Somatic POLE Research Articles

42MO Interpreting somatic POLE mutations in endometrial cancer emerging from comprehensive genomic profiling

42MO Interpreting somatic POLE mutations in endometrial cancer emerging from comprehensive genomic profiling

Somatic POLE Mutation and Ultra-Hypermutated Genotype in a De Novo High-Grade, Isocitrate Dehydrogenase Wild-Type Glioma: Treatment Implications.

Pembrolizumab leads to a durable response in ultra-hypermutated, high-grade, glioma.

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.

A novel somatic mutation in POLE exonuclease domain associated with ultra-mutational signature and MMR deficiency in endometrial cancer: a case report

BackgroundDefect in proofreading exonuclease activity of polymerases epsilon and delta (Pols ε and δ) leads to mutagenesis and genomic instability and has been described in several cancer types. Somatic POLE exonuclease domain mutations (EDMs) have been reported in 7–12% endometrial cancers (ECs) and defined a subgroup of endometrial cancers with ultrahigh somatic mutation frequencies, high tumor infiltrated lymphocytes and favorable outcomes.Case presentationHerein, we presented a novel somatic mutation in POLE exonuclease domain associated with ultra-mutational signature and MMR deficiency in endometrial cancer. A novel POLE EDM (p.T278K) was found by a 11-gene NGS panel. The MSS status detected by the MSI test was inconsistent with the dMMR status by IHC. The loss of MSH6 expression in the tumor could be interpreted by the two nonsense mutations (p.E1234* and p.E1322*) of the MSH6 gene which may lead to truncated proteins. The T278K mutation was pathogenic identified by a 602-gene NGS panel with 27.3% of C > A substitution, 0.6% of indels, 0.6% of C > G substitution and a high TMB of 203.8 mut/Mb.ConclusionsWe report an endometrial cancer patient harbored a novel somatic POLE T278K mutation. This mutation was a novel pathogenic POLE EDM should be considered as “POLE (ultramutated)” in clinical practice for the molecular classification of EC.

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

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

HGG-41. Glioma oncogenesis in the constitutional mismatch repair deficiency (CMMRD) syndrome

Abstract PURPOSE: Constitutional Mismatch Repair Deficiency (CMMRD) is a cancer predisposition due to bi-allelic mutations in one of the four main mismatch repair (MMR) genes (PMS2, MSH2, MSH6 or MLH1) associated with early onset of cancers, especially glioblastomas (GBM). Our aim was to decipher the molecular specificities of gliomas occurring in this context. METHODS: A comprehensive analysis of clinical, histopathological and genomic data (whole exome sequencing) was performed for 12 children with a CMMRD for which we had available frozen brain tumor material (10 GBM and 2 anaplastic astrocytomas). RESULTS: Eight patients harbored an ultra-mutated phenotype with more than 100 somatic non synonymous (NS) SNV/Mb. No correlation was observed between the number of mutation and sex, age, overall survival or mutated MMR gene. POLE and POLD1 exonuclease domain driver somatic mutations were described for eight and one patients respectively. The 4/12 tumors without POLE somatic mutation did not show the classical ultra-hypermutation pattern. All patients with POLE mutation had already more than 20 NS SNV/Mb (median 40, [range 23-114]) suggesting that the hypermutation phenomenon started before the appearance of the somatic POLE mutation. The mutational signatures of the tumors, dominated by the MMR signatures, were not modified after the onset of the POLE mutation when analyzing the different mutation bursts. Specific recurrent somatic mutations were observed in SETD2 (9/12), TP53 (9/12), NF1 (9/12), EPHB2 (8/12), and DICER1 (7/12). Only half of the tumors overexpressed PDL1 by immunohistochemistry and this overexpression was not associated with a higher tumor mutation burden. CONCLUSION: CMMRD-associated gliomas have a specific oncogenesis that does not trigger usual pathways and mutations seen in sporadic pediatric or adult GBM. Frequent alterations in other pathways (e.g. MAPK or DNA-PK pathway) may suggests the use of other targeted therapies aside from PD1 inhibitors.

