POLE Mutations Research Articles

QPOLE: A Quick, Simple, and Cheap Alternative for POLE Sequencing in Endometrial Cancer by Multiplex Genotyping Quantitative Polymerase Chain Reaction.

Detection of 11 pathogenic variants in the POLE gene in endometrial cancer (EC) is critically important to identify women with a good prognosis and reduce overtreatment. Currently, POLE status is determined by DNA sequencing, which can be expensive, relatively time-consuming, and unavailable in hospitals without specialized equipment and personnel. This may hamper the implementation of POLE-testing in clinical practice. To overcome this, we developed and validated a rapid, low-cost POLE hotspot test by a quantitative polymerase chain reaction (qPCR) assay, QPOLE. Primer and fluorescence-labeled 5'-nuclease probe sequences of the 11 established pathogenic POLE mutations were designed. Three assays, QPOLE-frequent for the most common mutations and QPOLE-rare-1 and QPOLE-rare-2 for the rare variants, were developed and optimized using DNA extracted from formalin-fixed paraffin-embedded tumor tissues. The simplicity of the design enables POLE status assessment within 4-6 hours after DNA isolation. An interlaboratory external validation study was performed to determine the practical feasibility of this assay. Cutoffs for POLE wild-type, POLE-mutant, equivocal, and failed results were predefined on the basis of a subset of POLE mutants and POLE wild-types for the internal and external validation. For equivocal cases, additional DNA sequencing is recommended. Performance in 282 EC cases, of which 99 were POLE-mutated, demonstrated an overall accuracy of 98.6% (95% CI, 97.2 to 99.9), a sensitivity of 95.2% (95% CI, 90.7 to 99.8), and a specificity of 100%. After DNA sequencing of 8.8% equivocal cases, the final sensitivity and specificity were 96.0% (95% CI, 92.1 to 99.8) and 100%. External validation confirmed feasibility and accuracy. QPOLE is a qPCR assay that is a quick, simple, and reliable alternative for DNA sequencing. QPOLE detects all pathogenic variants in the exonuclease domain of the POLE gene. QPOLE will make low-cost POLE-testing available for all women with EC around the globe.

Prognostic Impact of Pathologic Features in Molecular Subgroups of Endometrial Carcinoma.

The molecular characterization of endometrial carcinoma (EC) has recently been included in the ESGO/ESTRO/ESP guidelines. The study aims to evaluate the impact of integrated molecular and pathologic risk stratification in the clinical practice and the relevance of pathologic parameters in predicting prognosis in each EC molecular subgroup. ECs were classified using immunohistochemistry and next-generation sequencing into the four molecular classes: POLE mutant (POLE), mismatch repair deficient (MMRd), p53 mutant (p53abn), and no specific molecular profile (NSMP). According to the WHO algorithm, 219 ECs were subdivided into the following molecular subgroups: 7.8% POLE, 31% MMRd, 21% p53abn, 40.2% NSMP. Molecular classes as well as ESGO/ESTRO/ESP 2020 risk groups were statistically correlated with disease-free survival. Considering the impact of histopathologic features in each molecular class, stage was found to be the strongest prognostic factor in MMRd ECs, whereas in the p53abn subgroup, only lymph node status was associated with recurrent disease. Interestingly, in the NSMP tumor, several histopathologic features were correlated with recurrence: histotype, grade, stage, tumor necrosis, and substantial lymphovascular space invasion. Considering early-stage NSMP ECs, substantial lymphovascular space invasion was the only independent prognostic factor. Our study supports the prognostic importance of EC molecular classification and demonstrated the essential role of histopathologic assessment in patients' management.

Abstract 1515: Tumor molecular profiling and spatial transcriptomics to assess colorectal cancer heterogeneity and microenvironment

Abstract Introduction: CRC is the second leading cause of cancer mortality in the United States. Clinical factors and molecular characteristics may impact therapeutics and prognosis. Scientific studies have uncovered significant aberrations, including critical genes and pathways. Despite these efforts, few studies have utilized similar sample sizes to TCGA, heterogeneous populations and integrative clinical data to compare their reported genomic changes, even though little is known about the tumor microenvironment and its significance for colorectal tumorigenesis. Methods: Clinical and DNA genomic data from 262 colorectal tumor/normal DNA samples were obtained for whole exome sequencing analysis from the Oncology Research Information Exchange Network and two colorectal tissue samples for Spatial Transcriptomics profiling from the USC colorectal cancer Moonshot project. Whole Exome Sequencing data analysis was used for identifying genetic alterations. Samples were stratified based on microsatellite instability, age group, anatomical subsites, and histological subtypes. Emphasis was given to comparing molecular characteristics among Hispanic/Latino patients since their reported higher mortality and cancer disparities. Mutation frequencies from our cohort were compared to those from the TCGA. An ongoing spatial transcriptomics analysis consists in processing two fresh-frozen tissue samples through the visium spatial gene expression library using standard short-read sequencing. Results: Tumor molecular profiling revealed 20 genes significantly mutated. We found differences in gene mutation frequencies among our samples, highlighting APC (60%), TP53 (56%), TTN (49%), and KRAS (44%) gene mutations. In comparison with TCGA data, in addition to the expected APC, TP53, PIK3CA, and KRAS mutations, we found frequent mutations in POLE and TTN. To assess the basis for the considerably different mutation rates, we evaluated MSI. Among the 262 tumors, 41 (16%) had high levels of MSI, 2 (1%) had low levels of MSI (MSI-L), and 215 (82%) were microsatellite stable (MSS). Cancer heterogeneity was reported among tumors with different characteristics such as age group, anatomical subsite, and histological subtypes. The ongoing spatial transcriptomics analysis that will be ready before the annual meeting will identify cellular populations and map immunological and inflammatory signals in the microenvironment. Conclusions: Our study reveals the impact of molecularly profiling tumors using considerable sample sizes and stratified samples in several subgroups. Our study adds important information about tumor heterogeneity and the tumor microenvironment in colorectal cancer by including our spatial transcriptomics data analysis. Citation Format: Enrique Velazquez Villarreal, Seeta Rajpara, Yuxin Jin, Jing Qian, Bohan Zhang, Brigette Waldrup, Donna Loza, Heinz-Josef Lenz, David W. Craig, John D. Carpten. Tumor molecular profiling and spatial transcriptomics to assess colorectal cancer heterogeneity and microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1515.

