POLE Exonuclease Domain Research Articles

Application of novel algorithm on a retrospective series to implement the molecular classification for endometrial cancer

IntroductionThe study aimed to validate the Betella algorithm, focusing on molecular analyses exclusively for endometrial cancer patients, where molecular classification alters risk assessment based on ESGO/ESTRO/ESP 2020 guidelines. Materials and methodsConducted between March 2021 and March 2023, the retrospective research involved endometrial cancer patients undergoing surgery and comprehensive molecular analyses. These included p53 and mismatch repair proteins immunohistochemistry, as well as DNA sequencing for POLE exonuclease domain. We applied the Betella algorithm to our population and evaluated the proportion of patients in which the molecular analysis changed the risk class attribution. ResultsOut of 102 patients, 97 % obtained complete molecular analyses. The cohort exhibited varying molecular classifications: 10.1 % as POLE ultra-mutated, 30.3 % as mismatch repair deficient, 11.1 % as p53 abnormal, and 48.5 % as non-specified molecular classification. Multiple classifiers were present in 3 % of cases. Integrating molecular classification into risk group calculation led to risk group migration in 11.1 % of patients: 7 moved to lower risk classes due to POLE mutations, while 4 shifted to higher risk due to p53 alterations. Applying the Betella algorithm, we can spare the POLE sequencing in 65 cases (65.7 %) and p53 immunochemistry in 17 cases (17.2 %). ConclusionIn conclusion, we externally validated the Betella algorithm in our population. The application of this new proposed algorithm enables assignment of the proper risk class and, consequently, the appropriate indication for adjuvant treatment, allowing for the rationalization of the resources that can be allocated otherwise, not only for the benefit of settings with low resources, but of all settings in general.

Discovery of recessive effect of human polymerase δ proofreading deficiency through mutational analysis of POLD1-mutated normal and cancer cells

Constitutional heterozygous pathogenic variants in the exonuclease domain of POLE and POLD1, which affect the proofreading activity of the corresponding polymerases, cause a cancer predisposition syndrome characterized by increased risk of gastrointestinal polyposis, colorectal cancer, endometrial cancer and other tumor types. The generally accepted explanation for the connection between the disruption of the proofreading activity of polymerases epsilon and delta and cancer development is through an increase in the somatic mutation rate. Here we studied an extended family with multiple members heterozygous for the pathogenic POLD1 variant c.1421T>C p.(Leu474Pro), which segregates with the polyposis and cancer phenotypes. Through the analysis of mutational patterns of patient-derived fibroblasts colonies and de novo mutations obtained by parent-offspring comparisons, we concluded that heterozygous POLD1 L474P just subtly increases the somatic and germline mutation burden. In contrast, tumors developed in individuals with a heterozygous mutation in the exonuclease domain of POLD1, including L474P, have an extremely high mutation rate (>100 mut/Mb) associated with signature SBS10d. We solved this contradiction through the observation that tumorigenesis involves somatic inactivation of the wildtype POLD1 allele. These results imply that exonuclease deficiency of polymerase delta has a recessive effect on mutation rate.

Abstract 3889: Genomic landscape of gynecologic cancers with poor prognosis in Japan, an analysis of the national database of comprehensive genomic profiling tests

Abstract Objective: Comprehensive genomic profiling (CGP) tests were approved in Japan in June 2019 under universal health insurance coverage for cancer patients who have finished (or almost finished) standardized treatment. All the data are collected to the “Center for Cancer Genomics and Advanced Therapeutics (C-CAT)” under written informed consent. Up to date, over 60,000 CGP data have been deposited with a 99.7% consent rate. This study aims to analyze mutational landscape of gynecologic cancers, especially with poor prognosis, by using this database. Methods: We analyzed CGP datasets from 3,006 gynecologic cancer patients using FoundationOne® CDx (F1CDx), the most commonly tested in Japan. This study was ethically approved by our institutional ethics committee (#2021341G) and the Information Utilization Review Board of C-CAT (#CDU2022-026N). Results: Endometrial cancers (n=561) showed high rates of tumor mutational burden-high (TMB-H) (13.9%) and MSI-high (MSI-H) (10.8%), especially in endometrioid carcinomas. POLE exonuclease mutations were less frequent (1.4%) compared to TCGA data (7%), while TP53 mutations were more common (54.4% vs. 29% in TCGA), suggesting the impact of prognosis of the molecular subtypes. Other frequently mutated genes included PIK3CA (41%), PTEN (35%), ARID1A (31%) and KRAS (26%). In 839 cervical cancers, TMB-H prevalence was 14.2%, with low MSI-H at 1.5%. TMB-H was significantly higher in squamous cell carcinomas (20.6%) than adenocarcinomas (8.3%). Frequent mutations included PIK3CA (32%), STK11 (20%), TP53 (20%), KRAS (14%), and CDKN2A (11%). TP53 mutations were significantly higher than TCGA data (<5%), particularly in mucinous carcinomas (60%), indicating a prevalence of HPV-independent tumors with poorer prognosis. Actionable mutations were generally more frequent in adenocarcinomas than in squamous cell carcinomas, including KRAS (32.2%), TP53 (29.4%), CDKN2A (18.3%), and ERBB2 (16.7%). In 1,606 ovarian cancers, the ratio of TMB-H was 5.0%, and MSI-H was 1.2%. TMB-H rates varied between 3.3-6.5% across histological types, while the ratio of MSI-H was significantly lower in serous carcinomas (0.3%) than non-serous carcinomas. Pathogenic mutations in the POLE exonuclease domain were rare in cervical (0.0%) and ovarian (0.2%) cancers. Conclusion: The C-CAT database offers insights into the mutational landscape of various cancers and histological subtypes, especially those with poor prognosis, highlighting the unmet needs for drug development in these gynecologic cancers. Citation Format: Qian Xi, Hidenori Kage, Miho Ogawa, Asami Matsunaga, Akira Nishijima, Kenbun Sone, Kei Kawana, Katsutoshi Oda. Genomic landscape of gynecologic cancers with poor prognosis in Japan, an analysis of the national database of comprehensive genomic profiling tests [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3889.