EPCO-03. GLIOMA ONCOGENESIS IN THE CONSTITUTIONAL MISMATCH REPAIR DEFICIENCY (CMMRD) SYNDROME

Abstract PURPOSE Constitutional Mismatch Repair Deficiency (CMMRD) is a cancer predisposition due to bi-allelic mutations in one of the four main mismatch repair (MMR) genes (PMS2, MSH2, MSH6 or MLH1) associated with early onset of cancers, especially glioblastomas (GBM). Our aim was to decipher the molecular specificities of gliomas occurring in this context. METHODS A comprehensive analysis of clinical, histopathological and genomic data (whole exome sequencing) was performed for 12 children with a CMMRD for which we had available frozen brain tumor material (10 GBM and 2 anaplastic astrocytomas). RESULTS Eight patients harbored an ultra-mutated phenotype with more than 100 somatic non synonymous (NS) SNV/Mb. No correlation was observed between the number of mutation and sex, age, overall survival or mutated MMR gene. POLE and POLD1 exonuclease domain driver somatic mutations were described for eight and one patients respectively. The 4/12 tumors without POLE somatic mutation did not show the classical ultra-hypermutation pattern. All patients with POLE mutation had already more than 20 NS SNV/Mb (median 40NS SNV/Mb, [range 23-114]) suggesting that the hypermutation phenomenon started before the appearance of the somatic POLE mutation. The mutational signatures of the tumors, dominated by the MMR signatures, were not modified after the onset of the POLE mutation when analyzing the different mutation bursts. Specific recurrent somatic mutations were observed in SETD2 (9/12), TP53 (9/12), NF1 (9/12), EPHB2 (8/12), and DICER1 (7/12). Only half of the tumors overexpressed PDL1 by immunohistochemistry and this overexpression was not associated with a higher tumor mutation burden. CONCLUSION CMMRD-associated gliomas have a specific oncogenesis that does not trigger usual pathways and mutations seen in sporadic pediatric or adult GBM. Frequent alterations in other pathways (e.g. MAPK or DNA-PK pathway) may suggests the use of other targeted therapies aside from PD1 inhibitors.

Clinical and epigenetic features of colorectal cancer patients with somatic POLE proofreading mutations

BackgroundMutations in the POLE gene result in an ultra-hypermutated phenotype in colorectal cancer (CRC); however, the molecular characterisation of epigenetic alterations remains unclear. We examined the genetic and epigenetic profiles of POLE-mutant CRC to elucidate the clinicopathological features of the associated genetic and epigenetic alterations.ResultsTumour tissues (1,013) obtained from a cohort of patients with CRC were analysed to determine associations between the proofreading domain mutations of POLE with various clinicopathological variables, microsatellite instability (MSI) status, BRAF and KRAS mutations, and the methylation status of key regions of MLH1, MGMT, and SFRP2 promoters by calculating the methylation scores (range 0–6). Only four cases (0.4%) exhibited pathogenic POLE hotspot mutations (two p.P286R [c.857C > G], one p.V411L [c.1231G > C], and p.S459F [c.1376C > T] each), which were mutually exclusive to BRAF and KRAS mutations and MSI. CRC patients were divided into four subgroups: patients with POLE mutations (POLE, 0.4%, n = 4), patients with both MSI and extensive methylation in MLH1 (MSI-M, 2.9%, n = 29), patients with MSI but no extensive methylation in MLH1 (MSI-U, 3.6%, n = 36), and patients without MSI (non-MSI, 93.2%, n = 944). The POLE group was younger at diagnosis (median 52 years, P < 0.0001), with frequent right-sided tumour localisation (frequency of tumours located in the right colon was 100%, 93.1%, 36.1%, and 29.9% in POLE, MSI-M, MSI-U, and non-MSI, respectively; P < 0.0001), and was diagnosed at an earlier stage (frequency of stages I–II was 100%, 72.4%, 77.8%, and 46.6% in POLE, MSI-M, MSI-U, and non-MSI, respectively, P < 0.0001). The mean methylation score in POLE was not different from that in MSI-U and non-MSI, but the methylation signature was distinct from that of the other subgroups. Additionally, although the examined number of POLE-mutant tumours was small, the number of CD8-positive cells increased in tumours with partial methylation in the MLH1 gene.ConclusionsCRC patients with POLE proofreading mutations are rare. Such mutations are observed in younger individuals, and tumours are primarily located in the right colon. Diagnosis occurs at an earlier stage, and distinct epigenetic alterations may be associated with CD8 cell infiltration.

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

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

Ultra-mutated colorectal cancer patients with POLE driver mutations exhibit distinct clinical patterns.

