POLE Mutations Research Articles

Practical lessons learned from real-world implementation of the molecular classification for endometrial carcinoma

ObjectivesThis study aimed to explore the practical organisational aspects and difficulties in the implementation of the molecular classification of endometrial carcinoma (EC), and to demonstrate its potential impact in prognostic risk group classification. MethodsWe conducted a multicentre, retrospective cohort study of 230 patients with EC diagnosed between 2019 and 2022. Sample processing, clinicopathological, treatment and follow-up data were collected. Molecular classification was obtained by p53 and mismatch repair proteins immunohistochemistry, and POLE next-generation sequencing. ResultsImplementation was achieved through centralization of molecular analysis. In practice, it was possible to optimise turnaround times of complete integrative reports for hysterectomy specimens to a median time of 18 workdays. If genetic study was started in endometrial biopsies before surgery, 82.0% were available at the time of multidisciplinary tumour board, compared to 8.4% if performed in hysterectomy. ECs were classified as follows: 37.8% no specific molecular profile, 31.7% p53 abnormal, 24.3% mismatch repair deficient, and 6.1% POLE mutant. Integration of these results with traditional clinicopathologic factors led to a change in prognostic risk group in 15 (6.5%) patients, most being initially allocated to high-intermediate (n = 8) and low (n = 5) risk groups. Eight patients changed to a higher risk, and 7 to a lower risk group, whereas 2 remained in the same group. ConclusionsCentralization of EC molecular classification is a feasible option for countries with limited resources. Optimization of workflows may be achieved by earlier analysis in biopsies and prioritisation of patients whose results imply changes in risk group classification.

High-grade Serous Carcinoma can Show Squamoid Morphology Mimicking True Squamous Differentiation.

Tubo-ovarian high-grade serous carcinoma (HG-SC) and ovarian endometrioid carcinoma (EC) can show overlapping morphologic features, such as glandular and solid patterns. The differential diagnosis of these subtypes is thus sometimes difficult. The existence of "squamous differentiation" tends to lead to a diagnosis of EC rather than HG-SC. We noticed that HG-SC can contain a "squamoid component," but its nature has been poorly investigated. This study was thus established to clarify the nature of this "squamoid component" in HG-SC by investigating its frequency and immunohistochemical features. We reviewed hematoxylin and eosin-stained slides of 237 primary untreated cases of tubo-ovarian HG-SC and identified 16 cases (6.7%) of HG-SC with "squamoid component." An immunohistochemical staining panel (CK5/6, CK14, CK903, p40, p63, WT1, ER, and PgR) was used to analyze all of these 16 cases. We also selected 14 cases of ovarian EC with "squamous differentiation" as a control. The "squamoid component" in HG-SC was completely p40-negative and showed significantly lower expression of CK5/6, CK14, CK903, and p63 than the "squamous differentiation" in EC. The immunophenotype of the "squamoid component" in HG-SC was concordant with the conventional HG-SC component (WT1-positive/ER-positive). Furthermore, all 16 tumors were confirmed to be truly "HG-SC" by the findings of aberrant p53 staining pattern and/or WT1/p16 positivity, and the lack of mismatch repair deficiency and POLE mutation. In conclusion, HG-SC can on rare occasions show a "squamoid component" mimicking "squamous differentiation." However, the "squamoid component" in HG-SC does not represent true "squamous differentiation." The "squamoid component" is one part of the morphologic spectrum of HG-SC, which should be interpreted carefully for the differential diagnosis of HG-SC and EC. An immunohistochemical panel including p40, p53, p16, and WT1 is a useful adjunct to achieve a correct diagnosis.

Germline de novo mutations in families with Mendelian cancer syndromes caused by defects in DNA repair

DNA repair defects underlie many cancer syndromes. We tested whether de novo germline mutations (DNMs) are increased in families with germline defects in polymerase proofreading or base excision repair. A parent with a single germline POLE or POLD1 mutation, or biallelic MUTYH mutations, had 3-4 fold increased DNMs over sex-matched controls. POLE had the largest effect. The DNMs carried mutational signatures of the appropriate DNA repair deficiency. No DNM increase occurred in offspring of MUTYH heterozygous parents. Parental DNA repair defects caused about 20–150 DNMs per child, additional to the ~60 found in controls, but almost all extra DNMs occurred in non-coding regions. No increase in post-zygotic mutations was detected, excepting a child with bi-allelic MUTYH mutations who was excluded from the main analysis; she had received chemotherapy and may have undergone oligoclonal haematopoiesis. Inherited DNA repair defects associated with base pair-level mutations increase DNMs, but phenotypic consequences appear unlikely.

