POLE Exonuclease Domain Mutations Research Articles

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

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

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.

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<0.001) in the context of different clinical ordering patterns for EC (0.9% of LB cohort vs 3.1% of TB cohort; P<0.001) and CRC (7.5% of LB cohort vs 12.6% of TB cohort; P<0.001). Median TB TMB for pPOLE+ samples was 157.5 mut/mb, compared to 3.5 for the cohort overall (P<0.001). Similarly in LB, median pPOLE+ bTMB was 165.6 vs 2.5 overall (P<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<0.001). Notably, 6.8% of pPOLE+ cases had TMB<10, which was associated median pPOLE VAF of 3.5%, compared with 25.7% among pPOLE samples with TMB≥10 (P<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.

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.

Prospective molecular classification of endometrial carcinomas: institutional implementation, practice, and clinical experience

The comprehensive genomic analysis of endometrial carcinoma (EC) by The Cancer Genome Atlas (TCGA) led to the discovery of four distinct and prognostically significant molecular subgroups. Molecular classification has the potential to improve risk-stratification when integrated with clinicopathologic features and has recently been included in national and international patient management EC guidelines. Thus, the adoption of molecular classification into routine pathologic and clinical practice is likely to grow significantly in the upcoming years. Establishing an efficient and standardized workflow for performing molecular classification on ECs, and reporting both the molecular and histologic findings in an integrative manner, is imperative. Here we describe our effort to implement rapid and routine molecular classification on all ECs diagnosed at our institution. To this effect, we performed immunohistochemistry as a surrogate marker for identifying genetic and/or epigenetic alterations in DNA mismatch repair (e.g., MLH1, PMS2, MSH6, MSH2), and TP53 genes. In addition, we have developed and employed a single-gene POLE SNaPshot assay, which is a rapid and analytically sensitive method for detecting select POLE exonuclease domain mutations (EDMs). We report our molecular testing workflow and integrative reporting system as well as the clinicopathologic and molecular features of 310 ECs that underwent routine molecular classification at our institution. The 310 ECs were molecularly classified as follows: 15 (5%) POLE mutant (POLEmut), 79 (25%) mismatch repair-deficient (MMRd), 135 (44%) no specific molecular profile (NSMP), and 81 (26%) p53 abnormal (p53abnl). This work provides an initial framework for implementing routine molecular classification of ECs.

Clinical impact of genetic alterations of CTNNB1 in patients with grade 3 endometrial endometrioid carcinoma

To identify prognostic factors in patients with grade 3 (high‐grade) endometrial endometrioid carcinoma, we evaluated the spectrum of genomic alterations and examined whether previously reported molecular subtypes of endometrial carcinoma were adapted to clinical outcome prediction. Seventy‐five Japanese patients with grade 3 endometrial endometrioid carcinoma, who underwent a potentially curative resection procedure between 1997 and 2018 at the National Cancer Center Hospital, were included. We classified the patients into four risk groups of the disease based on the Proactive Molecular Risk Classifier for Endometrial Cancer. Genomic alterations in PTEN, ARID1A, TP53, and PIK3CA were detected in more than 30% of the patients. Overall survival and recurrence‐free survival of patients with genomic alterations in CTNNB1 were poorer than those of patients with wild‐type CTNNB1 (p = 0.006 and p = 0.004, respectively). Compared with that of alterations prevalent in Caucasians, the frequency of genomic alterations in POLE and TP53 was higher in our study than in The Cancer Genome Atlas dataset (p = 0.01 and p = 0.01, respectively). The tendency for recurrence‐free survival in the POLE exonuclease domain mutation group was better than that in the TP53 mutation and mismatch repair‐deficient groups (p = 0.08 and p = 0.07, respectively), consistent with the Proactive Molecular Risk Classifier for Endometrial Cancer risk classifier definition. The CTNNB1 mutation is a potential novel biomarker for the prognosis of patients with grade 3 endometrial endometrioid carcinoma, and prognosis classification using Proactive Molecular Risk Classifier for Endometrial Cancer may help screen Japanese patients with the disease.

