Comparison between a Luminex-based multiplex kit with a sequence-specific oligonucleotide probe and next-generation sequencing for the detection of POLE oncogenic mutations in endometrial cancer

Comparison between a Luminex-based multiplex kit with a sequence-specific oligonucleotide probe and next-generation sequencing for the detection of POLE oncogenic mutations in endometrial cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Comparison between a Luminex-based multiplex kit with a sequence-specific oligonucleotide probe and next-generation sequencing for the detection of POLE oncogenic mutations in endometrial cancer Mayumi Kobayashi Kato, Takayuki Kawai, Hideki Okada, Takuya Kondo, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5540131/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: In endometrial cancer, detection of oncogenic mutations in the polymerase epsilon ( POLE ) gene is crucial for accurate staging according to the 2023 International Federation of Gynecology and Obstetrics classification and for minimizing overtreatment. However, POLE sequencing is expensive, time-consuming, and often inaccessible in settings without specialized equipment. We developed a novel multiplex kit for the detection of POLE mutations using a Luminex (xMAP) assay in a single reaction. The aim of this study was to evaluate the accuracy of the multiplex kit for routine clinical samples and compare it with that of conventional next-generation sequencing (NGS). Methods : Hysterectomy specimens and endometrial biopsies were collected at the National Cancer Center Hospital between 1999 and 2023. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissues. Both the Luminex (xMAP)-based multiplex kit and NGS targeting all POLE exons were used. Concordance was assessed using Cohen’s kappa. Results: Of the 502 samples, 432 were hysterectomy specimens and 70 were biopsies. In the surgical samples, both the Luminex (xMAP)-based kit and NGS detected 52 POLE mutations (12.0%) with perfect concordance (κ=1.000). In the biopsies, 33 POLE mutations were identified using both methods, with complete concordance. Notably, the Luminex (xMAP)-based kit successfully analyzed all 28 samples that failed NGS quality control and detected four cases with POLE mutations. Conclusions: The Luminex (xMAP)-based kit demonstrates high concordance with NGS for the detection of POLE mutations. With further external validation, this kit could become a reliable and accessible alternative to NGS. Biological sciences/Cancer Health sciences/Medical research endometrial cancer POLE mutation Luminex (xMAP) assay next-generation sequencing concordance rate Figures Figure 1 Introduction In the new era of molecular classification of endometrial cancer (EC), the International Federation of Gynecology and Obstetrics (FIGO) recommended incorporation of molecular subtypes (polymerase epsilon [ POLE ] mutated [ POLE -mut], mismatch repair [MMR] deficiency [MMR-D], no specific molecular profile [NSMP], and p53-abnormality [p53abn]) into the 2023 revision of EC staging 1 . In addition, POLE -mut EC confined to the uterus and POLE -mut EC with cervical invasion were downstaged to stage IA (stage IA m ) regardless of lymph vascular space invasion, because they are associated with an excellent clinical outcome and a very low risk of relapse 2 . The POLE -mut group is the most upstream branching group according to the steps for molecular classification based on the 2020 WHO classification code 3 . Because the other three groups (MMR-D, NSMP, and p53abn) cannot be determined unless the POLE mutation status is correctly evaluated, it is necessary to perform tests for the detection of these mutations, even though POLE -mut EC accounts for only approximately 10% of all ECs 4 . However, the POLE mutation status can only be assessed using DNA sequencing methods such as next-generation sequencing (NGS) or Sanger sequencing, without any immunohistochemical (IHC) markers. These methods are expensive and time-consuming and require specialized equipment; these are factors that prevent widespread use of methods for the detection of POLE mutations, unlike IHC for the detection of MMR-D and p53abn. Luminex (xMAP) technology is used for multiplex molecular testing in a single well and requires only a small amount of tumor-derived DNA 5 , 6 . The MEBGEN RASKET-B KIT, which uses the Luminex (xMAP) assay to detect the BRAF mutation (V600E) and 48 different RAS mutations in colorectal cancer, has received regulatory approval for diagnostic use in Japan, has become widely used in daily practice, and is recognized as a testing platform 7 . Using this technique, we developed a new Luminex (xMAP)-based multiplex kit that simultaneously detects 25 POLE pathogenic mutations, including the five most common mutations (P286R, V411L, S297F, S459F, and A456P) 8 . The remaining 20 mutations include those determined to be pathogenic by the POLE pathogenic score and those suspected to be pathogenic by the COSMIC and cBioPortal databases, all of which have been linked to clinical information with a favorable prognosis in previous literature 8 – 10 . The aim of this study was to investigate the degree of concordance between the novel Luminex (xMAP)-based kit and NGS for the detection of POLE mutations in EC samples. If the robustness and feasibility of this multiplex kit using routine clinical samples are proven, this new method can be used as an alternative to NGS for POLE testing. Material and Methods Study design We identified consecutive patients with histologically confirmed EC whose samples were collected at the National Cancer Center Hospital between 1999 and 2023. In addition to surgical specimens, endometrial biopsy specimens were used in this study. A pathologist (H. Y.) blinded to the prognostic information selected cases for endometrial biopsy; this biopsy cohort was the same as that used in our previous study on the accuracy of molecular classification in preoperative materials 11 . Patients with multiple cancers who received neoadjuvant chemotherapy, did not have formalin-fixed paraffin-embedded (FFPE) samples, or had tissue samples of insufficient quality were excluded. Each case was reviewed by at least two gynecological pathologists, and the pathological diagnoses were validated in accordance with the 2020 World Health Organization tumor classification 12 . Clinicopathological information, including age and tumor stage (as defined by FIGO 2023 with molecular classification), was retrospectively collected for every patient. During their initial visit to our hospital, all patients provided informed consent for the utilization of samples in the research. The data derived from the samples collected after acquisition of informed consent are summarized on the hospital’s website. Patients retained the right to withdraw their presumed consent at any time. Only patients whose consent was not revoked were included in this study. The Institutional Review Board of the National Cancer Center Research Institute approved the study (2017 − 331). DNA preparation and NGS DNA was extracted from 530 FFPE samples of endometrial tumor tissue using a QIAamp DNA FFPE tissue kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany) 2 , 10 , 11 . All FFPE samples of endometrial tumor tissue obtained by hysterectomy as well as FFPE samples obtained via endometrial biopsy were successfully analyzed in our cohort. Fifty nanograms of DNA extracted from tumor tissues were utilized for library construction with the Ion AmpliSeq™ Custom Panel (Thermo Fisher Scientific, Waltham, MA, USA). For the identification of oncogenic mutations in POLE , the same criteria used in previously published studies were used 13 – 16 . Genomic data from paired biopsy and hysterectomy specimens were obtained using methods detailed in the prior study 11 . In some cases, the tumor areas were excised through macrodissection to enhance the concentration of tumor cells within the surgical specimens. In contrast, entire sections were used without dissection for all biopsy specimens. The purity of DNA derived from hysterectomy and biopsy specimens was measured using Nanodrop and Invitrogen Qubit assays. Both DNAs had A260/A280 ≥ 1.8, with high purity. Luminex (xMAP) assay The Luminex (xMAP) assay was conducted using DNA obtained from the Ion AmpliSeq custom panel. Twenty-five POLE mutations involving exon 9 codon 286 (P286R, P286S, P286L, P286H, P286C), codon 297 (S297F, S297Y), codon 278 (T278M), codon 295 (M295R), exon 11 codon 367 (F367S, F367C), codon 368 (D368Y), exon12 codon 369 (W369*), exon 13 codon 411 (V411L), codon 424 (L424V, L424l), codon 426 (A426V), codon 436 (P436R), codon 411 (P411L), codon 444 (M444K), exon 14 codon 456 (A456P), codon 457 (T457M), codon 459 (S459F, S459del), and codon 465 (A465V) were analyzed using Luminex (xMAP) technology. Assays using a pre-production POLE mutation detection kit (MBL, Tokyo, Japan) were performed according to the manufacturer’s instructions. The procedure took approximately 4.5 h (Supplementary Fig. 1). Statistical analysis Data are presented as percentages of patients, 95% confidence intervals (CIs), or hazard ratios (HRs). Cohen’s kappa (κ) coefficient was used to assess concordance between the results of NGS and those of the Luminex assay 17 . Kappa statistics of < 0, 0.00–0.20, 0.21–0.40, 0.41–0.60, 0.61–0.80, and 0.81–1.00 represented “no,” “slight,” “fair,” “moderate,” “substantial,” and “almost perfect” levels of agreement, respectively 18 . Percent agreement was also used to assess concordance between the results of NGS and those of the Luminex assay. R version 4.3.1 (R Foundation for Statistical Computing, Viena, Austria) was used to perform the binomial test. Relapse-free survival (RFS) was determined from the date of the initial surgical procedure to the date of death from any cause or the first recurrence. Overall survival (OS) was calculated from the date of the initial surgery to the date of death from any cause. Survivors were censored at the cut-off date (September 9, 2024). A P -value of < 0.05 was considered statistically significant. All statistical analyses were analyzed using JMP version 8.0.1 (SAS Institute Inc., Cary, North Carolina, United States). Results Specimen analysis The consort diagram for this study is shown in Fig. 1 . In total, 588 specimens were collected from 525 patients with histologically confirmed EC at our hospital, and FFPE tissue samples were obtained. A total of 530 samples were subjected to NGS; these included 458 surgical specimens and 72 biopsy specimens. After exclusion of 28 cases that did not pass quality control (QC) for NGS, 502 samples (432 surgical specimens and 70 biopsy specimens) were investigated to determine the concordance between the Luminex assay and NGS for POLE mutation detection. Frequency of POLE mutations Table 1 shows the frequencies of all POLE mutations detected by the Luminex assay and NGS in the 432 surgical specimens from patients with EC. The Luminex multiplex kit and NGS detected 52 POLE mutations (12.0%). The most frequent mutations were P286R (30.8%, 16/52), V411L (23.1%, 12/52), and A456P (17.3%, 9/52), followed by S297F (3.9%, 2/52) and P436R (5.8%, 3/52). Table 1. Frequency of all POLE mutations detected in patients with endometrial carcinoma by Luminex assay and NGS (n = 52) POLE status Number of cases (n) Proportion among 52 cases (%) POLE mutation 52 Exon 9 mutations P286R 16 30.8 S297F 3 5.8 P286C 1 1.9 S297Y 1 1.9 Exon 11 mutations F367C 1 1.9 Exon 13 mutations V411L 12 23.1 P436R 3 5.8 M444K 1 1.9 A426V 1 1.9 Exon 14 mutations A456P 9 17.3 S459F 2 3.9 S459del 1 1.9 A465V 1 1.9 Abbreviations: NGS, next-generation sequencing; POLE, polymerase epsilon gene Characteristics of 52 patients with POLE mutations The clinical characteristics and pathological data of the 52 patients with POLE -mutated EC are summarized in Table 2. Total hysterectomy and bilateral salpingo-oophorectomy were performed for each patient. Of the 52 patients, 40 (76.9%) underwent full lymph node dissection, with the exception of those with stage IA disease who presented with low-grade EC (FIGO 2008). In the latter group, lymph nodes were sampled, and the number of lymph nodes removed varied from 2 to 11 (median, 5). Moreover, 44 of 52 cases (84.6%) were categorized as stage IA according to FIGO 2023 with molecular classification. Among the 52 cases with POLE mutations, the proportion of grade 3 EC was the second highest, following grade 1 EC. Approximately 