POLE mutations, which lead to an ultramutated phenotype in colorectal cancer (CRC), have been reported as a promising marker in immunotherapy. We performed sequencing of CRC cases in Zhejiang University (ZJU) and extracted obtainable data from recently published results, including The Cancer Genome Atlas (TCGA), Japanese studies and clinical trials, to present clinical patterns of POLE driver‐mutated CRC and reveal its heterogeneity. The rate of somatic POLE driver mutations has been reported as 2.60% (ZJU cohort), 1.50% (TCGA cohort), 1.00% (Japan cohort), and 1.00% (Lancet cohort). POLE driver mutations show a clearly increased mutation burden (mean TMB: 217.98 mut/Mb in ZJU; 203.13 mut/Mb in TCGA). Based on pooled data, more than 70.00% of patients with POLE driver mutations were diagnosed before they were 55 years old and at an early disease stage (Stage 0–II >70.00%), and more than 70.00% were male. Among Asian patients, 68.40% developed POLE driver mutations in the left‐side colon, whereas 64.00% of non‐Asian patients developed them in the right‐side colon (p < 0.01). The top three amino acid changes due to POLE driver mutations are P286R, V411L, and S459F. Investigators and physicians should ascertain the heterogeneity and clinical patterns of POLE driver mutations to be better equipped to design clinical trials and analyze the data.

Germline RAD54L with somatic POLE defect implicated in Hypermutation phenotype: case report

BackgroundColorectal cancer is one of the most frequent causes of death among cancer patients. Hypermutated CRC is an extraordinary case of cancer, but curable if detected at early stages. However, the mechanism for developing a hypermutated CRC remains unclear. An association between RAD54L germline mutation and POLE exonuclease domain hypermutated cancer has not been reported before.Case presentationWe present a rare case of a 41-year-old Chinese female with a right-sided colon adenocarcinoma who harboured a (p.P286R) POLE somatic mutation. Genomic analysis was performed using the Illumina HiSeq Sequencing platform, which, identified MSS tumour with a (c.1093_1169 + 15dup) germline mutation in RAD54L gene and tumour mutation burden of 377.0 Muts/Mb. Based on our report a new mechanism for developing hypermutated colon cancer has been conjectured through a novel RAD54L_POLE DSBR pathway.ConclusionThis report highlights the clinical importance of next-generation sequencing technology in diagnosing rare tumours and investigating novel mechanisms for developing exceptional genetic diseases.

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

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

High mutational burden in colorectal carcinomas with monoallelic POLE mutations: absence of allelic loss and gene promoter methylation.

Hypermutator-type colorectal carcinomas are microsatellite-stable and have point mutations of the exonuclease domain of the DNA polymerase ε or δ genes (POLE and POLD1, respectively), and an ultrahigh tumor mutational burden (TMB). These tumors may be associated with enhanced antitumor immunity and preferentially afflict younger patients, but this notion awaits validation by accrual of further cases for detailed correlative phenotypic and molecular study. We performed POLE and POLD1 exonuclease domain Sanger sequencing of 271 unselected colorectal carcinomas. We identified two microsatellite-stable tumors with somatic POLE p.P286R variants, both with ultrahigh TMBs as demonstrated by whole exome sequencing. A POLE p.V411L was found in another two microsatellite-stable tumors with ultrahigh TMBs. Two of these four tumors were from young patients (<50 years old, nonsyndromic), and there was seen a prominent T-cell infiltration in three of them. Furthermore, a somatic POLE p.A465T was found in a Lynch-associated tumor, which, hypothetically, might have enhanced TMB (which was the highest of all). In two tumors, a somatic POLE p.V411L and a POLD1 p.E279K, respectively, were found only focally, and TMBs were low. It is commonly assumed that compromise of one allele is sufficient, but this has not been specifically addressed. Therefore, resequencing of the POLE or POLD1 mutations was done with DNA from tumor cells isolated by laser-capture microdissection. This demonstrated that the mutations were monoallelic, and there was no evidence of a "second hit", neither by allelic loss (allelotyping with polymorphic microsatellite markers), nor by promoter methylation (Pyromark CpG assays). Taken together, including at least the more common DNA polymerase mutations in NGS panels allows for straightforward identification of hypermutator-type colorectal carcinomas which often may be "immunoreactive". This is important at least in young patients or when a metastasizing stage of disease has been reached and immune-checkpoint therapy enters deliberation.