Prognostic relevance of the molecular classification in high-grade endometrial cancer for patients staged by lymphadenectomy and without adjuvant treatment

IntroductionThe clinical role of the molecular endometrial cancer (EC) classification has not been fully explored in patients staged with lymphadenectomy or without adjuvant treatment, conditions that could potentially moderate the prognostic value of the classification. We aimed to evaluate the clinical outcome of the molecular subgroups in patients with high-grade EC staged by lymphadenectomy and those without adjuvant treatment. MethodsDNA-sequencing for the detection of pathogenic POLE-exonuclease domain mutations and immunohistochemistry for mismatch repair (MMR) proteins and p53 expression were performed on 412 high-grade EC from the Danish Gynaecological Cancer Database (2005–2012) to classify them as POLE-ultramutated (POLEmut), MMR-deficient (MMRd), p53-mutant (p53abn), or no specific molecular profile (NSMP). Patients with stage IV or residual disease after surgery were excluded. Kaplan-Meier method, log-rank test and Cox proportional hazard models were used for analysis. ResultsMolecular analysis was successful in 367 EC; 251 patients had undergone lymphadenectomy. Five-year recurrence rates in this subgroup of patients was 36.7% for women with p53abn EC, 0.0% for POLEmut EC, 13.4% for MMRd EC and 42.9% for NSMP EC (p < 0.001). Similar results were observed among stage IA-IB patients. Among patients without adjuvant treatment (n = 264), none with POLEmut EC (n = 26) had a recurrence. ConclusionThe molecular EC classification has strong prognostic value, independent of clinicopathological factors, also among high-grade EC patients staged by lymphadenectomy and those without adjuvant treatment. The unfavourable prognosis of early-stage p53abn EC is not due to undetected lymph node metastasis, and the indolent behaviour of POLEmut EC is independent of adjuvant treatment.

Molecular and Pathology Features of Colorectal Tumors and Patient Outcomes Are Associated with Fusobacterium nucleatum and Its Subspecies animalis.

Fusobacterium nucleatum (F. nucleatum) activates oncogenic signaling pathways and induces inflammation to promote colorectal carcinogenesis. We characterized F. nucleatum and its subspecies in colorectal tumors and examined associations with tumor characteristics and colorectal cancer-specific survival. We conducted deep sequencing of nusA, nusG, and bacterial 16s rRNA genes in tumors from 1,994 patients with colorectal cancer and assessed associations between F. nucleatum presence and clinical characteristics, colorectal cancer-specific mortality, and somatic mutations. F. nucleatum, which was present in 10.3% of tumors, was detected in a higher proportion of right-sided and advanced-stage tumors, particularly subspecies animalis. Presence of F. nucleatum was associated with higher colorectal cancer-specific mortality (HR, 1.97; P = 0.0004). This association was restricted to nonhypermutated, microsatellite-stable tumors (HR, 2.13; P = 0.0002) and those who received chemotherapy [HR, 1.92; confidence interval (CI), 1.07-3.45; P = 0.029). Only F. nucleatum subspecies animalis, the main subspecies detected (65.8%), was associated with colorectal cancer-specific mortality (HR, 2.16; P = 0.0016), subspecies vincentii and nucleatum were not (HR, 1.07; P = 0.86). Additional adjustment for tumor stage suggests that the effect of F. nucleatum on mortality is partly driven by a stage shift. Presence of F. nucleatum was associated with microsatellite instable tumors, tumors with POLE exonuclease domain mutations, and ERBB3 mutations, and suggestively associated with TP53 mutations. F. nucleatum, and particularly subspecies animalis, was associated with a higher colorectal cancer-specific mortality and specific somatic mutated genes. Our findings identify the F. nucleatum subspecies animalis as negatively impacting colorectal cancer mortality, which may occur through a stage shift and its effect on chemoresistance.

Expression of the cancer-associated DNA polymerase ε P286R in fission yeast leads to translesion synthesis polymerase dependent hypermutation and defective DNA replication

Somatic and germline mutations in the proofreading domain of the replicative DNA polymerase ε (POLE-exonuclease domain mutations, POLE-EDMs) are frequently found in colorectal and endometrial cancers and, occasionally, in other tumours. POLE-associated cancers typically display hypermutation, and a unique mutational signature, with a predominance of C > A transversions in the context TCT and C > T transitions in the context TCG. To understand better the contribution of hypermutagenesis to tumour development, we have modelled the most recurrent POLE-EDM (POLE-P286R) in Schizosaccharomyces pombe. Whole-genome sequencing analysis revealed that the corresponding pol2-P287R allele also has a strong mutator effect in vivo, with a high frequency of base substitutions and relatively few indel mutations. The mutations are equally distributed across different genomic regions, but in the immediate vicinity there is an asymmetry in AT frequency. The most abundant base-pair changes are TCT > TAT transversions and, in contrast to human mutations, TCG > TTG transitions are not elevated, likely due to the absence of cytosine methylation in fission yeast. The pol2-P287R variant has an increased sensitivity to elevated dNTP levels and DNA damaging agents, and shows reduced viability on depletion of the Pfh1 helicase. In addition, S phase is aberrant and RPA foci are elevated, suggestive of ssDNA or DNA damage, and the pol2-P287R mutation is synthetically lethal with rad3 inactivation, indicative of checkpoint activation. Significantly, deletion of genes encoding some translesion synthesis polymerases, most notably Pol κ, partially suppresses pol2-P287R hypermutation, indicating that polymerase switching contributes to this phenotype.