Abstract 4542: Development of a one-step molecular classifier for endometrial carcinoma using an amplicon-based gene panel and next generation sequencing technology

Abstract Objectives: Molecular classification of endometrial carcinoma (EC) is now recommended by the WHO, ESGO/ESTRO/ESP and NCCN guidelines. The pragmatic molecular classification tool, ProMisE, identifies four molecular subtypes based on next generation sequencing (NGS) for the detection of somatic pathogenic POLE mutations, and immunohistochemistry for mismatch repair and p53 proteins. ProMisE provides valuable prognostic and predictive information to direct care, however, multiple molecular results are often received from different time periods and/or from different centers which can cause delays. Therefore, we developed a classifier that relies on a single DNA-based NGS test with the goal of recapitulating the prognostic value of ProMisE by producing concordant results. Methods: Formalin fixed paraffin embedded (FFPE) EC tumor DNA was sequenced using the clinically validated Imagia Canexia Health Find ItTM amplicon-based next generation sequencing gene panel assay. The 38 gene panel assessed single nucleotide variants (SNVs), indels (insertions and deletions), gene amplification and microsatellite instability (MSI) using 21 MSI loci. To compare to the original ProMisE classifier, we identified somatic mutations in POLE, TP53 and MSI-High or MS-Stable for molecular classification. Molecular subtypes assigned by both classifiers were assessed for concordance metrics and Kaplan-Meier survival curves. Results: The one-step NGS molecular classifier assessed 165 unique patient FFPE EC samples that had previously been assessed by ProMisE. There were 152/165 cases that were concordant between molecular subtype assignment from the original ProMisE and the one-step NGS assay with a kappa statistic of 0.88 and accuracy of 0.92. There were 13 samples that were discordant (original classifications: 3 POLE, 3 MMRd, 2 p53abn, 5 NSMP/p53wt), which will be reviewed in detail. Results were concordant in 14 of 15 cases (93%) when both diagnostic biopsy and hysterectomy specimens were tested. Molecular subtypes maintained their associations with clinical outcomes (progression free survival, disease specific survival, overall survival). Conclusion: The one-step NGS molecular classifier demonstrates high concordance with the original ProMisE classifier including between biopsy and hysterectomy samples. This shows that reliable molecular testing could be obtained from time of first diagnosis. Prognostic value of this new one-step classification tool is maintained. Appropriate interpretation of results is critical, including limiting POLE mutation assignment to a confirmed list of pathogenic mutations and correct order of segregation for ECs with more than one molecular feature. Further validation is needed in a larger cohort to implement into standard of care testing. Citation Format: Melissa K. McConechy, Amy Jamieson, Amy Lum, Samuel Leung, Adrian Kense, Sonal Brahmbhatt, Ka Mun Nip, Ebru Baran, Dilmi Perera, Kurt Yakimovich, Ruth Miller, C. Blake Gilks, David Huntsman, Jessica N. McAlpine. Development of a one-step molecular classifier for endometrial carcinoma using an amplicon-based gene panel and next generation sequencing technology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4542.

Abstract 4253: Pan-cancer analysis of XPO1 R749Q mutations across 217,570 patients reveals association with high tumor mutational burden and therapy resistance