80% patients did not receive postoperative adjuvant therapy based on our hospital regulations. None of the patients with early-stage EC, except those with carcinosarcoma, received adjuvant therapy, including chemotherapy or radiotherapy 19 . Two patients experienced recurrence after the primary surgery, although neither died of EC. Interestingly, the recurrence site in these patients was the para-aortic lymph nodes. Both patients underwent surgical resection, with 1 out of 11 and 2 out of 18 positive para-aortic lymph nodes, respectively. Since then, the patients showed no evidence of disease. The follow-up period for all patients varied between 6 and 135 months, with a median duration of 47 months. The 5-year RFS and OS rates for patients with POLE mutations were 92.8% and 100%, respectively. The patients’ characteristics and types of POLE mutations in the biopsy specimens have been described in our previous study 11 . Table 2. Characteristics of 52 patients with POLE-mutated endometrial cancer as defined by the Luminex assay or NGS Category [n = 52] (%) Clinicopathological parameters Age (median, range) 54 ( 38–80 ) Histological types Endometrioid carcinoma 42 ( 80.8% ) Grade 1 22 ( 42.3% ) Grade 2 4 ( 7.7% ) Grade 3 16 ( 30.8% ) Carcinosarcoma 6 ( 11.5% ) Serous carcinoma 1 ( 1.9% ) Clear cell carcinoma 1 ( 1.9% ) Mixed carcinoma 2 ( 3.8% ) Histological grade 1 Low 26 ( 50.0% ) High 26 ( 50.0% ) Stage (FIGO 2023 with molecular classification) IA POLEmut 44 ( 84.6% ) IIIA2 1 ( 1.9% ) IIIC1ii 1 ( 1.9% ) IIIC2ii 6 ( 11.5% ) Myometrial invasion None 12 ( 23.1% ) <1/2 25 ( 48.1% ) ≧1/2 15 ( 28.8% ) Lymph vascular space invasion None/ focal 38 ( 73.1% ) Substantial 14 ( 26.9% ) Cervical stromal invasion Negative 45 ( 86.5% ) Positive 7 ( 13.5% ) Concordance between the Luminex kit and NGS The rate of concordance between results obtained with the Luminex kit and those obtained with NGS for the 432 surgical specimens was 100.0% (95% confidence interval [CI]: 99.1–100.0%); this was a “perfect” level of agreement (κ = 1.000) (Table 3a). Similarly, the concordance rate for 70 biopsy specimens was 100.0% (κ = 1.000) (Table 3b). For surgical and biopsy specimens confirmed to be POLE mutation-positive by both the Luminex assay and NGS, the rate of concordance between the Luminex assay and NGS for each genotype in the overall population was 100% (Supplementary Table 1). The detected somatic mutations showed > 4% variant allele frequency by NGS. All these mutations could be detected by Luminex; therefore, the sensitivity of detection was considered equivalent to that of NGS. Table 3. Concordance of POLE mutations detecetd with Luminex assay versus next generation sequencing (A) Surgical specimens (n = 432) Category POLE mutation status Next generation sequencing (POLE mutaiton) Total Positive Negative Luminex assay Positive 52 0 52 Negative 0 380 380 Total 52 380 432 Overall percent agreement 100.0% (95%CI, 99.1%-100.0%) Positive percent agreement 100.0% (95%CI, 93.2%-100.0%) Negative percent agreement 100.0% (95%CI, 99.0%-100.0%) (B) Biopsy specimens (n = 70) Category POLE mutation status Next generation sequencing (POLE mutaiton) Total Positive Negative Luminex assay Positive 33 0 33 Negative 0 37 37 Total 33 37 70 Overall percent agreement 100.0% (95%CI, 94.9%-100.0%) Positive percent agreement 100.0% (95%CI, 89.4%-100.0%) Negative percent agreement 100.0% (95%CI, 90.5%-100.0%) Concordance between surgical and biopsy specimens for the POLE mutation status Sixty-one biopsy specimens paired with hysterectomy specimens were obtained after exclusion of two cases with insufficient sample quality for NGS. The concordance rate between the surgical and biopsy specimens assessed for the POLE mutation status using the Luminex kit was 100.0% (κ = 1.000) (Table 4). Table 4. Concordance of POLE status determined by Luminex assay between pairs of surgical specimen and biopsy specimen (n = 61) Category POLE mutation status Surgical specimen (POLE mutation) Total Positive Negative Biopsy specimen Positive 26 0 26 Negative 0 35 35 Total 26 35 61 Overall percent agreement 100.0% (95%CI, 94.1%-100.0%) Positive percent agreement 100.0% (95%CI, 86.8%-100.0%) Negative percent agreement 100.0% (95%CI, 90.0%-100.0%) Evaluation of cases with insufficient sample quality for NGS This study excluded 28 cases (26 surgical and two biopsy specimens) that did not pass QC for NGS. On the other hand, with the Luminex kit, it was possible to evaluate the POLE mutation status in all 28 cases; four cases were positive for POLE mutations, all of which were confirmed by subsequent Sanger sequencing. The primer sequences used for Sanger sequencing are listed in Supplementary Table 2. There were three P286R mutations and one V411L mutation, and all four had good prognoses. Table 5 Comparison of POLE testing methods Characteristics Luminex FoundationOne ® CDx QPOLE [9] Technology Multiplex PCR, a bead-based multiplex platform that uses fluorescence to detect mutations. High-throughput capture-based sequencing that can detect a wide array of mutations across multiple genes. Multiplex qPCR Sensitivity High, capable of detecting mutations when present in more than 4% of total DNA. Very high, capable of detecting mutations at low frequencies. High, typically detects mutations present in more than 1–5% of total DNA. Covered POLE mutations 25 All mutations detected on the exons 11 Detecting undefined mutations No Yes No Concordance to results of NGS 100% - 98.6% [9] DNA requirement 50-100ng/case 50-1000ng/case 30ng/case Procedure and effort A single tube/medium A single tube/hard Multi-tube/easy Turn around time 4.5 hours 2 weeks 4-6 hours Cost Low High Low Bioinformatics and data interpretation Simple Complex Simple High-throughput Up to 96 samples Up to 64 samples Up to 32 samples Quality manegment NanoDrop Qubit NanoDrop Expandability and customization High Low High Abbreviations: DNA; deoxyribonucleic acid, NGS; next generation sequencing, PCR;polymerase chain reaction, qPCR;quantitative polymerase chain reaction Discussion In this study, we presented a multiplex PCR and probe-based detection kit that simultaneously detects 25 pathogenic POLE mutations using the Luminex (xMAP) assay as an alternative to DNA sequencing. Our study demonstrated excellent concordance between the multiplex PCR and probe-based detection kit and conventional NGS. Even when DNA from older FFPE blocks was used, comparison of the Luminex assay with NGS showed an overall agreement of 100% (95%CI: 99.1%-100.0%, 94.9%-100.0%, respectively) for both surgical and biopsy specimens. Our method ensures that the assessment of POLE mutations is feasible and acceptable in many laboratories worldwide, and it is expected that this will lead to the spread of molecular classification and the prevention of overtreatment for POLE -mut EC. ECs with POLE -exonuclease domain mutations (EDM) in exons 9, 11, 13, and 14 have been internationally recognized to qualify as POLE -mut EC. In the present study, the five most common mutations, P286R, S297F, V411L, A456P, and S459F, accounted for approximately 80% of all POLE -mut ECs. It has been reported that these five mutations account for more than 95% POLE -mut ECs 8 , and the revised WHO classification system for EC recommends analyses of these “hotspot” POLE mutations 3 . However, in addition to these five mutations, many POLE mutations are associated with a favorable prognosis, and the classification of these less frequent mutations is currently challenging. The newly developed Luminex (xMAP) assay appears to have significant advantages over the previously reported POLE test (Table 5). In 2023, a quantitative polymerase chain reaction (qPCR) assay, QPOLE , was developed to detect 11 mutations: the five most common mutations (P286R, S297F, V411L, A456P or S459F) plus M295R, F367S, D368Y, L424I, P436R, and M444K, all of which have been reported to have a POLE-score of ≥ 4 and an excellent clinical outcome 8 , 9 , 20 . QPOLE achieved an overall accuracy of 98.6%, a sensitivity of 95.2%, and a specificity of 100%. In QPOLE , PCR must be performed for each mutation in each tube. In contrast, the Luminex kit can measure all 25 mutations simultaneously in one tube. In addition, the accuracy of the Luminex kit was 100% in this study, equivalent to that of QPOLE . Similar to QPOLE , the Luminex (xMAP) assay enables POLE status assessment within 4.5 h, significantly shortening the turnaround time from the 2 weeks required for NGS. Moreover, it is inexpensive and can be performed using only small amounts of DNA, and it does not require interpretation by a molecular biologist, unlike NGS. Furthermore, the Luminex kit has sufficient reliability, and methods of QC/quality assurance have already been established. We believe that the Luminex (xMAP)-based multiplex kit is more suitable for routine clinical use because it processes multiple samples. To develop this kit, we selected 25 POLE mutations, including the abovementioned ones. According to the COSMIC and cBioPortal databases, T278M, P286S, P286L, P286H, S297Y, L424V, A426V, P441L, T457M, and A465V are suspected pathogenic mutations, and there are many reports of good prognosis 21 – 27 . Moreover, our previous study on genetic alterations in Japanese patients with POLE -EDM EC demonstrated that P286C, F367C, W369*, and S459del were newly detected, and that the prognosis of patients with these mutations was extremely favorable, similar to that of patients with other POLE -EDM 10 . Thus, our new kit was used to investigate 25 mutations in exons 9, 11, 13, and 14. With the Luminex (xMAP)-based kit, there is a possibility that rare pathogenic variants of POLE will be identified in the future. In this study, five of 52 POLE -mut cases (9.6%) showed rare variants, and all showed excellent prognoses. Therefore, a validation cohort for these rare variants is urgently required. Two key limitations of our study are its small sample size and retrospective design in a single institution. Our new kit included POLE mutations that were not present in The Cancer Genome Atlas or POLE -score = 3. These mutations were associated with a good prognosis and could be considered pathogenic variants of POLE ; however, in future, it will be necessary to collect specimens from multiple institutions and perform external validation. In our study, 28 cases failed to pass QC for NGS, and all 28 could be tested using the Luminex kit, with POLE mutations detected in four. If this kit is validated, it may become more useful than NGS for detecting POLE mutations. Conclusion In this study, we developed a new multiplex kit that can detect 25 POLE pathogenic mutations simultaneously using the Luminex (xMAP) assay. Our study demonstrated excellent concordance between the multiplex kit and conventional NGS for both surgical and biopsy specimens. The Luminex multiplex kit can be a powerful tool that surpasses NGS in evaluating the POLE mutation status rapidly and easily, leading to accurate prognosis prediction using FIGO 2023 with molecular classification and preventing overtreatment for POLE -mut EC. Declarations Acknowledgments The authors thank Yuka Asami, Kengo Hiranuma, Hitoshi Ichikawa, Sachiyo Mitani, Maiko Matsuda, Yoko Shimada, other physicians and staff members of the National Cancer Center, and other hospitals for their assistance and support. We express our gratitude to Editage (https://www.editage.jp) for assisting us with English language editing. This study was supported by the Japan Agency for Medical Research and Development (23ama221520h0001), Grant-in-Aid for Young Scientists (B) (20K18207 and 19K16572), Grant-in-Aid for Scientific Research (B) (20H03695), Grant-in-Aid for Scientific Research (C) (20K09636), BRIDGE (programs for bridging the gap between R&D and the ideal society [Society 5.0] and generating economic and social value), and the National Cancer Center Research and Development Fund (2022-A-20, 2023-J-2, NCC Biobank, and NCC Core Facility). The funder played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript. A competing interests statement All authors declare no financial competing interests. Author contributions M. K. K., K. S., H. Y., and T. K. designed the study. M. K. K. wrote the first draft of the manuscript. K.S., T.K., T.S., M.Y., D.H., and H.Y. contributed to the data analysis and interpretation and assisted with manuscript preparation. All other authors contributed to data collection, interpretation, and critical manuscript