POLE mutations improve the prognosis of endometrial cancer via regulating cellular metabolism through AMF/AMFR signal transduction

BackgroundThe morbidity and mortality of endometrial tumors, a common type of malignant cancer in women, have increased in recent years. POLE encodes the DNA polymerase ε, which is responsible for the leading strand DNA replication. Somatic mutations of POLE have been acknowledged in numerous cancers, resulting in the accumulation of DNA errors, leading to ultra-mutated tumors. Mutations in the exonuclease domain of POLE have been reported to improve progression-free survival in endometrial cancer. However, the potential relationship and underlying mechanism between POLE mutations and the prognosis of endometrial cancer patients remains unclear.MethodsThe whole exome sequencing data, RNA sequencing data, and clinical information were obtained from the TCGA database and employed for the analyses in this study. The detailed mutational information was analyzed using whole exome sequencing data and the mutated genes were shown with OncoPlot. The survival curves and cox proportional hazards regression analysis were used to accessed patient prognosis, the association of clinical characteristics and prognosis. Differentially expressed genes were analyzed by the edgeR R/Bioconductor package, then the GSEA Pre-ranked tool was used for Gene Set Enrichment Analysis (GSEA) to estimate the function of genes. Expression values were clustered using hierarchical clustering with Euclidean distance and ward linkage by the dendextend R package.ResultsPOLE mutational status was proven to be an independent prognostic factor for endometrial cancer patients. Patients with somatic POLE mutations presented a favorable prognosis. POLE mutations regulated glycolysis and cytokine secretion, affecting cell metabolism and immune response. Autocrine motility factor (AMF)/PGI and AMFR/gp78 exhibited higher expression levels in POLE mutant patients. The comprehensive high expressions of AMFR/gp78 and low expression of POLE were associated with the favorable prognosis of endometrial cancer patients.ConclusionsThis study showed the POLE mutations a vital factor in endometrial cancer patients, leading to a higher expression of AMF/PGI and AMFR/gp78. These results suggested comprehensive consideration of the POLE mutations, expression of AMF/PGI and AMFR/gp78 may provide a more feasible and effective approach for the treatment of endometrial cancer, which might improve the prognosis.

Clinical outcomes of patients with POLE mutated endometrioid endometrial cancer

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

Somatic POLE proofreading domain mutations by next generation sequencing to predict outcomes in stage II colorectal cancer.

559 Background: Stage II colorectal cancers (CRC) exhibit unique molecular heterogeneity and patients with somatic POLE mutations are defined as a distinct tumor subgroup. The aim of this study was to clarify the characteristics and prognostic effect of somatic POLE proofreading domain mutations by Next Generation Sequencing in stage II CRC. Methods: Recurrent patients were 1:1 matched to patient with no recurrence for stage II CRC patients diagnosed between 2008 and 2013 in Fudan University Shanghai Cancer Center (FUSCC). All patients were pathologically confirmed and received radical resection. Microsatellite instability status and POLE mutation status were determined by NGS using ColonCore panel (Burning Rock, Guangzhou, China). Groups based on NGS-POLE mutation status were compared in terms of patient demographics and pathologic features using chi-squared tests. Survival curves were plotted using the Kaplan-Meier method. Results: In total, 245 stage II CRC patients were enrolled. POLE mutations were detected in 25 (10.2%) of 245 stage II CRC, while only 9 (3.7%) was located in proofreading domain (NGS-POLE EDM-mutations) and other 16 were found in non-proofreading domain (non-NGS-POLE mutations). Compared to cases with wild-type POLE, cases with NGS-POLE EDM-mutations were more prone to be microsatellite instability-high (MSI-H) (3 of 9 [33.3%] vs. 23 of 236 [9.7%], p = 0.024), younger at diagnosis (median 46 years vs. 62 years, p &lt; 0.001), and more frequently with right-sided tumor location (6 of 9 [66.7%] vs. 54 of 236 [22.9%]; p = 0.003). All tumors with NGS-POLE EDM-mutations presented with a surprisingly high tumor mutation burden (TMB) (median 145.2 per Mb). Patients with NGS-POLE EDM-mutations have favorable 5-year disease-free survival (DFS) compared with those with non-NGS-POLE EDM-mutations and wild-type POLE (100% vs. 62.5% vs. 52.5%, p = 0.037). Though there is DFS difference between patients with non-NGS-POLE EDM-mutations and wild-type POLE, the difference was not statistically significant (p = 0.412). Conclusions: Stage II CRC patients with NGS-POLE EDM-mutation identify a special cancer subset with better immune environment predicting excellent outcomes.