Abstract Background: XPO1 is a nuclear export receptor responsible for exporting >200 proteins out of the nucleus, including many cancer-related proteins. We previously reported recurrent hotspot mutations occurring in XPO1 in cancer and identified XPO1 R749Q variant only in solid tumors. To determine the prevalence of XPO1 mutations across cancer types, we performed a large-scale genomic analysis of 217,570 patients with cancer to identify and characterize XPO1 variants from real-world patient tumors. Methods: Solid tumor samples representing 14 cancer types were submitted to Caris Life Sciences (Phoenix, AZ) for Next-generation sequencing of DNA (592 genes or WES). TMB-High (defined as >10 mt/MB) and dMMR/MSI-H was tested by IHC/NGS. We engineered CRISPR knock-in HCT116 and LS-174T colon cancer isogenic cell lines bearing XPO1 R749Q mutations and Stochastic Optical Reconstruction Microscopy (STORM) analysis was performed. Structural modeling was performed with published XPO1 structures by mapping XPO1 mutations from the MSKCC-IMPACT dataset. Results: A total of 96 patients with XPO1 R749Q mutations were identified (50% occurred in endometrial cancer and 23% occurred in colorectal cancer). TMB-H was observed in 92% of XPO1 R749Q mt tumors (9% were dMMR/MSI-H), while 86% of XPO1 R749Q mt tumors were POLE-mutated within the endonuclease domain. Median variant allele frequency for XPO1 R749Q mt and POLE mt were 25% and 29%, respectively. STORM imaging revealed that XPO1 R749Q mutant cells had significant localization of XPO1 in the cytoplasm compared to XPO1 WT cells, especially at the outside edge of the nuclear pores. Structural modeling predicted that XPO1 R749Q mt affected the regulatory H9-loop of XPO1 favoring increased shuttling and retention in the cytoplasm. Mass spectrometry analysis of nuclear and cytoplasmic fractionated proteomes confirmed that XPO1 R749Q mt cells had increased export of proteins compared to XPO1 WT cells. A library screen of >200 FDA-approved drugs revealed a strong therapeutic resistance of XPO1 R749Q cells, especially to chemotherapies used in the treatment of colon cancer. XPO1 inhibition with selinexor synergized with chemotherapy in XPO1 R749Q mt cells in vitro and overcame resistance to irinotecan in vivo in xenograft mice models. Conclusion: This study sheds novel insights into the role of nuclear export in cancers. Specifically, XPO1 R749Q mutations are enriched in TMB-H endometrial and colon cancers and increase nuclear export of key proteins that confer resistance to therapies using DNA-damaging agents. Our current work aims to identify novel therapies that can overcome resistance to DNA-damaging therapies seen in XPO1 mutant cells, such as immune checkpoint inhibitor (ICI) therapy given the co-occurrence with POLE mutations and recent reports of high level of response to ICI therapy in TMB-H and POLE mt colorectal cancers. Citation Format: Tulasigeri M. Totiger, Yasmine Baca, Wannasiri Chiraphapphaiboon, Sana Chaudhry, Skye Montoya, Monika Chojnacka, Gabriel Gaidosh, Jumana Afaghani, Maurizio Affer, Christopher D. Armstrong, Ryan Notti, Jenna Zabroski, Jacob Jahn, Vindhya Nawaratne, Ramiro Verdun, Hai Dang Nguyen, Chadi Nabhan, Thomas J. Herzog, Phil Walker, Andrew Elliott, Emil Lou, Wafik S. El-Deiry, Edith Mitchell, Jose Antonio Rodriguez, Justin Taylor. Pan-cancer analysis of XPO1 R749Q mutations across 217,570 patients reveals association with high tumor mutational burden and therapy resistance. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4253.

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

Abstract BACKGROUND: Pathogenic POLE exonuclease domain mutations (pPOLE) undermine mismatch error correction during DNA replication, causing somatic ultramutation and response to immunotherapy. We examined the pan-cancer landscape of POLE mutations and applied a POLE-specific variant classification model. METHODS: Comprehensive genomic profiling was performed during clinical care. Mutational signature calling was performed via decomposition using the 96-feature single-base substitution COSMIC reference signatures. A POLE-specific classification model encompassing mutation position in the exonuclease domain, TMB, presence of POLE signature, absence of other signatures, germline frequency, and other features was applied to identify pPOLE mutations causative of ultramutation. RESULTS: POLE mutation status was evaluated in 458,437 samples (425,520 tissue biopsies (TB) and 32,917 liquid biopsies (LB)). One or more POLE alterations, including pathogenic alterations and variants of unknown significance (VUS), were detected in 3.8% of samples. 19,470 total alterations were identified, 84.8% of which were missense substitutions. Application of the POLE-specific classification model identified 35 unique pathogenic variants, many of which were VUS prior to this study. 749 samples harbored a pPOLE, more than half (56.6%) of which were either p.P286R (n=245) or p.V411L (n=179). pPOLE were found in 1.4% (199/13,688) of endometrial cancers (EC) and 0.5% (270/55,981) of colorectal cancers (CRC) and were rarer in a long tail of other malignancies. The overall pPOLE rate was significantly lower in LB than TB (0.02% vs 0.17%, P<0.001) in the context of different clinical ordering patterns for EC (0.9% of LB cohort vs 3.1% of TB cohort; P<0.001) and CRC (7.5% of LB cohort vs 12.6% of TB cohort; P<0.001). Median TB TMB for pPOLE+ samples was 157.5 mut/mb, compared to 3.5 for the cohort overall (P<0.001). Similarly in LB, median pPOLE+ bTMB was 165.6 vs 2.5 overall (P<0.001). MSI-H or an MMR-associated signature was found in 17.5% of samples with pPOLE, most commonly in neurologic malignancies (75%, 27/36). Median TMB of samples with both pPOLE and MMRD was 2.4-fold higher than those with pPOLE alone (337.6 vs 139.4; P<0.001). Notably, 6.8% of pPOLE+ cases had TMB<10, which was associated median pPOLE VAF of 3.5%, compared with 25.7% among pPOLE samples with TMB≥10 (P<0.001). This pattern suggests that TMB is underestimated when tumor purity is near the limit of detection for the assay. CONCLUSIONS: pPOLE were seen in both TB and LB across cancer types. The high rate of passenger mutations underscores the utility of this POLE-specific variant classification model. Because TMB can be underestimated when tumor purity is near the limit of detection for the assay, accurate detection and classification of pPOLE is critical for identifying patients who may benefit from immunotherapy. Citation Format: Rachel B. Keller, James Haberberger, Tyler Janovitz, Alexa B. Schrock, Hanna Tukachinsky, Lei Zhong, Douglas A. Mata, Lyle V. Lopez, Zoe Fleischmann, Radwa Sharaf, Ethan S. Sokol, Garrett M. Frampton, Nimesh R. Patel, Douglas I. Lin, Geoff R. Oxnard, Erik A. Williams, Julia A. Elvin, Brennan Decker. POLE-specific variant classification strategy is critical for identifying patients who may benefit from immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 305.