review. All authors have approved the final version of the manuscript and agreed to be accountable for all aspects of the study. Questions related to the accuracy or integrity of the study were investigated and resolved. Data availability statements The datasets generated and analysed during the current study are not publicly available due to the sensitive nature of the questions asked in this study but are available from the corresponding author on reasonable request. Code availability statements The underlying code for this study is not publicly available but may be made available for proprietary reasons. Ethical approval statement Written informed consent was obtained from all patients. This study was approved by the Institutional Review Board of the National Cancer Center Research Institute. References Berek, J. S. et al. FIGO staging of endometrial cancer: 2023. Int J Gynaecol Obstet 162, 383–394, doi: 10.1002/ijgo.14923 (2023). Kobayashi-Kato, M. et al. Utility of the revised FIGO2023 staging with molecular classification in endometrial cancer. Gynecol Oncol 178, 36–43, doi: 10.1016/j.ygyno.2023.09.011 (2023). Cancer, I. A. f. R. o. WHO Classification of Tumours Editorial Board. Female Genital Tumors. (2020). Consortium, R. R. Refining adjuvant treatment in endometrial cancer based on molecular features: the RAINBO clinical trial program. Int J Gynecol Cancer 33, 109–117, doi: 10.1136/ijgc-2022-004039 (2022). Bando, H. et al. Simultaneous identification of 36 mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA in a single reaction by multiplex assay kit. BMC Cancer 13, 405, doi: 10.1186/1471-2407-13-405 (2013). Yoshino, T. et al. Clinical Validation of a Multiplex Kit for RAS Mutations in Colorectal Cancer: Results of the RASKET (RAS KEy Testing) Prospective, Multicenter Study. EBioMedicine 2, 317–323, doi: 10.1016/j.ebiom.2015.02.007 (2015). Taniguchi, H. et al. Clinical Validation of Newly Developed Multiplex Kit Using Luminex xMAP Technology for Detecting Simultaneous RAS and BRAF Mutations in Colorectal Cancer: Results of the RASKET-B Study. Neoplasia 20, 1219–1226, doi: 10.1016/j.neo.2018.10.004 (2018). Leon-Castillo, A. et al. Interpretation of somatic POLE mutations in endometrial carcinoma. J Pathol 250, 323–335, doi: 10.1002/path.5372 (2020). Van den Heerik, A. et al. QPOLE: A Quick, Simple, and Cheap Alternative for POLE Sequencing in Endometrial Cancer by Multiplex Genotyping Quantitative Polymerase Chain Reaction. JCO Glob Oncol 9, e2200384, doi: 10.1200/GO.22.00384 (2023). Asami, Y. et al. Utility of molecular subtypes and genetic alterations for evaluating clinical outcomes in 1029 patients with endometrial cancer. Br J Cancer 128, 1582–1591, doi: 10.1038/s41416-023-02203-3 (2023). Kato, M. K. et al. Excellent concordance of the molecular classification between preoperative biopsy and final hysterectomy in endometrial carcinoma. Gynecol Oncol 190, 139–145, doi: 10.1016/j.ygyno.2024.08.016 (2024). Breiley JD GM, W. C. the 2020 World Health Organization tumor classification . (Wiley, 2016). Hirose, S. et al. Genomic alterations in STK11 can predict clinical outcomes in cervical cancer patients. Gynecol Oncol 156, 203–210, doi: 10.1016/j.ygyno.2019.10.022 (2020). Kuno, I. et al. TP53 mutants and non-HPV16/18 genotypes are poor prognostic factors for concurrent chemoradiotherapy in locally advanced cervical cancer. Sci Rep 11, 19261, doi: 10.1038/s41598-021-98527-2 (2021). Murakami, N. et al. Distribution of genetic alterations in high-risk early-stage cervical cancer patients treated with postoperative radiation therapy. Sci Rep 11, 10567, doi: 10.1038/s41598-021-90139-0 (2021). Takayanagi, D. et al. Comparative Analysis of Genetic Alterations, HPV-Status, and PD-L1 Expression in Neuroendocrine Carcinomas of the Cervix. Cancers (Basel) 13, doi: 10.3390/cancers13061215 (2021). Agresti, A. Categorical Data Analysis . (John Wiley and Sons, 1990). Landis, J. R. & Koch, G. G. The measurement of observer agreement for categorical data. Biometrics 33, 159–174 (1977). Sasada, S. et al. Baseline risk of recurrence in stage I-II endometrial carcinoma. J Gynecol Oncol 29, e9, doi: 10.3802/jgo.2018.29.e9 (2018). McAlpine, J. N. et al. Evaluation of treatment effects in patients with endometrial cancer and POLE mutations: An individual patient data meta-analysis. Cancer 127, 2409–2422, doi: 10.1002/cncr.33516 (2021). Meng, B. et al. POLE exonuclease domain mutation predicts long progression-free survival in grade 3 endometrioid carcinoma of the endometrium. Gynecol Oncol 134, 15–19, doi: 10.1016/j.ygyno.2014.05.006 (2014). Shinbrot, E. et al. Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication. Genome Res 24, 1740–1750, doi: 10.1101/gr.174789.114 (2014). Parra-Herran, C. et al. Molecular-based classification algorithm for endometrial carcinoma categorizes ovarian endometrioid carcinoma into prognostically significant groups. Mod Pathol 30, 1748–1759, doi: 10.1038/modpathol.2017.81 (2017). Zou, Y. et al. Frequent POLE1 p.S297F mutation in Chinese patients with ovarian endometrioid carcinoma. Mutat Res 761, 49–52, doi: 10.1016/j.mrfmmm.2014.01.003 (2014). Palles, C. et al. Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas. Nat Genet 45, 136–144, doi: 10.1038/ng.2503 (2013). Billingsley, C. C. et al. Polymerase varepsilon (POLE) mutations in endometrial cancer: clinical outcomes and implications for Lynch syndrome testing. Cancer 121, 386–394, doi: 10.1002/cncr.29046 (2015). Stenzinger, A. et al. Mutations in POLE and survival of colorectal cancer patients–link to disease stage and treatment. Cancer Med 3, 1527–1538, doi: 10.1002/cam4.305 (2014). Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1.tiff SupplementaryTable1.xlsx SupplementaryTable2.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5540131","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":386827176,"identity":"3d4e34b1-a2b5-43d3-ad1b-4813d1f59f99","order_by":0,"name":"Mayumi Kobayashi Kato","email":"","orcid":"","institution":"National Cancer Center Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Mayumi","middleName":"Kobayashi","lastName":"Kato","suffix":""},{"id":386827177,"identity":"b9963f1a-1ac1-49c3-810c-2c526deb2c0d","order_by":1,"name":"Takayuki Kawai","email":"","orcid":"","institution":"National Cancer Center Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Takayuki","middleName":"","lastName":"Kawai","suffix":""},{"id":386827178,"identity":"7093a2bf-d405-4322-b368-e30d44d1a2cd","order_by":2,"name":"Hideki Okada","email":"","orcid":"","institution":"Medical \u0026 Biological Laboratories Co., Ltd. (MBL)","correspondingAuthor":false,"prefix":"","firstName":"Hideki","middleName":"","lastName":"Okada","suffix":""},{"id":386827179,"identity":"87b34ae5-ae14-4a67-92bb-b27a14e98af7","order_by":3,"name":"Takuya Kondo","email":"","orcid":"","institution":"Medical \u0026 Biological Laboratories Co., Ltd. (MBL)","correspondingAuthor":false,"prefix":"","firstName":"Takuya","middleName":"","lastName":"Kondo","suffix":""},{"id":386827180,"identity":"cfbbd958-6e04-4463-9636-a196a2b169b7","order_by":4,"name":"Tetsuro Shiraishi","email":"","orcid":"","institution":"National Cancer Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tetsuro","middleName":"","lastName":"Shiraishi","suffix":""},{"id":386827181,"identity":"12bb5a32-0566-4de9-9199-377c134f3e6d","order_by":5,"name":"Maiko Yamaguchi","email":"","orcid":"","institution":"National Cancer Center Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Maiko","middleName":"","lastName":"Yamaguchi","suffix":""},{"id":386827182,"identity":"80fd5750-3551-4e75-bef6-bb91d3a77024","order_by":6,"name":"Daiki Higuchi","email":"","orcid":"","institution":"National Cancer Center Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Daiki","middleName":"","lastName":"Higuchi","suffix":""},{"id":386827183,"identity":"93257d7a-b472-4018-a51a-a8303b0f3ee6","order_by":7,"name":"Masaaki Komatsu","email":"","orcid":"","institution":"National Cancer Center Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Masaaki","middleName":"","lastName":"Komatsu","suffix":""},{"id":386827184,"identity":"8d536b5b-9218-4434-b4e3-b04b48d6c0c3","order_by":8,"name":"Ryuji Hamamoto","email":"","orcid":"","institution":"National Cancer Center Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Ryuji","middleName":"","lastName":"Hamamoto","suffix":""},{"id":386827185,"identity":"2e3ad0be-5972-4970-9d5c-f2d322b608ea","order_by":9,"name":"Koji Matumoto","email":"","orcid":"","institution":"Showa University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Koji","middleName":"","lastName":"Matumoto","suffix":""},{"id":386827186,"identity":"8723f15a-c9d1-47be-99ef-97c27485e301","order_by":10,"name":"Yasuhisa Terao","email":"","orcid":"","institution":"Juntendo University Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yasuhisa","middleName":"","lastName":"Terao","suffix":""},{"id":386827187,"identity":"c28324f4-fa98-41fa-b6b4-0cd7f356ed0d","order_by":11,"name":"Tomoyasu Kato","email":"","orcid":"","institution":"National Cancer Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tomoyasu","middleName":"","lastName":"Kato","suffix":""},{"id":386827188,"identity":"6f28a028-7f79-46b6-8028-6044eeb15e88","order_by":12,"name":"Takashi Kohno","email":"","orcid":"","institution":"National Cancer Center Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Takashi","middleName":"","lastName":"Kohno","suffix":""},{"id":386827189,"identity":"68251c0e-5b5b-4f67-948e-b44a1dd06254","order_by":13,"name":"Mitsuya Ishikawa","email":"","orcid":"","institution":"National Cancer Center Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mitsuya","middleName":"","lastName":"Ishikawa","suffix":""},{"id":386827190,"identity":"740818a0-539c-4003-aedd-b807003b33de","order_by":14,"name":"Kouya Shiraishi","email":"","orcid":"","institution":"National Cancer Center Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Kouya","middleName":"","lastName":"Shiraishi","suffix":""},{"id":386827191,"identity":"f68a44b6-924a-499b-8e82-37118e40d3f9","order_by":15,"name":"Hiroshi Yoshida","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYBACA2YIncAPE2FsIFaLJEwlYS1QOsHgALEOM2fnffiYp6Iuz/h288MPDL9sGJhnE7DGspnd2JjnzOFiszvHjCUY+9IYGOcQsM/gMBubNG/bgcRtN3LYGBh7DjMwzkggSktd4uYZJGphTtwgAdTC8IM4LcyGc84cTpxxI81YIrEhjYewX84fY3zwpqIusX9G8sMPH/7YyBkSCjEQYOKBsRLbGHgMZxDWwcD4A878w8AgL0GEllEwCkbBKBhRAAB2zUEw76fuIgAAAABJRU5ErkJggg==","orcid":"","institution":"National Cancer Center Hospital","correspondingAuthor":true,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Yoshida","suffix":""}],"badges":[],"createdAt":"2024-11-28 07:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5540131/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5540131/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":71056124,"identity":"ff4f15d5-638b-4d49-a0dc-c2863a943010","added_by":"auto","created_at":"2024-12-10 16:14:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":29591,"visible":true,"origin":"","legend":"\u003cp\u003eConsort diagram for this study.\u003cstrong\u003e \u003c/strong\u003eIn total, 588 specimens were collected from 525 patients with histologically confirmed endometrial cancer at our hospital.\u003c/p\u003e\n\u003cp\u003eNCCH: National Cancer Center Hospital, NGS: next-generation sequencing, \u003cem\u003ePOLE\u003c/em\u003e: polymerase epsilon gene\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5540131/v1/1f6689b43da817632c2df992.png"},{"id":71058945,"identity":"b24da5b9-4237-424e-9306-197da65d81c6","added_by":"auto","created_at":"2024-12-10 16:38:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":713335,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5540131/v1/3e93f2fa-c165-4b7d-b434-3aa772663e3c.pdf"},{"id":71056752,"identity":"395dacff-221a-4300-a3fc-73c13efc2576","added_by":"auto","created_at":"2024-12-10 16:22:43","extension":"tiff","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":878246,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.tiff","url":"https://assets-eu.researchsquare.com/files/rs-5540131/v1/08abdcfbe3699ed1fa76f021.tiff"},{"id":71057637,"identity":"a62cc7a9-42ed-423e-98b1-b5421877c4cf","added_by":"auto","created_at":"2024-12-10 16:30:43","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":10184,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5540131/v1/54cc0d1ead911ff47e0d7b2a.xlsx"},{"id":71058868,"identity":"cdeaa620-5c74-42a4-ae4f-7784832d361d","added_by":"auto","created_at":"2024-12-10 16:38:49","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":10305,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5540131/v1/e4fd8719413069daae776ce8.