Germline TP53 and MSH6 mutations implicated in sporadic triple-negative breast cancer (TNBC): a preliminary study

BackgroundGermline BRCA1/2 prevalence is relatively low in sporadic triple-negative breast cancer (TNBC). We hypothesized that non-BRCA genes may also have significant germline contribution to Chinese sporadic TNBC, and the somatic mutational landscape of TNBC may vary between ethnic groups. We therefore conducted this study to investigate germline and somatic mutations in 43 cancer susceptibility genes in Chinese sporadic TNBC.Patients and methodsSixty-six Chinese sporadic TNBC patients were enrolled in this study. Germline and tumor DNA of each patient were subjected to capture-based next-generation sequencing using a 43-gene panel. Standard bioinformatic analysis and variant classification were performed to identify deleterious/likely deleterious germline mutations and somatic mutations. Mutational analysis was conducted to identify significantly mutated genes.ResultsDeleterious/likely deleterious germline mutations were identified in 27 (27/66, 40.9%) patients. Among the 27 patients, 9 (9/66, 13.6%) were TP53 carriers, 5 (5/66, 7.6%) were MSH6 carriers, and 5 (5/66, 7.6%) were BRCA1 carriers. Somatic mutations were identified in 64 (64/66, 97.0%) patients. TP53 somatic mutations occurred in most of the patients (45/66, 68.2%) and with highest mean allele frequency (28.1%), while NF1 and POLE were detected to have the highest mutation counts.ConclusionsOur results supported our hypotheses and suggested great potentials of TP53 and MSH6 as novel candidates for TNBC predisposition genes. The high frequency of somatic NF1 and POLE mutations in this study showed possibilities for clinical benefits from androgen-blockade therapies and immunotherapies in Chinese TNBC patients. Our study indicated necessity of multi-gene testing for TNBC prevention and treatment.

Abstract 1426: Targeted sequencing of colorectal cancer in search for prognostic biomarkers

Abstract Introduction Extensive genetic studies of recent years using next generation sequencing have clarified a comprehensive registry of genetic alteration in colorectal cancer (CRC). However, the clinical impacts of these genetic alterations have not fully been investigated, except for a better response to immune checkpoint blockade and favorable prognosis in patients with microsatellite instability (MSI) cancer. In this study, we investigated the effects of common genetic alterations on survival in CRC patients using large-scale mutation profiling. Methods We performed targeted-capture sequencing to common driver genes (n=128) in CRC in a total of 544 patients with stage I (n = 36), II (n = 236) and III (n = 272) CRC. All patients were treated at a single institute between 2004 and 2016 and clinically well-annotated. Genome-wide copy numbers, fusion genes and MSI were also interrogated using baits for 1,605 SNPs, 7 introns and 18 microsatellites, respectively. RT-PCR was performed for the detection of PTPRK-RSPO3 fusion gene. Results The median age at diagnosis was 65 (32-95) with a median observation period of 1250 days. Of the 156 patients with recurrence, 15, 61 and 80 patients had stage I, II and III diseases, respectively. APC, TP53, KRAS, and PIK3CA represented the most frequently mutated, while common focal copy number alterations involved PTEN, SMAD4, SMAD2, FGFR1 and IGF2. Novel fusion genes including PTPRK-RSPO3 were detected in only 7 (1.5%) samples. Fifty-five patients had more than 20 mutations, comprising a group of hypermutated CRC. Among these, 44 were thought to have MSI based on the microsatellite analysis, while the remaining 11 were explained by POLE mutations affecting the proof reading domain. CRCs with POLE mutations were diagnosed earlier in life, with the median age of 49 (35-66). The frequency of CRCs with MSI was significantly lower than previous reports from the western population. In contrast to mutation frequency, copy number alterations were infrequent, which most hypermutated tumors showed Genomic Instability Index (GII) below 0.2. FBXW7 mutation positively affected tumor free survival. BRAF mutation, SMAD4 mutation, or existence of fusion genes did not have any effect on prognosis. In accordance with previous reports, only 4 patients (9.1%) with MSI had recurrence, confirming a better prognosis of MSI-positive patients. Similarly, only one of the 11 patients (9.1%) with POLE mutation had recurrence. The low relapse rate was also observed among an additional cohort of 819 patients, which included 6 POLE mutated cases, none of whom had recurrence, suggesting that POLE mutation was associated with a favorable prognosis. Conclusions We revealed the landscape of driver mutations in patients with stage I, II and III CRC with their implication in clinical outcomes. In addition to FBXW7 mutations, the MSI status and somatic POLE mutations are significantly associated with a better prognosis. Citation Format: Yoshikage Inoue, Nobuyuki Kakiuchi, Kenichi Yoshida, Yusuke Shiozawa, Kenichi Chiba, Tetsuichi Yoshizato, Yuichi Shiraishi, Satoru Miyano, Satoshi Nagayama, Yoshiharu Sakai, Seishi Ogawa. Targeted sequencing of colorectal cancer in search for prognostic biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1426.

