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In chromatin replication, faithful recycling of histones from parental DNA to replicated strands is essential for maintaining epigenetic information across generations. A previous experiment has revealed that disrupting interactions between the N-terminal tail of Mcm2, a subunit in DNA replication machinery, and a histone H3/H4 tetramer perturb the recycling. However, the molecular pathways and the factors that regulate the ratio recycled to each strand and the destination location are yet to be revealed. Here, we performed molecular dynamics simulations of yeast DNA replication machinery, an H3/H4 tetramer, and replicated DNA strands. The simulations demonstrated that histones are recycled via Cdc45-mediated and unmediated pathways without histone chaperones, as our in vitro biochemical assays supported. Also, RPA binding regulated the ratio recycled to each strand, whereas DNA bending by Pol ε modulated the destination location. Together, the simulations provided testable hypotheses, which are vital for elucidating the molecular mechanisms of histone recycling., (© 2024. The Author(s).)

The purpose of this study is to investigate the characteristics and significance of tertiary lymphoid structures (TLSs) in endometrial cancer (EC) based on molecular subtypes. A total of 220 patients with EC were retrospectively enrolled, including 20 with polymerase epsilon ultramutated (POLE-mut), 63 with mismatch repair deficient, 32 with p53 abnormal, and 105 with no specific molecular profile. The presence and maturity of TLSs were determined by immunohistochemical markers (CD3, CD20, CD21, and Bcl6). Disease-free survival served as the endpoint event. TLSs were found in 91 out of 220 patients (41.1%), with 68 located in peritumoral tissues and 37 exhibiting well-formed germinal center structures. The presence and different maturity of TLSs were closely associated with tumor-infiltrating lymphocytes and the programmed cell death ligand-1 expression. Moreover, TLSs displayed heterogeneity across different molecular subtypes. Notably, the TLSs, tumor-infiltrating lymphocytes, and expression of the programmed cell death ligand-1 were significantly enriched in POLE-mut EC. Multivariate logistic regression analysis showed the presence of TLSs (odds ratio: 3.483, 95% CI: 1.044-11.623, P = 0.042) as a potential predictor of POLE-mut EC. Kaplan-Meier survival curves revealed that molecular subtypes significantly stratified prognosis in patients with EC (P = 0.002), whereas TLSs did not. Multivariate Cox regression analysis indicated that The International Federation of Gynecology and Obstetrics stage and Ki-67 expression were independent prognostic factors affecting disease-free survival in patients with EC, and TLSs were not included. In conclusion, TLSs in EC exhibit heterogeneity based on molecular subtypes, necessitating further exploration to determine their clinical application value., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 by the International Society of Gynecological Pathologists.)

C-to-T transitions in CpG dinucleotides are the most prevalent mutations in human cancers and genetic diseases. These mutations have been attributed to deamination of 5-methylcytosine (5mC), an epigenetic modification found on CpGs. We recently linked CpG>TpG mutations to replication and hypothesized that errors introduced by polymerase ε (Pol ε) may represent an alternative source of mutations. Here we present a new method called polymerase error rate sequencing (PER-seq) to measure the error spectrum of DNA polymerases in isolation. We find that the most common human cancer-associated Pol ε mutant (P286R) produces an excess of CpG>TpG errors, phenocopying the mutation spectrum of tumors carrying this mutation and deficiencies in mismatch repair. Notably, we also discover that wild-type Pol ε has a sevenfold higher error rate when replicating 5mCpG compared to C in other contexts. Together, our results from PER-seq and human cancers demonstrate that replication errors are a major contributor to CpG>TpG mutagenesis in replicating cells, fundamentally changing our understanding of this important disease-causing mutational mechanism., (© 2024. The Author(s).)

Objective: The 2023 International Federation of Gynecology and Obstetrics classification with molecular classification shows superior discriminatory ability compared to staging systems lacking molecular data. However, the accuracy of endometrial biopsy data in molecular classification remains uncertain. This study aimed to assess the concordance of molecular classifications between preoperative biopsy and hysterectomy to predict prognosis before surgical staging., Methods: Endometrial biopsies and corresponding hysterectomy specimens were collected at the National Cancer Center Hospital between 2012 and 2023. Immunohistochemistry for p53 and mismatch repair (MMR) proteins and next-generation sequencing of all exons of polymerase epsilon (POLE) were performed. Given the limited number of POLE mut cases in prior studies, we prepared a POLE mut-enriched cohort. Cohen's kappa estimates were used to determine concordance for molecular and clinicopathological subgroup assignments., Results: Among 70 patients classified into four molecular subtype groups, 33 exhibited POLE mutations, 15 showed loss of MMR protein expression, 13 had p53-abnormality, and 9 had no specific molecular profile. Concordance between biopsy and hysterectomy specimens was 100% (κ = 1.000). In contrast, histological types and grades between biopsy and surgical specimens showed moderate and substantial agreement (κ = 0.420 and κ = 0.780, respectively)., Conclusions: Molecular subtypes were completely consistent with those derived from surgical specimens, demonstrating high concordance between preoperative and postoperative molecular classifications. This suggests that endometrial biopsies could reliably predict prognosis. Future studies should investigate how biopsy-based molecular profiling influences treatment planning and patient outcomes., Competing Interests: Declaration of competing interest The authors state that there are no conflicts of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Research groups have identified 4 groups [polymerase epsilon (POLE) mutant, mismatch repair-deficient, p53-abnormal, and no specific molecular profile)] reflecting the Tumor Cancer Genomic Atlas Research Network subgroups in endometrial carcinomas, improving the clinical applicability of molecular classification. We have analyzed the histopathologic and prognostic characteristics of our cases based on the ProMisE classification, supported by growing data on recommended treatment regimens. The study included 118 cases of endometrial carcinoma diagnosed between 2016 and 2020, which underwent mismatch repair and p53 immunohistochemistry. Next-generation sequencing was performed for POLE mutation analysis, dividing the cases into 4 subgroups. The histopathologic and clinical characteristics of these groups were then analyzed statistically. Four cases(3.4%) were classified as POLE mutant, 31 (26.3%) as mismatch repair-deficient, 22 (18.6%) as p53 mutant, and 61 (51.7%) as no specific molecular profile. We categorized 118 patients with endometrial carcinoma into low (n=43), intermediate (n=28), high-intermediate (n=21), high (n=22), and advanced metastatic (n=4) risk groups regardless of the molecular subtypes of their disease. When we reclassified all cases according to the molecular subtypes of endometrial carcinoma only the risk group of 3 (2.5%) cases changed. Using the new algorithm we designed, after narrowing down the number of patients, the microcystic, elongated, and fragmented pattern of invasion was revealed as an independent prognostic factor that reduces overall survival time (hazard ratio: 16.395, 95% CI: 2.140-125.606, P =0.007). In conclusion, using the new algorithm we have designed, and by identifying patients for whom molecular classification could alter risk groups, we observed that molecular tests can be utilized more efficiently in populations with limited economic resources and, in doing so, we discovered a new morphologic marker with prognostic significance., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

DNA polymerase ε (Polε) is an essential replicative polymerase consisting of Pol2, Dpb2, Dpb3, and Dpb4 subunits and has not been explored in the pathogenic yeast Candida albicans. C. albicans is accountable for >40% of deaths due to systemic candidiasis per year worldwide. Genome plasticity is one of the adaptive mechanisms associated with virulence, and as it is associated with DNA polymerase function, this study explored the role of Polε in genome stability and pathogenesis of C. albicans. POL2 and DPB2 are haploinsufficient, but DPB3 and DPB4 are dispensable for cell survival in diploid C. albicans . However, unlike in Saccharomyces cerevisiae , loss of any or both of the nonessential subunits or defective interaction between the two resulted in slow growth and temperature-sensitive phenotypes. Knockout strains of C. albicans (dpb3 ΔΔ and dpb4 ΔΔ and dpb3 ΔΔ dpb4 ΔΔ) also exhibited sensitivity to genotoxic agents and delayed cell cycle progression. Reduced processive DNA synthesis and increased rate of mutagenesis were observed in dpb3 and dpb4 null strains. Whole-genome sequencing further confirmed the accumulation of indels and SNPs majorly in the intergenic repeat regions of the chromosomes of dpb3 ΔΔ dpb4 ΔΔ. Polε-defective strains were constitutively filamentous and non-pathogenic in mice models of systemic candidiasis. Altogether, this study showed that the function of the Dpb3-Dpb4 subcomplex is critical for fungal morphogenesis and virulence besides its role as a structural component of Polε in DNA replication and genome stability; thus, their interacting interface may be targeted to develop antifungal drugs., Importance: This study explored the role of DNA polymerase epsilon, especially its non-essential structural subunits in Candida albicans biology. Apart from their role in DNA replication and genome stability, the Dpb3-Dpb4 subcomplex regulates morphological switching and virulence. Since the defective strain is locked in filamentous form and is avirulent, the complex may be targeted for anti-fungal drug development., Competing Interests: The authors declare no conflict of interest.