Corded and Hyalinized and Spindled Endometrioid Endometrial Carcinoma: A Clinicopathologic and Molecular Analysis of 9 Tumors Based on the TCGA Classifier.

Corded and hyalinized and spindled carcinomas are rare variants of endometrioid carcinoma (EC) characterized by cords of low-grade epithelial cells (±spindle cells) within a hyalinized stroma or spindled epithelial cells, respectively, that merge with conventional low-grade EC. Due to their "biphasic" morphology, these tumors are often misdiagnosed as carcinosarcoma. The clinicopathologic features including mismatch repair protein (PMS2 and MSH6) and p53 immunohistochemical expression and POLE mutational status of 9 corded and hyalinized and spindled endometrial ECs were evaluated and classified into The Cancer Genome Atlas (TCGA) based molecular subgroups. Beta-catenin immunohistochemistry was performed as a surrogate for CTNNB1 mutational status. The mean age at diagnosis was 49 years (range: 34 to 68 y) with staging information available for 6 patients: stage IA (n=1), stage IB (n=1), stage II (n=2), stage IIIA (n=1), stage IIIC1 (n=1). A prominent corded and hyalinized component was present in 7 ECs comprising 15% to 80% of the tumor with a minor (5% to 15%) spindled morphology in 5. Two additional tumors were composed of a low-grade spindled component comprising 25% to 30% of the neoplasm. Tumors were grade 1 (n=3), grade 2 (n=5), and grade 2 to 3 (n=1) and squamous differentiation was identified in 8/9. All tumors had preserved expression of mismatch repair proteins with 8 showing a p53 wild-type phenotype including the grade 2 to 3 EC; 1 grade 2, stage IB tumor exhibited a mutant pattern of expression. All (n=7) but 1 tumor demonstrated nuclear beta-catenin expression in the glandular, squamous, and corded or spindled components. POLE exonuclease domain mutations were absent in all tumors. Based on our findings, corded and hyalinized EC and EC with spindle cells are usually low grade, low stage, and present at a younger age and exhibit squamous differentiation at an increased frequency compared to typical EC. Unlike carcinosarcomas, which frequently harbor TP53 mutations, these tumors usually exhibit wild-type p53 and nuclear beta-catenin expression, indicative of underlying CTNNB1 mutations. According to the TCGA subgroups of endometrial carcinoma, the majority of corded and hyalinized and spindled EC appear to fall into the copy number low ("no specific molecular profile") subgroup.

Unraveling the genomic landscape of colorectal cancer through mutational signatures.

Colorectal cancer, along with most other cancer types, is driven by somatic mutations. Characteristic patterns of somatic mutations, known as mutational signatures, arise as a result of the activities of different mutational processes. Mutational signatures have diverse origins, including exogenous and endogenous sources. In the case of colorectal cancer, the analysis of mutational signatures has elucidated specific signatures for classically associated DNA repair deficiencies, namely mismatch repair (leading to microsatellite instability), base excision repair (due to MUTYH or NTHL1 mutations), and polymerase proofreading (due to POLE and POLD1 exonuclease domain mutations). Additional signatures also play a role in colorectal cancer, including those related to normal aging and those associated with gut microbiota, as well as a number of signatures with unknown etiologies. This chapter provides an overview of the current knowledge of mutational signatures, with a focus on colorectal cancer and on the recently reported signatures in physiologically normal and inflammatory bowel disease-affected somatic colon tissues.

Endometrial Carcinomas with a "Serous" Component in Young Women Are Enriched for DNA Mismatch Repair Deficiency, Lynch Syndrome, and POLE Exonuclease Domain Mutations.