Abstract IA021: Novel genomic features of POLE-mutant tumors: the utility for tumor classification and identification of new driver alleles

Abstract Alterations in the exonuclease domain of POLE drive the development of ultramutated endometrial tumors with better prognosis. A hallmark of these tumors is the accumulation of G>T mutations in AGA sequences; however, understanding of their genomic features beyond the trinucleotide motifs is limited. We applied a novel computational framework to the whole-exome sequencing data of 524 endometrial tumors reported by The Cancer Genome Atlas network to analyze the extended DNA sequence context of ultramutation. We found that the presence of POLE driver alleles is associated with an increased frequency of G>T mutations in polypurine tracts. Sequences containing three consecutive purines, including the classic AGA context, are only moderately mutable, but the mutability increased abruptly as the tract length reached six or more purines. Moreover, mutations showed a strong preference for certain positions within the tracts, creating a characteristic quantifiable pattern. Using this signature, we developed a machine learning classifier to identify tumors with hitherto unknown POLE drivers and validated two new drivers, POLE-E978G and POLE-S461L, by functional assays in yeast. Unlike previously known pathogenic variants, the E978G substitution affects the DNA polymerase rather than exonuclease domain of POLE. We further show that the extended genomic signature of POLE ultramutation is shared by tumors with POLD1 drivers. These findings uncover new mechanisms of ultramutation and highlight the limitations of the current focus on POLE exonuclease domain variants when stratifying patients. Citation Format: Daria Ostroverkhova, Kathryn Tyryshkin, Annette K. Beach, Elizabeth A. Moore, Yosef Masoudi-Sobhanzadeh, Stephanie R. Barbari, Igor B Rogozin, Konstantin V Shaitan, Anna R Panchenko, Polina V. Shcherbakova. Novel genomic features of POLE-mutant tumors: the utility for tumor classification and identification of new driver alleles [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA021.

Abstract B014: Patient-derived organoid models for drug screening in molecular subtypes of endometrial cancer

Abstract Introduction: The lack of reliable pre-clinical models has significantly impeded the progress of drug discovery for specific molecular subtypes of endometrial cancer. This study aimed to develop patient-derived organoid models for use in preclinical screening of novel and repurposed therapies in endometrial cancer molecular subtypes. Method: Endometrial cancer tissue (n=27) and ascites fluid (n=1) were collected for this study (ethics approval SESLHD HREC 2021_ETH11975 and 19/001) from patients of various subtypes (17 endometrioid, 3 high grade serous, 2 carcinosarcoma, 2 clear cell, 3 mixed) undergoing treatment at the Royal Hospital for Women and Prince of Wales Private Hospital in Sydney, Australia. Tumor cells were isolated, embedded in 70% Matrigel and cultured in defined culture medium to form organoids. Molecular subtyping of organoids and matched parental tissue was undertaken by mismatch repair (MMR) and p53 immunohistochemistry (IHC), and sequencing of POLE exonuclease domain mutations (EDMs). A unique matched two-dimensional (2D) cell line and three-dimensional (3D) organoid culture derived from ascites isolated from a high grade serous endometrial cancer patient was established. These molecularly stratified pre-clinical models were then used to test drug activity. As a proof of principle, response to paclitaxel and carboplatin was undertaken. Result: 20 organoids representing all major molecular subtypes were established (71% success rate), including one derived from ascites fluid. Organoids are presented with either a cystic or solid structure that highly resembles the morphology of the original tissue. Organoid culture can faithfully retain key features including MMR, p53 status as well as POLE EDMs of the parental tissue. The drug screening results identified contrasting responses of 2D and 3D models obtained from the same patient tissue, highlighting the importance of selecting appropriate pre-clinical models for drug development and testing. Conclusion: This study demonstrated that patient-derived organoids can successfully recapitulate the molecular characterization of endometrial cancer. Molecularly defined organoids represent a promising preclinical resource for drug screening in endometrial cancer. In addition, the model structure should be taken into account while conducting drug screening. Citation Format: Dongli Liu, Elyse Powell, King Man Wan, Ramanand Athavale, Christine Loo, Caroline E. Ford. Patient-derived organoid models for drug screening in molecular subtypes of endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B014.