Abstract 4615: Differential immune characteristics and anti-PD-1-induced reinvigoration capacity of tumor-infiltrating lymphocytes according to ProMisE classifier in endometrial cancer

Abstract Endometrial cancer (EC), one of the most common gynecological malignancies, has been gradually increasing worldwide. The Cancer Genome Atlas endometrial collaborative project discovered 4 distinct prognostic genomic subtypes, and the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) was developed: POLE-mutated, mismatch repair deficiency (MMR-D), non-specific molecular profile (NSMP), and p53-abnormal (p53abn). In the era of immunotherapy, immune checkpoint inhibitors (ICIs) become a definite management of EC, both as single agent or in combination with other targeted agents. However, the characteristics of the tumor immune microenvironment according to molecular classification in EC are not well known. This study was conducted to identify distinct immune properties of tumor infiltrating lymphocytes (TILs) according to molecular classification in EC and find optimal therapeutic targets in each molecular subtypes. We isolated peripheral blood mononuclear cells (PBMCs) and TILs from 81 patients with EC. The expression of immune checkpoint receptors and T-cell transcription factors of TILs were examined using flow cytometry. Sixty-six patients were evaluated according to the ProMisE algorithm based on sequencing for the presence of POLE mutations, immunohistochemistry for MMR proteins and p53 (POLE-mut, n=6; MMR-D, n=17; p53abn, n=20; NSMP, n=23). Tumors were categorized into one of the four molecular subtypes. TILs were ex vivo stimulated with anti-CD3 in the presence of anti-PD-1, and their proliferation was assessed to predict ICI-induced reinvigoration capacity of CD8 TILs. Comparing T-cell transcription factors (TCF-1 and TOX), CD8 TILs showed higher expression of TOX, and lower expression of TCF-1. When comparing the immune characteristics of TILs according to molecular subtypes, Tregs of POLE-mut showed significantly lower expression of CTLA-4 and Ki-67 than that of MMR-D. In addition, CD8 TILs of MMR-D showed higher expression of immune checkpoint receptors, including PD-1, CTLA-4, and TIM-3, and lower expression of TCF-1 than those of p53abn. CD8+TILs of p53abn showed significantly higher expression of CD226 than those of MMR-D. In particular, the frequency of antigen reactive CD39+CD103+CD8 T cells significantly lower in p53abn than in MMR-D. CD8 TILs of NSMP showed higher expression of TOX than those of MMR-D. In ex vivo T cell proliferation assay (n=49), 80.0% POLE-mut (4 of 5), 46.2% MMR-D (6 of 13), 35.7% p53abn (5 of 14) and 35.36% NSMP (6 of 17) showed enhanced proliferation of CD8 TILs upon anti-PD-1 treatment. In endometrial cancer, TILs showed distinct immune properties according to ProMisE classification. Further study is needed to develop optimal treatment targets for the p53abn and NSMP subtype, which is non-immunogenic and hardly reinvigorated by ICIs. Citation Format: Jung Chul Kim, Junsik Park, Yong Jae Lee, Sunghoon Kim, Sang Wun Kim, Jung-Yun Lee. Differential immune characteristics and anti-PD-1-induced reinvigoration capacity of tumor-infiltrating lymphocytes according to ProMisE classifier in endometrial cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4615.

Integrated clinicopathologic and molecular analysis of endometrial carcinoma: Prognostic impact of the new ESGO-ESTRO-ESP endometrial cancer risk classification and proposal of histopathologic algorithm for its implementation in clinical practice.

The European Society of Gynecologic Oncology/European Society of Radiation Therapy and Oncology/European Society of Pathology (ESGO/ESTRO/ESP) committee recently proposed a new risk stratification system for endometrial carcinoma (EC) patients that incorporates clinicopathologic and molecular features. The aim of the study is to compare the new ESGO/ESTRO/ESP risk classification system with the previous 2016 recommendations, evaluating the impact of molecular classification and defining a new algorithm for selecting cases for molecular analysis to assign the appropriate risk class. The cohort included 211 consecutive EC patients. Immunohistochemistry and next-generation sequencing were used to assign molecular subgroups of EC: POLE mutant (POLE), mismatch repair deficient (MMRd), p53 mutant (p53abn), and no specific molecular profile (NSMP). Immuno-molecular analysis was successful in all cases, identifying the four molecular subgroups: 7.6% POLE, 32.2% MMRd, 20.9% p53abn, and 39.3% NSMP. The recent 2020 guidelines showed a 32.7% risk group change compared with the previous 2016 classification system: the reassignment is due to POLE mutations, abnormal p53 expression, and a better definition of lymphovascular space invasion. The 2020 system assigns more patients to lower-risk groups (42.2%) than the 2016 recommendation (25.6%). Considering the 2020 risk classification system that includes the difference between "unknown molecular classification" and "known," the integration of molecular subgroups allowed 6.6% of patients to be recategorized into a different risk class. In addition, the use of the proposed algorithm based on histopathologic parameters would have resulted in a 62.6% reduction in molecular analysis, compared to applying molecular classification to all patients. Application of the new 2020 risk classification integrating clinicopathologic and molecular parameters provided more accurate identification of low-and high-risk patients, potentially allowing a more specific selection of patients for post-operative adjuvant therapy. The proposed histopathologic algorithm significantly decreases the number of tests needed and could be a promising tool for cost reduction without compromising prognostic stratification.

Relationship between molecular markers and lymphadenectomy and lymphovascular space invasion in endometrial cancer.