Background: Traditional classification tools for endometrial carcinoma (EC), such as DNA sequencing, immunohistochemistry (IHC), or PCR, are cumbersome and time-consuming. Large next-generation sequencing (NGS) panels have simplified testing but are expensive. In this study, we propose a concise NGS panel as an effectively viable approach for classifying EC., Materials and Methods: We retrospectively enrolled a consecutive EC cohort of hysterectomy with bilateral salpingo-oophorectomy from Fudan University Shanghai Cancer Center between 2020 and 2022. A 46-gene NGS panel was utilized to identify POLE exonuclease domain mutations, microsatellite instability-high (MSI-H), TP53 mutations, and other clinically relevant targets., Results: Tumor tissue samples from 331 EC patients were evaluated, with 284 (85.8%) cases classified as endometrioid endometrial carcinoma. The median follow-up time was 32.6 months (n = 303), during which 23 patients experienced recurrence or disease progression. Using the concise NGS panel, patients were stratified into four molecular subgroups according to the World Health Organization classification criteria: POLE mut (n = 47; 14.2%), mismatch repair deficiency (dMMR) (n = 79; 23.9%), non-specific molecular profile (n = 148; 44.7%), and abnormal p53 expression (p53 abn) (n = 57; 17.2%). POLE mut displayed the most favorable prognosis, while p53 abn had the worst prognosis (P < 0.001). The concordance between NGS and IHC was 91.8% (269/293) for detecting MMR status and 65.3% (201/308) for detecting p53 status. Patients detected solely by NGS had significantly worse prognosis than those detected solely by IHC, indicating higher accuracy of the NGS panel. With the molecular subtyping information, adjuvant treatment plans for 19.6% of patients could potentially be altered, mainly concentrated in the POLE mut and p53 abn subtypes. This panel also aids targeted therapy and poly (ADP-ribose) polymerase (PARP) inhibitor-related gene mutation detection, as well as auxiliary genetic screening., Conclusion: Our study demonstrates that the concise NGS panel is an effective 'one-stop' strategy for precisely classifying EC with high clinical availability., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: The treatment for stage IB grade 3 endometrioid endometrial adenocarcinoma is challenging with variable practice. Molecular characterization may help identify adjuvant therapy strategies beyond stage. We aimed to better understand the molecular features of these tumors by characterizing them by ProMisE classification, mutational signature, and commonly mutated genes., Methods: Patients with stage IB grade 3 EEC at two institutions were included. Immunohistochemistry and whole exome sequencing were performed on archival FFPE tissue sections to determine ProMisE classification. Personal Cancer Genome Reporter was used for somatic variant annotation, and mutational signatures were generated based on COSMIC single base substitution mutational signatures., Results: 46 patients were included with variable adjuvant treatment. Nine patients recurred (19.6%), most with extra-abdominal disease (n = 5, or 55.6%). 10 had POLE mutations (21.7%), 18 were MMR deficient (39.1%), 6 had abnormal p53 (13.0%), and 12 were p53 wildtype (26.1%). There were no recurrences in the POLE subgroup. A dominant mutational signature was identified in 38 patients: 17 SBS5 signature (44.7%), 10 SBS15 or SBS44 signature (26.3%), 7 SBS10a or SBS10b signature (18.4%), 3 SBS14 signature (7.9%), and 1 SBS40 signature (2.6%). The six patients that recurred had a SBS5 signature. Frequently mutated genes included ARID1A (n = 30, 65%), PTEN (n = 28, 61%), MUC16 (n = 27, 59%), and PIK3CA (n = 25, 54%)., Conclusions: This comprehensive evaluation found a molecularly diverse cohort of tumors, despite the same histology, stage and grade. Mutational signature SBS5 correlated with a high risk of recurrence. Further refining of endometrial cancer classification may enable more precise patient stratification and personalized treatment approaches., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In eukaryotes, the leading strand DNA is synthesized by Polε and the lagging strand by Polδ. These replicative polymerases have higher processivity when paired with the DNA clamp PCNA. While the structure of the yeast Polε catalytic domain has been determined, how Polε interacts with PCNA is unknown in any eukaryote, human or yeast. Here we report two cryo-EM structures of human Polε-PCNA-DNA complex, one in an incoming nucleotide bound state and the other in a nucleotide exchange state. The structures reveal an unexpected three-point interface between the Polε catalytic domain and PCNA, with the conserved PIP (PCNA interacting peptide)-motif, the unique P-domain, and the thumb domain each interacting with a different protomer of the PCNA trimer. We propose that the multi-point interface prevents other PIP-containing factors from recruiting to PCNA while PCNA functions with Polε. Comparison of the two states reveals that the finger domain pivots around the [4Fe-4S] cluster-containing tip of the P-domain to regulate nucleotide exchange and incoming nucleotide binding., (© 2024. The Author(s).)

Introduction: Pathogenic mutations in POLE/POLD1 lead to decreased fidelity of DNA replication, resulting in a high tumor mutational burden (TMB-H), defined as TMB ≥ 10 mut/Mb, independent of deficient mismatch repair (dMMR) and microsatellite instability high (MSI-H) status., Methods: De-identified records of patients with colorectal cancer (CRC) profiled with the Tempus xT assay (DNA-seq of 595-648 genes at 500×) were identified from the Tempus Database., Results: Among 9136 CRC samples profiled, the frequency of POLE/POLD1 genomic alterations was 2.4% (n = 217). Copy number loss was the most common genomic alteration (64%, n = 138) of POLE/POLD1, followed by copy number amplifications (18%, n = 40) and short variant mutations (18%, n = 39). The POLE/POLD1 mutated group presented with a higher frequency of TMB-H phenotype relative to wild type (WT; 22% vs. 9%, P < .001), with a median TMB of 127 mut/Mb in the TMB-H POLE/POLD1 subset. The TMB showed a dramatic contrast between POLE/POLD1 short variant mutations as compared to the group with copy number alterations, with a TMB of 159 mut/Mb vs 15 mut/Mb, respectively. Thus, the short variant mutations represented the so-called ultra-hypermutated phenotype. The POLE/POLD1 mutated group, as compared to WT, exhibited a higher rate of coexisting mutations, including APC, ALK, ATM, BRCA2, and RET mutations., Conclusion: Patients with POLE/POLD1 mutations exhibited significant differences across immunological markers (ie, TMB, MMR, and MSI-H) and molecular co-alterations. Those with short variant mutations represented 18% of the POLE/POLD1 cohort and 0.4% of the total cohort examined. This group of patients had a median TMB of 159 mut/Mb (range 34-488), representing the ultra-hypermutated phenotype. This group of patients is important to identify given the potential for exceptional response to immune checkpoint inhibitors., (© The Author(s) 2024. Published by Oxford University Press.)