A cost-effective, machine learning-based new unified risk-classification score (NU-CATS) for patients with endometrial cancer

IntroductionTreatment for endometrial cancer (EC) is increasingly guided by molecular risk classifications. Here, we aimed at using machine learning (ML) to incorporate clinical and molecular risk factors to optimize risk assessment. MethodsThe Cancer Genome Atlas-Uterine Corpus Endometrial Carcinoma (n = 596), Memorial Sloan Kettering-Metastatic Events and Tropisms (n = 1315) and the American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange (n = 4561) datasets were used to identify genetic alterations and clinicopathological features. Software packages including Keras, Pytorch, and Scikit Learn were tested to build artificial neural networks (ANNs) with a binary output as either intra-abdominal metastatic progression (‘1’) vs. non-metastatic (‘0’). ResultsBlack patients with EC have worse prognosis than White patients, adjusting for TP53 or POLE mutation status. Over 75% of Black patients carry TP53 mutations as compared to approximately 40% of White patients. Older age is associated with an increasing likelihood of TP53 mutation, high risk histology, and distant metastasis. For patients above age 70, 91% of Black and 60% of White EC patients carry TP53 mutations. A ML-based New Unified classifiCATion Score (NU-CATS) that incorporates age, race, histology, mismatch repair status, and TP53 mutation status showed 75% accuracy in prognosticating intra-abdominal progression. A higher NU-CATS is associated with an increasing risk of having positive pelvic or para-aortic lymph nodes and distant metastasis. NU-CATS was shown to outperform Leiden/TransPORTEC model for estimating risk of FIGO Stage I/II disease progression and survival in Black EC patients. ConclusionThe NU-CATS, a ML-based, cost-effective algorithm, incorporates diverse clinicopathologic and molecular variables of EC and yields superior prognostication of the risk of nodal involvement, distant metastasis, disease progression, and overall survival.

Harmonized molecular classification; assessment of a single-test ProMisE NGS tool

ObjectivesDespite recommendations for integrating molecular classification of endometrial cancers (EC) into pathology reporting and clinical management, uptake is inconsistent. To assign ProMisE subtype, all molecular components must be available (POLE mutation status, mismatch repair (MMR) and p53 immunohistochemistry (IHC)) and often these are assessed at different stages of care and/or at different centres resulting in delays in treatment. We assessed a single-test DNA-based targeted next generation sequencing (NGS) molecular classifier (ProMisE NGS), comparing concordance and prognostic value to the original ProMisE classifier. MethodsDNA was extracted from formalin-fixed paraffin embedded (FFPE) ECs that had previously undergone ProMisE molecular classification (POLE sequencing, IHC for p53 and MMR). DNA was sequenced using the clinically validated Imagia Canexia Health Find It™ amplicon-based NGS gene panel assay to assess for pathogenic POLE mutations (unchanged from original ProMisE), TP53 mutations (in lieu of p53 IHC), and microsatellite instability (MSI) (in lieu of MMR IHC),with the same order of segregation as original ProMisE used for subtype assignment. Molecular subtype assignment of both classifiers was compared by concordance metrics and Kaplan-Meier survival statistics. ResultsThe new DNA-based NGS molecular classifier (ProMisE NGS) was used to determine the molecular subtype in 164 ECs previously classified with ProMisE. 159/164 cases were concordant with a kappa statistic of 0.96 and an overall accuracy of 0.97. Prognostic differences in progression-free, disease-specific and overall survival between the four molecular subtypes were observed for the new NGS classifier, recapitulating the survival curves of the original ProMisE classifier. ProMisE NGS was 100% concordant between matched biopsy and hysterectomy samples. ConclusionProMisE NGS is feasible on standard FFPE material, demonstrates high concordance with the original ProMisE classifier and maintains prognostic value in EC. This test has the potential to facilitate implementation of molecular classification of EC at the time of first diagnosis.

HGG-26. GENOMIC AND IMMUNE ANALYSIS OF PRIMARY REPLICATION-REPAIR DEFICIENT (RRD) GLIOMAS REVEALS THREE SUBGROUPS WITH DISTINCT DRIVERS AND RESPONSE TO IMMUNOTHERAPY: AN IRRDC REPORT

Abstract BACKGROUND Replication-repair deficiency (RRD) caused by germline/somatic defects in mismatch repair (MMRD) and/or polymerase-proofreading genes (PPD) drives 5-10% of gliomas in children, adolescents, and young adults (CAYA). Although RRD-gliomas harbour high mutation-burden (TMB), the basis of their heterogenous biology, clinical behavior, and response to immune-checkpoint inhibitors (ICI) is unknown. METHODS We analyzed the genome (whole exome, low-coverage genome), methylome, transcriptome (bulk, single-nuclei), and the immune-microenvironment of RRD-gliomas in a large cohort of IRRDC patients and correlated these with clinical outcomes and response to ICI. RESULTS Gliomas in 202 CAYA-patients uniformly harbored hypermutation and genomic microsatellite-instability. Median TMB was 297-mutations/megabase, with frequent mutations in TP53 (90%), ATRX (85%), RAS/MAPK (80%) and IDH1/2 (15%). MMRD (but not PPD) mutational signatures contributed to the enrichment of driver mutations in POLE, IDH1, and TP53 while common pediatric mutations (K27M, G34R/V and BRAF;p.V600E) not driven by MMRD signatures were absent. Paired analyses suggested acquisition of novel variants driven by the mutational signatures that also impacted the immune microenvironment. Multi-omic analyses classified RRD-gliomas into three subgroups: RRD1 (MMRD+PPD; 60%), RRD2 (MMRD-only; 23%), and RRD3 (MMRD+IDH1/2; 16%). All RRD1-gliomas were glioblastomas with earlier age of onset, enrichment in CMMRD, arose at diverse locations including the posterior-fossa, classified in proximity to methylation-RTK1-subclass, exhibited balanced copy number profiles, harbored the highest TMB (median: 409-mutations/megabase), and immunogenic PPD signatures. Conversely, RRD3-gliomas were frequent in Lynch syndrome, presented at an older age, with predominant localization in the forebrain, more complex genomic instability, clustered in proximity to IDH1-gliomas, and harbored lower TMB (median: 33-mutations/megabase). RRD1-gliomas revealed the highest immune infiltrates with significantly improved median post-ICI survival of 52-months versus <12-months for RRD2/3 (p<0.0001). CONCLUSIONS The distinct genomic subgroups of RRD-gliomas can explain their diverse clinical outcomes, highlighting the need for developing subgroup-specific, immune-directed treatment approaches for these patients.