Exploring Co-occurring POLE Exonuclease and Non-exonuclease Domain Mutations and Their Impact on Tumor Mutagenicity.

Somatic POLE ExoD driver mutations cause proofreading deficiency that induces high TMB. This study suggests a novel modifier role for POLE variants in POLE ExoD-driven tumors, associated with ultra-high TMB. These data, in addition to future functional studies, may inform tumor classification, therapeutic response, and patient outcomes.

Prognosis of Stage I Endometrial Cancer According to the FIGO 2023 Classification Taking into Account Molecular Changes.

Optimum risk stratification in an early stage of endometrial cancer (EC) combines molecular and clinicopathological features. The purpose of the study was to determine the prognostic value of molecular classification and traditional pathological factors in a sample group of patients with stage I EC according to the FIGO 2023 criteria, to achieve a more personalized approach to patient care and treatment. The immunohistochemistry for p53 and mismatch repair (MMR) proteins, and DNA sequencing for POLE exonuclease domain and clinicopathological parameters, including disease disease-free survival (DFS) and overall survival (OS) in 139 patients, were analyzed. It has been shown that the independent recurrence risk factors are stage IC (p < 0.001), aggressive histological types EC (p < 0.001), and the presence of p53abn protein immunoexpression (p = 0.009). Stage IC (p = 0.018), aggressive histological types EC (p = 0.025) and the presence of p53abn protein immunoexpression (p = 0.010) were all significantly associated with lower 5-year OS rates. Our research studies confirm that the molecular category corresponds to a different prognosis in clinical stage I EC according to the new 2023 FIGO classification, with POLEmut cases presenting the best outcomes and p53abn cases showing the worst outcomes. Beyond the previous routine clinicopathological assessment, the new EC staging system represents an important step toward improving our ability to stratify IC stage EC risk.

Genomic Landscape of Endometrial, Ovarian, and Cervical Cancers in Japan from the Database in the Center for Cancer Genomics and Advanced Therapeutics.

This study aimed to comprehensively clarify the genomic landscape and its association with tumor mutational burden-high (TMB-H, ≥10 mut/Mb) and microsatellite instability-high (MSI-H) in endometrial, cervical, and ovarian cancers. We obtained genomic datasets of a comprehensive genomic profiling test, FoundationOne® CDx, with clinical information using the "Center for Cancer Genomics and Advanced Therapeutics" (C-CAT) database in Japan. Patients can undergo the tests only after standardized treatments under universal health insurance coverage. Endometrial cancers were characterized by a high frequency of TMB-H and MSI-H, especially in endometrioid carcinomas. The lower ratio of POLE exonuclease mutations and the higher ratio of TP53 mutations compared to previous reports suggested the prognostic effects of the molecular subtypes. Among the 839 cervical cancer samples, frequent mutations of KRAS, TP53, PIK3CA, STK11, CDKN2A, and ERBB2 were observed in adenocarcinomas, whereas the ratio of TMB-H was significantly higher in squamous cell carcinomas. Among the 1606 ovarian cancer samples, genomic profiling of serous, clear cell, endometrioid, and mucinous carcinomas was characterized. Pathogenic mutations in the POLE exonuclease domain were associated with high TMB, and the mutation ratio was low in both cervical and ovarian cancers. The C-CAT database is useful for determining the mutational landscape of each cancer type and histological subtype. As the dataset is exclusively collected from patients after the standardized treatments, the information on "druggable" alterations highlights the unmet needs for drug development in major gynecological cancers.