Patients carrying mutations in polymerase epsilon/polymerase delta have shown positive responses to immune checkpoint inhibitors. Yet, prospective trials exploring the efficacy in those with polymerase epsilon/polymerase delta mutations are still lacking. A phase II clinical trial was initiated to evaluate the efficacy of toripalimab, a humanized IgG4K monoclonal antibody to human PD-1, in patients with advanced solid tumors with unselected polymerase epsilon/polymerase delta mutations but without microsatellite instability-high. A total of 15 patients were enrolled, 14 of whom were assessed for treatment efficacy. There was a 21.4% overall response rate, with a disease control rate of 57.1%. The median overall survival and median progression-free survival were 17.9 (95% CI 13.5-not reach) months and 2.5 (95% CI 1.4-not reach) months, respectively. For patients with exonuclease domain mutations, the objective response rate was 66.7% (2/3), with a disease control rate of 66.7% (2/3). For those with non-exonuclease domain mutations, the rates were 9.1% (1/11) and 54.5% (6/11), respectively. Notably, patients with PBRM1 gene mutations exhibited a high response rate to toripalimab at 75.0% (3/4). This study showed that neither the exonuclease domain mutations nor non-exonuclease domain mutations could fully predict the efficacy of immunotherapy, urging the need for more investigations to clarify potential immune sensitization differences within polymerase epsilon/polymerase delta mutation variants., (© 2024. The Author(s).)

Among the 4 molecular subgroups of endometrial carcinoma, the p53 abnormal (copy number high) subgroup has the worst prognosis; however, the histologic characteristics of this subgroup are not well established. Also, it is not well established whether low-grade tumors can belong to the p53 abnormal molecular subgroup and if so, what is the prognostic significance of the p53-mutated molecular subgroup in low-grade tumors. In the current study, we included 146 p53-mutated endometrial carcinomas and performed molecular subgrouping either based on a combination of immunohistochemical studies for p53 and MMR protein expression and POLE mutation testing (81 cases) or based on array-based and sequencing-based technologies (65 cases). We excluded cases that belonged to the POLE mutant or MSI molecular subgroups and only studied p53 abnormal (molecular subgroup) endometrial carcinomas (125 cases). In 71 cases, the molecular subgroup was determined by a combination of immunohistochemical studies and POLE mutation testing, and in 54 cases by array-based and sequencing-based methods. We reviewed 1 to 2 representative digital slides from each case and recorded the morphologic characteristics as well as clinical, treatment, and survival follow-up data. Overall, 47 cases were classified as endometrioid carcinoma, 55 serous carcinoma, and 23 other histotypes. Eight cases were FIGO 1, 21 were FIGO 2, and 91 were FIGO 3. A significant proportion of the cases (24.2%) were histologically classified as low-grade (FIGO 1 or 2) endometrioid carcinoma. There was no morphologic characteristic that showed prognostic implication. There was no significant difference in survival among different histotypes (P=0.60). There was no significant difference in survival among low-grade endometrioid (FIGO 1 or 2) versus high-grade (FIGO 3) tumors (P=0.98). Early-stage (stage I), low-grade tumors showed no significant survival advantage over early-stage, high-grade tumors (P=0.16) and this was more evident in FIGO 2 tumors. Although not statistically significant, the FIGO 2 tumors showed a trend toward worse survival than FIGO 3 tumors. Among the cases with available treatment data, more patients with early-stage high-grade tumors received adjuvant treatment, compared to patients with early-stage low-grade tumors, possibly explaining this trend (P=0.03). In conclusion, the findings of our study suggest that low-grade p53 abnormal endometrioid endometrial carcinomas (especially FIGO 2 tumors) have an aggressive course, with a prognosis similar to high-grade tumors. Furthermore, our study suggests that patients who had early-stage low-grade p53 abnormal disease might have been undertreated because of the "low-grade" histotype., Competing Interests: The authors declare no conflict of interest., (Copyright © 2024 by the International Society of Gynecological Pathologists.)

The molecular classification of endometrial carcinoma defines four main groups: polymerase‑ɛ(PolE) gene mutated, microsatellite unstable (MSI), p53 abnormal tumors and tumors with no specific molecular profile (NSMP). This classification provides significant insights into the prognosis and therapeutic decisions. Each group exhibits unique genetic profiles identified through immunohistochemistry and molecular diagnostics, enabling personalized treatment. The identification of these molecular signatures necessitates precise analytical methods, selected based on the local circumstances at each site. The approach to molecular classification highlights the critical role of pathology in the diagnosis and emphasizes the necessity of collaboration between the clinic and pathology., (© 2024. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Objective: The Cancer Genome Atlas (TCGA) project identified favorable prognosis regarding the ultra-mutated endometrial cancer (EC) subtype linked to polymerase epsilon gene (POLE) mutations. This study investigated POLE mutations in EC of Indian patients., Methods: This retrospective analytical study was conducted between January 2016 and January 2023 at the Government Medical College, Kozhikode, and the MVR Cancer Center, Kozhikode, Kerala. Sanger sequencing of POLE gene exons 9 and 13 in 151 EC patients was carried out to analyze the relationship between mutations and epidemiological factors, clinicopathologic features, and treatment outcomes., Results: Among 151 cases enrolled, 39 were unique POLE-mutated cases. Significant associations were high-grade tumors, myometrial invasion >50%, and Lymph-vascular space invasion (LVSI). The median follow-up was 40 months (95% confidence interval [CI], 34-46). A lower mean disease-specific survival (DSS) of 51.7 months (95% CI, 43.7-59.6) was noted in the POLE-mutated group compared with 72.11 months (95% CI, 67.60-76.62) for the POLE wild-type. A statistically significant hazard ratio (HR) of 2.683 for DSS in the POLE-mutated group was noted. In advanced stages (FIGO stages II-IV), a nine-fold HR for DSS and overall survival (OS) compared with POLE wild-type was identified. After controlling for treatment effects using Cox proportional HR, advanced-stage POLE-mutated tumors had a significantly higher HR of 8.67 for DSS compared with POLE-wild-type tumors of the same stage., Conclusion: This study identified a unique set of POLE mutations in Indian EC patients associated with poor prognosis, which were particularly pronounced in advanced stages. Advanced stage of presentation, type of POLE mutations, and possibly ethnicity are predictors of adverse outcomes in POLE-mutated EC. The present study highlights ethnicity as a determinant of phenotypic expression of genetic change., (© 2024 International Federation of Gynecology and Obstetrics.)

The eukaryotic replisome is assembled around the CMG (CDC45-MCM-GINS) replicative helicase, which encircles the leading-strand DNA template at replication forks. When CMG stalls during DNA replication termination, or at barriers such as DNA-protein crosslinks on the leading strand template, a second helicase is deployed on the lagging strand template to support replisome progression. How these 'accessory' helicases are targeted to the replisome to mediate barrier bypass and replication termination remains unknown. Here, by combining AlphaFold structural modelling with experimental validation, we show that the budding yeast Rrm3 accessory helicase contains two Short Linear Interaction Motifs (SLIMs) in its disordered N-terminus, which interact with CMG and the leading-strand DNA polymerase Polε on one side of the replisome. This flexible tether positions Rrm3 adjacent to the lagging strand template on which it translocates, and is critical for replication termination in vitro and Rrm3 function in vivo. The primary accessory helicase in metazoa, RTEL1, is evolutionarily unrelated to Rrm3, but binds to CMG and Polε in an analogous manner, revealing a conserved docking mechanism for accessory helicases in the eukaryotic replisome., (© 2024. The Author(s).)

Colorectal cancer (CRC) is one of the most common malignant carcinoma worldwide. DNA polymerase epsilon 2, accessory subunit (POLE2) participates in DNA replication, repair, and cell cycle control, but its association with CRC development remains unclear. In the present study, the differentially expressed genes (DEGs) in CRC were screened from bioinformatics analysis based on GEO database. RT-qPCR was used to assess mRNA expression. CCK-8 and colony formation assays were applied for the evaluation of cell proliferation. Wound healing and transwell assays were used to detect cell migration and invasion. Protein levels were determined by Western blotting assay. We found that POLE2 was highly expressed in CRC tissues and cell lines. Inhibition of POLE2 suppressed the proliferation, migration and invasion of CRC cells. Mechanistically, Wnt/β-catenin signaling pathway was inactivated by inhibition of POLE2. Activation of Wnt/β-catenin pathway can reverse the function of POLE2 knockdown on CRC cells. In vivo studies demonstrated that POLE2 silencing could notably inhibit the growth of tumors, which was consistent with the results in vitro . In conclusion, we found POLE2 as a novel oncogene in CRC, providing a potential therapeutic or diagnostic target in CRC.