BIOL-18. NEWLY DEVELOPED REPLICATION REPAIR DEFICIENT (RRD) MOUSE MODELS PROVIDE INSIGHTS INTO MEDULLOBLASTOMA/GLIOMAGENESIS AND RESPONSE TO IMMUNOTHERAPY

Abstract Replication Repair Deficiency (RRD), caused by germline monoallelic (Lynch Syndrome) or biallelic (Constitutional Mismatch Repair Deficiency, CMMRD) mutations in MMR genes, is present in 5-10% of glioblastomas in children, adolescents, and young adults. RRD glioblastomas are chemoradiation-resistant, but respond favorably to immune checkpoint inhibition (ICI). Representative immunocompetent animal models are urgently needed for 3 recently identified subgroups based on specific somatically-acquired mutations, survival, and immunotherapy response (RRD1: MMRD with POLE mutations, RRD2: MMRD associated with TP53 mutations, and RRD3: MMRD harboring IDH1 mutations). Using germline mutations and brain-specific Cre-drivers, we genetically engineered mouse models that recapitulate each human RRD-subgroup. RRD1 (Nestin- and Olig2-Cre+/ Msh2LoxP/LoxP/PoleS459F/+ and LSL-PoleP286R/+): CMMRD-like Nestin-Cre-driven mice develop posterior-fossa glioma-like or medulloblastoma (MB)-like tumors at ~2.7 months. Olig2-Cre-driven mice display hemispheric gliomas at ~10 months, suggesting distinct cell-of-origin. RRD2 (Nestin-Cre+/Trp53LoxP/LoxP and Msh2LoxP/LoxP or Mlh1-/-): CMMRD-like tumors develop in heterogenous locations at ~4.5 months (p<0.0001), classifying primarily as MB-like in hindbrain, and glioma-like in other brain regions. Strikingly, germline Mlh1 tumors occur earlier than Nestin-Cre-driven RRD2 tumors, indicating early developmental mutation accumulation in CMMRD-patients. Lynch-like RRD1/2 mice succumb exclusively to gliomas >13 months (p<0.0001). RRD3 (Olig2-Cre+/Msh2LoxP/LoxP/Trp53LoxP/LoxP/LSL-Idh1R132H/+): brain tumors occur later and are hemispheric. These observations recapitulate human data, where CMMRD-patients develop glioblastoma/MB earlier than Lynch-patients (8.6 vs. 14-years; p<0.0001), and posterior-fossa glioblastoma/MB presents earlier than hemispheric gliomas (p=0.04). Additionally, tumor onset and location vary (RRD1: 7.6-years, RRD2: 8.3-years, hemispheric/posterior-fossa; RRD3: 12-years, hemispheric; p=0.005). In both mice and humans, RRD1 exhibits ultra-hypermutation, high immune infiltration, and response to ICI, whereas RRD2 harbors lower mutational burden, are immune-cold, and ICI-monotherapy resistant. Temporal dynamics of RRD tumor development is currently being tracked by serial MRI to define biologically relevant time points. Our models accurately mimic the human condition and provide unique insights into RRD tumorigenesis, allowing optimization of subgroup-tailored therapeutic approaches.

Comparative study of next generation sequencing and immunohistochemistry on molecular classification of endometrial carcinoma

Objective: To investigate the differences in molecular classification of endometrial carcinoma (EC) between various technical methods and to explore molecular classification schemes suitable for Chinese population. Methods: The study used a comprehensive scheme of next generation sequencing (NGS) and immunohistochemistry for molecular classification of 254 EC cases that were obtained at Department of Pathology, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China from April 2021 to March 2022. According to the recommended threshold of Sanger sequencing which was approximate-20% variant allele fraction (VAF), NGS data were extracted to simulate the results of Sanger sequencing. Results: The 254 EC patients had a mean age of 51 years (range, 24 to 89 years). Combination of POLE (9-14 exons), TP53 total exons and microsatellite instability (MSI) detection was a better single scheme than NGS alone, while combination of MSI fragment analysis and conventional immunohistochemistry was the best solution and seemed best aligned with TCGA data and recent studies. POLE ultramuted type, mismatch repair defect type, TP53 mutant type and non-specific molecular characteristic type accounted for 11.4% (29/254), 31.5% (80/254), 22.4% (57/254) and 34.6% (88/254) of the cases, respectively. If Sanger sequencing was adopted for POLE and TP53 detection, the frequencies of these EC types were 9.1% (23/254), 31.5% (80/254), 12.9% (33/254) and 46.6% (118/254), respectively, with greatly increasing non-specific molecular characteristics cases. If POLE was detected by Sanger sequencing and others by immunohistochemistry, they were 9.1% (23/254), 42.2% (92/218), 13.8% (35/254) and 40.9% (105/254), respectively, with increasing the false positive rates of the mismatch repair defect group. Conclusions: Small and medium-sized NGS panels with MSI detection is a better solution than NGS alone. Sanger sequencing is currently available for POLE mutation detection, which is not sensitive enough for TP53 mutation detection, and seems equivalent to the efficiency of TP53 by immunohistochemistry. Further optimization of small and medium-sized NGS panels covering MSI detection and POLE and TP53 full exons may be the best choice for the future to meet national conditions.