Endometrial carcinoma (EC), as described by Bokhman, has historically been classified as Type I (low-grade, hormone-dependant, young patients, good prognosis) or Type II (high-grade, hormone-independent, older patients, poor prognosis). This classification is no longer pragmatic, however, as EC is a much more heterogeneous disease. Four molecular subtypes of EC were identified by The Cancer Genome Atlas (TCGA), and subsequent studies have demonstrated its utility in predicting prognosis. While endometrial serous carcinoma (ESC), the prototypical Type II EC, largely occurs in older women, younger women with ESC were not accounted for in the Bokhman model and were underrepresented in the TCGA study. We hypothesized that a subset of ESCs in young patients do not represent bona fide serous carcinomas but rather high-grade endometrioid carcinomas mimicking a serous phenotype. We identified ESCs and mixed endometrioid/serous carcinomas in women <60 years (n=37), and analyzed their clinical, morphologic, immunohistochemical, and molecular characteristics. Sixteen percent showed mismatch repair deficiency (MMR-D) and 11% were diagnosed with Lynch syndrome. Additionally, 16% of cases tested harbored a hotspot POLE exonuclease domain mutation (POLE-EDM). Morphologically, 47% of tumors showed confirmatory endometrioid features, including atypical hyperplasia, a low-grade endometrioid carcinoma component, or squamous differentiation. Clinically, the overall survival in patients with MMR-D and POLE-EDM was significantly better than that of patients without these features (P=0.0329). In conclusion, ESCs in young patients comprise a heterogeneous group of tumors, demonstrating diverse clinical, immunohistochemical, morphologic, and molecular features which have implications for prognosis and adjuvant therapy.

Clinicopathological characteristics and prognostic value of POLE mutations in endometrial cancer: A systematic review and meta-analysis.

Background:The aim of this meta-analysis was to assess the clinicopathological features and to confirm prognostic value of POLE exonuclease domain mutations (EDM) in endometrial carcinoma patients.Methods:The PubMed, Web of Science, the data of China National Knowledge Infrastructure, and Wan fang Medical Network were systematically searched for relevant articles without a cut-off date. The keywords for the search were “endometrial cancer,” “endometrial carcinoma,” “EC,” “POLE mutations,” “POLE exonuclease domain mutations,” “POLE-mutant,” “clinical characteristics” “prognostic.” Pooled hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs) were calculated by using Review manager 5.3 and Stata 14.0 statistical software.Results:Six cohort studies assessing 179 EC patients with POLE EDMs were included. The results indicated a favorable progression-free survival in POLE-mutant patients (HR = 0.32; 95% CI: = [0.09–1.18]). Furthermore, the overall survival was great in patients with POLE-mutant (HR = 0.68; 95% CI = [0.41–1.13]). It was shown that a significantly higher incidence of POLE mutations with Federation of International of Gynecologists and Obstetricians (FIGO) I group compared to FIGO II-IV group (pooled ORs: 0.34, 95% CI: [0.12–0.94], P = .04), POLE-mutant EC was not significantly associated with histology (OR = 0.56,95% CI: 0.29–1.23), tumor grade (OR = 1.22,95% CI:0.85–1.74), lymph-vascular space invasion (OR = 0.40,95% 0.06–2.42), depth of myometrial invasion (OR = 0.70,95% CI: 0.41–1.18), lymph node status (OR = 0.41, 95% 0.04–4.50), and European Society for Medical Oncology risk groups (OR = 0.68,95% CI: 0.37–1.26).Conclusion:This meta-analysis has confirmed POLE EDMs may serve as a predictive biomarker of favorable prognosis. Further studies are needed to explore the appropriate clinical utility of POLE EDMs in EC.

Clinicopathological and molecular characterisation of 'multiple-classifier' endometrial carcinomas.