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparison between a Luminex-based multiplex kit with a sequence-specific oligonucleotide probe and next-generation sequencing for the detection of POLE oncogenic mutations in endometrial cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the new era of molecular classification of endometrial cancer (EC), the International Federation of Gynecology and Obstetrics (FIGO) recommended incorporation of molecular subtypes (polymerase epsilon [\u003cem\u003ePOLE\u003c/em\u003e] mutated [\u003cem\u003ePOLE\u003c/em\u003e-mut], mismatch repair [MMR] deficiency [MMR-D], no specific molecular profile [NSMP], and p53-abnormality [p53abn]) into the 2023 revision of EC staging \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In addition, \u003cem\u003ePOLE\u003c/em\u003e-mut EC confined to the uterus and \u003cem\u003ePOLE\u003c/em\u003e-mut EC with cervical invasion were downstaged to stage IA (stage IA\u003csub\u003em\u003c/sub\u003e) regardless of lymph vascular space invasion, because they are associated with an excellent clinical outcome and a very low risk of relapse \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ePOLE\u003c/em\u003e-mut group is the most upstream branching group according to the steps for molecular classification based on the 2020 WHO classification code \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Because the other three groups (MMR-D, NSMP, and p53abn) cannot be determined unless the \u003cem\u003ePOLE\u003c/em\u003e mutation status is correctly evaluated, it is necessary to perform tests for the detection of these mutations, even though \u003cem\u003ePOLE\u003c/em\u003e-mut EC accounts for only approximately 10% of all ECs \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, the \u003cem\u003ePOLE\u003c/em\u003e mutation status can only be assessed using DNA sequencing methods such as next-generation sequencing (NGS) or Sanger sequencing, without any immunohistochemical (IHC) markers. These methods are expensive and time-consuming and require specialized equipment; these are factors that prevent widespread use of methods for the detection of \u003cem\u003ePOLE\u003c/em\u003e mutations, unlike IHC for the detection of MMR-D and p53abn.\u003c/p\u003e \u003cp\u003eLuminex (xMAP) technology is used for multiplex molecular testing in a single well and requires only a small amount of tumor-derived DNA \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The MEBGEN RASKET-B KIT, which uses the Luminex (xMAP) assay to detect the \u003cem\u003eBRAF\u003c/em\u003e mutation (V600E) and 48 different \u003cem\u003eRAS\u003c/em\u003e mutations in colorectal cancer, has received regulatory approval for diagnostic use in Japan, has become widely used in daily practice, and is recognized as a testing platform \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Using this technique, we developed a new Luminex (xMAP)-based multiplex kit that simultaneously detects 25 \u003cem\u003ePOLE\u003c/em\u003e pathogenic mutations, including the five most common mutations (P286R, V411L, S297F, S459F, and A456P) \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. The remaining 20 mutations include those determined to be pathogenic by the \u003cem\u003ePOLE\u003c/em\u003e pathogenic score and those suspected to be pathogenic by the COSMIC and cBioPortal databases, all of which have been linked to clinical information with a favorable prognosis in previous literature \u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe aim of this study was to investigate the degree of concordance between the novel Luminex (xMAP)-based kit and NGS for the detection of \u003cem\u003ePOLE\u003c/em\u003e mutations in EC samples. If the robustness and feasibility of this multiplex kit using routine clinical samples are proven, this new method can be used as an alternative to NGS for \u003cem\u003ePOLE\u003c/em\u003e testing.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eWe identified consecutive patients with histologically confirmed EC whose samples were collected at the National Cancer Center Hospital between 1999 and 2023. In addition to surgical specimens, endometrial biopsy specimens were used in this study. A pathologist (H. Y.) blinded to the prognostic information selected cases for endometrial biopsy; this biopsy cohort was the same as that used in our previous study on the accuracy of molecular classification in preoperative materials \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePatients with multiple cancers who received neoadjuvant chemotherapy, did not have formalin-fixed paraffin-embedded (FFPE) samples, or had tissue samples of insufficient quality were excluded. Each case was reviewed by at least two gynecological pathologists, and the pathological diagnoses were validated in accordance with the 2020 World Health Organization tumor classification \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Clinicopathological information, including age and tumor stage (as defined by FIGO 2023 with molecular classification), was retrospectively collected for every patient. During their initial visit to our hospital, all patients provided informed consent for the utilization of samples in the research. The data derived from the samples collected after acquisition of informed consent are summarized on the hospital\u0026rsquo;s website. Patients retained the right to withdraw their presumed consent at any time. Only patients whose consent was not revoked were included in this study. The Institutional Review Board of the National Cancer Center Research Institute approved the study (2017\u0026thinsp;\u0026minus;\u0026thinsp;331).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA preparation and NGS\u003c/h3\u003e\n\u003cp\u003eDNA was extracted from 530 FFPE samples of endometrial tumor tissue using a QIAamp DNA FFPE tissue kit according to the manufacturer\u0026rsquo;s instructions (Qiagen, Hilden, Germany) \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. All FFPE samples of endometrial tumor tissue obtained by hysterectomy as well as FFPE samples obtained via endometrial biopsy were successfully analyzed in our cohort. Fifty nanograms of DNA extracted from tumor tissues were utilized for library construction with the Ion AmpliSeq\u0026trade; Custom Panel (Thermo Fisher Scientific, Waltham, MA, USA). For the identification of oncogenic mutations in \u003cem\u003ePOLE\u003c/em\u003e, the same criteria used in previously published studies were used \u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Genomic data from paired biopsy and hysterectomy specimens were obtained using methods detailed in the prior study \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In some cases, the tumor areas were excised through macrodissection to enhance the concentration of tumor cells within the surgical specimens. In contrast, entire sections were used without dissection for all biopsy specimens. The purity of DNA derived from hysterectomy and biopsy specimens was measured using Nanodrop and Invitrogen Qubit assays. Both DNAs had A260/A280\u0026thinsp;\u0026ge;\u0026thinsp;1.8, with high purity.\u003c/p\u003e\n\u003ch3\u003eLuminex (xMAP) assay\u003c/h3\u003e\n\u003cp\u003eThe Luminex (xMAP) assay was conducted using DNA obtained from the Ion AmpliSeq custom panel. Twenty-five \u003cem\u003ePOLE\u003c/em\u003e mutations involving exon 9 codon 286 (P286R, P286S, P286L, P286H, P286C), codon 297 (S297F, S297Y), codon 278 (T278M), codon 295 (M295R), exon 11 codon 367 (F367S, F367C), codon 368 (D368Y), exon12 codon 369 (W369*), exon 13 codon 411 (V411L), codon 424 (L424V, L424l), codon 426 (A426V), codon 436 (P436R), codon 411 (P411L), codon 444 (M444K), exon 14 codon 456 (A456P), codon 457 (T457M), codon 459 (S459F, S459del), and codon 465 (A465V) were analyzed using Luminex (xMAP) technology. Assays using a pre-production \u003cem\u003ePOLE\u003c/em\u003e mutation detection kit (MBL, Tokyo, Japan) were performed according to the manufacturer\u0026rsquo;s instructions. The procedure took approximately 4.5 h (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as percentages of patients, 95% confidence intervals (CIs), or hazard ratios (HRs). Cohen\u0026rsquo;s kappa (κ) coefficient was used to assess concordance between the results of NGS and those of the Luminex assay \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Kappa statistics of \u0026lt;\u0026thinsp;0, 0.00\u0026ndash;0.20, 0.21\u0026ndash;0.40, 0.41\u0026ndash;0.60, 0.61\u0026ndash;0.80, and 0.81\u0026ndash;1.00 represented \u0026ldquo;no,\u0026rdquo; \u0026ldquo;slight,\u0026rdquo; \u0026ldquo;fair,\u0026rdquo; \u0026ldquo;moderate,\u0026rdquo; \u0026ldquo;substantial,\u0026rdquo; and \u0026ldquo;almost perfect\u0026rdquo; levels of agreement, respectively \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Percent agreement was also used to assess concordance between the results of NGS and those of the Luminex assay. R version 4.3.1 (R Foundation for Statistical Computing, Viena, Austria) was used to perform the binomial test. Relapse-free survival (RFS) was determined from the date of the initial surgical procedure to the date of death from any cause or the first recurrence. Overall survival (OS) was calculated from the date of the initial surgery to the date of death from any cause. Survivors were censored at the cut-off date (September 9, 2024). A \u003cem\u003eP\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. All statistical analyses were analyzed using JMP version 8.0.1 (SAS Institute Inc., Cary, North Carolina, United States).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eSpecimen analysis\u003c/h2\u003e\n \u003cp\u003eThe consort diagram for this study is shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. In total, 588 specimens were collected from 525 patients with histologically confirmed EC at our hospital, and FFPE tissue samples were obtained. A total of 530 samples were subjected to NGS; these included 458 surgical specimens and 72 biopsy specimens. After exclusion of 28 cases that did not pass quality control (QC) for NGS, 502 samples (432 surgical specimens and 70 biopsy specimens) were investigated to determine the concordance between the Luminex assay and NGS for \u003cem\u003ePOLE\u003c/em\u003e mutation detection.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFrequency of\u003c/strong\u003e \u003cstrong\u003ePOLE\u003c/strong\u003e \u003cstrong\u003emutations\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTable 1 shows the frequencies of all \u003cem\u003ePOLE\u003c/em\u003e mutations detected by the Luminex assay and NGS in the 432 surgical specimens from patients with EC. The Luminex multiplex kit and NGS detected 52 \u003cem\u003ePOLE\u003c/em\u003e mutations (12.0%). The most frequent mutations were P286R (30.8%, 16/52), V411L (23.1%, 12/52), and A456P (17.3%, 9/52), followed by S297F (3.9%, 2/52) and P436R (5.8%, 3/52).