A deep learning model for the prediction of microsatellite instability and pathogenic POLE mutations in colorectal cancer using histopathologic images.

3543 Background: Immunotherapy has brought about a landmark change in anti-tumor treatment in the past years. High microsatellite instability (MSI-H) is now the only clinically approved biomarker predicting response to immunotherapy in CRC. Increasing evidence suggests that POLE mutations in the exonuclease domain could drive an ultra-mutational phenotype and improve the treatment outcomes of ICI in solid tumors. In this study, we set out to apply a deep learning model using H&E-stained, formalin-fixed, paraffin-embedded (FFPE) whole slide images (WSIs) of CRC primary tumors. Methods: The deep learning model is developed and validated through five-fold cross-validation using WSI of primary tumors from 506 CRC patients and externally validated using 52 WSIs from a prospective cohort. The microsatellite status, tumor mutation burden (TMB) and POLE genotype were determined by next-generation sequencing (NGS). Patients with MSS status and a low TMB (＜20Mutations/Mb) were admitted to the MSS group, and CRCs with a POLE mutation which was defined as an oncogenic mutation referring to the POLE functional mutation list at OncoKB( POLE (oncokb.org) were admitted to the POLE mutant group. Clustering-constrained-attention multiple-instance learning (CLAM) model is employed as the base model, and we conduct the model ensemble by performing a large-scale hyper-parameter search, selecting five models with the highest value in one of the performance metrics, including the AURoC, accuracy, precision, recall, and f1 score, and finally averaging the predictions of the five models. Results: The internal dataset included 237 MSS, 142 MSI-H, and 127 POLE mutant CRC. The three groups had significant differences in primary location (p ＜ 0.0001), histology (p ＜ 0.0001), tumor differentiation (p = 0.002), tumor stage (p ＜ 0.0001), Crohn's-like reaction (p ＜ 0.0001) and tumor growth pattern (p = 0.001). The cross-validation performance of the ensemble model (M E) in the internal dataset achieves an AURoC of 0.944 for three-way classification task (POLE vs. MSI-H vs. MSS) and 0.940 for two-way classification task (POLE & MSI-H vs. MSS) which were superior to the performance of each single CLAM model. To demonstrate the generalizability of the deep learning model, a domestic perspective cohort consisting of 20 MSS, 17 MSI-H, and 15 POLE mutant CRC H&E images were used to validate the external performance. And the M E retained robust performance on the external dataset, with an AURoC of 0.904 for three-way classification task and 0.836 for two-way classification task. Conclusions: A CLAM-based deep learning model could directly predict the MSI-H and POLE mutation from histological images that could be used to stratify CRC patients for immunotherapy with faster turnaround time and lower costs compared with traditional sequencing methods.

Risk reclassification of endometrial cancer according to molecular profiling: UK single centre experience.

e17628 Background: Endometrial cancer (EC) ranks second among gynaecological malignancies worldwide. Traditionally, adjuvant treatment recommendation was based on risk stratification based on clinicopathological data. The Cancer Genome Atlas (TCGA) defined four distinct and prognostically significant molecular subgroups. Recent international guidelines recommend integrating molecular classification into risk stratification. The aim of our study was to evaluate the distribution of molecular groups in our population and its impact on treatment decision. Methods: Retrospective cohort study including all newly diagnosed endometrial cancer cases treated at the Royal Marsden Hospital between July 2020 and May 2022. Tumour specimens underwent next generation sequencing (NGS) of a two hundred gene panel including POLD1, POLE; MMR proteins and TP53; concordance for the latter two assessed also by immunohistochemistry (IHC). Risk stratification according to ESMO guidelines and following molecular classification was reviewed for each case and its potential impact on treatment decision. Results: 121 patients were included. POLE mutated tumours comprised 14 cases (11.6%), MSI hypermutated 24 cases (19.8 %), copy number high 36 cases (29.8%), copy number low 45 cases (37.2%) and 2 cases (1.6%) where classified as NOS (not otherwise specified). According to clinicopathological data, 39 tumours were categorised as low risk, 14 tumours as intermediate risk, 15 tumours as high-intermediate risk, 39 tumours as high risk and 14 were advanced/metastatic. According to final prognostic group when molecular profile was integrated 8.3 % (n = 10) of cases were reclassified: 8 cases were down staged (2 high, 3 high-intermediate, 3 intermediate) to low-risk category due to POLE mutations. 2 cases were upstaged from high-intermediate to high-risk category (copy number high tumours). There were no shifts noted in the low risk (based on clinicopathological data) prognostic group. One further case in the high-risk category (stage III, gr3 endometrioid tumour with substantial LVSI) was found to be POLE mutated and may be reclassified as low risk. For the 10 cases that were reclassified, adjuvant treatment recommendations may have been adjusted according to the most recent ESMO guidelines. Conclusions: Molecular classification refines traditional risk classification of EC and may change treatment recommendation in a non-negligible proportion of patients. Efforts should be made to implement molecular analysis in routine clinical practice to guide appropriate treatment.

Durvalumab + tremelimumab in biologically selected advanced/metastatic solid tumors.

