Exonuclease Domain Mutations Research Articles

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.

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.

Increased somatic mutation burdens in normal human cells due to defective DNA polymerases

Mutation accumulation in somatic cells contributes to cancer development and is proposed as a cause of aging. DNA polymerases Pol ε and Pol δ replicate DNA during cell division. However, in some cancers, defective proofreading due to acquired POLE/POLD1 exonuclease domain mutations causes markedly elevated somatic mutation burdens with distinctive mutational signatures. Germline POLE/POLD1 mutations cause familial cancer predisposition. Here, we sequenced normal tissue and tumor DNA from individuals with germline POLE/POLD1 mutations. Increased mutation burdens with characteristic mutational signatures were found in normal adult somatic cell types, during early embryogenesis and in sperm. Thus human physiology can tolerate ubiquitously elevated mutation burdens. Except for increased cancer risk, individuals with germline POLE/POLD1 mutations do not exhibit overt features of premature aging. These results do not support a model in which all features of aging are attributable to widespread cell malfunction directly resulting from somatic mutation burdens accrued during life.

Opposite effects of cytomegalovirus UL54 exonuclease domain mutations on acyclovir and cidofovir susceptibility

Acyclovir has weak activity against human cytomegalovirus (CMV). Despite some efficacy as prophylaxis, more potent anti-CMV drugs are preferred. Acyclovir resistance of CMV has been little studied. The viral UL97 kinase phosphorylates acyclovir, and cross-resistance of ganciclovir-resistant mutants is documented. However, UL54 exonuclease domain mutants may confer ganciclovir and cidofovir resistance by a mechanism that does not apply to acyclovir as an obligate chain terminator. To test for differential susceptibilities, 11 exonuclease domain mutants were tested for their 50% inhibitory concentrations (EC50s) of acyclovir in comparison with cidofovir. The 5 mutants with the highest cidofovir EC50s (>10-fold increased over wild type) all had acyclovir EC50s less than 20% of wild type. The relatively common N408K mutant had an acyclovir EC50 of 6 μM, comparable to that reported for wild type varicella-zoster virus. Several foscarnet-resistant UL54 mutants outside the exonuclease domains, some with low-grade ganciclovir/cidofovir cross-resistance, showed various degrees of acyclovir resistance. Based on these in vitro data, acyclovir may become a therapeutic option when a highly cidofovir-resistant exonuclease mutation is present without a simultaneous mutation in UL97.

Mismatch Repair Deficiency as a Predictive and Prognostic Biomarker in Molecularly Classified Endometrial Carcinoma.

Simple SummaryWe studied mismatch repair (MMR) deficiency as a predictive and prognostic biomarker in endometrial carcinoma. MMR deficiency was associated with poor outcome only when p53 aberrant and polymerase-ϵ mutant tumors were excluded from the MMR proficient subgroup, in accordance with molecular classification based on The Cancer Genome Atlas. MMR deficiency was associated with an increased risk of death in the absence of various clinicopathologic risk factors, but the outcome was not worsened when such risk factors were present. The proportion of pelvic relapses and lymphatic dissemination, defined as primary lymph node involvement or relapses in regional lymph nodes, were higher in the MMR deficient subgroup. In conclusion, the effect of MMR deficiency on the outcome of endometrial carcinoma depends on how MMR proficiency is defined. MMR deficiency is associated with an increased risk of death in the absence of established risk factors and a unique pattern of disease spread.The aggressiveness of mismatch repair (MMR) deficient endometrial carcinomas was examined in a single institution retrospective study. Outcomes were similar for MMR proficient (n = 508) and deficient (n = 287) carcinomas, identified by immunohistochemistry. In accordance with molecular classification based on The Cancer Genome Atlas (TCGA), tumors with abnormal p53 staining or polymerase-ϵ exonuclease domain mutation were excluded from the MMR proficient subgroup, termed as “no specific molecular profile” (NSMP). Compared with NSMP (n = 218), MMR deficiency (n = 191) was associated with poor disease-specific survival (p = 0.001). MMR deficiency was associated with an increased risk of cancer-related death when controlling for confounders (hazard ratio 2.0). In the absence of established clinicopathologic risk factors, MMR deficiency was invariably associated with an increased risk of cancer-related death in univariable analyses (hazard ratios ≥ 2.0). In contrast, outcomes for MMR deficient and NSMP subgroups did not differ when risk factors were present. Lymphatic dissemination was more common (p = 0.008) and the proportion of pelvic relapses was higher (p = 0.029) in the MMR deficient subgroup. Our findings emphasize the need for improved triage to adjuvant therapy and new therapeutic approaches in MMR deficient endometrial carcinomas.