\u0026nbsp;\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"427\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" height=\"61\" class=\"xl74\" style=\"width: 427px;\"\u003eTable 1. Frequency of all POLE\u0026nbsp;mutations detected in patients with endometrial carcinoma by Luminex assay and NGS (n = 52)\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"69\" class=\"xl63\"\u003ePOLE\u0026nbsp;status\u003c/td\u003e\n \u003ctd class=\"xl69\"\u003eNumber of cases\u003cbr\u003e\u0026nbsp;(n)\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl75\"\u003eProportion among 52 cases\u003cbr\u003e\u0026nbsp;(%)\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl65\"\u003ePOLE\u0026nbsp;mutation\u003c/td\u003e\n \u003ctd class=\"xl64\"\u003e52\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl64\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl70\" colspan=\"2\"\u003e Exon 9 mutations\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl64\"\u003eP286R\u003c/td\u003e\n \u003ctd class=\"xl64\"\u003e16\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl77\"\u003e30.8\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl64\"\u003eS297F\u003c/td\u003e\n \u003ctd class=\"xl64\"\u003e3\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl77\"\u003e5.8\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl64\"\u003eP286C\u003c/td\u003e\n \u003ctd class=\"xl64\"\u003e1\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl64\"\u003e1.9\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl64\"\u003eS297Y\u003c/td\u003e\n \u003ctd class=\"xl64\"\u003e1\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl64\"\u003e1.9\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl71\" colspan=\"2\"\u003e Exon 11 mutations\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl64\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl64\"\u003eF367C\u003c/td\u003e\n \u003ctd class=\"xl64\"\u003e1\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl64\"\u003e1.9\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl71\" colspan=\"2\"\u003e Exon 13 mutations\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl64\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl64\"\u003eV411L\u003c/td\u003e\n \u003ctd class=\"xl68\"\u003e12\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl64\"\u003e23.1\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl64\"\u003eP436R\u003c/td\u003e\n \u003ctd class=\"xl68\"\u003e3\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl64\"\u003e5.8\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl64\"\u003eM444K\u003c/td\u003e\n \u003ctd class=\"xl68\"\u003e1\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl64\"\u003e1.9\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl64\"\u003eA426V\u003c/td\u003e\n \u003ctd class=\"xl68\"\u003e1\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl64\"\u003e1.9\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl71\" colspan=\"2\"\u003e Exon 14 mutations\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl64\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl64\"\u003eA456P\u003c/td\u003e\n \u003ctd class=\"xl68\"\u003e9\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl64\"\u003e17.3\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl64\"\u003eS459F\u003c/td\u003e\n \u003ctd class=\"xl68\"\u003e2\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl64\"\u003e3.9\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl64\"\u003eS459del\u003c/td\u003e\n \u003ctd class=\"xl68\"\u003e1\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl64\"\u003e1.9\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"23\" class=\"xl63\"\u003eA465V\u003c/td\u003e\n \u003ctd class=\"xl67\"\u003e1\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl63\"\u003e1.9\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" height=\"52\" class=\"xl72\" style=\"width: 427px;\"\u003eAbbreviations: NGS, next-generation sequencing; POLE, \u0026nbsp;polymerase epsilon gene\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003cbr\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristics of 52 patients with\u003c/strong\u003e \u003cstrong\u003ePOLE\u003c/strong\u003e \u003cstrong\u003emutations\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe clinical characteristics and pathological data of the 52 patients with \u003cem\u003ePOLE\u003c/em\u003e-mutated EC are summarized in Table 2. Total hysterectomy and bilateral salpingo-oophorectomy were performed for each patient. Of the 52 patients, 40 (76.9%) underwent full lymph node dissection, with the exception of those with stage IA disease who presented with low-grade EC (FIGO 2008). In the latter group, lymph nodes were sampled, and the number of lymph nodes removed varied from 2 to 11 (median, 5). Moreover, 44 of 52 cases (84.6%) were categorized as stage IA according to FIGO 2023 with molecular classification. Among the 52 cases with \u003cem\u003ePOLE\u003c/em\u003e mutations, the proportion of grade 3 EC was the second highest, following grade 1 EC. Approximately 80% patients did not receive postoperative adjuvant therapy based on our hospital regulations. None of the patients with early-stage EC, except those with carcinosarcoma, received adjuvant therapy, including chemotherapy or radiotherapy \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Two patients experienced recurrence after the primary surgery, although neither died of EC. Interestingly, the recurrence site in these patients was the para-aortic lymph nodes. Both patients underwent surgical resection, with 1 out of 11 and 2 out of 18 positive para-aortic lymph nodes, respectively. Since then, the patients showed no evidence of disease. The follow-up period for all patients varied between 6 and 135 months, with a median duration of 47 months. The 5-year RFS and OS rates for patients with \u003cem\u003ePOLE\u003c/em\u003e mutations were 92.8% and 100%, respectively. The patients\u0026rsquo; characteristics and types of \u003cem\u003ePOLE\u003c/em\u003e mutations in the biopsy specimens have been described in our previous study \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eTable 2. Characteristics of 52 patients with POLE-mutated endometrial cancer as defined by the Luminex assay or NGS\u003c/p\u003e\u0026nbsp;\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"527\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" height=\"42\" class=\"xl79\"\u003eCategory\u003c/td\u003e\n \u003ctd colspan=\"5\" class=\"xl80\" dir=\"LTR\"\u003e[n = 52] (%)\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"7\" class=\"xl64\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl65\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl66\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl66\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl67\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl67\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl68\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl67\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl67\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\" colspan=\"4\" dir=\"LTR\"\u003eClinicopathological parameters\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" class=\"xl72\" dir=\"LTR\"\u003eAge (median, range)\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e54\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e38\u0026ndash;80\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"5\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" class=\"xl72\" dir=\"LTR\"\u003eHistological types\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl72\" dir=\"LTR\"\u003eEndometrioid carcinoma\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e42\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e80.8%\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003eGrade 1\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e22\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e42.3%\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003eGrade 2\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e4\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e7.7%\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003eGrade 3\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e16\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e30.8%\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl72\" dir=\"LTR\"\u003eCarcinosarcoma\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e6\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e11.5%\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl72\" dir=\"LTR\"\u003eSerous carcinoma\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e1\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e1.9%\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl72\" dir=\"LTR\"\u003eClear cell carcinoma\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e1\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e1.9%\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl72\" dir=\"LTR\"\u003eMixed carcinoma\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e2\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e3.8%\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"5\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" class=\"xl72\"\u003eHistological grade\u003csup\u003e1\u003c/sup\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl72\" dir=\"LTR\"\u003eLow\u0026nbsp;\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e26\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e50.0%\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl72\" dir=\"LTR\"\u003eHigh\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e26\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e50.0%\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"5\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" class=\"xl72\"\u003eStage \u0026nbsp;(FIGO 2023 with molecular classification)\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003eIA \u003csub\u003ePOLEmut\u003c/sub\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e44\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e84.6%\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003eIIIA2\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e1\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e1.9%\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003eIIIC1ii\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e1\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e1.9%\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003eIIIC2ii\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e6\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e11.5%\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"5\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" class=\"xl72\" dir=\"LTR\"\u003eMyometrial invasion\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl75\"\u003eNone\u003c/td\u003e\n \u003ctd class=\"xl75\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e12\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e23.1%\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl75\"\u003e\u0026lt;1/2\u003c/td\u003e\n \u003ctd class=\"xl75\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e25\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e48.1%\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e≧1/2\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e15\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e28.8%\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"5\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"3\" class=\"xl73\"\u003eLymph vascular space invasion\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003eNone/ focal\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e38\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e73.1%\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003eSubstantial\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e14\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e26.9%\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"5\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl72\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl69\" colspan=\"3\" dir=\"LTR\"\u003eCervical stromal invasion\u003c/td\u003e\n \u003ctd class=\"xl77\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl77\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl77\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl76\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl72\" dir=\"LTR\"\u003eNegative\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e45\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e86.5%\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl69\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl76\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl72\" dir=\"LTR\"\u003ePositive\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e7\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e(\u003c/td\u003e\n \u003ctd class=\"xl74\"\u003e13.5%\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e)\u003c/td\u003e\n \u003ctd class=\"xl71\" dir=\"LTR\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003ch3\u003eConcordance between the Luminex kit and NGS\u003c/h3\u003e\n\u003cp\u003eThe rate of concordance between results obtained with the Luminex kit and those obtained with NGS for the 432 surgical specimens was 100.0% (95% confidence interval [CI]: 99.1\u0026ndash;100.0%); this was a \u0026ldquo;perfect\u0026rdquo; level of agreement (\u0026kappa;\u0026thinsp;=\u0026thinsp;1.000) (Table 3a). Similarly, the concordance rate for 70 biopsy specimens was 100.0% (\u0026kappa;\u0026thinsp;=\u0026thinsp;1.000) (Table 3b). For surgical and biopsy specimens confirmed to be \u003cem\u003ePOLE\u003c/em\u003e mutation-positive by both the Luminex assay and NGS, the rate of concordance between the Luminex assay and NGS for each genotype in the overall population was 100% (Supplementary Table 1). The detected somatic mutations showed\u0026thinsp;\u0026gt;\u0026thinsp;4% variant allele frequency by NGS. All these mutations could be detected by Luminex; therefore, the sensitivity of detection was considered equivalent to that of NGS.