, and (3) a high grade. However, in the context of cytology, the morphological characteristics of this subtype remain unknown. DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues was subjected to next-generation sequencing analysis and categorised according to the TCGA classifications. Genomic mutation, tumour mutation burden (TMB), and microsatellite instability were also assessed. Cytological specimens of resected uteri obtained using the Papanicolaou method were histologically separated into three types. Seven out of 112 patients (6%) with endometrial cancer were diagnosed with the POLEmut-subtype between January 2019 and August 2021. Tumour giant cells were observed in three cases (43%) on histology and cytology. TIL and/or peritumoral lymphocytes with inflammatory cells were detected in five cases (71%) on histology and three cases (43%) on cytology. Cases in which these three characteristics were observed on both cytology and histology may have belonged to the POLEmut-subtype. There were no cases in which these characteristics were absent on histology but present on cytology. TMB tended to be higher in cases when the three characteristics were observed in both cytological and histological findings. Preoperative endometrial cytology highlighted the characteristics of the POLEmut-subtype in the histological analysis of resected uterine specimens and has the potential to play an important role in treatment decisions.

Fast and reliable Sanger POLE sequencing protocol in FFPE tissues of endometrial cancer.

The new classification of endometrial carcinoma (EC) requires molecular interpretation of somatic polymerase epsilon (POLE) exonuclease domain mutations. The identification of pathogenic mutations within the POLE gene defines the important subtype of ultramutated tumours ("POLE-ultramutated") with specified prognostic and predictive utility. POLE somatic mutations are present in 7-12% of ECs, usually high-grade tumours with aggressive appearance. Molecular analysis of the POLE gene can be performed using a qPCR test, the Sanger sequencing method, a next generation sequencing (NGS) panel test and also in situ hybridisation (IHC) assay. We describe our current approach of identification of POLE mutations using Sanger sequencing technology, which is still the most robust, accurate and fast technique to sequence DNA. We present a reliable protocol for Sanger sequencing of the entire sequence coding exonuclease domain of POLE - exons 9, 10, 11, 12, 13 and 14 (codons 268-491) with 5-10 nucleotides in exon/intron boundaries (reference sequences: NM_006231.4, NP_006222.2). The protocol has been optimized for formalin-fixed, paraffin-embedded (FFPE) EC tissues. The method developed in our laboratory allows better diagnosis of patients with EC according to current standards.

Incorporating Molecular Classification When Stratifying the Survival Risk of Patients with High-Grade Endometrial Carcinomas.

Assessing survival risk in patients with high-grade endometrial carcinomas has remained challenging. We aimed to investigate the distribution of molecular subtypes and assess their prognostic role in a large cohort of 355 patients with high-grade endometrial carcinoma. Molecular classification was determined using DNA polymerase epsilon (POLE) sequencing as well as immunohistochemical staining for p53 and mismatch repair (MMR) proteins. Endometrial carcinomas were stratified into four subtypes: POLE ultramutated, MMR-deficient, non-specific molecular profile (NSMP), and p53-mutant. This study included 177 and 178 patients with endometrioid and non-endometrioid carcinomas, respectively. Forty-two patients (11.8%) were categorized as POLE ultramutated, 106 (29.9%) as MMR-deficient, 128 (36.1%) as p53-mutant, and 79 (22.2%) as NSMP. Patients of different molecular subtypes had distinct survival times; molecular classification, but not histotype, was significantly associated with survival outcomes. When incorporating molecular classification into the stratification model, 52 patients (15.5%) switched risk groups, with 40 (11.9%) shifting to a lower risk for having a POLE mutation and 12 (3.6%) shifting to a higher risk owing to p53-mutant status. Molecular classification may provide more accurate prognostic information among patients with high-grade endometrial carcinomas and improve their stratification for purposes of clinical management.

359P Analysis of TMB and tumor microenvironment in Polymerase epsilon (POLE) deficient colorectal cancer

POLE (DNA Polymerase Epsilon, Catalytic Subunit) is a protein coding gene. This gene encodes the catalytic subunit of DNA polymerase epsilon, involved in DNA repair and chromosomal DNA replication. Mutations in this gene, especially related to proofreading defects in the POLE polymerase (POLE pd), have been associated with hypermutated genomes and sensitive response by checkpoint blocking immunotherapies for colorectal cancer (CRC). The profile of Tumor mutation burden (TMB) and tumor microenvironment between POLE mutation(POLEmut) and POLE wildtype(POLEwt) CRC is not clearly elaborated.