2604 Background: Tremelimumab (T) in combination with Durvalumab (D) is approved by the FDA for the treatment of unresectable hepatocellular carcinoma and metastatic non–small cell lung cancer. We prospectively examined its clinical activity in a cohort of biologically-selected advanced/metastatic solid tumors from the MOST Plus clinical trial (NCT NCT02029001). Methods: Adult patients (pts) previously treated by at least one prior line but immunotherapy-naive were eligible if their tumor harbored TMB-H (>10mut/Mb) and/or MSI/dMMR (by PCR and IHC tests), and/or PD1/PD-L1/CTLA4 amplification and/or POLD1 or POLE mutation. T was administered for up to 4 cycles (75mg, IV, Q4W) and D (1500mg, IV, Q4W) until disease progression, unacceptable toxicity or patient/investigator decision. Tumor response was assessed as per RECIST 1.1 and adverse events reported according to NCI-CTCAE v4.03. Results: 70 patients were enrolled (24 male, median age: 65 years [33-88], MSI n=56, TMB-H n=10, PDL1 and PDL2 ampl. n=1, POLE mut. n=1, MSH6 mut. n=1, MSH2 mut. n=1). The median number of prior lines of systemic therapy was 1 (range: 0-4). Main tumor types were colorectal (44%), endometrial (19%), and stomach (7%) carcinomas. As of October 2022, overall response (ORR) rate was 52.8% (21.4% CR, 31.4% PR). With a median follow-up of 37 months, median PFS and OS were 8.4 months [95% CI: 4.4- 22.1] and 37.2 months [95% CI: 10.4-NE], respectively. Median duration response of response was not reached. Overall, 24.3% of patients experienced at least one serious treatment-related (TR) adverse event. Two TR-deaths were reported (haemophagocytic lymphohistiocytosis and nephritis). ORR, median PFS and OS are reported. Conclusions: T+D is a highly efficient combination in biologically selected tumors leading to a high complete response rate. Enrolment is still ongoing, to further evaluate the utility of a maintenance treatment, i.e the optimal treatment duration. Clinical trial information: NCT02029001 . [Table: see text]

Targeted Treatment of Advanced Endometrial Cancer: Focus on Pembrolizumab.

Endometrial cancer (EC) accounts for 2% of all new cancers. Advanced forms have a poor prognosis with barely 17% 5-year survival. The last few years improved our knowledge of EC with a new molecular classification derived from The Cancer Genome Atlas (TCGA). They are now divided between POLE mutant, Microsatellite Instability High (MSI-H) or deficient in Mismatch Repair System (dMMR), TP53 mutant and no specific molecular profile. Until now, treatments for advanced EC have included conventional platinum-based chemotherapy or hormonotherapy. The revolution in oncology represented by the advent of immune checkpoints inhibitors (ICI) has also led to a major advance in the management of recurrent and metastatic EC. Pembrolizumab, a well-known anti PD-1, has firstly been approved as monotherapy in the second-line setting for dMMR/MSI-H advanced EC. More recently, a combination of lenvatinib with pembrolizumab offered a new effective option in the second line setting irrespectively of the MMR status, giving a new opportunity for these patients who had no actual standard of care before. This combination is currently being evaluated as frontline therapy. Despite exciting results, the main problem in identifying solid biomarkers remains unresolved and further investigations are required. New original combinations of pembrolizumab with other drugs including chemotherapy, poly ADPribose polymerase inhibitors (PARP-i) or tyrosine kinase inhibitors are being tested and promise exciting new therapeutic evolutions in a close future.

Comprehensive analysis of the mutational status of early colon cancer: Real world data from the AIO ColoPredict registry study.

e15535 Background: The comprehensive analysis of the mutational status of colon cancer (CC) may identify targetable alterations also in early stages. This may open up potential beneficial treatment choices, e.g. immunotherapy in microsatellite instable (MSI) tumors. So far, there is few data regarding the distribution of molecular alterations in CC in a real world setting in Germany. Methods: We analyzed clinical and molecular data from our Colopredict Plus (CPP) registry trial including CC patients (pts.) stage I-III. Samples were tested for MS status by immunohistochemistry and for gene mutations (MT) using a 71 gene panel comprised for CC. Molecular data was correlated with clinicopathologic parameters. Disease free Survival (DFS) was estimated by the Kaplan-Meier method. Results: Data from 2746 pts. was included. Female: 1476/2746 (53,8 %), median age: 72 yrs (range: 22-100 yrs), right sided tumor: 1607/2746 (58,5 %), stage distribution I/II/III/NA: 414 (15,1 %)/1198 (43,6 %)/1076 (39,2 %)/58 (2,1 %). MSI/MSS: 544(19,8%)/2202/ (80,2%). A KRAS mutation was observed in 846/2746 (30,8 %). The most frequent hotspot MT were codon 12 KRAS MT with p.G12D (27%), p.G12V (19%), p.G12A (6%), and p.G12C (6%) followed by codon 13 MT with p.G13D (18%). The subtypes p.G12D, p.G12V, and p.Q61K were significantly more frequent in the right colon. BRAF MT was seen in 560/2746 (20,4%). Observed rare MT were: POLE (76/2746 (2,8 %), PTEN 188/2746 (6,8 %), and EGFR (21/2746; 0,8%). MSI CC were more frequent in the right colon and associated with BRAF MT (346/560; 61,8 %), and also MT in BRCA1 (35/53; 66 %), BRCA2 (119/171; 69,6 %), POLE (45/76 (59,2 %), PTEN 107/188 (56,9 %), and EGFR (18/21, 85,7%). In contrast, TP53 MT (1223/2746 (44,5 %)) were associated with MSS (1090/2746 (49,5 %)). 3 yrs DFS in MSI BRAF WT vs. MSS BRAF MT pts. was 0,75 (0,67 – 0,81 vs 0,52 (0,36 – 0,65) (p =0,11). DFS did not differ in specific KRAS MT variants. Conclusions: We here show a comprehensive analysis of genetic alterations in early CC in a prospective real world cohort. As expected, MSI was correlated with female sex, right sided primary tumor and BRAF MT. Interestingly, we also found a strong correlation of BRCA1 MT, BRCA2 MT, EGFR MT, POLE MT, and PTEN MT with presence of MSI while MSS was associated with TP53 MT. Broad molecular characterization of early CC may help individualize treatment strategies in the future.