Somatic POLE exonuclease domain mutations are early events in sporadic endometrial and colorectal carcinogenesis, determining driver mutational landscape, clonal neoantigen burden and immune response.

Genomic instability, which is a hallmark of cancer, is generally thought to occur in the middle to late stages of tumourigenesis, following the acquisition of permissive molecular aberrations such as TP53 mutation or whole genome doubling. Tumours with somatic POLE exonuclease domain mutations are notable for their extreme genomic instability (their mutation burden is among the highest in human cancer), distinct mutational signature, lymphocytic infiltrate, and excellent prognosis. To what extent these characteristics are determined by the timing of POLE mutations in oncogenesis is unknown. Here, we have shown that pathogenic POLE mutations are detectable in non‐malignant precursors of endometrial and colorectal cancer. Using genome and exome sequencing, we found that multiple driver mutations in POLE‐mutant cancers show the characteristic POLE mutational signature, including those in genes conventionally regarded as initiators of tumourigenesis. In POLE‐mutant cancers, the proportion of monoclonal predicted neoantigens was similar to that in other cancers, but the absolute number was much greater. We also found that the prominent CD8+ T‐cell infiltrate present in POLE‐mutant cancers was evident in their precursor lesions. Collectively, these data indicate that somatic POLE mutations are early, quite possibly initiating, events in the endometrial and colorectal cancers in which they occur. The resulting early onset of genomic instability may account for the striking immune response and excellent prognosis of these tumours, as well as their early presentation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Abstract 1756: Genomic reclassification of endometrial carcinoma predicts drug response and facilitates refinement of current management based on PDX-guided efficacy outcomes

Abstract Endometrial cancer (EC) is the most common gynecological malignancy among American women. Steadily increasing numbers of newly diagnosed cases and deaths emphasize the necessity of improving current management strategies. Rather than the commonly known two types of EC based on histological classification, the Cancer Genome Atlas (TCGA) Research Network recently reported a reclassification that categorizes EC into four subtypes based on genomic characterization. Molecular similarities between these subtypes and other cancers, including colorectal, ovarian, and breast cancers, were identified. But to date, no prospective validation has delivered an improved understanding on how to translate these findings to the clinic in the form of treatments adapting genomic guidance. Objectives: To evaluate drug response according to TCGA classification using a panel of twenty-seven established endometrial cancer patient-derived xenografts (EC-PDXs). Methods: Twenty-seven EC-PDXs representing various stages of disease and histological subtypes were orthotopically transplanted and propagated over multiple generations. Upon thorough characterization including histology, metastatic status, and molecular features, EC-PDXs were further categorized into the four TCGA molecular subtypes on the basis of somatic copy number variations (CNV), microsatellite instability (MSI), and POLE mutations. A PDX clinical trial (PCT) was performed to re-evaluate current standard treatments in comparison to therapeutic agents adopted from treatment regimens for other cancer types based on their molecular similarities. Results: EC-PDXs established from patients with recurrent disease exhibited minimal activity to first-line chemotherapeutic drugs, as expected, but remained sensitive to other second-line drugs. None of the tested drugs exhibited sufficient activity in POLE EC-PDXs. MSI EC-PDXs were found to be less sensitive to platinum-based treatment and resistant to 5-FU as reported for MSI colorectal cancer. CNV-high EC-PDXs were more sensitive to drugs adopted from current ovarian and breast cancer treatment regimens. Conclusions: This is the first study that demonstrates the utility of TCGA classification for treatment guidance based on a PCT. Patients with recurrent and persistent disease are in desperate need of new therapeutic options. Our findings open up the possibility to utilize drugs which are currently used in the treatment of other cancers for the treatment of EC based on molecular similarities. In addition, this is the first study that successfully demonstrated the use of molecular classification to enable the prediction of drug response, and thus may facilitate the refinement of current EC management in a more precise and personalized way. Citation Format: Chieh-Hsiang Yang, David K. Gaffney, Theresa L. Werner, Elke A. Jarboe, Jason Gertz, Katherine E. Varley, Matthew Peterson, Margit M. Janat-Amsbury. Genomic reclassification of endometrial carcinoma predicts drug response and facilitates refinement of current management based on PDX-guided efficacy outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1756. doi:10.1158/1538-7445.AM2017-1756