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"618\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" height=\"45\" class=\"xl73\" style=\"width: 618px;\"\u003eTable 3. Concordance of POLE\u0026nbsp;mutations detecetd with Luminex assay versus next generation sequencing\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"6\" class=\"xl73\" style=\"width: 120px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\" style=\"width: 141px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\" style=\"width: 128px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\" style=\"width: 128px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl73\" style=\"width: 101px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" height=\"25\" class=\"xl76\" style=\"width: 618px;\"\u003e(A) Surgical specimens (n = 432)\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" height=\"70\" class=\"xl77\"\u003eCategory\u003c/td\u003e\n \u003ctd rowspan=\"2\" class=\"xl78\"\u003ePOLE\u0026nbsp;mutation\u003cbr\u003e\u0026nbsp;status\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl79\"\u003eNext generation sequencing\u003cbr\u003e(POLE\u0026nbsp;mutaiton)\u003c/td\u003e\n \u003ctd rowspan=\"2\" class=\"xl81\"\u003eTotal\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"25\" class=\"xl64\"\u003ePositive\u003c/td\u003e\n \u003ctd class=\"xl64\"\u003eNegative\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"25\" class=\"xl65\"\u003eLuminex assay\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003ePositive\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e52\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e0\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e52\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"25\" class=\"xl67\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003eNegative\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e0\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e380\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e380\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"25\" class=\"xl67\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003eTotal\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e52\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e380\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e432\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" height=\"25\" class=\"xl68\"\u003eOverall percent agreement\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl66\"\u003e100.0% (95%CI, 99.1%-100.0%)\u003c/td\u003e\n \u003ctd class=\"xl67\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"25\" class=\"xl68\" colspan=\"2\"\u003ePositive percent agreement\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl66\"\u003e100.0% (95%CI, 93.2%-100.0%)\u003c/td\u003e\n \u003ctd class=\"xl67\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"25\" class=\"xl69\" colspan=\"2\"\u003eNegative percent agreement\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl63\"\u003e100.0% (95%CI, 99.0%-100.0%)\u003c/td\u003e\n \u003ctd class=\"xl70\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"25\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" height=\"23\" class=\"xl76\" style=\"width: 618px;\"\u003e(B) Biopsy specimens (n = 70)\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" height=\"70\" class=\"xl77\"\u003eCategory\u003c/td\u003e\n \u003ctd rowspan=\"2\" class=\"xl78\"\u003ePOLE\u0026nbsp;mutation\u003cbr\u003e\u0026nbsp;status\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl79\"\u003eNext generation sequencing\u003cbr\u003e(POLE\u0026nbsp;mutaiton)\u003c/td\u003e\n \u003ctd rowspan=\"2\" class=\"xl81\"\u003eTotal\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"25\" class=\"xl64\"\u003ePositive\u003c/td\u003e\n \u003ctd class=\"xl64\"\u003eNegative\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"25\" class=\"xl65\"\u003eLuminex assay\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003ePositive\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e33\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e0\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e33\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"25\" class=\"xl67\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003eNegative\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e0\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e37\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e37\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"25\" class=\"xl67\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003eTotal\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e33\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e37\u003c/td\u003e\n \u003ctd class=\"xl66\"\u003e70\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" height=\"25\" class=\"xl68\"\u003eOverall percent agreement\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl66\"\u003e100.0% (95%CI, 94.9%-100.0%)\u003c/td\u003e\n \u003ctd class=\"xl67\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"25\" class=\"xl68\" colspan=\"2\"\u003ePositive percent agreement\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl74\"\u003e100.0% (95%CI, 89.4%-100.0%)\u003c/td\u003e\n \u003ctd class=\"xl71\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"25\" class=\"xl69\" colspan=\"2\"\u003eNegative percent agreement\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl75\"\u003e100.0% (95%CI, 90.5%-100.0%)\u003c/td\u003e\n \u003ctd class=\"xl72\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eConcordance between surgical and biopsy specimens for the\u003c/strong\u003e \u003cstrong\u003ePOLE\u003c/strong\u003e \u003cstrong\u003emutation status\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSixty-one biopsy specimens paired with hysterectomy specimens were obtained after exclusion of two cases with insufficient sample quality for NGS. The concordance rate between the surgical and biopsy specimens assessed for the \u003cem\u003ePOLE\u003c/em\u003e mutation status using the Luminex kit was 100.0% (\u0026kappa;\u0026thinsp;=\u0026thinsp;1.000) (Table 4).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"691\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" class=\"xl74\" style=\"width: 59.0657%;\"\u003eTable 4. Concordance of POLE status determined by Luminex assay between pairs of surgical specimen and biopsy specimen (n = 61)\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" class=\"xl77\" style=\"width: 11.4877%;\"\u003eCategory\u003c/td\u003e\n \u003ctd rowspan=\"2\" class=\"xl78\" style=\"width: 13.498%;\"\u003ePOLE\u0026nbsp;mutation\u003cbr\u003e\u0026nbsp;status\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl75\" style=\"width: 24.4113%;\"\u003eSurgical specimen\u003cbr\u003e(POLE\u0026nbsp;mutation)\u003c/td\u003e\n \u003ctd rowspan=\"2\" class=\"xl79\" style=\"width: 9.6688%;\"\u003eTotal\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd class=\"xl64\" style=\"width: 12.2535%;\"\u003ePositive\u003c/td\u003e\n \u003ctd class=\"xl64\" style=\"width: 12.2535%;\"\u003eNegative\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd class=\"xl65\" style=\"width: 11.4877%;\"\u003eBiopsy specimen\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 13.498%;\"\u003ePositive\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 12.2535%;\"\u003e26\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 12.2535%;\"\u003e0\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 9.6688%;\"\u003e26\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd class=\"xl67\" style=\"width: 11.4877%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl66 \" style=\"width: 13.498%;\"\u003eNegative\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 12.2535%;\"\u003e0\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 12.2535%;\"\u003e35\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 9.6688%;\"\u003e35\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd class=\"xl67\" style=\"width: 11.4877%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 13.498%;\"\u003eTotal\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 12.2535%;\"\u003e26\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 12.2535%;\"\u003e35\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 9.6688%;\"\u003e61\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" class=\"xl68\" style=\"width: 24.9856%;\"\u003eOverall percent agreement\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl66\" style=\"width: 24.4113%;\"\u003e100.0% (95%CI, 94.1%-100.0%)\u003c/td\u003e\n \u003ctd class=\"xl67\" style=\"width: 9.6688%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd class=\"xl68\" colspan=\"2\" style=\"width: 24.9856%;\"\u003ePositive percent agreement\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl72\" style=\"width: 24.4113%;\"\u003e100.0% (95%CI, 86.8%-100.0%)\u003c/td\u003e\n \u003ctd class=\"xl70\" style=\"width: 9.6688%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd class=\"xl69\" colspan=\"2\" style=\"width: 24.9856%;\"\u003eNegative percent agreement\u003c/td\u003e\n \u003ctd colspan=\"2\" class=\"xl73\" style=\"width: 24.4113%;\"\u003e100.0% (95%CI, 90.0%-100.0%)\u003c/td\u003e\n \u003ctd class=\"xl71\" style=\"width: 9.6688%;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch3\u003eEvaluation of cases with insufficient sample quality for NGS\u003c/h3\u003e\n\u003cp\u003eThis study excluded 28 cases (26 surgical and two biopsy specimens) that did not pass QC for NGS. On the other hand, with the Luminex kit, it was possible to evaluate the \u003cem\u003ePOLE\u003c/em\u003e mutation status in all 28 cases; four cases were positive for \u003cem\u003ePOLE\u003c/em\u003e mutations, all of which were confirmed by subsequent Sanger sequencing. The primer sequences used for Sanger sequencing are listed in Supplementary Table 2. There were three P286R mutations and one V411L mutation, and all four had good prognoses.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\" width=\"784\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd height=\"33\" class=\"xl63\" colspan=\"2\" style=\"width: 361px;\"\u003eTable 5 Comparison of POLE\u0026nbsp;testing methods\u003c/td\u003e\n \u003ctd class=\"xl63\" style=\"width: 221px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd class=\"xl63\" style=\"width: 202px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"21\" class=\"xl73\" style=\"width: 159px;\"\u003eCharacteristics\u003c/td\u003e\n \u003ctd class=\"xl72\" style=\"width: 202px;\"\u003eLuminex\u003c/td\u003e\n \u003ctd class=\"xl72\" style=\"width: 221px;\"\u003eFoundationOne\u003csup\u003e\u0026reg;\u003c/sup\u003eCDx\u003c/td\u003e\n \u003ctd class=\"xl72\" style=\"width: 202px;\"\u003eQPOLE [9]\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"94\" class=\"xl67\" style=\"width: 159px;\"\u003eTechnology\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 202px;\"\u003eMultiplex PCR, a bead-based multiplex platform that uses fluorescence to detect mutations.\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 221px;\"\u003eHigh-throughput capture-based sequencing that can detect a wide array of mutations across multiple genes.\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 202px;\"\u003eMultiplex qPCR\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"58\" class=\"xl67\" style=\"width: 159px;\"\u003eSensitivity\u003c/td\u003e\n \u003ctd class=\"xl68\" style=\"width: 202px;\"\u003eHigh, capable of detecting mutations when present in more than 4% of total DNA.\u003c/td\u003e\n \u003ctd class=\"xl68\" style=\"width: 221px;\"\u003eVery high, capable of detecting mutations at low frequencies.\u003c/td\u003e\n \u003ctd class=\"xl68\" style=\"width: 202px;\"\u003eHigh, typically detects mutations present in more than 1\u0026ndash;5% of total DNA.