Clinical, pathologic, and genomic hallmarks of KRAS-amplified non-small cell lung cancer.

9118 Background: KRAS-mutant non-small cell lung cancers (NSCLCs) have unique clinicopathologic and genomic features, and novel therapies are in development to target many KRAS-mutant tumor types. Whether KRAS amplification also defines a unique, potentially targetable, molecular subset of NSCLC is currently unknown. Methods: Clinicopathologic and genomic data were abstracted from multiple independent cohorts of pts whose cancers underwent tumor genomic profiling at the Dana-Farber Cancer Institute (Cohort 1), AACR Project GENIE v.13, The Cancer Genome Atlas (TCGA), and 212 other studies (Cohort 2). Patient (pt) outcomes were examined according to KRAS amplification status ( KRAS amplified vs KRAS non-amplified). Whole transcriptome sequencing and quantitative proteomics data from TCGA and the Cancer Cell Line Encyclopedia (CCLE) were used to correlate KRAS amplification with KRAS RNA levels and protein expression. Results: Among 15,341 pts with NSCLC, KRAS amplification was identified in 355 (2.3%) cases (median number of copies [range]: 11 [7-87]); the prevalence was similar in adenocarcinomas and squamous cell carcinomas. Compared to KRAS non-amplified cases, pts with KRAS-amplified NSCLC were more likely to be men (52.4% vs 45.6%, p = 0.01), have a history of smoking (82.1% vs 64.2%, p < 0.01), and higher median pack-years (35 vs 23.5, p < 0.01). However, median age was similar between the two groups (66 vs 66, p = 0.88). KRAS amplification was also associated with higher aneuploidy (median fraction of genome altered 30.4% vs 14.3%, p < 0.01), tumor mutational burden (p < 0.01 across different platforms), and increased median PD-L1 expression (20% vs 5%, p = 0.01). Of the 355 NSCLC samples with KRAS amplification, 152 (43%) had no concurrent oncogene driver mutations. In unbiased mutation enrichment analyses, KRAS-amplified tumors were enriched in concurrent mutations in KRAS, PALB2, POLE, and SLC34A2 (q < 0.1), while KRAS non-amplified tumors were enriched for oncogenic EGFR mutations (q < 0.1). Transcriptomic and proteomic profiling from the TCGA and CCLE cohorts demonstrated that KRAS amplification was associated with significantly increased KRAS mRNA (p < 0.01) and protein expression (p < 0.01), compared to KRAS non-amplified samples. In pts with available clinical outcomes data in the combined cohort (N = 9,335), the median overall survival (OS) from the date of diagnosis was significantly shorter in KRAS-amplified vs KRAS non-amplified cases (adjusted HR: 1.40, p < 0.01). KRAS amplification was confirmed to confer significantly worse survival outcomes in pts with both KRAS wild-type (N = 6,695, adjusted HR 1.37, p = 0.02) and KRAS-mutant (N = 2,640, adjusted HR 1.34, p = 0.02) NSCLC. These results were independently replicated in Cohort 1 and Cohort 2. Conclusions: KRAS amplification defines a novel molecular subset of NSCLC characterized by distinct clinicopathologic and genomic features and worse survival.

Clinical utility of plasma ctDNA sequencing monitoring and tumor molecular profile exploration in endometrial carcinoma: A Caribbean prospective cohort from Martinique.