Recommendations for the classification of germline variants in the exonuclease domain of POLE and POLD1

BackgroundGermline variants affecting the proofreading activity of polymerases epsilon and delta cause a hereditary cancer and adenomatous polyposis syndrome characterized by tumors with a high mutational burden and a specific mutational spectrum. In addition to the implementation of multiple pieces of evidence for the classification of gene variants, POLE and POLD1 variant classification is particularly challenging given that non-disruptive variants affecting the proofreading activity of the corresponding polymerase are the ones associated with cancer. In response to an evident need in the field, we have developed gene-specific variant classification recommendations, based on the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) criteria, for the assessment of non-disruptive variants located in the sequence coding for the exonuclease domain of the polymerases.MethodsA training set of 23 variants considered pathogenic or benign was used to define the usability and strength of the ACMG/AMP criteria. Population frequencies, computational predictions, co-segregation data, phenotypic and tumor data, and functional results, among other features, were considered.ResultsGene-specific variant classification recommendations for non-disruptive variants located in the exonuclease domain of POLE and POLD1 were defined. The resulting recommendations were applied to 128 exonuclease domain variants reported in the literature and/or public databases. A total of 17 variants were classified as pathogenic or likely pathogenic, and 17 as benign or likely benign.ConclusionsOur recommendations, with room for improvement in the coming years as more information become available on carrier families, tumor molecular characteristics and functional assays, are intended to serve the clinical and scientific communities and help improve diagnostic performance, avoiding variant misclassifications.

Comparison of Molecular and Clinicopathologic-Based Classifications and the Transition of Adjuvant Treatment Mode for Early-Stage Endometrial Cancer

Due to the division in classification strategies based on different molecular and clinicopathologic guidelines, the current emergence of multiple molecular typing methods greatly challenges the traditional classification-guided hierarchical treatment model. The early-stage ECs who underwent a total hysterectomy and comprehensive molecular analyses were analyzed consecutively between May 2021 and December 2022. All enrolled patients were performed with immunohistochemistry for lymph-vascular space invasion (LVSI), p53, and mismatch repair (MMR) proteins, NGS-panel Sanger sequencing for POLE exonuclease domain, and TP53. The Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) classifier and the ESGO/ESTRO/ESP guidelines with and without molecular character were respectively employed to reclassify the enrolled patients. Categorical variables of the risk-group shift were compared by Pearson's χ2 or two-sided Fisher's exact test. A total of 81 early-stage ECs patients were enrolled. Molecular analyses identified four subgroups across the 81 ECs: 9 (11%) POLE mut, 22 (27.1%) MMRd, 38 (46.9%) NSMP, and 12 (14.8%) p53 abn. Compared with ESGO/ESTRO/ESP 2016 classifier, 26 (32.1%) and 23 (28.4%) patients in ESGO/ESTRO/ESP 2020 cohort with and without molecular classification, respectively, were risk-group downshifted (p>0.05). Ten (12.3%) patients were upshifted in ESGO/ESTRO/ESP 2020 molecular classification comparing to clinical classifier. Remarkably, two patients demonstrated discordance between the ProMisE and TCGA classifiers since the different sequences of classification strategies. On the other hand, 48 of 81 patients received adjuvant radiotherapy, and 12 patients received external beam radiation therapy (EBRT). According to the final molecular test, eight of 12 were classified into low and intermediate risk. The treatment of endometrial cancer is in a period of transition from the clinicopathologic-based model to the era of molecular precision. Discordance between different classifiers and overtreatment remain in clinical practice. Therefore, we should be cautious about using molecular typing to guide adjuvant treatment decisions until it is finally validated in prospective trials.

The association of progesterone receptor and abnormal p53 expression in endometrioid endometrial cancer.

e17611 Background: Based on the TCGA endometrial cancer study results, a clinically applicable classification that identifies distinct subgroups with prognostic signature, the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE), has been built and validated. It shows that those with abnormal expression of p53 (p53abn) are the group with the worst prognosis. We attempted to explore the association of p53abn and progesterone receptor (PR) in endometrioid endometrial cancer. Methods: We included 397 consecutive endometrial cancer cases of endometrioid histology with adequate tumor tissue in the formalin-fixed, paraffin-embedded (FFPE) block and available follow-up information. This study was granted by Chang Gung Medical Foundation IRB 201702242B0D001. Sanger sequencing to assess the POLE exonuclease domain hotspot mutations of exon 9-14 and immunohistochemical staining on FFPE tumor tissue sections for p53, ER, PR, and mismatch repair proteins were performed. Immunohistochemical p53 expression of cancer cell nucleus was recorded as completely negative (0%), weakly positive (1-59%), and diffusely strong positive (&gt;60%). Tumors with weak positive p53 staining were defined as p53 wild type (p53wt), while the other two were defined as p53 abnormal (p53abn). PR scores were calculated by multiplying the average staining intensity by the tumor cell nucleus stained percentage. We arbitrarily defined a progesterone receptor score of 100 or higher as positive (PR+). Progression-free and endometrial cancer-specific overall survivals starting at the date of diagnosis were evaluated using Kaplan-Meier method and compared by log-rank test between groups. Results: PR was positive in 82% of all studied cases. Of the 48 cases with p53abn tumor, 35(73%) were PR (+). With a median follow-up of 74 months, the progression-free survival and cancer-specific survival of patients with PR+ tumors (N = 310) were 93% and 96.8%, respectively, compared with 75% and 85.7% of those with PR- tumors (N = 70) (both p &lt; 0.001, log-rank test). Among those with P53abn, PR expression was associated with a favorable progression-free survival ( p = 0.081) and better cancer-specific survival ( p = 0.044). POLEmut, POLE mutated; dMMR, deficient mismatch repair PR+, progesterone receptor score ≥100, ER+, estrogen receptor score ≥100; p53abn, abnormal p53 expression. MMR was undetermined in 32 cases, PR in 17 cases, ER in 17 cases, and p53 was undetermined in 10 cases. POLE status was detected in all cases. Cases with POLE wild type, proficient mismatch repair, progesterone score &lt; 100, estrogen score &lt; 100 and wild type p53 were not listed in this table. Conclusions: Our study showed that incorporating PR into prognostic molecular markers for endometrioid endometrial cancer might provide further risk stratification. [Table: see text]