Cancer de l’endomètre de stade localisé : la perspective d’une désescalade thérapeutique guidée par la classification moléculaire

In Europe, endometrial cancer is the fourth most common cancer among women. The majority of patients are diagnosed at a localized stage. For these patients, the standard of care is based on an hysterectomy with salpingo oophorectomy±lymph node staging. Through the assessment of histopathologic features, risk groups are determined: low, intermediate, high-intermediate, and high risk. Adjuvant strategies are guided by these risk groups. While the prognosis of low-risk and high-risk is well known, that of intermediate and high-intermediate risk is more heterogeneous, and the therapeutic index of adjuvant treatments is more questionable. Several trials (PORTEC [Post Operative Radiation Therapy in Endometrial Carcinoma] I, GOG [Gynecologic Oncology Group] 99, ASTEC [A Study in the Treatment of Endometrial Cancer] EN.5, PORTEC II, Sorbe et al trial) have assessed observation, vaginal cuff brachytherapy and/or pelvic external beam radiotherapy in this population. Vaginal cuff brachytherapy reduces the local recurrence rate, and pelvic external beam radiotherapy the pelvic recurrence rate. However, no benefit in terms of overall survival or occurrence of distant metastases is highlighted. Compared to observation, brachytherapy and above all external beam radiotherapy are associated with an increased morbidity, and with a decreased quality of life. In order to improve the therapeutic ratio and to optimize medico-economic decisions, therapeutic de-escalation strategies, based on the molecular profiles, are emerging in clinical trials, and in the recommendations for the management of intermediate and high-intermediate risk endometrial cancers. The four main molecular profiles highlighted by the genomic analyzes of The Cancer Genome Atlas (TCGA) – POLE (polymerase epsilon) mutation, non-specific molecular profile, MMR (MisMatch repair) deficiency, and p53 mutation – but also the quantification of lymphovascular space invasion (absent, focal or substantial), and the assessment of L1CAM (L1 cell adhesion molecule) overexpression represent growing concerns. Thus, the use of molecular-integrated risk profile to determine the best adjuvant treatment represent a major way to personalize adjuvant treatment of endometrial cancers, with therapeutic de-escalation opportunity for around half of the high-intermediate risks. However, in the absence of prospective data, inclusion in clinical trials assessing molecular profile-based treatment remains the best therapeutic opportunity.