e17625 Background: A higher incidence of agressive endometrial cancer (EC) has been reported amongst the Martinican population. Indeed, there are respectively 25%, 12% and 3 % of high grade serous carcinoma, carcinosarcoma and clear cell uterine carcinoma, all with poor prognosis. Genomic stratification helps to improve the management of patients with EC. Indeed, localized EC molecular classification discriminated the benefit of adjuvant therapy according to molecular subgroups. However, collecting tissue samples in EC remains challenging. The aim of our study was to assess the clinical utility of ctDNA monitoring in EC and tumor tissue molecular profiling in high grade non endometrioid EC. Methods: We conducted a prospective monocentric study including high grade non endometrioid EC patients enrolled in the EndoNGS trial (NCT04932525). Each patient underwent molecular analysis to define the tumor molecular landscape. A comparative analysis “tumor-germline” including microsatellite instability and panel gene sequencing was then performed. ctDNA was profiled using dPCR and RT PCR. Results: From May 2020 to September 2021, 25 patients with high grade non endometriod EC were included. Median age at diagnosis was 72 years (60-88). FIGO stage at diagnosis was I, II, III and IV in 28%, 4%, 32% and 24% respectively. 10 patients out 25 received radiochemotherapy. Only 6 underwent exclusive radiotherapy and 13 received adjuvant platinum chemotherapy addjuvant to surgery. All tumor had MSS and none harbored a POLE mutation. 19 patients underwent molecular profiling according to a panel of 80 genes. The analysis identified 30 mutations: 22 pathogenic missenses, 3 nonsenses and 5 frameshift mutations. A TP53 mutation was observed in 89.5 % of cases. We identified 26% hotspot mutations. The other mutations are infrequent or not described in TGCA database. TP53 mutation analysis reported 15 missenses, one frameshift and one nonsense mutation. Moreover 88% of TP53 mutated tumors were affected by biallelic inactivation. In this population, variants in PIK3CA represent 26% compared to 42% reported from TGCA database and none PPP2R1A and FBXW7 mutations have been detected compared to 32% and 24% for PPP2R1A and FBXW7 respectively. ctDNA abondance was correlated to the stage. 10 patients had positive ctDNA at diagnosis (9 were stage III and 1 stage IV). In 3 patients, ctDNA became negative during therapy (radiochemotherapy). During course of disease, 15 relapsed, (with a median 12 month relapse free period) amongst which 10 had positive ctDNA. Conclusions: We reported for the first time the molecular profiling of aggressive EC in a homogenous carribbean cohort from Martinique. Overall, our analysis demonstrates that genomic profiling with a large panel of ctDNA in localized EC is reliable and feasible; ctDNA monitoring was associated to early relapse.

Integration of clinical sequencing and immunohistochemistry for the molecular classification of endometrial carcinoma

PurposeUsing next generation sequencing (NGS), The Cancer Genome Atlas (TCGA) found that endometrial carcinomas (ECs) fall under one of four molecular subtypes, and a POLE mutation status, mismatch repair (MMR) and p53 immunohistochemistry (IHC)-based surrogate has been developed. We sought to retrospectively classify and characterize a large series of unselected ECs that were prospectively subjected to clinical sequencing by utilizing clinical molecular and IHC data. Experimental designAll patients with EC with clinical tumor-normal MSK-IMPACT NGS from 2014 to 2020 (n = 2115) were classified by integrating molecular data (i.e., POLE mutation, TP53 mutation, MSIsensor score) and MMR and p53 IHC results. Survival analysis was performed for primary EC patients with upfront surgery at our institution. ResultsUtilizing our integrated approach, significantly more ECs were molecularly classified (1834/2115, 87%) as compared to the surrogate (1387/2115, 66%, p < 0.001), with an almost perfect agreement for classifiable cases (Kappa 0.962, 95% CI 0.949–0.975). Discrepancies were primarily due to TP53 mutations in p53-IHC-normal ECs. Of the 1834 ECs, most were of copy number (CN)-high molecular subtype (40%), followed by CN-low (32%), MSI-high (23%) and POLE (5%). Histologic and genomic variability was present amongst all molecular subtypes. Molecular classification was prognostic in early- and advanced-stage disease, including early-stage endometrioid EC. ConclusionsThe integration of clinical NGS and IHC data allows for an algorithmic approach to molecularly classifying newly diagnosed EC, while overcoming issues of IHC-based genetic alteration detection. Such integrated approach will be important moving forward given the prognostic and potentially predictive information afforded by this classification.

In-silico analysis of TCGA data showing multiple POLE-like favourable subgroups overlapping with TP53 mutated endometrial cancer: Implications for clinical practice in low and middle-income countries

IntroductionThe Cancer Genome Atlas cohort of endometrial carcinoma (TCGA-UCEC) includes almost 40% TP53-mutants encompassing missense and truncated variants. TCGA revealed ‘POLE’, characterized by POLE gene bearing exonuclease domain mutation (EDM), as the prognostically best molecular profile. The worst profile was characterized by TP53-mutated Type 2 cancer requiring adjuvant therapy having cost implications in low-resource settings. We aimed to find more ‘POLE-like’ favourable subgroups by searching TCGA cohort, especially within TP53 mutated risk group, that could eventually avoid adjuvant treatment in resource-poor settings. MethodOur study was an in-silico survival analysis performed on the TCGA-UCEC dataset using SPSS statistical package. TP53 and POLE mutations, microsatellite instability (MSI), time-to-event and clinicopathological parameters were compared among 512 endometrial cancer cases. Deleterious POLE-mutations were identified by Polyphen2. Progression free survival was studied using Kaplan-Meier plots keeping original ‘POLE’ as comparator. ResultIn presence of wild type (WT)-TP53, other deleterious POLE-mutations behaved like POLE-EDM. Only truncated and not missense TP53 benefitted from POLE/MSI overlap. However, TP53 missense mutation, Y220C, was found to be as favourable as ‘POLE’. Overlapping POLE, MSI and WT-TP53 also performed favourably. Truncated TP53 overlapped with POLE and/or MSI, TP53 Y220C alone and, WT-TP53 overlapped with POLE and MSI both, were named ‘POLE-like’ for prognostically behaving like the comparator ‘POLE’. ConclusionObesity being a lesser frequent event in low and middle-income countries (LMICs), relative proportion of women with lower BMI and Type 2 endometrial cancers may be high. Identification of ‘POLE-like’ groups may facilitate therapeutic de-escalation in some TP53-mutated cases - a novel option. Instead of 5% (POLE-EDM), potential beneficiary would then comprise 10% (POLE-like) of TCGA-UCEC.