Relationship between pathologic parameters, surgical parameters, or lymph node status with oncologic outcomes is not fully elucidated in endometrial cancer (EC). We want to investigate the molecular classification of uterine cancer in the Turkish population and its relationship between lymphadenectomy and lymph node metastasis. In this study, 100 patients' clinical and pathologic data diagnosed with EC were analyzed. Pathologic and molecular parameters were investigated and compared them with clinical parameters. According to the molecular analysis, 16 patients (16%) had p53 mutation, 3 patients (3%) were classified as POLE mutant group, 38 (38%) patients in the MSI group, and the remaining 43 patients (43%) into the no specific mutation profile (NSMP) group. Lymph node metastasis rate was significantly higher in copy number high (CNH) group compared to the others. In the CNH group, 29 of 437 (6.6%) dissected lymph nodes had metastasis. The median OS was the highest in the POLE group (72months) and lowest in the CNH group (36months). Endometrial cancer patients showed significantly different overall and disease-free survival according to the molecular subtypes and it was consistent with the literature, Lymph node metastasis risk was the highest in CNH group. MSI status is important for the lymph node metastasis risk but not all abnormalities, especially PMS2 and MLH1 expression changes showed the highest risk.

Clinical characteristics and prognostic characterization of endometrial carcinoma: a comparative analysis of molecular typing protocols

BackgroundEndometrial carcinoma (EC) is one of the most common gynecological malignancies in China and globally, accounting for the fourth-prevalent cancer in women. Although numerous studies have confirmed prognostic value of The Cancer Genome Atlas (TCGA) molecular subgroups, it is unclear how they are combined with histological features. The main objective of this study was to compare ProMisE and TCGA classification for the rapid and accurate prediction of prognosis within EC patients, together with the provision of a revised strategy for individualized diagnosis and treatment of patients.MethodsWithin this study, 70 patients with EC from Beijing Tsinghua Changgeng Hospital (affiliated to Tsinghua University) were retrospectively examined between July 2015 and December 2021. Samples were processed for determination of clinical markers, together with ProMisE and TCGA classification.ResultsComparative analysis across four TCGA types (POLE, Low-CN, High-CN, and MSI-H) and age, was statistically significant (χ²= 7.000, p = 0.029). There was no significant difference observed among the four TCGA types and FIGO stage, vascular invasion and depth of invasion, or lymph node metastasis and tumor area. There was no significant association between the expression of Vimentin, Ki-67, PTEN, MSH2, PAX-8, β-catenin, CD10, ER, PR, P16, MLH1, and PMS2 with the four TCGA types. In addition, p63 expression (χ²= 11.09, p = 0.029) and p53 expression (χ²= 11.585, p = 0.005) were statistically significant. Numerous models demonstrated that patients with POLE mutations and low-CN had higher progression free survival (PFS) and overall survival (OS), whereas those with high-CN had lowest values. The log-rank test revealed that the survival rate of PR-positive and ER-positive patients was significantly higher (p < 0.001).ConclusionOverall, these results can be of additional benefit for clinical applications, in comparison to the ProMisE classification method. In addition, PR, ER, vascular infiltration, hyperlipidemia and atherosclerosis were found to be the key factors affecting EC prognosis.

Prognosis and Characterization of Microenvironment in Cervical Cancer Influenced by Fatty Acid Metabolism-Related Genes.

Increasing evidence suggests that diverse activation patterns of metabolic signalling pathways may lead to molecular diversity of cervical cancer (CC). But rare research focuses on the alternation of fatty acid metabolism (FAM) in CC. Therefore, we constructed and compared models based on the expression of FAM-related genes from the Cancer Genome Atlas by different machine learning algorithms. The most reliable model was built with 14 significant genes by LASSO-Cox regression, and the CC cohort was divided into low-/high-risk groups by the median of risk score. Then, a feasible nomogram was established and validated by C-index, calibration curve, net benefit, and decision curve analysis. Furthermore, the hub genes among differential expression genes were identified and the post-transcriptional and translational regulation networks were characterized. Moreover, the somatic mutation and copy number variation landscapes were depicted. Importantly, the specific mutation drivers and signatures of the FAM phenotypes were excavated. As a result, the high-risk samples were featured by activated de novo fatty acid synthesis, epithelial to mesenchymal transition, angiogenesis, and chronic inflammation response, which might be caused by mutations of oncogenic driver genes in RTK/RAS, PI3K, and NOTCH signalling pathways. Besides the hyperactivity of cytidine deaminase and deficiency of mismatch repair, the mutations of POLE might be partially responsible for the mutations in the high-risk group. Next, the antigenome including the neoantigen and cancer germline antigens was estimated. The decreasing expression of a series of cancer germline antigens was identified to be related to reduction of CD8 T cell infiltration in the high-risk group. Then, the comprehensive evaluation of connotations between the tumour microenvironment and FAM phenotypes demonstrated that the increasing risk score was related to the suppressive immune microenvironment. Finally, the prediction of therapy targets revealed that the patients with high risk might be sensitive to the RAF inhibitor AZ628. Our findings provide a novel insight for personalized treatment in CC.

Molecular genetic sequencing of neuroendocrine tumors of the gastrointestinal tract.