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"37\" class=\"xl67\" style=\"width: 159px;\"\u003eCovered\u0026nbsp;POLE\u0026nbsp;mutations\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 202px;\"\u003e25\u003c/td\u003e\n \u003ctd class=\"xl68\" style=\"width: 221px;\"\u003eAll mutations detected on the exons\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 202px;\"\u003e11\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"44\" class=\"xl67\" style=\"width: 159px;\"\u003eDetecting undefined mutations\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 202px;\"\u003eNo\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 221px;\"\u003eYes\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 202px;\"\u003eNo\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"39\" class=\"xl67\" style=\"width: 159px;\"\u003eConcordance to results of NGS\u003c/td\u003e\n \u003ctd class=\"xl71\" style=\"width: 202px;\"\u003e100%\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 221px;\"\u003e-\u003c/td\u003e\n \u003ctd class=\"xl70\" style=\"width: 202px;\"\u003e98.6% [9]\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"19\" class=\"xl67\" style=\"width: 159px;\"\u003eDNA requirement\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 202px;\"\u003e50-100ng/case\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 221px;\"\u003e50-1000ng/case\u003c/td\u003e\n \u003ctd class=\"xl68\" style=\"width: 202px;\"\u003e30ng/case\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"19\" class=\"xl67\"\u003eProcedure and effort\u003c/td\u003e\n \u003ctd class=\"xl69\"\u003eA single tube/medium\u003c/td\u003e\n \u003ctd class=\"xl69\"\u003eA single tube/hard\u003c/td\u003e\n \u003ctd class=\"xl69\"\u003eMulti-tube/easy\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"19\" class=\"xl67\" style=\"width: 159px;\"\u003eTurn around time\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 202px;\"\u003e4.5 hours\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 221px;\"\u003e2 weeks\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 202px;\"\u003e4-6 hours\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"19\" class=\"xl67\" style=\"width: 159px;\"\u003eCost\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 202px;\"\u003eLow\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 221px;\"\u003eHigh\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 202px;\"\u003eLow\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"39\" class=\"xl67\" style=\"width: 159px;\"\u003eBioinformatics and data interpretation\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 202px;\"\u003eSimple\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 221px;\"\u003eComplex\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 202px;\"\u003eSimple\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"19\" class=\"xl67\" style=\"width: 159px;\"\u003eHigh-throughput\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 202px;\"\u003eUp to 96 samples\u003c/td\u003e\n \u003ctd class=\"xl68\" style=\"width: 221px;\"\u003eUp to 64 samples\u003c/td\u003e\n \u003ctd class=\"xl68\" style=\"width: 202px;\"\u003eUp to 32 samples\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"19\" class=\"xl67\" style=\"width: 159px;\"\u003eQuality manegment\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 202px;\"\u003eNanoDrop\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 221px;\"\u003eQubit\u003c/td\u003e\n \u003ctd class=\"xl66\" style=\"width: 202px;\"\u003eNanoDrop\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"39\" class=\"xl65\" style=\"width: 159px;\"\u003eExpandability and customization\u003c/td\u003e\n \u003ctd class=\"xl64\" style=\"width: 202px;\"\u003eHigh\u003c/td\u003e\n \u003ctd class=\"xl64\" style=\"width: 221px;\"\u003eLow\u003c/td\u003e\n \u003ctd class=\"xl64\" style=\"width: 202px;\"\u003eHigh\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" height=\"39\" class=\"xl74\" style=\"width: 784px;\"\u003eAbbreviations: DNA; deoxyribonucleic acid, NGS; next generation sequencing, PCR;polymerase chain reaction, qPCR;quantitative polymerase chain reaction\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we presented a multiplex PCR and probe-based detection kit that simultaneously detects 25 pathogenic \u003cem\u003ePOLE\u003c/em\u003e mutations using the Luminex (xMAP) assay as an alternative to DNA sequencing. Our study demonstrated excellent concordance between the multiplex PCR and probe-based detection kit and conventional NGS. Even when DNA from older FFPE blocks was used, comparison of the Luminex assay with NGS showed an overall agreement of 100% (95%CI: 99.1%-100.0%, 94.9%-100.0%, respectively) for both surgical and biopsy specimens. Our method ensures that the assessment of \u003cem\u003ePOLE\u003c/em\u003e mutations is feasible and acceptable in many laboratories worldwide, and it is expected that this will lead to the spread of molecular classification and the prevention of overtreatment for \u003cem\u003ePOLE\u003c/em\u003e-mut EC.\u003c/p\u003e \u003cp\u003eECs with \u003cem\u003ePOLE\u003c/em\u003e-exonuclease domain mutations (EDM) in exons 9, 11, 13, and 14 have been internationally recognized to qualify as \u003cem\u003ePOLE\u003c/em\u003e-mut EC. In the present study, the five most common mutations, P286R, S297F, V411L, A456P, and S459F, accounted for approximately 80% of all \u003cem\u003ePOLE\u003c/em\u003e-mut ECs. It has been reported that these five mutations account for more than 95% \u003cem\u003ePOLE\u003c/em\u003e-mut ECs \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, and the revised WHO classification system for EC recommends analyses of these \u0026ldquo;hotspot\u0026rdquo; \u003cem\u003ePOLE\u003c/em\u003e mutations \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, in addition to these five mutations, many \u003cem\u003ePOLE\u003c/em\u003e mutations are associated with a favorable prognosis, and the classification of these less frequent mutations is currently challenging.\u003c/p\u003e \u003cp\u003eThe newly developed Luminex (xMAP) assay appears to have significant advantages over the previously reported \u003cem\u003ePOLE\u003c/em\u003e test (Table\u0026nbsp;5). In 2023, a quantitative polymerase chain reaction (qPCR) assay, \u003cem\u003eQPOLE\u003c/em\u003e, was developed to detect 11 mutations: the five most common mutations (P286R, S297F, V411L, A456P or S459F) plus M295R, F367S, D368Y, L424I, P436R, and M444K, all of which have been reported to have a POLE-score of \u0026ge;\u0026thinsp;4 and an excellent clinical outcome \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eQPOLE\u003c/em\u003e achieved an overall accuracy of 98.6%, a sensitivity of 95.2%, and a specificity of 100%. In \u003cem\u003eQPOLE\u003c/em\u003e, PCR must be performed for each mutation in each tube. In contrast, the Luminex kit can measure all 25 mutations simultaneously in one tube. In addition, the accuracy of the Luminex kit was 100% in this study, equivalent to that of \u003cem\u003eQPOLE\u003c/em\u003e. Similar to \u003cem\u003eQPOLE\u003c/em\u003e, the Luminex (xMAP) assay enables \u003cem\u003ePOLE\u003c/em\u003e status assessment within 4.5 h, significantly shortening the turnaround time from the 2 weeks required for NGS. Moreover, it is inexpensive and can be performed using only small amounts of DNA, and it does not require interpretation by a molecular biologist, unlike NGS. Furthermore, the Luminex kit has sufficient reliability, and methods of QC/quality assurance have already been established. We believe that the Luminex (xMAP)-based multiplex kit is more suitable for routine clinical use because it processes multiple samples.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo develop this kit, we selected 25 \u003cem\u003ePOLE\u003c/em\u003e mutations, including the abovementioned ones. According to the COSMIC and cBioPortal databases, T278M, P286S, P286L, P286H, S297Y, L424V, A426V, P441L, T457M, and A465V are suspected pathogenic mutations, and there are many reports of good prognosis \u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25 CR26\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Moreover, our previous study on genetic alterations in Japanese patients with \u003cem\u003ePOLE\u003c/em\u003e-EDM EC demonstrated that P286C, F367C, W369*, and S459del were newly detected, and that the prognosis of patients with these mutations was extremely favorable, similar to that of patients with other \u003cem\u003ePOLE\u003c/em\u003e-EDM \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Thus, our new kit was used to investigate 25 mutations in exons 9, 11, 13, and 14. With the Luminex (xMAP)-based kit, there is a possibility that rare pathogenic variants of \u003cem\u003ePOLE\u003c/em\u003e will be identified in the future. In this study, five of 52 \u003cem\u003ePOLE\u003c/em\u003e-mut cases (9.6%) showed rare variants, and all showed excellent prognoses. Therefore, a validation cohort for these rare variants is urgently required.\u003c/p\u003e \u003cp\u003eTwo key limitations of our study are its small sample size and retrospective design in a single institution. Our new kit included \u003cem\u003ePOLE\u003c/em\u003e mutations that were not present in The Cancer Genome Atlas or \u003cem\u003ePOLE\u003c/em\u003e-score\u0026thinsp;=\u0026thinsp;3. These mutations were associated with a good prognosis and could be considered pathogenic variants of \u003cem\u003ePOLE\u003c/em\u003e; however, in future, it will be necessary to collect specimens from multiple institutions and perform external validation. In our study, 28 cases failed to pass QC for NGS, and all 28 could be tested using the Luminex kit, with \u003cem\u003ePOLE\u003c/em\u003e mutations detected in four. If this kit is validated, it may become more useful than NGS for detecting \u003cem\u003ePOLE\u003c/em\u003e mutations.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we developed a new multiplex kit that can detect 25 \u003cem\u003ePOLE\u003c/em\u003e pathogenic mutations simultaneously using the Luminex (xMAP) assay. Our study demonstrated excellent concordance between the multiplex kit and conventional NGS for both surgical and biopsy specimens. The Luminex multiplex kit can be a powerful tool that surpasses NGS in evaluating the \u003cem\u003ePOLE\u003c/em\u003e mutation status rapidly and easily, leading to accurate prognosis prediction using FIGO 2023 with molecular classification and preventing overtreatment for \u003cem\u003ePOLE\u003c/em\u003e-mut EC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Yuka Asami, Kengo Hiranuma, Hitoshi Ichikawa, Sachiyo Mitani, Maiko Matsuda, Yoko Shimada, other physicians and staff members of the National Cancer Center, and other hospitals for their assistance and support. We express our gratitude to Editage (https://www.editage.jp) for assisting us with English language editing. This study was supported by the Japan Agency for Medical Research and Development (23ama221520h0001), Grant-in-Aid for Young Scientists (B) (20K18207 and 19K16572), Grant-in-Aid for Scientific Research (B) (20H03695), Grant-in-Aid for Scientific Research (C) (20K09636), BRIDGE (programs for bridging the gap between R\u0026amp;D and the ideal society [Society 5.0] and generating economic and social value), and the\u0026nbsp;National Cancer Center Research and Development Fund (2022-A-20, 2023-J-2, NCC Biobank, and NCC Core Facility). The funder played no role in study design, data collection, analysis and interpretation of data, or the writing of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA competing interests statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no financial competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM. K. K., K. S., H. Y., and T. K. designed the study. M. K. K. wrote the first draft of the manuscript. K.S., T.K., T.S., M.Y., D.H., and H.Y. contributed to the data analysis and interpretation and assisted with manuscript preparation. All other authors contributed to data collection, interpretation, and critical manuscript review. All authors have approved the final version of the manuscript and agreed to be accountable for all aspects of the study. Questions related to the accuracy or integrity of the study were investigated and resolved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are not publicly available due to the sensitive nature of the questions asked in this study but are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe underlying code for this study is not publicly available but may be made available for proprietary reasons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from all patients. This study was approved by the Institutional Review Board of the National Cancer Center Research Institute.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBerek, J. S. \u003cem\u003eet al.