Alteration in molecular properties during establishment and passaging of endometrial carcinoma patient-derived xenografts

Patient-derived xenograft (PDX) tumor models are known to maintain the genomic and phenotypic profiles, including the histopathological structures, of the parental tumors. On the other hand, unique enrichment of single-nucleotide variants or copy number aberrations has been reported in several types of tumors. However, an understanding of endometrial carcinoma PDXs is limited. The purpose of the present study was to clarify the presence or absence of the molecular properties of endometrial carcinomas in PDXs passaged up to eight times. Established PDXs of endometrioid carcinomas maintained their histopathological characteristics, but those of carcinosarcomas predominantly consisted of sarcomatous components when compared to the parental tumors. Alterations in the proportion of cells with positive/negative immunohistochemical staining for estrogen receptor, PTEN, PAX8, and PAX2 were observed, whereas the proportions of cells with AE1/AE3, TP53, ARID1A, PMS2, and MSH6 staining were unchanged. Variants of cancer-associated genes were compared between PDXs and parental tumors. Mutations in POLE and a frameshift deletion in BRCA1 were observed in the parental tumor tissue in each of the six cases, and additional genomic alterations, which were not apparently related to histopathological and immunohistochemical alterations, were found in the PDXs of these cases. The genomic and phenotypic alterations observed between endometrial carcinoma PDXs and parental tumors were partly associated with endometrial cancer-specific characteristics related to cellular differentiation and gene mutations.

Genomic landscape and its prognostic significance in stage III colorectal cancer: JCOG1506A1, an ancillary of JCOG0910.

Large-scale genomic sequencing of colorectal cancers has been reported mainly for Western populations. Differences by stage and ethnicity in the genomic landscape and their prognostic impact remain poorly understood. We investigated 534 Japanese stage III colorectal cancer samples from the Phase III trial, JCOG0910. Targeted-capture sequencing of 171 potentially colorectal cancer-associated genes was performed, and somatic single-nucleotide variants and insertion-deletions were determined. Hypermutated tumors were defined as tumors with MSIsensor score >7 and ultra-mutated tumors with POLE mutations. Genes with alterations associated with relapse-free survival were analyzed using multivariable Cox regression models. In all patients (184 right-sided, 350 left-sided), mutation frequencies were TP53, 75.3%; APC, 75.1%; KRAS, 43.6%; PIK3CA, 19.7%; FBXW7, 18.5%; SOX9, 11.8%; COL6A3, 8.2%; NOTCH3, 4.5%; NRAS, 4.1%; and RNF43, 3.7%. Thirty-one tumors were hypermutated (5.8%; 14.1% right-sided, 1.4% left-sided). Modest associations were observed: poorer relapse-free survival was seen with mutant KRAS (hazard ratio 1.66; p = 0.011) and mutant RNF43 (2.17; p = 0.055), whereas better relapse-free survival was seen with mutant COL6A3 (0.35; p = 0.040) and mutant NOTCH3 (0.18; p = 0.093). Relapse-free survival tended to be better for hypermutated tumors (0.53; p = 0.229). In conclusion, the overall spectrum of mutations in our Japanese stage III colorectal cancer cohort was similar to that in Western populations, but the frequencies of mutation for TP53, SOX9, and FBXW7 were higher, and the proportion of hypermutated tumors was lower. Multiple gene mutations appeared to impact relapse-free survival, suggesting that tumor genomic profiling can support precision medicine for colorectal cancer.

Investigation of the Relationship Between Immunohistochemical Mismatch Repair (MMR) Protein Expression and Prognostic Parameters in Endometrial Carcinomas

Objective: Molecular classification has been the most important development in endometrial carcinomas in recent years. This classification divides tumors into four groups: (i) POLE mutant group, (ii) microsatellite instable (MSI) group, (iii) high copy number group (P53 mutation), and (iv) low copy number group. Among these groups, POLE and MSI groups stand out with their better prognosis and potential to benefit from immune-control inhibitor therapy. In our study, we aimed to compare the prognostic parameters of patients with and without nuclear expression loss in MMR proteins (MLH-1, PMS-2, MSH-2, MSH-6) by immunohistochemical (IHC) method. Methods: Between 2017 and 2020, 80 patients who were diagnosed with endometrial carcinoma in hysterectomy material and whose MMR proteins were evaluated as IHC were included in the study. Patients with and without MMR loss were compared in terms of tumor size, histological grade (HD), depth of myometrial invasion, lymphovascular invasion (LVI), and cervical involvement. Results: Loss of any of the MMR proteins was present in 37 cases (46.3%), while 43 cases (53.7%) had no loss. When the cases were compared in terms of loss of MMR protein nuclear expression, 45.9% (17/37) of the cases with loss and 27.9% (12/43) of the cases without loss had histologic grade III (P = 0.03). There was no statistically significant difference between the two groups in terms of myometrium 1/2 external invasion, cervical stromal involvement, and LVI. Conclusion: Approximately half of the patients in our study lost at least one of the MMR proteins. The most common losses were MLH-1 and PMS-2. The HD of patients with loss of nuclear expression of MMR proteins tended to be statistically significantly higher than those without loss.