Abstract CT021: PD-1 blockade in solid tumors with defects in polymerase epsilon

Abstract Context: Polymerase epsilon (POLE) gene missense hotspot mutations can generate pathogenic (p) proofreading defects resulting in hypermutated genomic profiles. Aim: Determine the prevalence, genomic consequences and immunotherapy sensitivity of advanced POLE mutated tumors according to mutation site, primary tumor and tumor mutational burden (TMB). Results: Pan-Cancer TCGA &amp; MSKCC databases genomic analyses found a prevalence of non-pathogenic POLE mutations (POLEnp) of 3.4% with median TMB of 11 mutations/Megabase (mt/Mb, IQR 3-34). Pathogenic POLE mutations (POLEp) prevalence was 0.4% with median TMB of 215 mt/Mb (IQR 107-324), predominantly in colorectal and endometrial cancers. Prevalence dropped to 0.1% in metastatic cancers. We assessed prospectively the efficacy of PD-1 blockade in mismatch repair proficient advanced solid tumors harboring POLE missense mutations (phase II ASCe Nivolumab trial; NCT03012581). Variants were categorized prospectively by a molecular board as POLEp, POLEnp or Variants with Unknown significance (VUS). The primary endpoint was the Overall Response Rate (ORR) at 12 weeks according to RECIST 1.1, and secondary endpoints included survival analyses according to POLE variants pathogenicity. Among 61 screened patients, 21 were eligible and 20 received Nivolumab and 19 were assessable for response (table 1). The 12-week ORR was 37% for patients harboring POLEp and VUS and resulted in major survival improvement compared to POLEnp patients (HR=0.1 ; CI95% 0.02-0.7); see results in Table 1. Among patients POLEp tumors, while higher TMB was not predictive of response, higher proportion of POLE-related mutational signature correlated with improved benefit. In silico exonucleasic POLE domain analyses confirmed that all POLEp and 2 VUS clustered in the DNA binding or the Catalytic site. Recategorizing the VUS according to the location within the exonucleasic domain improved the prediction of survival outcomes. Impact: This study gives new insights on how DNA repair defects, mutational burden and signatures sensitize to PD-1 blockade and may offer emerging tumor agnostic biomarkers for benefit to checkpoint blockade. POLE variant pathogenicity All(N=21) POLEnp(N=5) VUS(N=4) POLEp(N=12) Age, years ± SD 57 ± 16 64 ± 10 56 ± 16 54 ± 17 Sex, Male (%) 12 (57) 5 (100) 2 (50) 5 (42) PS (ECOG)=1 (%) 16 (75) 4 (80) 2 (50) 10 (83) Primary tumor Colorectal 9 (43) 2 (40) 2 (50) 5 (42) Endometrial 6 (29) 0 (0) 0 (0) 6 (50) Gastric 2 (9) 2 (40) 0 (0) 0 (0) Glial 1 (5) 0 (0) 0 (0) 1 (8) Biliary tract 1 (5) 0 (0) 1 (25) 0 (0) Pancreas 2 (9) 1 (20) 1 (25) 0 (0) Number of previous treatments 2.4 ± 2 5 ± 2 1.8 ± 1 1.5 ± 1 TMB (mt/Mb, Min-Max)(N=16) 36.2 (2-385) 5 (4-9) 3 (2-4) 114 (25-385) ORR at 12 weeks (CR+PR) 37%(N=7/19) 0%(N=0/5) 50%(N=2/4) 46%(5/10) DCR at 12 weeks (CR+PR+SD) 58%(N=11/19) 0%(N=0/5) 75%(N=3/4) 80%(8/10) Median Progresssion-Free survival (months) 5.6 2.3 10.3vs POLEnp: HR=0.2 IC95% 0.1-0.7 Median Overall Survival (months) 9.1 5.0 Not Reachedvs POLEnp:HR=0.1 IC95% 0.02-0.7 Citation Format: Benoît Rousseau, Ivan Bieche, Eric Pasmant, Nadim Hamzaoui, Nicolas Leulliot, Lucas Michon, Aurelien de Reynies, Mike Foote, Julien Masliah-Planchon, Magali Svrcek, Romain Cohen, Victor Simmet, Paule Augereau, David Malka, Antoine Hollebecque, Damien Pouessel, Carlos Gomez-Roca, Rosine Guimbaud, Amandine Bruyas, Marielle Guillet, Muriel Duluc, Sophie Cousin, Christelle de la Fourchardiere, Frederic Rolland, Sandrine Hiret, Esma Saada-Bouzid, Olivier Bouche, Thierry Andre, Diane Pannier, Farid El Hajbi, Stephane Oudard, Christophe Tournigand, Jean-Charles Soria, Drew Gerber, Dennis Stephens, Michelle Lamandola-Essel, Steven B Maron, Bill Diplas, Guillem Argiles, Asha Krishnan, Neil Segal, Andrea Cercek, Nathalie Hoog-Labouret, Frederic Legrand, Clotide Simon, Assia Lamrani-Ghaouti, Luis A. Diaz, Pierre Saintigny, Sylvie Chevret, Aurelien Marabelle. PD-1 blockade in solid tumors with defects in polymerase epsilon [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT021.

Abstract 5751: Comprehensive analysis of POLE/POLD1 variants, MMR deficient/MSI, and tumor mutational burden in Chinese population