Objectives: The characterization of DNA polymerase epsilon (POLE) mutations has transformed the classification of endometrial endometrioid carcinomas (EECs), highlighting the need for efficient identification methods. This study aims to examine the relationship between distinct morphologic features-namely, squamous morules and squamous differentiation (SD), as well as β-catenin expression-and the POLE mutation status in endometrial cancer (EC)., Methods: Our study included 35 POLE-mutated (POLEmut) EC cases and 395 non-POLEmut EEC cases., Results: Notably, we observed no presence of morules in POLEmut cases, while SD was identified in 20% of instances. Conversely, morules and SD were identified in 12.7% and 26.1% of non-POLEmut EC cases, respectively, with morules consistently linked to a POLE wild-type status. The nuclear β-catenin expression is typically absent in tumors with wild-type POLE (wt-POLE) status., Conclusions: Our findings suggest that the presence of either morules or nuclear β-catenin expression in EEC could practically rule out the presence of POLE mutations. These morphologic and immunohistochemical features can be used as preliminary screening tools for POLE mutations, offering significant savings in time and resources and potentially enhancing clinical decision-making and patient management strategies. However, further validation in larger, multi-institutional studies is required to fully understand the implications of these findings on clinical practice., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background and Objectives: Recent molecular analyses have shown that the driver genetic mutations of meningiomas were associated with the anatomic location. Among these, POLR2A mutation is common among lesions in the skull base, mainly in the cerebellopontine angle (CPA). The objective of this study was to investigate the efficacy of POLR2A mutation as a prognostic marker for CPA meningiomas., Methods: We retrospectively analyzed the clinical data of 70 patients who had World Health Organization grade I CPA meningiomas. Somatic DNA was analyzed by Sanger sequencing and microsatellite array to examine for NF2 , AKT1 , KLF4 , SMO , and POLR2A mutations and 22q loss. Genetic and clinical parameters were analyzed to identify the factors related with tumor recurrence., Results: We detected clearly the clinical features of the CPA cases with POLR2A mutation. Compared with cases without POLR2A mutation, cases with POLR2A mutation had more meningothelial type ( P = 6.9 × 10 -4 ), and higher rate of recurrence ( P = .04). We found that the poor prognostic factors associated with the recurrence of CPA meningiomas were POLR2A mutation ( P = .03, hazard ratio [HR] 9.38, 95% CI 1.26-70.0) and subtotal resection (STR) ( P = 5.1 × 10 -4 , HR 63.1, 95% CI 6.09-655.0). In addition, in the group that underwent STR, POLR2A mutation was a poor prognostic factor associated with tumor recurrence ( P = .03, HR 11.1, 95% CI 1.19-103.7)., Conclusion: POLR2A mutation and STR were the poor prognostic markers associated with the recurrence of CPA meningioma. For CPA meningioma cases that underwent STR, only POLR2A mutation was a poor prognostic factor. Detecting POLR2A mutation may be a cost-effective, easy, and useful marker for prognostication., (Copyright © Congress of Neurological Surgeons 2024. All rights reserved.)

Context.—: Molecular stratification of endometrial carcinoma provides more accurate prognostic information than traditional clinicopathologic features. However, because next-generation sequencing is typically recommended for polymerase tifilon (POLE) mutation detection, the practical application of a test based on molecular stratification is limited in the clinical setting., Objective.—: To evaluate a polymerase chain reaction (PCR)-based assay for POLE mutation detection in endometrial carcinoma., Design.—: We developed a PCR-based technology called Dalton Mutation Identifier Technology (Dalton-MIT) that targets 9 mutation sites within POLE exons. Endometrial carcinoma specimens from 613 patients were tested for POLE mutations. Correlations between POLE mutations and patient clinicopathologic characteristics and prognosis were analyzed., Results.—: PCR detection data showed that the incidence rate of POLE mutation was 11.4% (70 of 613). Patients with POLE mutations had better clinicopathologic characteristics and prognosis than those with non-POLE mutations. Comparison between Dalton-MIT and next-generation sequencing in 59.5% (365 of 613) of specimens showed that the sensitivity of Dalton-MIT for detecting POLE pathogenic mutations was 100%, the specificity was 99.3%, the Youden index was .993, and the κ value was .981 (P < .001)., Conclusions.—: Our data demonstrate that POLE mutation detection by Dalton-MIT correlates with next-generation sequencing. This suggests that Dalton-MIT represents a promising alternative assay for detecting POLE mutations and will facilitate the wider application of molecular stratification tools for endometrial carcinoma in the clinic., (© 2024 College of American Pathologists.)

The clinical success of PARP1/2 inhibitors (PARPi) prompts the expansion of their applicability beyond homologous recombination deficiency. Here, we demonstrate that the loss of the accessory subunits of DNA polymerase epsilon, POLE3 and POLE4, sensitizes cells to PARPi. We show that the sensitivity of POLE4 knockouts is not due to compromised response to DNA damage or homologous recombination deficiency. Instead, POLE4 loss affects replication speed leading to the accumulation of single-stranded DNA gaps behind replication forks upon PARPi treatment, due to impaired post-replicative repair. POLE4 knockouts elicit elevated replication stress signaling involving ATR and DNA-PK. We find POLE4 to act parallel to BRCA1 in inducing sensitivity to PARPi and counteracts acquired resistance associated with restoration of homologous recombination. Altogether, our findings establish POLE4 as a promising target to improve PARPi driven therapies and hamper acquired PARPi resistance., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Based on findings from The Cancer Genome Atlas and the Proactive Molecular Risk Classifier for Endometrial Cancer algorithm, endometrial carcinoma can now be stratified into 4 prognostically distinct subgroups based on molecular alterations and immunohistochemical (IHC) aberrations. In this study, we describe the de novo adoption and clinical reporting of prognostic subgroup classification based on next-generation sequencing (NGS) and IHC analyses of all endometrial carcinoma resections at a single institution, framed by the Exploration, Preparation, Implementation, and Sustainment model. Results from the first 13 months show 188 tumors underwent analysis by a combination of IHC and a medium-sized (56 analyzed genes) NGS-based assay. All cases were assigned as either POLE ( POLE -mutated) (5.3%), mismatch repair deficient (27.7%), no specific molecular profile (45.7%), or p53 abnormal (21.3%) inclusive of multiple-classifier cases. NGS-based analysis revealed additional distinctions among the subgroups, including reduced levels of PI3K pathway activation in the p53 abnormal subgroup, an increased rate of CTNNB1 activating mutation in the no specific molecular profile subgroup, and lower TP53 mutation variant allele frequencies in POLE and mismatch repair deficient subgroups compared with the p53 abnormal subgroup. Overall, we describe the testing protocol, reporting, and results of a combination of NGS and IHC to prospectively prognosticate endometrial carcinomas at a single tertiary care center., Competing Interests: The authors declare no conflict of interest., (Copyright © 2023 by the International Society of Gynecological Pathologists.)

Introduction: The 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 Methods: Conducted 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., Results: Out 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 %)., Conclusion: In 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The diagnosis of synchronous carcinomas, involving both the endometrium and ovaries, is not a rare finding in gynecologic pathology and represents a challenge with implications on tumor staging and therapeutic decision-making. A mono-institutional series of 11 metastatic and 6 paired synchronous endometrial and ovarian carcinomas were reviewed by 2 expert pathologists based on previously published histopathologic criteria. The series was investigated for DNA mismatch repair proteins, p53, and POLE status and was subject to DNA-based next-generation sequencing targeting 67 cancer-related genes. Out of 17 pairs, 16 featured the same histotype (10 endometrioid, 4 serous high-grade, and 2 clear cells). By using WHO 2020 criteria, 11 couples of tumors were confirmed as metastatic and 6 couples were confirmed as independent. Based on next-generation sequencing analysis, 16 of 17 cases (11 metastatic and 5 independent) of our series showed evidence of a clonal relationship between endometrial and ovarian carcinomas. In metastatic cases, the adverse outcome was associated with nonendometrioid/high-grade endometrioid histotype and with the p53-abnormal molecular subtype. Four cases originally fulfilling clinicopathological criteria of independent endometrial and ovarian carcinomas were clonally related, low-grade endometrioid histotype and POLE -mut, mismatch repair deficient, and no specific molecular profile molecular subtypes; no adverse event was recorded in this group. In summary, the molecular characterization of synchronous gynecologic carcinomas confirms their clonal origin in most cases. However, the results of our study point out that the clinical behavior of these tumors seems to be determined by the presence of high-risk WHO 2020 histologic criteria and molecular features (i.e. p53-abnormal), rather than the monoclonal origin., Competing Interests: The authors declare no conflict of interest., (Copyright © 2023 by the International Society of Gynecological Pathologists.)