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.

Abstract IA013: Endometrial carcinomas with a mutation in DNA polymerase epsilon: A pathologist view

Abstract POLE exonuclease domain mutations (referred to as ‘POLE mutations’) are found in a wide variety of human malignancies, including endometrial cancer (EC). In all tumor types, cancers with POLE mutations are characterized by an exceptionally high mutational burden and a specific mutational signature. Despite their so-called ‘ultramutated’ phenotype, patients with POLE-mutant cancers have significantly better outcomes compared to patients with POLE-wild-type tumors. This prognostic benefit has thus far been shown for early-stage EC and stage II/III colorectal cancers, and has been suggested for glioblastomas. I will discuss the gained insight into somatic POLE mutations, the underlying mechanism(s) by which these POLE mutations contribute to low risk of recurrence and the possible ways for clinical implementation. Citation Format: Tjalling Bosse. Endometrial carcinomas with a mutation in DNA polymerase epsilon: A pathologist view [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr IA013.

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.

Molecular characterization in the prediction of disease extent in endometrial carcinoma

ObjectivePatients with endometrial carcinoma are usually triaged to staging lymphadenectomy selectively based on estimated risk of lymphatic spread. The risk is generally assessed by the presence of uterine risk factors, but their preoperative and intraoperative identification remain a challenge. The objective of this study was to assess the capability of molecular classification, described by The Cancer Genome Atlas (TCGA), to predict the stage of endometrial carcinoma. Study designSequencing of polymerase-ε (POLE) and immunohistochemistry of mismatch repair (MMR) proteins and p53 were performed to stratify endometrial carcinomas into subgroups of POLE exonuclease domain mutation (EDM), MMR deficiency, abnormal p53 (p53 abn) and ‘no specific molecular profile’ (NSMP). NSMP was the reference subgroup for comparisons. Associations of molecular subgroups and uterine risk factors with stage were examined in univariable and multivariable analyses. ResultsSix hundred and four patients were included in the study. None of the POLE EDM tumours extended beyond the uterine cervix. In an unadjusted analysis, p53 abn was associated with increased risk for stage IIIC–IV disease [odds ratio (OR) 4.6, 95% confidence interval (CI) 2.3–9.2; p < 0.0005]. When controlling for uterine risk factors (histotype and grade, depth of myometrial invasion, tumour size, lymphovascular space invasion), p53 was not an independent predictor of advanced disease. In contrast, POLE EDM independently predicted local disease (OR 0.12, 95% CI 0.015–0.99; p = 0.049 for stage II–IV cancer). Of the molecular subgroups, p53 abn was most strongly associated with the presence of high-risk uterine factors (ORs between 2.2 and 19; p ≤ 0.010). ConclusionOf the TCGA-based molecular subgroups, POLE EDM independently predicted early-stage endometrial carcinoma. Although p53 abn was not an independent predictor of advanced disease, its association with uterine risk factors could allow utilization of molecular data in deciding the type of staging surgery if knowledge of uterine factors is deficient.

Clinicopathological significance of multiple molecular features in undifferentiated and dedifferentiated endometrial carcinomas

We investigated the clinicopathological significance of multiple molecular features in undifferentiated and dedifferentiated endometrial carcinomas (UDECs). Eighteen dedifferentiated endometrial carcinomas (DDECs) and three undifferentiated endometrial carcinomas (UECs) were collected. Polymerase-ε exonuclease domain mutations (POLE-EDM) were analysed by Sanger sequencing. SWI/SNF complex subunits, mismatch repair (MMR) proteins, p53, and PD-L1 were evaluated by immunohistochemistry. The SWI/SNF complex was inactivated in half of the UDECs; variably combined with deficient MMR (dMMR), POLE-EDM, or p53 aberrance. Deficiencies in BRG1 and ARID1A were mutually exclusive (p<0.05) in DDECs. ARID1A defects were mostly (8/9) associated with dMMR and typically occurred simultaneously in both endometrioid and dedifferentiated components, whereas BRG1 defects were less frequently (3/7) combined with dMMR and were only observed in dedifferentiated cells. Two-thirds of the UDECs displayed dMMR, mainly caused by the MLH1 promotor methylation. Mutant p53 immunostaining was detected in accordant or subclonal patterns. All three POLE-EDM UDEC patients had stage IA disease with either dMMR or p53 abnormality. Strong positive signals for PD-L1 were mainly detected in dMMR samples. BRG1 defects may likely trigger the progression of dedifferentiation in UDECs by superimposing the pre-existing driver events or by initiating UECs de novo, whereas ARID1A inactivation is subordinate and may likely be secondary to dMMR. The biological behaviours of BRG1-intact UDECs were evaluated according to The Cancer Genome Atlas molecular classification; their driver events require further analysis. Exact molecular subtypes can be helpful for clinical management and treatment decisions for patients with UDEC.