Abstract 305: POLE-specific variant classification strategy is critical for identifying patients who may benefit from immunotherapy

Abstract BACKGROUND: Pathogenic POLE exonuclease domain mutations (pPOLE) undermine mismatch error correction during DNA replication, causing somatic ultramutation and response to immunotherapy. We examined the pan-cancer landscape of POLE mutations and applied a POLE-specific variant classification model. METHODS: Comprehensive genomic profiling was performed during clinical care. Mutational signature calling was performed via decomposition using the 96-feature single-base substitution COSMIC reference signatures. A POLE-specific classification model encompassing mutation position in the exonuclease domain, TMB, presence of POLE signature, absence of other signatures, germline frequency, and other features was applied to identify pPOLE mutations causative of ultramutation. RESULTS: POLE mutation status was evaluated in 458,437 samples (425,520 tissue biopsies (TB) and 32,917 liquid biopsies (LB)). One or more POLE alterations, including pathogenic alterations and variants of unknown significance (VUS), were detected in 3.8% of samples. 19,470 total alterations were identified, 84.8% of which were missense substitutions. Application of the POLE-specific classification model identified 35 unique pathogenic variants, many of which were VUS prior to this study. 749 samples harbored a pPOLE, more than half (56.6%) of which were either p.P286R (n=245) or p.V411L (n=179). pPOLE were found in 1.4% (199/13,688) of endometrial cancers (EC) and 0.5% (270/55,981) of colorectal cancers (CRC) and were rarer in a long tail of other malignancies. The overall pPOLE rate was significantly lower in LB than TB (0.02% vs 0.17%, P&amp;lt;0.001) in the context of different clinical ordering patterns for EC (0.9% of LB cohort vs 3.1% of TB cohort; P&amp;lt;0.001) and CRC (7.5% of LB cohort vs 12.6% of TB cohort; P&amp;lt;0.001). Median TB TMB for pPOLE+ samples was 157.5 mut/mb, compared to 3.5 for the cohort overall (P&amp;lt;0.001). Similarly in LB, median pPOLE+ bTMB was 165.6 vs 2.5 overall (P&amp;lt;0.001). MSI-H or an MMR-associated signature was found in 17.5% of samples with pPOLE, most commonly in neurologic malignancies (75%, 27/36). Median TMB of samples with both pPOLE and MMRD was 2.4-fold higher than those with pPOLE alone (337.6 vs 139.4; P&amp;lt;0.001). Notably, 6.8% of pPOLE+ cases had TMB&amp;lt;10, which was associated median pPOLE VAF of 3.5%, compared with 25.7% among pPOLE samples with TMB≥10 (P&amp;lt;0.001). This pattern suggests that TMB is underestimated when tumor purity is near the limit of detection for the assay. CONCLUSIONS: pPOLE were seen in both TB and LB across cancer types. The high rate of passenger mutations underscores the utility of this POLE-specific variant classification model. Because TMB can be underestimated when tumor purity is near the limit of detection for the assay, accurate detection and classification of pPOLE is critical for identifying patients who may benefit from immunotherapy. Citation Format: Rachel B. Keller, James Haberberger, Tyler Janovitz, Alexa B. Schrock, Hanna Tukachinsky, Lei Zhong, Douglas A. Mata, Lyle V. Lopez, Zoe Fleischmann, Radwa Sharaf, Ethan S. Sokol, Garrett M. Frampton, Nimesh R. Patel, Douglas I. Lin, Geoff R. Oxnard, Erik A. Williams, Julia A. Elvin, Brennan Decker. POLE-specific variant classification strategy is critical for identifying patients who may benefit from immunotherapy [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 305.