651 Background: The objective of this work was to evaluate the molecular and genetic features of the NET gastrointestinal tract. Genomic sequencing of 30 tumor samples of the latest generation was carried out and the data obtained were evaluated. Methods: In total, 30 tumor blocks of patients with gastrointestinal NET were tested by the new generation sequencing (NGS) method. The following genes were analyzed: ATM, ATR, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CDK12, CHEK1, CHEK2, EPCAM, FANCL, MLH1, MSH2, NBN, NF1, PALB2, PMS2, RAD51B, RAD51C, RAD51D, RAD54L, STK11, TP53, POLE, KRAS, NRAS, BRAF, EGFR, ERBB2, PIK3CA, MET ex14, BAT25, BAT26, NR21, NR24, MONO27, KIT, PDGFRA, Pi3Ca. Preparation of libraries for sequencing was carried out using NimblGen SepCapEZ Choice. Sequencing was performed on the Illumina MiSeq device. Results: In the study cohort of patients whose tumor blocks were sent for examination by the NGS method, there were more women – 19 (63.3%) than men – 11 (36.7%). When analyzing the age of patients, it should be noted that among all age groups, elderly patients predominated (60-74 years according to the WHO classification, 2016) – 12 (40%). In the majority of patients – 10 (33.3%) – the primary tumor focus was localized in the pancreas, in 6 (20%) patients it was primarily affected stomach, 7 (23.3%) – small intestine. Other parts of the gastrointestinal tract were affected much less frequently: the colon – in 1 (3.3%) patients. According to the division according to the degree of differentiation of gastrointestinal NET10 tumors had a degree of differentiation G1 (33.3%), 16 -G3 (54.3%), 4– G3 (13.3%). In the tumor sample of a patient with gastric NET G1 (Ki 67 = 2%), POLE mutations were found: c.2276G &gt; A, p.R759H and CHEK2: c.470T &gt; C, p.I157T. Also, mutations MLH1: c.199G &gt; A, p.G67R BRCA1: c.1881 were found in the tumor sample of another patient with gastric NET G2 (Ki 67 = 7%). Conclusions: To improve the survival rates of patients, it is worth paying special attention to the molecular genetic study of the NET gastrointestinal tract. It is molecular genetic profiling that can become the basis for building more individualized algorithms for treating patients. The results of this study allow us to count on the fact that when conducting a study on a larger cohort of patients with gastrointestinal NET, it is possible to obtain results that will expand our knowledge of this nosology and allow us to supplement the already available information about prognosis factors and predictors of response to treatment.

Sentinel Lymph Node Biopsy in Uterine Cancer: Time for a Modern Approach.

Since the validation of the sentinel node technique (SLN) for vulvar cancer 20 years ago, this technique has been introduced in the management of operable cervical cancer and endometrial cancer. For cervical cancer a "one fits all" attitude has mainly been presented. However, this approach, consisting of a frozen section during the operation, can be discussed in some stages. We present and discuss the main option for each stage, as well as some secondary possibilities. For endometrial cancer, SLN is now the technique of choice for the nodal staging of low- and intermediate-risk groups. Some discussion exists for the high-risk group. We also discuss the impacts of using preoperatively the molecular classification of endometrial cancer. Patients with POLE or TP53 mutations could have different nodal staging. The story of SLN in uterine cancers is not finished. We propose a comprehensive algorithm of SLN in early cervical and endometrial cancers. However, several ongoing trials will give us important data in the coming years. They could substantially change these propositions.

Gene mutation profiling and clinical significances in patients with renal cell carcinoma

ObjectivesThe pathological mechanisms of patients with Renal Cell Carcinoma (RCC) remain defined. This study aimed to evaluate relationships between the landscape of gene mutations and their clinical significance in RCC patients. MethodsTissue and peripheral blood samples of 42 patients with RCC were collected and performed for the Next Generation Sequencing (NGS) with Geneseeq PrimeTM 425-gene panel probes. Their landscapes of gene mutation were analyzed. We also carried out an evaluation of Tumor-Node-Metastasis (TNM) staging, RENAL nephelometry score, surgery, and targeted drug treatment of patients. Then we compared the correlations of landscape in gene mutations and the prognosis. ResultsThe most common gene alternations, including BAP1, PBRM1, SETD2, CSF1R, NPM1, EGFR, POLE, RB1, and VHL genes, were identified in tissue and blood samples of 75% of patients. EGFR, POLE, and RB1 gene mutations frequently occurred in relapsed and metastatic patients. BAP1, CCND2, KRAS, PTPN11, ERBB2/3, JAK2, and POLE were presented in the patients with > 9 RENAL nephelometry score. Univariable analysis indicated that SETD2, BAP1, and PBRM1 genes were key factors for Disease-Free Survival (DFS). Multivariable analysis confirmed that mutated SETD1, NPM1, and CSF1R were critical factors for the Progression Free Survival (PFS) of RCC patients with target therapy. ConclusionsWild-type PBRM1 and mutated BAP1 in patients with RCC were strongly associated with the outcomes of the patient. The PFS of the patients with SETD2, NPM1, and CSF1R mutations were significantly shorter than those patients without variants.

Expression and significance of immune checkpoint B7-homolog 4 in endometrial cancer