Abstract Background: Mutations in genes encoding DNA polymerase epsilon (POLE) and delta 1 (POLD1) can lead to defects in the DNA replication, resulting in a hypermutated tumor phenotype, which might help identify potential responders to immunotherapy. This study aims to comprehensively investigate the different variants of POLE/POLD1 mutants and how it related to MSI/MMR status and TMB levels within a large Chinese population. Methods:Among 15640 Chinese patients who underwent NGS testing, the prevalence of POLE/POLD1 mutants was analyzed and the high frequencies of POLE/POLD1 variants as well as their relations with MMR/MSI and TMB were explored. Results: POLE and POLD1 mutations were highly distributed in endometrial cancer (11.1% and 9.3%), colorectal cancer (5.9% and 4.5%), and gastric cancer (4.6% and 3.3%). In our study, the mutation frequency of POLE was 3.4%, including p.Ala252Val (9.2%), p.Pro286Arg (2.7%), p.Val411Leu (1.3%), p.Asp601Glu (6.0%), p.Lys425Gln (2.9%) and p.Phe1849Val (1.8%). Variants of p.Ala252Val, p.Pro286Arg and p.Val411Leu displayed high TMB levels (median 71.4 muts/Mb, 236.7 muts/Mb and 220 muts/Mb, respectively) while variants of p.Asp601Glu, p.Lys425Gln, p.Phe1849Val and p.Asn2098His had a rather low TMB, ranging from 5.2-8.1 muts/Mb. It is worth mentioning that 37.5% of p. Pro286Arg and 87.5% of Val411Leu variants had concurrent loss-of-function mutations of MMR (MMRLOF), while MMRLOF rarely occurred in the other variants.For POLD1, the mutation frequency in Chinese patients was 2.8%, including p.Arg119His (18.8%), p.Glu57del (16.5%) and p.Asp987Thrfs (3.3%). p.Arg119His variants showed a high TMB level of 65.6 muts/Mb and most occurred in lung cancer. Variants of p.Asp987Thrfs, p.Pro116Hisfs and p.Arg19His had similar TMB levels (p&amp;gt;0.05), while much lower TMB levels was found in p.Glu57del (3.3 muts/Mb, p&amp;lt;0.001) compared to p.Arg119His.Compared with POLE/POLD1 wild-type (POLDE/POLD1-WT), the percentage of MSI-H samples in POLE/POLD1 mutants was much higher (14% vs 0.7, p&amp;lt;0.001). About 10% of POLE/POLD1 mutations carried MMRLOF mutations while it was barely observed in POLE/POLD1-WT (0.5%). The TMB levels of POLE/POLD1 mutations were significantly higher than that of the POLE/POLD1-WT (17.7 vs 2.9 muts/Mb, p&amp;lt;0.001). Furthermore, POLE/POLD1 mutations was found to be a main cause leading to high levels of TMB. Among samples with TMB &amp;gt;10 muts/Mb, 22.6% of them were POLE/POLD1 mutations. And in samples with TMB &amp;gt;100 muts/Mb, as high as 82% of samples were found carrying POLE/POLD1 mutations. Conclusion: POLE/POLD1 mutations were found in Chinese patients in multiple tumors. Different POLE/POLD1 variants were associated with different MMR status and TMB levels. In addition to MMR, POLE/POLD1 mutations might be another distinct means of inducing high TMB. Citation Format: Yongning Jia, Yaqun Xin, Hongling Yuan, Fei Pang, Fei Shan, Shuangxi Li, Honglin Zhu, Ziyu Li. Comprehensive analysis of POLE/POLD1 variants, MMR deficient/MSI, and tumor mutational burden in Chinese population [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5751.

Endometrial Cancer Management in Young Women.

Simple SummaryEndometrial cancer is a gynecological neoplasm characterized by a constant increase in incidence in highly developed countries. Due to the tendency towards late motherhood in these regions of the world, it is starting to affect more and more young women as well, often those who have not realized their maternity plans. At the same time, we note a very intensive development of research related to endometrial cancer, both in terms of diagnostics and new therapies. Therefore, nowadays, is there a sparing treatment option for young women?Endometrial cancer (EC) rarely develops in young women. Most cases are associated with known risk factors: BMI > 30, history of Polycystic Ovary Syndrome (PCOs), and race differentiation. The molecular EC classification based on The Cancer Genome Atlas Research Network divides these heterogeneous cancers into four types: Polymerase Epsilon Mutation (POLE), Microsatellite Instability (MSI), Copy Number Low (CNL), and Copy Number High (CNH). This division was introduced to allow for early assessment of neoplastic changes and clinical management, including targeted therapies. The basic technique for imaging endometrium changes is transvaginal sonography. Hysteroscopy is the standard for obtaining endometrial material for histological evaluation. The MRI result permits assessment of the extent of EC cancer infiltration. In young women who want to preserve fertility, apart from surgery, conservative management is often implemented after strict selection based on clinical and pathological data. This pharmacological treatment involves the administration of progestogens MPA (medroxyprogesterone acetate) and MA (megestrol acetate). The use of metformin may increase the effectiveness of such treatment. An alternative option is to apply progestogens locally—via the levonorgestrel-releasing intrauterine device. In addition to pharmacological treatment, hysteroscopic resection may be used—part of the uterine muscle adjacent to the pathologically changed endometrium may also undergo resection. An alternative is the administration of estrogen receptor modulators (e.g., SERMs) or aromatase inhibitors, or GnRH agonists.