Mutant POLQ and POLZ/REV3L DNA polymerases may contribute to the favorable survival of patients with tumors with POLE mutations outside the exonuclease domain

BackgroundMutations in the exonuclease domain of POLE, a DNA polymerase associated with DNA replication and repair, lead to cancers with ultra-high mutation rates. Most studies focus on intestinal and uterine cancers with POLE mutations. These cancers exhibit a significant immune cell infiltrate and favorable prognosis. We questioned whether loss of function of other DNA polymerases can cooperate to POLE to generate the ultramutator phenotype.MethodsWe used cases and data from 15 cancer types in The Cancer Genome Atlas to investigate mutation frequencies of 14 different DNA polymerases. We tested whether tumor mutation burden, patient outcome (disease-free survival) and immune cell infiltration measured by ESTIMATE can be attributed to mutations in POLQ and POLZ/REV3L.ResultsThirty six percent of colorectal, stomach and endometrial cancers with POLE mutations carried additional mutations in POLQ (E/Q), POLZ/REV3L (E/Z) or both DNA polymerases (E/Z/Q). The mutation burden in these tumors was significantly greater compared to POLE-only (E) mutant tumors (p < 0.001). In addition, E/Q, E/Z, and E/Q/Z mutant tumors possessed an increased frequency of mutations in the POLE exonuclease domain (p = 0.013). Colorectal, stomach and endometrial E/Q, E/Z, and E/Q/Z mutant tumors within TCGA demonstrated 100% disease-free survival, even if the POLE mutations occurred outside the exonuclease domain (p = 0.003). However, immune scores in these tumors were related to microsatellite instability (MSI) and not POLE mutation status. This suggests that the host immune response may not be the sole mechanism for prolonged disease-free survival of ultramutated tumors in this cohort.ConclusionResults in this study demonstrate that mutations in POLQ and REV3L in POLE mutant tumors should undergo further investigation to determine whether POLQ and REV3L mutations contribute to the ultramutator phenotype and favorable outcome of patients with POLE mutant tumors.

POLE Mutation Characteristics in a Chinese Cohort with Endometrial Carcinoma.

ObjectiveTo study the characteristics of polymerase epsilon (POLE) exonuclease domain mutations in Chinese patients with endometrial carcinoma (EC).MethodsThis study analyzed data from 529 patients with EC in The Cancer Genome Atlas (TCGA) and 467 EC patients evaluated at the Shanghai First Maternity and Infant Hospital (SFMIH). POLE mutation heterogeneity was analyzed in paired curettage and hysterectomy samples from 120 SFMIH patients. Sanger sequencing identified mutations in the POLE exonuclease domain, and correlations between POLE mutation status and various clinicopathological features were determined by chi-squared testing and Cohen’s kappa analysis, with Kaplan–Meier survival curves generated to assess correlations between POLE mutation status and overall survival (OS).ResultsThirty-five mutations were identified in 467 samples (7.5%), and novel mutations were detected in the SFMIH cohort. Compared to the TCGA cohort, the SFMIH cohort had fewer POLE mutations when matched by age (<60) and histology (endometrioid) (p < 0.001 and p = 0.010, respectively). In our study cohort, POLE mutations were significantly associated with adjuvant treatment (p = 0.029), and patients with POLE mutations who underwent chemoradiotherapy had a poor OS (p < 0.0001). Notably, shorter OS was significantly associated with POLE mutations in hysterectomy samples from patients aged >60 years or with stage I disease in the paired curettage-hysterectomy group.ConclusionThe significant difference in POLE mutation profiles between the TCGA and SFMIH cohorts, as well as the poor consistency between the curettage and hysterectomy samples, suggests that different parameters need to be applied to determine the prognosis of patients with EC in China.

POLE Mutation Spectra Are Shaped by the Mutant Allele Identity, Its Abundance, and Mismatch Repair Status

Human tumors with exonuclease domain mutations in the gene encoding DNA polymerase ε (POLE) have incredibly high mutation burdens. These errors arise in four unique mutation signatures occurring in different relative amounts, the etiologies of which remain poorly understood. We used CRISPR-Cas9 to engineer human cell lines expressing POLE tumor variants, with and without mismatch repair (MMR). Whole-exome sequencing of these cells after defined numbers of population doublings permitted analysis of nascent mutation accumulation. Unlike an exonuclease active site mutant that we previously characterized, POLE cancer mutants readily drive signature mutagenesis in the presence of functional MMR. Comparison of cell line and human patient data suggests that the relative abundance of mutation signatures partitions POLE tumors into distinct subgroups dependent on the nature of the POLE allele, its expression level, and MMR status. These results suggest that different POLE mutants have previously unappreciated differences in replication fidelity and mutagenesis.