Objective: To investigate the expression of B7 homolog 4 (B7-H4) and its clinical significance in endometrial cancer. Methods: A total of 833 patients with endometrial cancer admitted to Peking Union Medical College Hospital, Chinese Academy of Medical Sciences from 2009 to 2019, were enrolled. The expression of B7-H4, mismatch repair (MMR), p53, programmed cell death ligand 1 (PD-L1) protein, and CD8+ T lymphocyte density in endometrial cancer tissues were detected by the EnVision two-step method of immunohistochemical staining. First-generation sequencing (Sanger method) was used to determine molecular subtyping of endometrial cancer. The χ2 test was used to compare the differences in positive expression rate of B7-H4 protein in endometrial cancer tissues with different clinicopathological features and molecular subtyping, PD-L1 protein expression, and CD8+ T lymphocyte density. Survival analyses [including recurrence-free survival (RFS) and disease-specific survival (DSS)] were performed for 664 patients with follow-up time≥3 months, with a median follow-up time of 31 months (range: 4-121 months), and the Cox proportional hazards regression model was used to analyze the relevant factors affecting the prognosis of patients with endometrial cancer. Results: (1) The median age of 833 patients was 58 years (range: 25-88 years); pathological type: 595 with endometrioid carcinoma, 238 with non-endometrioid carcinoma; surgical-pathological staging: 542 cases at stage Ⅰ, 38 cases at stage Ⅱ, 173 cases at stage Ⅲ, and 45 cases at stage Ⅳ. Molecular subtyping was performed in 590 patients, including 50 with POLE mutation, 163 with mismatch repair defect (MMR-d) type, 246 with nospecific molecular change (NSMP) type, and 131 with p53 mutation subtype. (2) B7-H4 protein was expressed with brownish-yellow stainind in the cell membrane and cytoplasm of endometrial carcinoma, and the positivity rate of B7-H4 protein was 71.5% (596/833). The positivity rates of B7-H4 protein among patients with different age, surgical-pathological stage, tumor grade, pathological type, depth of muscular invasion, presence or absence of lymphovascular space invasion, and molecular subtype were significantly different (all P<0.05). The positivity rates of B7-H4 protein among patients with different PD-L1 protein expression and CD8+ T lymphocyte density were not significantly different (P>0.05). The 5-year RFS (83.9%) and DSS (87.3%) of B7-H4 protein-positive patients had an increasing trend compared with the 5-year RFS (77.2%) and DSS (78.1%) of B7-H4 protein-negative patients, but there were not statistically significant differences (P=0.053, P=0.083). (3) Univariate analysis showed that the 5-year RFS and DSS of patients with different age, tumor grade, surgical-pathological stage, pathological type, depth of muscular invasion, lymphovascular space invasion, and molecular subtype were significantly different (all P<0.01). There were no significant differences in 5-year RFS (P=0.184, P=0.113) and DSS (P=0.549, P=0.247) among patients with different CD8+ T lymphocyte density and PD-L1 protein expression. Further analysis according to molecular subtype, the results of CD8+ T lymphocyte density and PD-L1 protein expression showed that the 5-year RFS and DSS of B7-H4 protein-positive patients were higher than those of B7-H4 protein-negative patients with NSMP subtype, low density of CD8+ T lymphocyte and PD-L1 protein-negative endometrial carcinoma (all P<0.05), however, there was no significant difference in 5-year DSS between B7-H4 protein-positive patients and B7-H4 protein-negative patients with PD-L1 protein-negative endometrial cancer (P=0.060). Multivariate analysis showed that positive expression of B7-H4 protein was an independent factor for 5-year RFS (HR=0.27, 95%CI: 0.09-0.78, P=0.016) and DSS (HR=0.16, 95%CI: 0.05-0.58, P=0.005) in patients with NSMP subtype endometrial carcinoma. In patients with low-density CD8+ T lymphocytes endometrial cancer, positive expression of B7-H4 protein was an independent factor for 5-year RFS (HR=0.45, 95%CI: 0.26-0.80, P=0.006), but it was not an independent factor for 5-year DSS. In patients with PD-L1 protein-negative endometrial cancer, B7-H4 protein was not an independent factor for 5-year RFS. Conclusion: B7-H4 protein expressed highly in endometrial carcinoma tissues, and its high expression is closely related to clinicopathological features, molecular subtype of p53 mutant and NSMP, and the favorable prognosis of patients with low density of CD8+ T lymphocyte immunophenotype endometrial carcinoma.

Integration of Molecular Data in the Prognostic Stratification and Management of Endometrial Carcinoma

In the last years, the TCGA-based molecular classifier have been progressively integrated in the management of endometrial carcinoma. While molecular assays are increasingly available across pathology laboratories, the additional costs will expectedly be compensated by a reduction in overtreatments and a prevention of recurrences. The additional time might be shortened by assessing molecular markers on biopsy specimens. Retrospective data suggest that the molecular classifier will have a major impact of on the risk stratification, with many patients having their risk class down- or upstaged based on POLE mutations or p53 abnormalities, respectively. However, there are still several issues to be resolved, such as the prognostic value of the TCGA classifier in each FIGO stage, or the type of adjuvant treatment most suitable for each molecular group. Other issues regard the prognostic stratification of the mismatch repair-deficient and "no specific molecular profile" groups, which currently follows the same criteria; however, the former seems to be prognostically consistent regardless of FIGO grade and histotype, whereas the latter appears highly heterogeneous. Numerous clinical, histological, immunohistochemical and molecular markers have been proposed to refine the TCGA classification, but their prognostic value is still undefined. Hopefully, prospective data collected in the next years will help resolving these issues.

Association between tumor mutations and meningioma recurrence in Grade I/II disease.

Meningiomas are common intracranial tumors with variable prognoses not entirely captured by commonly used classification schemes. We sought to determine the relationship between meningioma mutations and oncologic outcomes using a targeted next-generation sequencing panel. We identified 184 grade I and II meningiomas with both >90 days of post-surgical follow-up and linked targeted next-generation sequencing. For mutated genes in greater than 5% of the sample, we computed progression-free survival Cox-regression models stratified by gene. We then built a multi-gene model by including all gene predictors with a p-value of less than 0.20. Starting with that model, we performed backward selection to identify the most predictive factors. ATM (HR = 4.448; 95% CI: 1.517-13.046), CREBBP (HR = 2.727; 95% CI = 1.163-6.396), and POLE (HR = 0.544; HR = 0.311-0.952) were significantly associated with alterations in disease progression after adjusting for clinical and pathologic factors. In the multi-gene model, only POLE remained a significant predictor of recurrence after adjusting for the same clinical covariates. Backwards selection identified recurrence status, resection extent, and mutations in ATM (HR = 7.333; 95% CI = 2.318-23.195) and POLE (HR = 0.413; 95% CI = 0.229-0.743) as predictive of recurrence. Mutations in ATM and CREBBP were associated with accelerated meningioma recurrence, and mutations in POLE were protective of recurrence. Each mutation has potential implications for treatment. The effect of these mutations on oncologic outcomes and as potential targets for intervention warrants future study.