Somatic mutations combined with clinical features can predict the postoperative prognosis of stage IIIA lung adenocarcinoma.

BackgroundPrognostic factors for stage IIIA lung adenocarcinoma (LUAD) are unclear. The current main treatment for stage IIIA LUAD is still controversial. Some Clinicians advocate synchronous chemoradiotherapy as the main treatment for stage IIIA LUAD. In contrast, some clinicians argue that there are still certain patients with stage IIIA LUAD who have a better postoperative prognosis. This study aimed to analyze preoperative factors as well as the association between somatic mutations and prognosis in stage IIIA LUAD [including overall survival (OS) time and the risk of postoperative recurrence].MethodsThis study retrospectively reviewed the data of patients with stage IIIA LUAD who underwent radical resection of lung cancer in the thoracic surgery department of Tianjin Chest Hospital from January 01, 2011 to September 30, 2016. All patients involved in the study provided written informed consent. The associations between OS and DFS and the clinical characteristics as well as somatic mutations of patients were analyzed separately. The Kaplan-Meier method was used for univariate analysis, and survival curves were drawn. Multivariate analysis was performed by the Cox regression model.ResultsFor univariate analysis, the prognostic factors of OS were the level of preoperative CYFRA21-1, the number of metastatic lymph node stations (NMLS), maximum tumor diameter, EGFR (epidermal growth factor receptor) classical base mutations, and the number of copies of POLE (polymerase epsilon) mutation (NCPM). Preoperative total protein level, preoperative CYFRA21-1 level, the number of metastatic lymph nodes (NMLN), maximum tumor diameter, the number of mutated genes (NMG) in tumor samples, TP53 mutations, and the number of copies of POLE mutation (NCPM) were associated with disease-free survival (DFS). The multivariate analysis showed that the preoperative CYFRA21-1 level, the number of metastatic lymph node stations (NMLS), and EGFR typical base mutations were independent prognostic factors of OS. The number of mutated genes (NMG), EGFR classical base mutations, preoperative NSE level, maximum tumor diameter, and the number of metastatic lymph node stations (NMLS) were independent prognostic factors for DFS.ConclusionsThe preoperative level of tumor markers, the number of metastatic lymph node stations, and EGFR typical base mutations are important factors for the prognosis of patients with resectable stage IIIA LUAD.

Clinicopathological and molecular characterization of high-grade endometrial carcinoma with POLE mutation: a single center study.

ObjectiveThe molecular classification system of endometrial carcinoma (EC) in ‘The Cancer Genome Atlas’ is widely acknowledged for its prognostic utility. Subsequently, more simplified classification system that incorporate DNA polymerase epsilon (POLE) exonuclease domain mutations, mismatch repair deficiencies (MMRd), and abnormal p53 (P53abn) has also demonstrated its clinical utility. These classifications helped identifying a ‘POLE ultramutated’ (POLEmut) category of patients, most of whom show excellent prognoses despite having high-grade ECs. We aimed to investigate the clinicopathological and molecular characteristics of high-grade ECs with POLEmut.MethodsWe investigated 414 patients with high-grade ECs (including endometrioid carcinomas grade 3, serous carcinomas, clear cell carcinomas, mixed carcinomas, undifferentiated and dedifferentiated carcinomas, and carcinosarcomas) by sequencing and immunohistochemical staining.ResultsForty-three tumors (10.4%) were classified as POLEmut, including 2 with new, possibly pathogenic POLE mutations at P286C and L424V. These patients had very good prognoses except for 1 with stage IV disease and residual tumor. Eleven patients in this group also had P53abn and 4 had MMRd; molecular analysis revealed that patients with synchronous POLE pathogenic mutation and other mutations had a POLEmut or MMRd phenotype; survival analysis found no difference in prognosis between these patient categories. The prognoses of patients in the POLEmut EC group were not significantly influenced by treatment or risk category.ConclusionsPatients with high-grade EC exhibiting POLEmut have very good clinical outcomes, and should be identified urgently in daily work owing to their conflicting morphology. Our findings also provide guidance on subclassifying ECs with poor histological appearance.