\u003c/em\u003e FIGO staging of endometrial cancer: 2023. Int J Gynaecol Obstet 162, 383\u0026ndash;394, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/ijgo.14923\u003c/span\u003e\u003cspan address=\"10.1002/ijgo.14923\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKobayashi-Kato, M. \u003cem\u003eet al.\u003c/em\u003e Utility of the revised FIGO2023 staging with molecular classification in endometrial cancer. Gynecol Oncol 178, 36\u0026ndash;43, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ygyno.2023.09.011\u003c/span\u003e\u003cspan address=\"10.1016/j.ygyno.2023.09.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCancer, I. A. f. R. o. WHO Classification of Tumours Editorial Board. Female Genital Tumors. (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eConsortium, R. R. Refining adjuvant treatment in endometrial cancer based on molecular features: the RAINBO clinical trial program. Int J Gynecol Cancer 33, 109\u0026ndash;117, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1136/ijgc-2022-004039\u003c/span\u003e\u003cspan address=\"10.1136/ijgc-2022-004039\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBando, H. \u003cem\u003eet al.\u003c/em\u003e Simultaneous identification of 36 mutations in KRAS codons 61 and 146, BRAF, NRAS, and PIK3CA in a single reaction by multiplex assay kit. BMC Cancer 13, 405, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/1471-2407-13-405\u003c/span\u003e\u003cspan address=\"10.1186/1471-2407-13-405\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoshino, T. \u003cem\u003eet al.\u003c/em\u003e Clinical Validation of a Multiplex Kit for RAS Mutations in Colorectal Cancer: Results of the RASKET (RAS KEy Testing) Prospective, Multicenter Study. \u003cem\u003eEBioMedicine\u003c/em\u003e 2, 317\u0026ndash;323, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ebiom.2015.02.007\u003c/span\u003e\u003cspan address=\"10.1016/j.ebiom.2015.02.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaniguchi, H. \u003cem\u003eet al.\u003c/em\u003e Clinical Validation of Newly Developed Multiplex Kit Using Luminex xMAP Technology for Detecting Simultaneous RAS and BRAF Mutations in Colorectal Cancer: Results of the RASKET-B Study. Neoplasia 20, 1219\u0026ndash;1226, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.neo.2018.10.004\u003c/span\u003e\u003cspan address=\"10.1016/j.neo.2018.10.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeon-Castillo, A. \u003cem\u003eet al.\u003c/em\u003e Interpretation of somatic POLE mutations in endometrial carcinoma. J Pathol 250, 323\u0026ndash;335, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/path.5372\u003c/span\u003e\u003cspan address=\"10.1002/path.5372\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan den Heerik, A. \u003cem\u003eet al.\u003c/em\u003e QPOLE: A Quick, Simple, and Cheap Alternative for POLE Sequencing in Endometrial Cancer by Multiplex Genotyping Quantitative Polymerase Chain Reaction. JCO Glob Oncol 9, e2200384, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1200/GO.22.00384\u003c/span\u003e\u003cspan address=\"10.1200/GO.22.00384\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsami, Y. \u003cem\u003eet al.\u003c/em\u003e Utility of molecular subtypes and genetic alterations for evaluating clinical outcomes in 1029 patients with endometrial cancer. Br J Cancer 128, 1582\u0026ndash;1591, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41416-023-02203-3\u003c/span\u003e\u003cspan address=\"10.1038/s41416-023-02203-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKato, M. K. \u003cem\u003eet al.\u003c/em\u003e Excellent concordance of the molecular classification between preoperative biopsy and final hysterectomy in endometrial carcinoma. Gynecol Oncol 190, 139\u0026ndash;145, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ygyno.2024.08.016\u003c/span\u003e\u003cspan address=\"10.1016/j.ygyno.2024.08.016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBreiley JD GM, W. C. \u003cem\u003ethe 2020 World Health Organization tumor classification\u003c/em\u003e. (Wiley, 2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHirose, S. \u003cem\u003eet al.\u003c/em\u003e Genomic alterations in STK11 can predict clinical outcomes in cervical cancer patients. Gynecol Oncol 156, 203\u0026ndash;210, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ygyno.2019.10.022\u003c/span\u003e\u003cspan address=\"10.1016/j.ygyno.2019.10.022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuno, I. \u003cem\u003eet al.\u003c/em\u003e TP53 mutants and non-HPV16/18 genotypes are poor prognostic factors for concurrent chemoradiotherapy in locally advanced cervical cancer. Sci Rep 11, 19261, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-021-98527-2\u003c/span\u003e\u003cspan address=\"10.1038/s41598-021-98527-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurakami, N. \u003cem\u003eet al.\u003c/em\u003e Distribution of genetic alterations in high-risk early-stage cervical cancer patients treated with postoperative radiation therapy. Sci Rep 11, 10567, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-021-90139-0\u003c/span\u003e\u003cspan address=\"10.1038/s41598-021-90139-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakayanagi, D. \u003cem\u003eet al.\u003c/em\u003e Comparative Analysis of Genetic Alterations, HPV-Status, and PD-L1 Expression in Neuroendocrine Carcinomas of the Cervix. Cancers (Basel) 13, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cancers13061215\u003c/span\u003e\u003cspan address=\"10.3390/cancers13061215\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgresti, A. \u003cem\u003eCategorical Data Analysis\u003c/em\u003e. (John Wiley and Sons, 1990).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLandis, J. R. \u0026amp; Koch, G. G. The measurement of observer agreement for categorical data. Biometrics 33, 159\u0026ndash;174 (1977).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSasada, S. \u003cem\u003eet al.\u003c/em\u003e Baseline risk of recurrence in stage I-II endometrial carcinoma. \u003cem\u003eJ Gynecol Oncol\u003c/em\u003e 29, e9, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3802/jgo.2018.29.e9\u003c/span\u003e\u003cspan address=\"10.3802/jgo.2018.29.e9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcAlpine, J. N. \u003cem\u003eet al.\u003c/em\u003e Evaluation of treatment effects in patients with endometrial cancer and POLE mutations: An individual patient data meta-analysis. Cancer 127, 2409\u0026ndash;2422, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/cncr.33516\u003c/span\u003e\u003cspan address=\"10.1002/cncr.33516\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng, B. \u003cem\u003eet al.\u003c/em\u003e POLE exonuclease domain mutation predicts long progression-free survival in grade 3 endometrioid carcinoma of the endometrium. Gynecol Oncol 134, 15\u0026ndash;19, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ygyno.2014.05.006\u003c/span\u003e\u003cspan address=\"10.1016/j.ygyno.2014.05.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShinbrot, E. \u003cem\u003eet al.\u003c/em\u003e Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication. Genome Res 24, 1740\u0026ndash;1750, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1101/gr.174789.114\u003c/span\u003e\u003cspan address=\"10.1101/gr.174789.114\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParra-Herran, C. \u003cem\u003eet al.\u003c/em\u003e Molecular-based classification algorithm for endometrial carcinoma categorizes ovarian endometrioid carcinoma into prognostically significant groups. Mod Pathol 30, 1748\u0026ndash;1759, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/modpathol.2017.81\u003c/span\u003e\u003cspan address=\"10.1038/modpathol.2017.81\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZou, Y. \u003cem\u003eet al.\u003c/em\u003e Frequent POLE1 p.S297F mutation in Chinese patients with ovarian endometrioid carcinoma. Mutat Res 761, 49\u0026ndash;52, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.mrfmmm.2014.01.003\u003c/span\u003e\u003cspan address=\"10.1016/j.mrfmmm.2014.01.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePalles, C. \u003cem\u003eet al.\u003c/em\u003e Germline mutations affecting the proofreading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas. Nat Genet 45, 136\u0026ndash;144, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/ng.2503\u003c/span\u003e\u003cspan address=\"10.1038/ng.2503\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBillingsley, C. C. \u003cem\u003eet al.\u003c/em\u003e Polymerase varepsilon (POLE) mutations in endometrial cancer: clinical outcomes and implications for Lynch syndrome testing. Cancer 121, 386\u0026ndash;394, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/cncr.29046\u003c/span\u003e\u003cspan address=\"10.1002/cncr.29046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStenzinger, A. \u003cem\u003eet al.\u003c/em\u003e Mutations in POLE and survival of colorectal cancer patients\u0026ndash;link to disease stage and treatment. Cancer Med 3, 1527\u0026ndash;1538, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/cam4.305\u003c/span\u003e\u003cspan address=\"10.1002/cam4.305\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"endometrial cancer, POLE mutation, Luminex (xMAP) assay, next-generation sequencing, concordance rate","lastPublishedDoi":"10.21203/rs.3.rs-5540131/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5540131/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eIn endometrial cancer,\u003cstrong\u003e \u003c/strong\u003edetection of oncogenic mutations in the polymerase epsilon (\u003cem\u003ePOLE\u003c/em\u003e) gene is crucial for accurate staging according to the 2023 International Federation of Gynecology and Obstetrics classification and for minimizing overtreatment. However, \u003cem\u003ePOLE\u003c/em\u003esequencing is expensive, time-consuming, and often inaccessible in settings without specialized equipment. We developed a novel multiplex kit for the detection of \u003cem\u003ePOLE\u003c/em\u003e mutations using a Luminex (xMAP) assay in a single reaction. The aim of this study was to evaluate the accuracy of the multiplex kit for routine clinical samples and compare it with that of conventional next-generation sequencing (NGS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Hysterectomy specimens and endometrial biopsies were collected at the National Cancer Center Hospital between 1999 and 2023. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissues. Both the Luminex (xMAP)-based multiplex kit and NGS targeting all \u003cem\u003ePOLE \u003c/em\u003eexons were used. Concordance was assessed using Cohen’s kappa.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eOf the 502 samples, 432 were hysterectomy specimens and 70 were biopsies. In the surgical samples, both the Luminex (xMAP)-based kit and NGS detected 52 \u003cem\u003ePOLE\u003c/em\u003e mutations (12.0%) with perfect concordance (κ=1.000). In the biopsies, 33 \u003cem\u003ePOLE\u003c/em\u003emutations were identified using both methods, with complete concordance. Notably, the Luminex (xMAP)-based kit successfully analyzed all 28 samples that failed NGS quality control and detected four cases with \u003cem\u003ePOLE\u003c/em\u003e mutations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThe Luminex (xMAP)-based kit demonstrates high concordance with NGS for the detection of \u003cem\u003ePOLE\u003c/em\u003e mutations. With further external validation, this kit could become a reliable and accessible alternative to NGS.\u003c/p\u003e","manuscriptTitle":"Comparison between a Luminex-based multiplex kit with a sequence-specific oligonucleotide probe and next-generation sequencing for the detection of POLE oncogenic mutations in endometrial cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-10 16:14:38","doi":"10.21203/rs.3.rs-5540131/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"96abffa0-fd01-4ea3-94b4-dbdb01e4f8ce","owner":[],"postedDate":"December 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":41222944,"name":"Biological sciences/Cancer"},{"id":41222945,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2024-12-10T16:14:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-10 16:14:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5540131","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5540131","identity":"rs-5540131","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}