Purpose: Molecular classification of endometrial cancer (EC) has become a promising information to tailor preoperatively the surgical treatment. We aimed to evaluate the rate of lymph node metastases (LNM) in patients with EC according to molecular profile., Methods: A systematic review and meta-analysis were performed according to PRISMA guidelines by searching in two major electronic databases (PubMed and Scopus), including original articles reporting lymph node metastases according to the molecular classification of EC as categorized in the ESGO-ESMO-ESP guidelines., Results: Fifteen studies enrolling 3056 patients were included. Pooled prevalence LNM when considering only patients undergoing lymph node assessment was 4% for POLE-mutated (95%CI: 0-12%), 22% for no specific molecular profile (95% CI: 9-39%), 23% for Mismatch repair-deficiency (95%CI: 10-40%) and 31% for p53-abnormal (95%CI: 24-39%)., Conclusions: The presence of LNM seems to be influenced by molecular classification. P53-abnormal group presents the highest rate of nodal involvement, and POLE-mutated the lowest., (© 2024. The Author(s), under exclusive licence to Federación de Sociedades Españolas de Oncología (FESEO).)

Genetic sequencing has revolutionized immunotherapy in colorectal cancer (CRC). Recent clinical trials have revealed a positive response to immunotherapy-based systemic therapies in CRC patient subgroups with microsatellite instability (MSI)-High or DNA polymerase epsilon (POLE) mutation. However, the unsatisfactory response rates was the major limitation in real-world practice of the precision immunotherapy in CRC. Adding photodynamic therapy (PDT) to systemic immunotherapy has showed synergetic anti-tumor effect by modulating tumor microenvironment, while the eligible patient's subgroups which would benefit from this combination remained equivocal. Here we reported a synchronous colorectal cancer patient with MSI-High and POLE mutation who had accelerated response in less than 2 cycles (42 days) of immunotherapy-based systemic therapies after tumor-directed PDT and has remained progression-free by far. This case enlightened the synergetic effect of PDT in immunotherapy-treated CRC patients, with the MSI and POLE-mutation status as predictors of survival benefits., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Gao, Ma, Shi, Yin, Zhu and Chen.)

Polymerase proofreading-associated polyposis (PPAP) is a rare disease with autosomal-dominant inheritance caused by germline variants in the POLE and POLD1 genes. PPAP has been reported to increase the risk of multiple cancers, including colon, duodenal, and endometrial cancers. Herein, we report a case in which multiple duodenal tumors led to the detection of a POLE mutation. A 43-year-old woman underwent esophagogastroduodenoscopy (EGD). Multiple duodenal tumors were detected, and all lesions were treated endoscopically. The patient had a history of multiple colorectal cancers and endometrial cancer along with a family history of cancer; hence, genetic testing was performed, and POLE variant, c.1270C > G (p.Leu424Val) was detected. Hereditary colorectal cancer syndromes should be considered in patients with colorectal cancer who have multiple cancers or a family history of cancer, and multigene panel sequencing is useful in confirming the diagnosis. In addition, duodenal tumors frequently coexist in patients with PPAP-carrying POLE variants, while the endoscopic treatment for duodenal tumors becomes safe and useful with several new approaches. Therefore, surveillance EGD is necessary in such patients for the early detection and treatment of duodenal tumors., (© 2024. Japanese Society of Gastroenterology.)

Recycling of parental histones is an important step in epigenetic inheritance. During DNA replication, DNA polymerase epsilon subunit DPB3/DPB4 and DNA replication helicase subunit MCM2 are involved in the transfer of parental histones to the leading and lagging strands, respectively. Single Dpb3 deletion (dpb3Δ) or Mcm2 mutation (mcm2-3A), which each disrupts one parental histone transfer pathway, leads to the other's predominance. However, the biological impact of the two histone transfer pathways on chromatin structure and DNA repair remains elusive. In this study, we used budding yeast Saccharomyces cerevisiae to determine the genetic and epigenetic outcomes from disruption of parental histone H3-H4 tetramer transfer. We found that a dpb3Δ mcm2-3A double mutant did not exhibit the asymmetric parental histone patterns caused by a single dpb3Δ or mcm2-3A mutation, suggesting that the processes by which parental histones are transferred to the leading and lagging strands are independent. Surprisingly, the frequency of homologous recombination was significantly lower in dpb3Δ, mcm2-3A and dpb3Δ mcm2-3A mutants, likely due to the elevated levels of free histones detected in the mutant cells. Together, these findings indicate that proper transfer of parental histones during DNA replication is essential for maintaining chromatin structure and that lower homologous recombination activity due to parental histone transfer defects is detrimental to cells., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Competing Interests: No potential conflict of interest relevant to this article was reported.

Background: POLE mutated endometrial carcinomas may represent a subspecific type of tumors harboring a more favorable prognosis. Grade 3 (G3 or high-grade) endometrioid endometrial carcinomas remain a clinical dilemma, with some tumors behaving as the low-grade counterparts and others presenting a more aggressive behavior., Objectives: To determine the association between POLE mutational status and the overall-survival (OS) and progression-free-survival (PFS) of patients with G3 endometrioid endometrial cancer (EC). We also aimed to determine the prevalence of POLE mutations in G3 endometrioid EC., Methods: We conducted a systematic review in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (PROSPERO No: CRD4202340008). We searched the following electronic databases: PubMed/Medline, EMBASE, Cochrane Library, Scopus, and Web of Science. For time-to-event data, the effect of POLE mutation in G3 EC was described using hazard ratios (HRs) and corresponding 95% confidence intervals (CIs). Individual patient data for each study was investigated if available from the study authors. If individual patient data were not available, information regarding time-to-event outcomes was extracted using an appropriate methodology. OS and PFS were analyzed using both one-stage and two-stage approaches, the Kaplan-Meier method, and Cox-proportional hazards models., Results: This systematic review and meta-analysis included 19 studies with 3092 patients who had high-grade endometrioid EC. Patients with POLE mutations had lower risks of death (HR = 0.36, 95% CI 0.26 to 0.50, I 2  = 0%, 10 trials) and disease progression (HR = 0.31, 95% CI 0.17 to 0.57, I 2  = 33%, 10 trials). The pooled prevalence of POLE mutation was 11% (95% CI 9 to 13, I 2  = 68%, 18 studies)., Conclusion: POLE mutations in high-grade endometrioid EC are associated with a more favorable prognosis with increased OS and PFS., Competing Interests: Declaration of competing interest Dr. Abu-Rustum received research funding paid to the institution from GRAIL. Memorial Sloan Kettering Cancer Center also has equity in GRAIL. All the other authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lymphoma is the most common malignant tumor arising from immune system. Recently, DNA polymerase epsilon subunit 2 (POLE2) was identified to be a tumor promotor in a variety of malignant tumors. However, the biological role of POLE2 in lymphoma is still largely unclear. In our present study, the expression patterns of POLE2 in lymphoma tissues were identified by immunohistochemistry (IHC) staining of human tissue microarray. Cell viability was determined by CCK-8 assay. Cell apoptosis and cycle distribution were evaluated by Annexin V and PI staining, respectively. Cell migration was analyzed by transwell assay. Tumor growth in vivo was observed by a xenograft model of mice. The potential signaling was explored by human phospho-kinase array and immunoblotting. POLE2 was significantly upregulated in human lymphoma tissues and cells. POLE2 knockdown attenuated the proliferation, migration capabilities of lymphoma cells, as well as induced cell apoptosis and cycle arrest. Moreover, POLE2 depletion impaired the tumor growth in mice. Furthermore, POLE2 knockdown apparently inhibited the activation of β-Catenin and downregulated the expression of Wnt/β-Catenin signaling-related proteins. POLE2 knockdown suppressed the proliferation and migration of lymphoma cells by inhibiting Wnt/β-Catenin signaling pathway. POLE2 may serve as a novel therapeutic target for lymphoma., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