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.

Homologous Recombination Repair Truncations Predict Hypermutation in Microsatellite Stable Colorectal and Endometrial Tumors.

INTRODUCTION:Somatic mutations in BRCA1/2 and other homologous recombination repair (HRR) genes have been associated with sensitivity to PARP inhibitors and/or platinum agents in several cancers, whereas hypermutant tumors caused by alterations in POLE or mismatch repair genes have demonstrated robust responses to immunotherapy. We investigated the relationship between somatic truncations in HRR genes and hypermutation in colorectal cancer (CRC) and endometrial cancer (EC).METHODS:We analyzed the mutational spectra associated with somatic BRCA1/2 truncations in multiple genomic cohorts (N = 2,335). From these results, we devised a classifier incorporating HRR genes to predict hypermutator status among microsatellite stable (MSS) tumors. Using additional genomic cohorts (N = 1,439) and functional in vivo assays, we tested the classifier to disambiguate POLE variants of unknown significance and identify MSS hypermutators without somatic POLE exonuclease domain mutations.RESULTS:Hypermutator phenotypes were prevalent among CRCs with somatic BRCA1/2 truncations (50/62, 80.6%) and ECs with such mutations (44/47, 93.6%). The classifier predicted MSS hypermutators with a cumulative true-positive rate of 100% in CRC and 98.0% in EC and a false-positive rate of 0.07% and 0.63%. Validated by signature analyses of tumor exomes and in vivo assays, the classifier accurately reassigned multiple POLE variants of unknown significance as pathogenic and identified MSS hypermutant samples without POLE exonuclease domain mutations.DISCUSSION:Somatic truncations in HRR can accurately fingerprint MSS hypermutators with or without known pathogenic exonuclease domain mutations in POLE and may serve as a low-cost biomarker for immunotherapy decisions in MSS CRC and EC.

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.

Interpretation of somatic POLE mutations in endometrial carcinoma.

Pathogenic somatic missense mutations within the DNA polymerase epsilon (POLE) exonuclease domain define the important subtype of ultramutated tumours (‘POLE‐ultramutated’) within the novel molecular classification of endometrial carcinoma (EC). However, clinical implementation of this classifier requires systematic evaluation of the pathogenicity of POLE mutations. To address this, we examined base changes, tumour mutational burden (TMB), DNA microsatellite instability (MSI) status, POLE variant frequency, and the results from six in silico tools on 82 ECs with whole‐exome sequencing from The Cancer Genome Atlas (TCGA). Of these, 41 had one of five known pathogenic POLE exonuclease domain mutations (EDM) and showed characteristic genomic alterations: C>A substitution > 20%, T>G substitutions > 4%, C>G substitutions < 0.6%, indels < 5%, TMB > 100 mut/Mb. A scoring system to assess these alterations (POLE‐score) was developed; based on their scores, 7/18 (39%) additional tumours with EDM were classified as POLE‐ultramutated ECs, and the six POLE mutations present in these tumours were considered pathogenic. Only 1/23 (4%) tumours with non‐EDM showed these genomic alterations, indicating that a large majority of mutations outside the exonuclease domain are not pathogenic. The infrequent combination of MSI‐H with POLE EDM led us to investigate the clinical significance of this association. Tumours with pathogenic POLE EDM co‐existent with MSI‐H showed genomic alterations characteristic of POLE‐ultramutated ECs. In a pooled analysis of 3361 ECs, 13 ECs with DNA mismatch repair deficiency (MMRd)/MSI‐H and a pathogenic POLE EDM had a 5‐year recurrence‐free survival (RFS) of 92.3%, comparable to previously reported POLE‐ultramutated ECs. Additionally, 14 cases with non‐pathogenic POLE EDM and MMRd/MSI‐H had a 5‐year RFS of 76.2%, similar to MMRd/MSI‐H, POLE wild‐type ECs, suggesting that these should be categorised as MMRd, rather than POLE‐ultramutated ECs for prognostication. This work provides guidance on classification of ECs with POLE mutations, facilitating implementation of POLE testing in routine clinical care. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

P53 immunohistochemistry is an accurate surrogate for TP53 mutational analysis in endometrial carcinoma biopsies.

