Validation of Modaplex POLE mutation assay in endometrial carcinoma.

The TCGA-based molecular classification of endometrial cancer has emerged as an important tool to stratify patients according to prognosis. A simplified scheme has been proposed, by using immunohistochemistry for p53, MSH6, and PMS2 and a molecular test for POLE mutations (NGS or Sanger sequencing, techniques that are not available in many centers worldwide). In this study, we validate a novel method that allows simultaneous analysis of multiple pathogenic POLE mutations. The Modaplex technology integrates polymerase chain reaction and capillary electrophoresis. The design of this study encompassed 4 different steps: (1) a retrospective-pilot phase, with 80 tumors, balancing the four molecular subgroups. (2) A retrospective phase of 25 tumors obtained between 2016 and 2020, and 30 tumors obtained between 2000 and 2015. (3) An inter-laboratory corssavalidation step with 19 cases (belonging to phases 1 and 2). (4) A prospective cohort of 123 tumors, of unknown POLE status, with simultaneous validation by Sanger sequencing. A total of 258 samples were analyzed. In the first and second phases, the test showed positive/negative predictive values of 100%, by correctly identifying POLE mutation status in 79/79 and 55/55 cases. Phase 3 showed 100% of inter-laboratory consistency. Phase 4 showed 16 positive samples out of the 123 prospective cases. Overall, the test has revealed sensitivity and specificity of 100%, identifying a total of 47 POLE-mutated tumors. We have shown that this technique allows faster and easier identification of multiple pathogenic POLE mutations with high robustness and confidence when comparing to other tests such as Sanger sequencing.

Evaluation of somatic mutations in cervicovaginal samples as a non-invasive method for the detection and molecular classification of endometrial cancer.

BACKGROUND: The incidence of endometrial cancer is increasing worldwide. While delays in diagnosis reduce survival, case molecular misclassification might be associated with under- and over-treatment. The objective of this study was to evaluate genetic alterations to detect and molecularly classify cases of endometrial cancer using non-invasive samples. METHODS: Consecutive patients with incident endometrial cancer (N = 139) and controls (N = 107) from a recent Spanish case-control study were included in this analysis. Overall, 339 cervicovaginal samples (out of which 228 were clinician-collected and 111 were self-collected) were analysed using a test based on next-generation sequencing (NGS), which targets 47 genes. Immunohistochemical markers were evaluated in 133 tumour samples. A total of 159 samples were used to train the detection algorithm and 180 samples were used for validation. FINDINGS: Overall, 73% (N = 94 out of 129 clinician-collected samples, and N = 66 out of 90 self-collected samples) of endometrial cancer cases had detectable mutations in clinician-collected and self-collected samples, while the specificity was 80% (79/99) for clinician-collected samples and 90% (19/21) for self-collected samples. The molecular classifications obtained using tumour samples and non-invasive gynaecologic samples in our study showed moderate-to-good agreement. The molecular classification of cases of endometrial cancer into four groups using NGS of both clinician-collected and self-collected cervicovaginal samples yielded significant differences in disease-free survival. The cases with mutations in POLE had an excellent prognosis, whereas the cases with TP53 mutations had the poorest clinical outcome, which is consistent with the data on tumour samples. INTERPRETATION: This study classified endometrial cancer cases into four molecular groups based on the analysis of cervicovaginal samples that showed significant differences in disease-free survival. The molecular classification of endometrial cancer in non-invasive samples may improve patient care and survival by indicating the early need for aggressive surgery, as well as reducing referrals to highly specialized hospitals in cancers with good prognosis. Validation in independent sets will confirm the potential for molecular classification in non-invasive samples. FUNDING: This study was funded by a competitive grant from Instituto de Salud Carlos III through the projects PI19/01835, PI23/00790, and FI20/00031, CIBERESP CB06/02/0073 and CIBERONC CB16/12/00231, CB16/12/00234 (Co-funded by European Regional Development Fund. ERDF: A way to build Europe). Samples and data were provided by Biobank HUB-ICO-IDIBELL, integrated into the Spanish Biobank Network, and funded by the Instituto de Salud Carlos III (PT20/00171) and by Xarxa de Bancs de Tumors de Catalunya (XBTC) sponsored by Pla Director d'Oncologia de Catalunya. This work was supported in part by the AECC, Grupos estables (GCTRA18014MATI). It also counts with the support of the Secretariat for Universities and Research of the Department of Business and Knowledge of the Generalitat de Catalunya, and grants to support the activities of research groups 2021SGR01354 and 2021SGR1112.

Evaluation of Somatic Mutations in Urine Samples as a Noninvasive Method for the Detection and Molecular Classification of Endometrial Cancer.

PURPOSE: Current diagnostic methods for endometrial cancer lack specificity, leading to many women undergoing invasive procedures. The aim of this study was to evaluate somatic mutations in urine to accurately discriminate patients with endometrial cancer from controls. EXPERIMENTAL DESIGN: Overall, 72 samples were analyzed using next-generation sequencing (NGS) with molecular identifiers targeting 47 genes. We evaluated urine supernatant samples from women with endometrial cancer (n = 19) and age-matched controls (n = 20). Cell pellets from urine and plasma samples from seven cases were sequenced; further, we also evaluated paired tumor samples from all cases. Finally, immunohistochemical markers for molecular profiling were evaluated in all tumor samples. RESULTS: Overall, we were able to identify mutations in DNA from urine supernatant samples in 100% of endometrial cancers. In contrast, only one control (5%) showed variants at a variant allele frequency (VAF) ≥ 2% in the urine supernatant samples. The molecular classification obtained by using tumor samples and urine samples showed good agreement. Analyses in paired samples revealed a higher number of mutations and VAF in urine supernatants than in urine cell pellets and blood samples. CONCLUSIONS: Evaluation of somatic mutations using urine samples may offer a user-friendly and reliable tool for endometrial cancer detection and molecular classification. The diagnostic performance for endometrial cancer detection was very high, and cases could be molecularly classified using these noninvasive and self-collected samples. Additional multicenter evaluations using larger sample sizes are needed to validate the results and understand the potential of urine samples for the early detection and prognosis of endometrial cancer.