The hereditary N363K POLE exonuclease mutant extends PPAP tumor spectrum to glioblastomas by causing DNA damage and aneuploidy in addition to increased mismatch mutagenicity.

The exonuclease domain of DNA polymerases epsilon's catalytic subunit (POLE) removes misincorporated nucleotides, called proofreading. POLE-exonuclease mutations cause colorectal- and endometrial cancers with an extreme burden of single nucleotide substitutions. We recently reported that particularly the hereditary POLE exonuclease mutation N363K predisposes in addition to aggressive giant cell glioblastomas. We knocked-in this mutation homozygously into human cell lines and compared its properties to knock-ins of the likewise hereditary POLE L424V mutation and to a complete proofreading-inactivating mutation (exo-null). We found that N363K cells have higher mutation rates as both L424V- or exo-null mutant cells. In contrast to L424V cells, N363K cells expose a growth defect, replication stress and DNA damage. In non-transformed cells, these burdens lead to aneuploidy but macroscopically normal nuclei. In contrast, transformed N363K cells phenocopy the enlarged and disorganized nuclei of giant cell glioblastomas. Taken together, our data characterize a POLE exonuclease domain mutant that not only causes single nucleotide hypermutation, but in addition DNA damage and chromosome instability, leading to an extended tumor spectrum. Our results expand the understanding of the polymerase exonuclease domain and suggest that an assessment of both the mutational potential and the genetic instability might refine classification and treatment of POLE-mutated tumors.

A novel somatic mutation in POLE exonuclease domain associated with ultra-mutational signature and MMR deficiency in endometrial cancer: a case report

BackgroundDefect in proofreading exonuclease activity of polymerases epsilon and delta (Pols ε and δ) leads to mutagenesis and genomic instability and has been described in several cancer types. Somatic POLE exonuclease domain mutations (EDMs) have been reported in 7–12% endometrial cancers (ECs) and defined a subgroup of endometrial cancers with ultrahigh somatic mutation frequencies, high tumor infiltrated lymphocytes and favorable outcomes.Case presentationHerein, we presented a novel somatic mutation in POLE exonuclease domain associated with ultra-mutational signature and MMR deficiency in endometrial cancer. A novel POLE EDM (p.T278K) was found by a 11-gene NGS panel. The MSS status detected by the MSI test was inconsistent with the dMMR status by IHC. The loss of MSH6 expression in the tumor could be interpreted by the two nonsense mutations (p.E1234* and p.E1322*) of the MSH6 gene which may lead to truncated proteins. The T278K mutation was pathogenic identified by a 602-gene NGS panel with 27.3% of C > A substitution, 0.6% of indels, 0.6% of C > G substitution and a high TMB of 203.8 mut/Mb.ConclusionsWe report an endometrial cancer patient harbored a novel somatic POLE T278K mutation. This mutation was a novel pathogenic POLE EDM should be considered as “POLE (ultramutated)” in clinical practice for the molecular classification of EC.

Distinct clinical pattern of colorectal cancer patients with POLE mutations: A retrospective study on real-world data.

Objective: Studies have demonstrated an association between somatic POLE exonuclease domain mutations (EDMs) and the prognosis of colorectal cancer (CRC). However, the prognostic value of POLE non-EDMs remains unclear. This retrospective study aimed to explore the possible relationships between POLE mutation subtypes and CRC prognosis. Methods: The 272 CRC patients from the First Affiliated Hospital of Zhengzhou University (ZZ cohort) and 499 CRC patients from The Cancer Genome Atlas database (TCGA cohort) were retrospectively collected. The cases were divided into subgroups based on POLE mutation sites and microsatellite instability (MSI) status. The continuous variables were compared among three subgroups with Kruskal-Wallis tests. Pairwise comparisons between three groups were performed by Bonferroni correction method, and adjusted p < 0.05 was considered statistically significant. The categorical variables were compared with Chi-square test and Fisher's exact test. The Kaplan-Meier curves and Cox regression models were conducted to evaluate prognostic values of POLE mutations. Results: In the ZZ cohort, POLE EDMs (2.6%) were significantly associated with younger age (p = 0.018) and localized in the left colon (p = 0.001). POLE non-EDMs were significantly associated with MSI-high status (p < 0.001) and localization in the right colon (p = 0.001). In the TCGA cohort, the tumor mutation burden (TMB) of both POLE EDM tumors (p < 0.001) and POLE non-EDM tumors (p < 0.001) was significantly higher than that of POLE wild-type (WT) tumors. A similar trend was observed in the ZZ cohort, although there were no significant differences. In the ZZ cohort, the POLE EDM group had higher progression-free survival (PFS) (p = 0.002) and overall survival (OS) (p = 0.042) than the POLE non-EDM group and POLE WT group. We also report one CRC patient harboring a germline POLE mutation who received camrelizumab and exhibited long-term stable disease. Conclusion: Both POLE-EDMs and POLE non-EDMs were associated with significantly increased TMB in CRC and may be biomarkers for CRC treatment and prognosis. Current evidence does not support an effect of POLE non-EDMs on PFS and OS. A significant association between POLE EDMs and improved PFS and OS may exist, but future studies with larger sample sizes are needed. Entire coding region of the POLE gene should be screened.