A Multiplex SNaPshot Assay is a Rapid and Cost-Effective Method for Detecting POLE Exonuclease Domain Mutations in Endometrial Carcinoma.

Determining the replicative DNA polymerase epsilon ( POLE) mutation status in endometrial carcinomas (ECs) has important clinical implications given that the majority of "ultramutated" tumors harboring pathogenic exonuclease domain mutations in POLE ( POLE mut) have a favorable prognosis, even among high-grade histotypes. Currently, there are no specific morphologic or immunophenotypic features that allow accurate detection of POLE mut tumors without molecular testing. Consequently, identifying POLE mut tumors has been challenging without employing costly and/or time-consuming DNA sequencing approaches. Here we developed a novel SNaPshot assay to facilitate routine and efficient POLE mutation testing in EC. The SNaPshot assay interrogates 15 nucleotide sites within exons 9, 11, 13, and 14 encoding the POLE exonuclease domain. The variant sites were selected based on recurrence, evidence of functional impact, association with high tumor mutation burden and/or detection in EC clinical outcome studies. Based on the pathogenic somatic variants reported in the literature, the assay is predicted to have a clinical sensitivity of 90% to 95% for ECs. Validation studies showed 100% specificity and sensitivity for the variants covered, with expected genotypic results for both the positive (n=11) and negative (n=20) patient controls on multiple repeat tests and dilution series. Analytic sensitivity was conservatively approximated at a 10% variant allele fraction (VAF), with documented detection as low as 5% VAF. As expected, the SNaPshot assay demonstrated greater sensitivity than Sanger sequencing for VAFs below 20%, an important characteristic for somatic mutation detection. Here we have developed and validated the first SNaPshot assay to detect hotspot POLE mutations. While next-generation sequencing and Sanger sequencing-based approaches have also been used to detect POLE mutations, a SNaPshot approach provides useful balance of analytical sensitivity, cost-effectiveness, and efficiency in a high-volume case load setting.

Protumorigenic Role of Elevated Levels of DNA Polymerase Epsilon Predicts an Immune-Suppressive Microenvironment in Clear Cell Renal Cell Carcinoma.

Increasing evidence indicates that DNA polymerase epsilon (POLE), which mediates DNA damage repair, is significantly associated with tumor prognosis. This study aimed to analyze POLE expression in tumor samples and its prognostic value for patients with clear cell renal cell carcinoma (ccRCC). We found significantly elevated POLE expression in ccRCC tissues compared with normal tissues of multiple independent cohorts. The POLE expression levels of 523 patients with ccRCC (The Cancer Genome Atlas RNA-seq data) and 179 patients with ccRCC with immunohistochemical data (Fudan University Shanghai Cancer Center) were analyzed to investigate the prognostic implications of POLE expression. Cox regression analyses were implemented to explore the effect of POLE expression on the prognosis of pan-cancer. These findings revealed that elevated POLE expression levels significantly correlated with shorter overall survival (p < 0.001, n = 701) of patients with ccRCC. These data indicate that POLE expression may serve as a prognostic biomarker for cancers. Although POLE mutations were not significantly associated with survival benefits conferred upon patients with ccRCC, a CD4+ T cell-regulated immune microenvironment was significantly activated. Moreover, we found that POLE expression in cancers significantly correlated with an immunosuppressive tumor microenvironment, higher intratumoral heterogeneity, and expression of immune checkpoint genes PDCD1, CTLA4, and CD86, possibly mediated via the JAK/STAT and Notch signaling pathways. In conclusion, the present study is the first to our knowledge to indicate that elevated POLE expression is significantly associated with poor survival and an immune-suppressive tumor microenvironment in ccRCC. These findings suggest that POLE can serve as a biomarker for guiding molecular diagnosis and facilitating the development of novel individual therapeutic strategies for patients with advanced ccRCC.