Endometrial carcinoma (EC) molecular classification based on four molecular subclasses identified in The Cancer Genome Atlas (TCGA) has gained relevance in recent years due to its prognostic utility and potential to predict benefit from adjuvant treatment. While most ECs can be classified based on a single classifier (POLE exonuclease domain mutations – POLEmut, MMR deficiency – MMRd, p53 abnormal – p53abn), a small but clinically relevant group of tumours harbour more than one molecular classifying feature and are referred to as ‘multiple‐classifier’ ECs. We aimed to describe the clinicopathological and molecular features of multiple‐classifier ECs with abnormal p53 (p53abn). Within a cohort of 3518 molecularly profiled ECs, 107 (3%) tumours displayed p53abn in addition to another classifier(s), including 64 with MMRd (MMRd–p53abn), 31 with POLEmut (POLEmut–p53abn), and 12 with all three aberrations (MMRd–POLEmut–p53abn). MMRd–p53abn ECs and POLEmut–p53abn ECs were mostly grade 3 endometrioid ECs, early stage, and frequently showed morphological features characteristic of MMRd or POLEmut ECs. 18/28 (60%) MMRd–p53abn ECs and 7/15 (46.7%) POLEmut–p53abn ECs showed subclonal p53 overexpression, suggesting that TP53 mutation was a secondary event acquired during tumour progression. Hierarchical clustering of TCGA ECs by single nucleotide variant (SNV) type and somatic copy number alterations (SCNAs) revealed that MMRd–p53abn tumours mostly clustered with single‐classifier MMRd tumours (20/23) rather than single‐classifier p53abn tumours (3/23), while POLEmut–p53abn tumours mostly clustered with single‐classifier POLEmut tumours (12/13) and seldom with single‐classifier p53abn tumours (1/13) (both p ≤ 0.001, chi‐squared test). Finally, the clinical outcome of patients with MMRd–p53abn and POLEmut–p53abn ECs [stage I 5‐year recurrence‐free survival (RFS) of 92.2% and 94.1%, respectively] was significantly different from single‐classifier p53abn EC (stage I RFS 70.8%, p = 0.024 and p = 0.050, respectively). Our results support the classification of MMRd–p53abn EC as MMRd and POLEmut–p53abn EC as POLEmut. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Rationale and design of the POLEM trial: avelumab plus fluoropyrimidine-based chemotherapy as adjuvant treatment for stage III mismatch repair deficient or POLE exonuclease domain mutant colon cancer: a phase III randomised study

Background10%–15% of early-stage colon cancers harbour either deficient mismatch repair (dMMR), microsatellite instability high (MSI-H) or POLE exonuclease domain mutations, and are characterised by high tumour mutational burden and increased lymphocytic infiltrate. Metastatic dMMR colon cancers are highly sensitive to immune checkpoint inhibition, and recent data show POLE-mutant tumours are similarly responsive. We are conducting a phase III randomised trial to determine if the addition of the anti-PD-L1 antibody avelumab following adjuvant chemotherapy improves disease-free survival (DFS) in patients with stage III dMMR/MSI-H or POLE mutant colon cancer and is a cost-effective approach for the UK National Health Service (NHS).MethodsWe are recruiting patients with completely resected, stage III colon cancer confirmed to have dMMR/MSI-H, locally or POLE exonuclease domain mutation on central testing. Eligible patients are randomised in a 1:1 ratio to standard fluoropyrimidine-based chemotherapy (capecitabine, oxaliplatin for 12 weeks or capecitabine for 24 weeks) or chemotherapy, followed by avelumab (10 mg/kg, 2 weekly for 24 weeks). Stratification is by chemotherapy received and MMR/MSI-H status. The primary endpoint is DFS. Secondary endpoints include overall survival, toxicity, quality of life and health resource use. The 3-year DFS rate in the control arm is expected to be ~75%. Avelumab is expected to improve the 3-year DFS rate by 12% (ie, 87%). Target accrual is 402 patients, which provides 80% power to detect an HR of 0.48 for DFS at a two-sided alpha of 0.05. This national, multicentre phase III trial is sponsored by the Royal Marsden NHS Foundation Trust and it is anticipated that approximately 40 centres in the UK will participate. This study opened to recruitment in August 2018.Trial registration numberNCT03827044

Atypical RAS Mutations in Metastatic Colorectal Cancer.

To describe the clinical and molecular features of metastatic colorectal cancers (mCRCs) bearing uncommon atypical RAS (At-RAS) mutations at codons other than 12, 13, 59, 61, 117, and 146. By exploiting five next-generation sequencing sources (Italian collaboration, Memorial Sloan Kettering Cancer Center, Samsung Medical Center, the Biomarker Research for Anti-EGFR Monoclonal Antibodies by Comprehensive Cancer Genomics (BREAC) study, and the Foundation Medicine database), we retrieved 175 At-RAS mutated cases. Molecular data were obtained from 163 samples from Memorial Sloan Kettering Cancer Center and the Foundation Medicine database. Clinical data were available for 27 At-RAS-positive and 467 negative cases from the Italian collaboration, Memorial Sloan Kettering Cancer Center, Samsung Medical Center, and the BREAC study. At-RAS mutations were identified in 163 (0.9%) of 18,270 mCRCs. Among 133 with evaluable microsatellite instability status, 11 (8%) were microsatellite instability high. POLE exonuclease domain mutations had higher frequency (7%) than expected and were found only in microsatellite-stable tumors with high tumor mutational burden (TMB). Overall, 17% (28 of 163) of At-RAS cases had TMB greater than 20 mutations/Mb. Co-occurring typical RAS/BRAF V600E mutations and NF1 mutations, presumed to cause RAS activation, were found in 30% and 12% of samples, respectively (up to 43% and 50%, respectively, in TMB-high samples). Patients with RAS/BRAF wild-type mCRC achieved a median overall survival (OS) of 42.1 months, whereas those harboring isolated At-RAS, typical RAS, or BRAF V600E mutations showed a median OS of 32.3, 30.0, and 17.9 months, respectively (P < .001). No significant OS difference (P = .240) was found between patients with At-RAS versus typical RAS-mutated mCRC. Only one of six patients evaluable for primary resistance to anti-epidermal growth factor receptors achieved tumor response. At-RAS mutations may be a marker for RAS pathway activation and can be associated with high co-occurrence of POLE exonuclease domain mutations.