DNA polymerase epsilon (Pol ε), a component of the core replisome, is involved in DNA replication. Although genetic defects of Pol ε have been reported to cause immunodeficiency syndromes, its role in haematopoiesis remains unknown. Here, we identified compound heterozygous variants (p.[Asp1131fs];[Thr1891del]) in POLE , encoding Pol ε catalytic subunit A (POLE1), in siblings with a syndromic form of severe congenital transfusion-dependent anaemia. In contrast to Diamond-Blackfan anaemia, marked reticulocytopenia or marked erythroid hypoplasia was not found. Their bone marrow aspirates during infancy revealed erythroid dysplasia with strongly positive TP53 in immunostaining. Repetitive examinations demonstrated trilineage myelodysplasia within 2 years from birth. They had short stature and facial dysmorphism. HEK293 cell-based expression experiments and analyses of patient-derived induced pluripotent stem cells (iPSCs) disclosed a reduced mRNA level of Asp1131fs-POLE1 and defective nuclear translocation of Thr1891del-POLE1. Analysis of iPSCs showed compensatory mRNA upregulation of the other replisome components and increase of the TP53 protein, both suggesting dysfunction of the replisome. We created Pole -knockout medaka fish and found that heterozygous fishes were viable, but with decreased RBCs. Our observations expand the phenotypic spectrum of the Pol ε defect in humans, additionally providing unique evidence linking Pol ε to haematopoiesis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Endometrial cancers with more than one molecular feature- POLE mutations (POLEmut), mismatch repair protein deficiency (MMRd), p53 abnormality (p53abn)-are called 'multiple classifiers'., Objective: To describe our cohort of multiple classifiers and to report the results of a review on their incidence and the techniques used to identify them., Methods: Multiple classifiers identified at the European Institute of Oncology, Milan, between April 2019 and Decmber 2022, were included. Clinicopathological, molecular characteristics, and oncologic outcomes were summarized and compared between single and multiple classifiers sharing common features. Studies on molecular classification of endometrial cancer were searched in the PubMed Database to collect data on the incidence of multiple classifiers and the techniques used for classification., Results: Among 422 patients, 48 (11.4%) were multiple classifiers: 15 (3.6%) POLEmut-p53abn, 2 (0.5%) POLEmut-MMRd, 28 (6.6%) MMRd-p53abn, and 3 (0.7%) POLEmut-MMRd-p53abn. MMRd-p53abn and MMRd differed in histotype (non-endometrioid: 14.8% vs 2.0%, p=0.006), grade (high-grade: 55.6% vs 22.2%, p=0.001), and MMR proteins expression, whereas they differed from p53abn in histotype (non-endometrioid: 14.8% vs 50.0%, p=0.006). POLEmut-p53abn and POLEmut differed only in grade (high-grade: 66.7% vs 22.7%, p=0.008), while they differed from p53abn in age (56.1 vs 66.7 years, p=0.003), stage (advanced: 6.7% vs 53.4%, p=0.001), and histotype (non-endometrioid: 6.7% vs 50.0%, p=0.002). Two (7.1%) patients with MMRd-p53abn, 4 (4.0%) with MMRd, and 25 (34.3%) with p53abn had a recurrence. No recurrences were observed in POLEmut-p53abn and POLEmut. TP53 sequencing allowed the detection of additional 7 (18.9%) multiple classifiers with normal p53 immunostaining. The incidence of multiple classifiers ranged from 1.8% to 9.8% in 10 published studies including >100 patients. When only p53 immunohistochemistry was performed, the highest incidence was 3.9%., Conclusions: The characteristics of POLEmut-p53abn resembled those of POLEmut, whereas MMRd-p53abn appeared to be intermediate between MMRd and p53abn. The high proportion of multiple classifiers may be related to the methods used for molecular classification, which included both p53 immunohistochemistry and TP53 sequencing., Competing Interests: Competing interests: None declared., (© IGCS and ESGO 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: The aim of this study is to delineate the molecular classification features within Chinese endometrial cancer (EC) patients and to evaluate the concurrence between two widely employed methods for diagnosing EC molecular subtypes., Methods: This retrospective observational cohort study encompassed 479 cases of EC for analysis. Utilizing next-generation sequencing (NGS) panels targeting POLE, TP53, and microsatellite instability (MSI) status, four subtypes [POLE ultramutated (POLE mut), MMR-deficient (MMRd), p53 abnormal (p53abn), and no specific molecular profile (NSMP)] were classified. Immunohistochemistry (IHC) was employed to ascertain the expression of p53 and MMR proteins., Results: Among the 479 patients, the distribution of EC subtypes was as follows: 28 (5.85%) POLE mut, 67 (13.99%) MMRd, 60 (12.53%) p53abn, and 324 (67.64%) NSMP. When compared to published findings on EC subtypes in the Caucasian population, our real-world data on Chinese ECs revealed a notably higher proportion of NSMP/CNL (copy number low). The evaluation of MSI/MMR status through NGS-based and IHC-based methods displayed substantial concordance (Kappa = 0.91). Slight discordance between the two techniques in identifying p53 abnormalities (Kappa = 0.83) might stem from TP53 truncating mutations, cytoplasmic p53 expression, null TP53 mutants, and well-documented challenges in interpreting p53 IHC., Conclusions: Chinese ECs exhibit distinctive molecular attributes. For accurate molecular subtyping of Chinese ECs, additional molecular markers that align with the Chinese population's characteristics should be incorporated into existing classifiers. The study's outcomes underscore a strong agreement between NGS and IHC in TP53/p53 detection and MSI assessment., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

POLE driver mutations in the exonuclease domain (ExoD driver) are prevalent in several cancers, including colorectal cancer and endometrial cancer, leading to dramatically ultra-high tumor mutation burden (TMB). To understand whether POLE mutations that are not classified as drivers (POLE Variant) contribute to mutagenesis, we assessed TMB in 447 POLE-mutated colorectal cancers, endometrial cancers, and ovarian cancers classified as TMB-high ≥10 mutations/Mb (mut/Mb) or TMB-low <10 mut/Mb. TMB was significantly highest in tumors with "POLE ExoD driver plus POLE Variant" (colorectal cancer and endometrial cancer, P < 0.001; ovarian cancer, P < 0.05). TMB increased with additional POLE variants (P < 0.001), but plateaued at 2, suggesting an association between the presence of these variants and TMB. Integrated analysis of AlphaFold2 POLE models and quantitative stability estimates predicted the impact of multiple POLE variants on POLE functionality. The prevalence of immunogenic neoepitopes was notably higher in the "POLE ExoD driver plus POLE Variant" tumors. Overall, this study reveals a novel correlation between POLE variants in POLE ExoD-driven tumors, and ultra-high TMB. Currently, only select pathogenic ExoD mutations with a reliable association with ultra-high TMB inform clinical practice. Thus, these findings are hypothesis-generating, require functional validation, and could potentially inform tumor classification, treatment responses, and clinical outcomes., Significance: 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., (© 2024 The Authors; Published by the American Association for Cancer Research.)