TP53 mutations are considered a surrogate biomarker of the serous-like 'copy number high' molecular subtype of endometrial carcinoma (EC). In ovarian carcinoma, p53 immunohistochemistry (IHC) accurately reflects mutational status with almost 100% specificity but its performance in EC has not been established. This study tested whether p53 IHC reliably predicts TP53 mutations identified by next-generation sequencing (NGS) in EC biopsy samples for all ECs and as part of a molecular classification algorithm after exclusion of cases harbouring mismatch repair defects (MMRd) or pathogenic DNA polymerase epsilon exonuclease domain mutations (POLEmut). A secondary aim assessed inter-laboratory variability in p53 IHC. From a total of 207 cases from five centres (37-49 cases per centre), p53 IHC carried out at a central reference laboratory was compared with local IHC (n = 164) and curated tagged-amplicon NGS TP53 sequencing results (n = 177). Following consensus review, local and central p53 IHC results were concordant in 156/164 (95.1%) tumours. Discordant results were attributable to both interpretive and technical differences in staining between the local and central laboratories. When results were considered as any mutant pattern versus wild-type pattern staining, however, there was disagreement between local and central review in only one case. The concordance between p53 IHC and TP53 mutation was 155/168 (92.3%) overall, and 117/123 (95.1%) after excluding MMRd and POLEmut EC. Three (3/6) discordant results were in serous carcinomas with complete absence of p53 staining but no detectable TP53 mutation. Subclonal mutant p53 IHC expression was observed in 9/177 (5.1%) cases, of which four were either MMRd or POLEmut. Mutant pattern p53 IHC was observed in 63/63 (100%) serous carcinomas that were MMR-proficient/POLE exonuclease domain wild-type. Optimised p53 IHC performs well as a surrogate test for TP53 mutation in EC biopsies, demonstrates excellent inter-laboratory reproducibility, and has high clinical utility for molecular classification algorithms in EC. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Analysis of 108 patients with endometrial carcinoma using the PROMISE classification and additional genetic analyses for MMR-D

ObjectivesTo apply the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) to a consecutive series of endometrial cancer (EC) patients diagnosed at a tertiary referral center and assign EC specimens to one of four molecular subgroups using immunohistochemistry (IHC) for p53/mismatch repair protein expression and sequencing for Polymerase Epsilon Exonuclease Domain Mutations (POLE-EDM). Mismatch Repair Deficient (MMR-D) cases were more thoroughly investigated to identify underlying somatic or germline genetic defects. MethodsHundred-and eight consecutive endometrial cancer patients, diagnosed between March 2017 and April 2019, were subjected to immunohistochemical and molecular analysis, according to ProMisE. IHC for p53 and the mismatch repair proteins (MLH1, PMS2, MSH6 and PMS2) was performed. All patients were also tested for POLE-EDM by Sanger sequencing. In addition, tumor and corresponding normal tissue of cases with abnormal MMR IHC were tested by PCR for microsatellite instability (MSI) (MSI analysis system, Promega). Hypermethylation of MLH1 promotor was tested with (methylation specific) multiplex ligation dependent probe amplification. MMR-D cases were subjected to germline mutation analysis of the mismatch repair genes, using next generation sequencing on MiSeq (Illumina) with the BRCA Hereditary Cancer MASTR Plus, (Multiplicom/Agilent), RNA mutation analysis and MLPA. ResultsFIGO classification was stage IA (n = 54), IB (n = 22) II(n = 8), III(n = 18) and IV(n = 6). Of the 33 patients with MMR-D on IHC (31%), 26 showed MLH1 promotor hypermethylation as the probable cause of MMR-D. The remaining 7 patients without MLH1 promotor hypermethylation were referred for germline analysis of Lynch syndrome. Six patients carried a pathogenic germline mutation in one of the mismatch repair genes: MSH6(n = 3), PMS2(n = 1), MLH1(n = 1) and MSH2 (n = 1). Pathogenic POLE-EDM were identified in 7 (6%) patients. Multiple molecular features (POLE-EDM + MMR-D or POLE-EDM + p53 abnormal) were observed in 4 patients (4%). A high concordance between MMR-D and microsatellite instability was observed in our cohort. In cases of a genetic defect in the MMR genes, we do note a large proportion of cases exhibiting microsatellite instability. On the contrary a hypermutation state, as seen in POLE EDM, does not result in accompanied phenotypic changes in MSI status. ConclusionThe ProMisE classification proved to be an efficient and easily implementable system. Future research should elucidate the precise biological and prognostic meaning of the cases with multiple molecular markers.

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.