Morphological, immunophenotypical and molecular features of hypermutation in colorectal carcinomas with mutations in DNA polymerase ε (POLE).

Colorectal carcinomas (CRC) with mismatch repair (MMR) deficiency have increased tumour mutation burden and respond to immune check-point inhibitor therapy. The Cancer Genome Atlas identified hypermutated CRCs with somatic mutations in DNA polymerase ε (POLE) with mutation burdens exceeding that of MMR-deficient CRCs. To identify the morphological, immunophenotypical and molecular features of POLE-mutated CRCs, 63 consecutive MMR-intact CRCs were evaluated by Sanger sequencing for POLE exonuclease domain mutations in exons 9, 11, 13 and 14 and confirmed by next-generation sequencing. Tumour immune microenvironment and IMMUNOSCORE®1 were assessed in POLE-mutated CRCs using immunohistochemistry to detect CD3+ /CD8+ tumour-infiltrating lymphocytes and compared to 59 non-POLE mutated MMR-intact CRC, 10 non-POLE mutated MMR-deficient CRCs and 223 normal colonic mucosa. A total of 4.8% CRC (four MMR-intact primary and one MMR-intact metastasis) harboured POLE mutations in amino acid 286 in exon 9 (p.P286R) or exon 13 (p.V411L). POLE-mutated CRCs arose in the transverse colon and rectum, were male-predominant, younger and showed increased tumour-infiltrating lymphocytes and immune cells at the tumour-stromal interface. The patient with metastatic POLE-mutated CRC was placed on PD-1 inhibitor treatment with marked and sustained response. These data indicate that POLE-mutated CRCs have hypermutated phenotypes despite MMR-intact status, with mutation burdens higher than that in microsatellite-unstable CRCs. Given the recent approval for treatment of microsatellite-unstable cancer with immune check-point inhibitors, assessment of POLE status may help to guide therapeutic decisions for hypermutated tumours with intact MMR that would otherwise be missed by routine testing.

Clinical outcomes of patients with POLE mutated endometrioid endometrial cancer

ObjectivesAssess outcomes of a clinical cohort of patients with endometrioid endometrial cancer (EEC) harboring somatic POLE exonuclease domain mutations (EDMs). MethodsPatients were consented to a protocol of tumor-normal massively parallel sequencing of 410–468 cancer related genes. EECs subjected to sequencing from 2014 to 2018 were reviewed. Tumors with somatic POLE EDMs were identified. EECs were assessed for microsatellite instability (MSI) using MSIsensor and immunohistochemical analysis for mismatch repair (MMR) proteins. ResultsOf the 451 EECs sequenced, 23 had a POLE EDM (5%): 20 primary and 3 recurrent tumors sequenced. Nineteen cases (83%) were stage I/II and 4 (17%) were stages III/IV. Thirteen EECs (57%) were of FIGO grades 1/2, 10 (43%) grade 3. All patients were treated with surgery and 17 (89%) received adjuvant therapy. Five (22%) demonstrated loss of DNA MMR protein expression, none were due to Lynch syndrome. MSIsensor scores were conclusive for 21 samples: 19 were microsatellite stable and 2 MSI-high. After median follow-up of 30 months, 4/23 (17%) developed recurrences: 3 with initial grade 3 stage I and 1 with grade 1 stage III disease. One patient with grade 2 stage IV EEC had progressive disease after treatment. ConclusionsPatients with POLE EDM EEC have been shown to have a favorable prognosis. In this real-world cohort of patients, de novo metastatic disease and recurrences in initially uterine-confined cases were observed. Further research is warranted before incorporating the presence of POLE EDM into decision-making regarding adjuvant therapy.