Alterations in the exonuclease domain of DNA polymerase ε cause ultramutated cancers. These cancers accumulate AGA>ATA transversions; however, their genomic features beyond the trinucleotide motifs are obscure. We analyze the extended DNA context of ultramutation using whole-exome sequencing data from 524 endometrial and 395 colorectal tumors. We find that G>T transversions in POLE-mutant tumors predominantly affect sequences containing at least six consecutive purines, with a striking preference for certain positions within polypurine tracts. Using this signature, we develop a machine-learning classifier to identify tumors with hitherto unknown POLE drivers and validate two drivers, POLE-E978G and POLE-S461L, by functional assays in yeast. Unlike other pathogenic variants, the E978G substitution affects the polymerase domain of Pol ε. We further show that tumors with POLD1 drivers share the extended signature of POLE ultramutation. These findings expand the understanding of ultramutation mechanisms and highlight peculiar mutagenic properties of polypurine tracts in the human genome., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Endometrial carcinoma (EC) is a prevalent gynecological tumor in women, and its treatment and prevention are significant global health concerns. The mutations in DNA polymerase ε (POLE) are recognized as key features of EC and may confer survival benefits in endometrial cancer patients undergoing anti-PD-1/PD-L1 therapy. However, the anti-tumor mechanism of POLE mutations remains largely elusive. This study demonstrates that the hot POLE P286R mutation impedes endometrial tumorigenesis by inducing DNA breakage and activating the cGAS-STING signaling pathway. The POLE mutations were found to inhibit the proliferation and stemness of primary human EC cells. Mechanistically, the POLE mutants enhance DNA damage and suppress its repair through the interaction with DNA repair proteins, leading to genomic instability and the upregulation of cytoplasmic DNA. Additionally, the POLE P286R mutant also increases cGAS level, promotes TBK1 phosphorylation, and stimulates inflammatory gene expression and anti-tumor immune response. Furthermore, the POLE P286R mutation inhibits tumor growth and facilitates the infiltration of cytotoxic T cells in human endometrial cancers. These findings uncover a novel mechanism of POLE mutations in antagonizing tumorigenesis and provide a promising direction for effective cancer therapy., (© 2024. The Author(s).)

Introduction: Patients with polymerase epsilon (POLE) mutation (POLEmut) subtype, MMR-deficient (MMR-d) subtype as classified by The Cancer Genome Atlas (TCGA), and a high tumor mutation burden (TMB-high) potentially benefit from immunotherapy. However, characteristics of the cytological morphology within these populations remain unknown., Methods: DNA extracted from formalin-fixed paraffin-embedded tissues was subjected to next-generation sequencing analysis. Genomic mutations related to gynecological cancers, TMB, and microsatellite instability were analyzed and were placed in four TCGA classification types. The following morphological cytological investigations were conducted on endometrial cancer using a liquid-based preparation method, prior to the commencement of initial treatment: (i) cytological backgrounds; (ii) differences between each count of neutrophils and lymphocytes as described below., Results: Insignificant differences in the cytological background patterns of TCGA groups and TMB status were found. Although there was no significant difference in neutrophil count (p = 0.955) in the TCGA groups, POLEmut and MMR-d had significantly higher lymphocyte counts than no specific molecular profile (NSMP) (p = 0.019 and 0.037, respectively); furthermore, p53mut also tended to be significant (p = 0.064). Lymphocyte counts in TMB-high were also significantly greater than TMB-low (p = 0.002). POLEmut showed a positive correlation between TMB levels and lymphocyte counts. For predicting patients with POLEmut plus MMR-d, lymphocyte counts demonstrated a superior diagnostic accuracy of area under the curve (AUC) (0.70, 95% CI: 0.57-0.84), with a cutoff value of 26 high-power field., Conclusion: Lymphocyte count using liquid-based cytology for patients with endometrial cancer may predict POLEmut plus MMR-d of TCGA groups and TMB-high in those who can benefit from immunotherapy., (© 2024 S. Karger AG, Basel.)

Background/aim: Among the four genomic subtypes of endometrial cancer, distinguishing between the DNA polymerase epsilon mutation (POLEmut) and no specific molecular profile (NSMP) subtypes requires genomic profiling owing to the lack of surrogate immunohistochemical markers. We have previously found that, histologically, the POLEmut-subtype exhibits surface epithelial slackening (SES). Therefore, to improve subtype identification, we aimed to extract cytological features corresponding to SES in POLEmut-subtype cervical cytology specimens., Materials and Methods: We analyzed 104 endometrial cancer cervical cytology specimens, with integrative diagnosis confirmation via histology, immunohistochemistry, and genomic profiling. Cytological features were evaluated for the presence of atypical glandular cells, atypical cell appearance in single cells and clusters, and cytological SES and the presence of tumor-infiltrating inflammatory cells in clusters., Results: Based on cervical cytology, the POLEmut- and p53mut-subtypes exhibited more frequent atypical cells in smaller clusters, giant tumor cells, and cytological SES patterns than the NSMP-subtype. Tumor-infiltrating lymphocytes were frequent in the POLEmut- and mismatch repair-deficient subtypes., Conclusion: Histologically-detected SES as well as other endometrial cancer features may be preserved in the atypical cell clusters observed in cervical cytology specimens. Cytological detection of SES and of smaller clusters of atypical cells and inflammatory cells with moderate atypia are suggestive of POLEmut-subtype. Integrative diagnosis including genomic profiling remains critical for diagnostic confirmation., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Errors made by DNA polymerases contribute to both natural variation and, in extreme cases, genome instability and its associated diseases. Recently, the importance of polymerase misincorporation in disease has been highlighted by the identification of cancer-associated polymerase variants with mutations in the exonuclease domain. A subgroup of these variants have a hypermutation phenotype in tumours, and when modelled in yeast, they show mutation rates in excess of that seen with polymerase with simple loss of proofreading activity. We have developed a bypass assay to rapidly determine the tendency of a polymerase to misincorporate in vitro. We have used the assay to compare misincorporation by wild-type, exonuclease-defective and two hypermutating human DNA polymerase ε variants, P286R and V411L. The assay clearly distinguished between the misincorporation rates of wild-type, exonuclease dead and P286R polymerases. However, the V411L polymerase showed misincorporation rate comparable to the exonuclease dead enzyme rather than P286R, suggesting that there may be some differences in the way that these variants cause hypermutation. Using this assay, misincorporation opposite a templated C nucleotide was consistently higher than for other nucleotides, and this caused predominantly C-to-T transitions. This is consistent with the observation that C-to-T transitions are commonly seen in DNA polymerase ε mutant tumours., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Genome topology is tied to R-loop formation and genome stability. However, the regulatory mechanism remains to be elucidated. By establishing a system to sense the connections between R-loops and genome topology states, we show that inhibiting DNA topoisomerase 1 (TOP1i) triggers the global increase of R-loops (called topoR-loops) and DNA damages, which are exacerbated in the DNA damage repair-compromised mutant atm. A suppressor screen identifies a mutation in POL2A, the catalytic subunit of DNA polymerase ε, rescuing the TOP1i-induced topoR-loop accumulation and genome instability in atm. Importantly we find that a highly conserved junction domain between the exonuclease and polymerase domains in POL2A is required for modulating topoR-loops near DNA replication origins and facilitating faithful DNA replication. Our results suggest that DNA replication acts in concert with genome topological states to fine-tune R-loops and thereby maintain genome integrity, revealing a likely conserved regulatory mechanism of TOP1i resistance in chemotherapy for ATM-deficient cancers., (© 2023. The Author(s).)

Pathogenic germline variants in the DNA polymerase genes POLE and POLD1 cause polymerase proofreading-associated polyposis, a dominantly inherited disorder with increased risk of colorectal carcinomas and other tumors. POLE/POLD1 variants may result in high somatic mutation and neoantigen loads that confer susceptibility to immune checkpoint inhibitors (ICIs). To explore the role of POLE/POLD1 germline variants in glioma predisposition, whole-exome sequencing was applied to leukocyte DNA of glioma patients from 61 tumor families with at least one glioma case each. Rare heterozygous POLE/POLD1 missense variants predicted to be deleterious were identified in glioma patients from 10 (16%) families, co-segregating with the tumor phenotype in families with available DNA from several tumor patients. Glioblastoma patients carrying rare POLE variants had a mean overall survival of 21 months. Additionally, germline variants in POLD1, located at 19q13.33, were detected in 2/34 (6%) patients with 1p/19q-codeleted oligodendrogliomas, while POLE variants were identified in 2/4 (50%) glioblastoma patients with a spinal metastasis. In 13/15 (87%) gliomas from patients carrying POLE/POLD1 variants, features of defective polymerase proofreading, e.g. hypermutation, POLE/POLD1-associated mutational signatures, multinucleated cells, and increased intratumoral T cell response, were observed. In a CRISPR/Cas9-derived POLE-deficient LN-229 glioblastoma cell clone, a mutator phenotype and delayed S phase progression were detected compared to wildtype POLE cells. Our data provide evidence that rare POLE/POLD1 germline variants predispose to gliomas that may be susceptible to ICIs. Data compiled here suggest that glioma patients carrying POLE/POLD1 variants may be recognized by cutaneous manifestations, e.g. café-au-lait macules, and benefit from surveillance colonoscopy., (© 2023. The Author(s).)