Abstract B018: Novel therapeutics for targeting the aberrant nuclear export machinery in colorectal cancer

Abstract XPO1 is a nuclear export receptor responsible for exporting many key proteins critical for cell survival from the nucleus to the cytoplasm. Recently, we identified a highly statistically significant hotspot mutation in XPO1 R749Q occurring in patients with colorectal cancer. Out of 96 patients identified to have XPO1 R749Q mutations, 27 (28%) were in colorectal cancer (CRC). 95% of XPO1 R749Q mutant CRC were tumor mutation burden-high (TMB-H; &amp;gt;10 mut/Mb) and 0 were mis-match repair deficient (dMMR)/micro satellite instability-high (MSI-H). Co-mutation analysis showed that 95% of XPO1 R749Q CRC were POLE mutated, which was significantly enriched over XPO1 WT tumors. To investigate whether XPO1 R749Q might play a role in CRC biology, we have generated two different isogenic colorectal cancer cell lines bearing the XPO1 R749Q mutation (HCT116 and LS174T) using CRISPR-CAS9 to better understand the role of this aberrant XPO1 mutation on nuclear export and tumorigenesis. Immunofluorescence (IF) and Stochastic Optical Reconstruction Microscopy (STORM) super-resolution experiments showed that R749Q mutant cells had significantly increased localization of XPO1 in the cytoplasm of the XPO1 R749Q mutant cells when compared to wildtype (XPO1 WT) cells, particularly at the edge of the nuclear pores of the XPO1 R749Q mutant cells suggesting increased nuclear export. Mass spectrometry analysis of nuclear and cytoplasmic fractionated proteins confirmed that XPO1 R749Q mutant cells had increased export of proteins from the nucleus compared to XPO1 WT cells. Structural modeling using published structures of XPO1 bound to Ran-GTP predicted that XPO1 R749Q mutation increased affinity of the regulatory H9-loop of XPO1 to Ran, thus favoring increased shuttling and retention in the cytoplasmic compartment. Therefore, we hypothesize that XPO1 R749Q may represent a novel mechanism of nuclear export alteration involved with kinetic tuning of global transport. Despite the increased nuclear export function, XPO1 R749Q cells did not show a proliferative advantage. We thus set out to test whether XPO1 R749Q might be enriched in cancer as an adaptive response to chemotherapy treatment. Using a chemical compound library of &amp;gt;200 FDA-approved cancer therapies we observed a strong therapeutic resistance of XPO1 R749Q cells relative to XPO1 WT cells, specifically to chemotherapies used in the treatment of colon cancer, such as irinotecan. However, these cells remained sensitive to treatment with the XPO1 inhibitor selinexor. Mice xenografted with XPO1 R749Q mutant HCT116 cells showed moderate tumor response to selinexor or irinotecan monotherapies but prolonged tumor responses to combination therapy. Recent clinical data has shown POLE mutations to be a biomarker for response to PD-1 immune checkpoint blockade, especially in CRC. Since our genetic data identify POLE mutations commonly co-occurring with XPO1 R749Q in colon cancer, we are planning to test the combination of PD-1 inhibitors and selinexor to target both these mutations, as well as CRC tumors without these mutations. Citation Format: Tulasigeri M. Totiger, Sana Chaudhry, Skye Montoya, Monika Chojnacka, Gabriel Gaidosh, Jumana Afaghani, Maurizio Affer, Jenna Zabroski, Jacob Jahn, Vindhya Nawaratne, Ryan Notti, Ramiro Verdun, Daniel Bilbao, Josean Rodriguez, Justin Taylor. Novel therapeutics for targeting the aberrant nuclear export machinery in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B018.

Endometrial serous carcinoma with extensive squamous differentiation mimicking primary endometrial squamous cell carcinoma: Clinicopathological and molecular analysis of a case with literature review

In the histological assessment of endometrial carcinoma, the presence of squamous differentiation is generally considered inconsistent with a serous histotype. Herein, we report a case of endometrial serous carcinoma with extensive squamous differentiation mimicking squamous cell carcinoma, with literature review.A 67-years-old-woman underwent hysterectomy, bilateral salpingo-oophorectomy, sentinel lymph node resection, omental and peritoneal biopsies, due to a 8cm endometrial mass. Histologically, the tumor showed squamous carcinoma features with positivity for squamous cell markers (p40, p63, cytokeratin-5/6, cytokeratin-34βE12) and negativity for Müllerian markers (PAX8, estrogen receptor, progesterone receptor). No immunohistochemical/molecular alterations typical ofendometrioid carcinoma (i.e., PTEN loss, mismatch repair deficiency, nuclear β-catenin expression,POLEmutation) were observed. P53 showed aberrant pattern and p16 showed basal positivity. The tumor infiltrated the myometrium full-thickness with extensive lymphovascular space invasion. Surprisingly, the sentinel lymph node metastasis showed overt serous carcinoma features. Additional sampling of the endometrial mass revealed a minor. In conclusion, serous carcinoma may show diffuse squamous differentiation. In the case of suspected primary endometrial squamous cell carcinoma, an extensive sampling is warranted.

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

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