Integrated Biomarker Analysis Reveals L1CAM as a Potential Stratification Marker for No Specific Molecular Profile High-Risk Endometrial Carcinoma.

Histopathologic assessment of high-risk endometrial cancer (EC) suffers from intersubject variability and poor reproducibility. The pragmatic classification in four molecular subgroups helps to overcome these limits, showing a significant prognostic value. The "no specific molecular profile" (NSMP) is the most heterogeneous EC subgroup, requiring further characterization to better guide its clinical management. DNA sequencing of POLE exonuclease domain and immunohistochemistry for PMS2, MSH6, and p53 were performed in order to stratify a cohort of 94 high-risk EC patients in the four molecular subgroups. Moreover, a panel of seven additional biomarkers was tested. Patients were found to be 16% POLE-mutated, 36% mismatch repair-deficient, 27% p53-abnormal, and 21% NSMP. In the multivariable model, molecular groups confirmed their significant association with disease-specific survival and progression-free survival, with p53-abnormal and NSMP endometrial cancer characterized by poor outcomes. Among the additional evaluated biomarkers, L1CAM was the only one with a significant prognostic value within the NSMP subgroup. NSMP/L1CAM-positive patients experienced the worst outcome and were "early-relapsing" after platinum-based chemotherapy, with a significantly shorter platinum-free interval compared to L1CAM-negative patients. L1CAM appears to be a promising candidate as a prognostic and predictive biomarker in the high-risk NSMP subgroup, which is actually known to lack specific molecular markers.

Nuclear β-Catenin Expression in the Context of Abnormal p53 Expression Indicates a Nonserous Histotype in Endometrial Carcinoma.

The interobserver reproducibility is poor for histotyping within the p53-abnormal molecular category of endometrial carcinomas (ECs); therefore, biomarkers that improve histologic classification are useful. β-catenin has been proposed to have prognostic significance in specific clinicopathologic and molecular contexts. The diagnostic utility for β-catenin expression patterns in determining the histotype of p53-abnormal ECs has not been well studied. We identified ECs molecularly classified as "p53-abnormal." The p53-abnormal classification was assigned when (1) no POLE exonuclease domain hotspot mutations identified, (2) mismatch-repair protein expression was retained, and (3) abnormal p53 expression (null or overexpression) was present. Morphology was re-reviewed and β-catenin immunohistochemistry was scored as abnormal (nuclear) or normal (membranous, non-nuclear). Eighty ECs were identified in the "p53-abnormal" category; 27 (33.75%) were uterine serous carcinomas, and 53 were of nonserous histotype: 28 uterine carcinosarcomas (35%), 16 endometrioid carcinomas (20%), 2 clear cell carcinomas (2.5%), and 7 high-grade EC with ambiguous morphology (8.75%). All 27 uterine serous carcinomas demonstrated membranous β-catenin staining. Of the 53 nonserous ECs, 11 (21%) showed abnormal β-catenin expression: 6 endometrioid carcinomas, 4 uterine carcinosarcoma, and 1 high-grade EC with ambiguous morphology. The specificity of abnormal β-catenin expression for nonserous EC is high (100%) but the sensitivity is low (21%) with positive and negative predictive values of 100% and 60%, respectively. Our data shows that abnormal β-catenin expression in the context of p53-abnormal EC is highly specific, but not sensitive, for nonserous ECs and may be of value as part of a panel in classifying high-grade EC, particularly to exclude uterine serous carcinoma when nuclear staining is present.

The Current and Evolving Role of Immunotherapy in Metastatic Colorectal Cancer.