Background: Histopathological examination is still the backbone for the diagnosis and treatment decision making in endometrial carcinoma (EC). The binary classification of EC into type 1 (mostly endometrioid) and type 2 (mostly serous), although still helpful, showed overlapping clinical, morphological and molecular features and was not very prognostic discriminatory for all subtypes of EC., Methods: Analysing the most recent studies dealing with the molecular classification of EC and the recommendations of the German S3-guidelines for EC., Results and Conclusion: Based on the comprehensive molecular study of The Cancer Genome Atlas Project (TCGA) four distinct molecular subtypes have been identified: EC with POLE mutation (POLEmut), with loss of mismatch repair proteins (MMR deficiency; dMMR), or with TP53 mutation (p53mut) and without any of these alterations, termed NSMP (no specific molecular profile). The molecular classification of EC presents a morphomolecular approach, based on histopathological evaluation (tumor diagnosis, subtyping, grading), immunohistochemistry (MMR, p53) and molecular analyses for POLE. The incorporation of this molecular classification is recommended for clinical use by the World Health Organisation (WHO) as well as many national guidelines and international societies. Due to the heterogeneity of NSMP-EC, which is the largest molecular group, additional research is indicated to further characterise these tumors., (© 2023. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Germline pathogenic variants in the exonuclease domain of the replicative DNA polymerase Pol ε encoded by the POLE gene, predispose essentially to colorectal and endometrial tumors by inducing an ultramutator phenotype. It is still unclear whether all the POLE alterations influence similar strength tumorigenesis, immune microenvironment, and treatment response. In this review, we summarize the current understanding of the mechanisms and consequences of POLE mutations in human malignancies; we highlight the heterogeneity of mutation rate and cancer aggressiveness among POLE variants, propose some mechanistic basis underlining such heterogeneity, and discuss novel considerations for the choice and efficacy of therapies of POLE tumors., (© 2023 Selves et al.)

Background: Germline 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., Methods: A 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., Results: Gene-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., Conclusions: Our 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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Oral squamous cell carcinoma is the most common malignant tumor in the head and neck at present, but the mechanism of its occurrence and development is still unclear, and there is still a lack of effective targeting drugs. The second major subunit of DNA polymerase (POLE2) has exonuclease activity and can catalyze the replication and modification of new chains. Our previous studies have found that it is associated with OSCC progression, but the mechanism is unclear.The expression of POLE2 in OSCC was detected by immunological method. The expression of POLE2 was inhibited in OSCC cells, and the biological function of the cells was detected by RT-PCR and Western Blot. Cell proliferation, apoptosis and migration were detected by colony formation, MTT, flow cytometry, wound healing and Transwell. The expression level of POLE2 gene in OSCC was significantly higher than that in normal tissues. In addition, the expression level of POLE2 gene was significantly different from the tumor type and prognosis. During the development of oral squamous cell carcinoma, silencing POLE2 inhibits the proliferation of oral cancer cells and promotes apoptosis. The results of animal experiments also support the positive correlation between POLE2 and OSCC progression. We further demonstrated that POLE2 can up-regulate the downregulation of apoptosis-related proteins such as Caspase3, CD40, CD40L, DR6, Fas, IGFBP-6, P21, and SMAC. In addition, POLE2 regulates OSCC progression by inhibiting the PI3K/AKT pathway. POLE2 is closely related to the progression of OSCC, and POLE2 may be a potential target for OSCC treatment., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The cancer syndrome polymerase proofreading-associated polyposis results from germline mutations in the POLE and POLD1 genes. Mutations in the exonuclease domain of these genes are associated with hyper- and ultra-mutated tumors with a predominance of base substitutions resulting from faulty proofreading during DNA replication. When a new variant is identified by gene testing of POLE and POLD1, it is important to verify whether the variant is associated with PPAP or not, to guide genetic counseling of mutation carriers. In 2015, we reported the likely pathogenic (class 4) germline POLE c.1373A > T p.(Tyr458Phe) variant and we have now characterized this variant to verify that it is a class 5 pathogenic variant. For this purpose, we investigated (1) mutator phenotype in tumors from two carriers, (2) mutation frequency in cell-based mutagenesis assays, and (3) structural consequences based on protein modeling. Whole-exome sequencing of two tumors identified an ultra-mutator phenotype with a predominance of base substitutions, the majority of which are C > T. A SupF mutagenesis assay revealed increased mutation frequency in cells overexpressing the variant of interest as well as in isogenic cells encoding the variant. Moreover, exonuclease repair yeast-based assay supported defect in proofreading activity. Lastly, we present a homology model of human POLE to demonstrate structural consequences leading to pathogenic impact of the p.(Tyr458Phe) mutation. The three lines of evidence, taken together with updated co-segregation and previously published data, allow the germline variant POLE c.1373A > T p.(Tyr458Phe) to be reclassified as a class 5 variant. That means the variant is associated with PPAP., (© 2023. The Author(s).)

DNA mismatch repair (MMR) in eukaryotes is believed to occur post-replicatively, wherein nicks or gaps in the nascent DNA strand are suggested to serve as strand discrimination signals. However, how such signals are generated in the nascent leading strand has remained unclear. Here we examine the alternative possibility that MMR occurs in conjunction with the replication fork. To this end, we utilize mutations in the PCNA interacting peptide (PIP) domain of the Pol3 or Pol32 subunit of DNA polymerase δ (Polδ) and show that these pip mutations suppress the greatly elevated mutagenesis in yeast strains harboring the pol3-01 mutation defective in Polδ proofreading activity. And strikingly, they suppress the synthetic lethality of pol3-01 pol2-4 double mutant strains, which arises from the vastly enhanced mutability due to defects in the proofreading functions of both Polδ and Polε. Our finding that suppression of elevated mutagenesis in pol3-01 by the Polδ pip mutations requires intact MMR supports the conclusion that MMR operates at the replication fork in direct competition with other mismatch removal processes and with extension of synthesis from the mispair by Polδ. Furthermore, the evidence that Polδ pip mutations eliminate almost all the mutability of pol2-4 msh2Δ or pol3-01 pol2-4 adds strong support for a major role of Polδ in replication of both the leading and lagging DNA strands., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Despite the process of DNA replication being mechanistically highly conserved, the location of origins of replication (ORI) may vary from one tissue to the next, or between rounds of replication in eukaryotes, suggesting flexibility in the choice of locations to initiate replication. Lists of human ORI therefore vary widely in number and location, and there are currently no methods available to compare them. Here, we propose a method of detection of ORI based on somatic mutation patterns generated by the mutator phenotype of damaged DNA polymerase epsilon (POLE)., Results: We report the genome-wide localization of constitutive ORI in POLE-mutated human tumors using whole genome sequencing data. Mutations accumulated after many rounds of replication of unsynchronized dividing cell populations in tumors allow to identify constitutive origins, which we show are shared with high fidelity between individuals and tumor types. Using a Smith-Waterman-like dynamic programming approach, we compared replication origin positions obtained from multiple different methods. The comparison allowed us to define a consensus set of replication origins, identified consistently by multiple ORI detection methods. Many DNA features co-localized with the consensus set of ORI, including chromatin loop anchors, G-quadruplexes, S/MARs, and CpGs. Among all features, the H2A.Z histone exhibited the most significant association., Conclusions: Our results show that mutation-based detection of replication origins is a viable approach to determining their location and associated sequence features., (© 2023. The Author(s).)

Pol2 is the leading-strand DNA polymerase in budding yeast. Here we describe an antagonism between its conserved POPS (Pol2 family-specific catalytic core peripheral subdomain) and exonuclease domain and the importance of this antagonism in genome replication. We show that multiple defects caused by POPS mutations, including impaired growth and DNA synthesis, genome instability, and reliance on other genome maintenance factors, were rescued by exonuclease inactivation. Single-molecule data revealed that the rescue stemmed from allowing sister replication forks to progress at equal rates. Our data suggest that balanced activity of Pol2's POPS and exonuclease domains is vital for genome replication and stability., (© 2023 Meng et al.; Published by Cold Spring Harbor Laboratory Press.)