Immunotherapy can be considered a therapeutic revolution in oncology, with great impact on many tumor types, such as melanoma and non-small cell lung cancer. However, in metastatic colorectal cancer, the benefits in terms of prolonged tumor control and high response rate are limited to the rare subgroup of tumors with high mutation burden - mostly tumors that harbor microsatellite instability (MSI) or a deficient mismatch repair system (dMMR), or tumor microsatellite stability and damaging mutations in the exonuclease domains of POLE or POLD. The KEYNOTE-028 uncontrolled phase II trial demonstrated an impressive antitumor activity of pembrolizumab in patients with treatmentrefractory Lynch-associated tumors, including colorectal cancer. Nivolumab with or without ipilimumab confirmed the efficacy of immune checkpoint inhibitors in patients with previously treated dMMR / MSI metastatic colorectal cancer. The recent KEYNOTE-177 phase III trial demonstrated that pembrolizumab significantly reduced the relative risk of disease progression or death and improved progression-free survival in patients with treatment-naive dMMR / MSI metastatic colorectal cancer in comparison with first-line chemotherapy with or without biologics. Unfortunately, current pharmacological strategies with immunotherapy have not been successful for most patients with microsatellite stable metastatic colorectal cancer. In this review, we critically appraise the applicability of immune checkpoint inhibitors in dMMR/MSI metastatic colorectal cancer. We also discuss the recent negative trials of immunotherapy combinations in microsatellite stabl.

Abstract 1504: Mouse model and human patient data suggest critical roles for Pten and p53 in suppressing POLE mutant tumor development

Abstract Mutations in the exonuclease domain of POLE are associated with tumors harboring very high mutation burdens. The mechanisms linking this significant mutation accumulation and tumor development remain poorly understood. Pole+/P286R;Trp53+/- mice showed accelerated cancer mortality compared to Pole+/P286R;Trp53+/+ mice. Cells from Pole+/P286R mice showed increased p53 activation, and subsequent loss of p53 permitted rapid growth, implicating canonical p53 loss of heterozygosity in POLE mutant tumor growth. Somewhat surprisingly, however, p53 status had no effect on tumor mutation burden or single base substitution signatures in POLE mutant tumors from mice or humans. Pten has important roles in maintaining genome stability. We find that PTEN mutations are highly enriched in human POLE mutant tumors, including many in POLE signature contexts. One such signature mutation, PTEN-F341V, was previously shown in a mouse model to specifically decrease nuclear Pten and lead to increased DNA damage. We found tumors in Pole+/P286R mice that spontaneously acquired PtenF341V mutations and were associated with significantly reduced nuclear Pten and elevated DNA damage. Further analysis in cells suggests the presence of the mutant polymerase triggers DNA damage in specific contexts. Taken together, our analysis supports the idea that POLE-mediated hypermutagenesis is necessary, but not entirely sufficient, for tumorigenesis. Disabling surveillance of nuclear DNA damage is a likely sufficient factor. Citation Format: Meijuan J. Sun, Vivian S. Park, Wesley D. Frey, Leonard G. Williams, Karl P. Hodel, Juliet D. Strauss, Sydney J. Wellens, James G. Jackson, Zachary F. Pursell. Mouse model and human patient data suggest critical roles for Pten and p53 in suppressing POLE mutant tumor development [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 1504.

HGG-41. Glioma oncogenesis in the constitutional mismatch repair deficiency (CMMRD) syndrome

Abstract PURPOSE: Constitutional Mismatch Repair Deficiency (CMMRD) is a cancer predisposition due to bi-allelic mutations in one of the four main mismatch repair (MMR) genes (PMS2, MSH2, MSH6 or MLH1) associated with early onset of cancers, especially glioblastomas (GBM). Our aim was to decipher the molecular specificities of gliomas occurring in this context. METHODS: A comprehensive analysis of clinical, histopathological and genomic data (whole exome sequencing) was performed for 12 children with a CMMRD for which we had available frozen brain tumor material (10 GBM and 2 anaplastic astrocytomas). RESULTS: Eight patients harbored an ultra-mutated phenotype with more than 100 somatic non synonymous (NS) SNV/Mb. No correlation was observed between the number of mutation and sex, age, overall survival or mutated MMR gene. POLE and POLD1 exonuclease domain driver somatic mutations were described for eight and one patients respectively. The 4/12 tumors without POLE somatic mutation did not show the classical ultra-hypermutation pattern. All patients with POLE mutation had already more than 20 NS SNV/Mb (median 40, [range 23-114]) suggesting that the hypermutation phenomenon started before the appearance of the somatic POLE mutation. The mutational signatures of the tumors, dominated by the MMR signatures, were not modified after the onset of the POLE mutation when analyzing the different mutation bursts. Specific recurrent somatic mutations were observed in SETD2 (9/12), TP53 (9/12), NF1 (9/12), EPHB2 (8/12), and DICER1 (7/12). Only half of the tumors overexpressed PDL1 by immunohistochemistry and this overexpression was not associated with a higher tumor mutation burden. CONCLUSION: CMMRD-associated gliomas have a specific oncogenesis that does not trigger usual pathways and mutations seen in sporadic pediatric or adult GBM. Frequent alterations in other pathways (e.g. MAPK or DNA-PK pathway) may suggests the use of other targeted therapies aside from PD1 inhibitors.