Highly Sensitive Microsatellite Instability and Immunohistochemistry Assessment in Endometrial Aspirates as a Tool for Cancer Risk Individualization in Lynch Syndrome.

Women with Lynch syndrome (LS) are at increased risk of endometrial cancer (EC), among other tumors, and are characterized by mismatch repair (MMR) deficiency and microsatellite instability (MSI). While risk-reducing gynecologic surgeries effectively decrease EC incidence, doubts arise regarding the appropriate timing of the surgery. We explored the usefulness of highly sensitive MSI (hs-MSI) assessment in endometrial aspirates for individualizing gynecologic surveillance in LS carriers. Ninety-three women with LS, 25 sporadic EC patients (9 MMR-proficient and 16 MMR-deficient), and 30 women with benign gynecologic disease were included in this study. hs-MSI was assessed in prospectively collected endometrial aspirates in 67 LS carriers, EC cases, and controls. MMR, PTEN, ARID1A, and PAX2 protein expression patterns were evaluated in the LS samples. Follow-up aspirates from 8 LS carriers were also analyzed. Elevated hs-MSI scores were detected in all aspirates from MMR-deficient EC cases (3 LS and 16 sporadic) and negative in aspirates from controls and MMR-proficient EC cases. Positive hs-MSI scores were also detected in all 4 LS aspirates reported as complex hyperplasia. High hs-MSI was also present in 10 of 49 aspirates (20%) from LS carriers presenting a morphologically normal endometrium, where MMR protein expression loss was detected in 69% of the samples. Interestingly, the hs-MSI score was positively correlated with MMR-deficient gland density and the presence of MMR-deficient clusters, colocalizing PTEN and ARID1A expression loss. High hs-MSI scores and clonality were evidenced in 2 samples collected up to 4 months before EC diagnosis; hs-MSI scores increased over time in 5 LS carriers, whereas they decreased in a patient with endometrial hyperplasia after progestin therapy. In LS carriers, elevated hs-MSI scores were detected in aspirates from premalignant and malignant lesions and normal endometrium, correlating with MMR protein loss. hs-MSI assessment and MMR immunohistochemistry may help individualize EC risk assessment in women with LS.

Generation and Integrated Analysis of Advanced Patient-Derived Orthoxenograft Models (PDOX) for the Rational Assessment of Targeted Therapies in Endometrial Cancer.

Clinical management of endometrial cancer (EC) is handicapped by the limited availability of second line treatments and bona fide molecular biomarkers to predict recurrence. These limitations have hampered the treatment of these patients, whose survival rates have not improved over the last four decades. The advent of coordinated studies such as The Cancer Genome Atlas Uterine Corpus Endometrial Carcinoma (TCGA_UCEC) has partially solved this issue, but the lack of proper experimental systems still represents a bottleneck that precludes translational studies from successful clinical testing in EC patients. Within this context, the first study reporting the generation of a collection of endometrioid-EC-patient-derived orthoxenograft (PDOX) mouse models is presented that is believed to overcome these experimental constraints and pave the way toward state-of-the-art precision medicine in EC. The collection of primary tumors and derived PDOXs is characterized through an integrative approach based on transcriptomics, mutational profiles, and morphological analysis; and it is demonstrated that EC tumors engrafted in the mouse uterus retain the main molecular and morphological features from analogous tumor donors. Finally, the molecular properties of these tumors are harnessed to assess the therapeutic potential of trastuzumab, a human epidermal growth factor receptor 2 (HER2) inhibitor with growing interest in EC, using patient-derived organotypic multicellular tumor spheroids and in vivo experiments.

Efficacy of CDK4/6 inhibitors in preclinical models of malignant pleural mesothelioma.

BACKGROUND: There is no effective therapy for patients with malignant pleural mesothelioma (MPM) who progressed to platinum-based chemotherapy and immunotherapy. METHODS: We aimed to investigate the antitumor activity of CDK4/6 inhibitors using in vitro and in vivo preclinical models of MPM. RESULTS: Based on publicly available transcriptomic data of MPM, patients with CDK4 or CDK6 overexpression had shorter overall survival. Treatment with abemaciclib or palbociclib at 100 nM significantly decreased cell proliferation in all cell models evaluated. Both CDK4/6 inhibitors significantly induced G1 cell cycle arrest, thereby increasing cell senescence and increased the expression of interferon signalling pathway and tumour antigen presentation process in culture models of MPM. In vivo preclinical studies showed that palbociclib significantly reduced tumour growth and prolonged overall survival using distinct xenograft models of MPM implanted in athymic mice. CONCLUSIONS: Treatment of MPM with CDK4/6 inhibitors decreased cell proliferation, mainly by promoting cell cycle arrest at G1 and by induction of cell senescence. Our preclinical studies provide evidence for evaluating CDK4/6 inhibitors in the clinic for the treatment of MPM.

Lung endothelial cells are sensitive to epsilon toxin from Clostridium perfringens.

The pore-forming protein epsilon toxin (Etx) from Clostridium perfringens produces acute perivascular edema affecting several organs, especially the brain and lungs. Despite the toxin evident effect on microvasculature and endothelial cells, the underlying molecular and cellular mechanisms remain obscure. Moreover, no Etx-sensitive endothelial cell model has been identified to date. Here, we characterize the mouse lung endothelial cell line 1G11 as an Etx-sensitive cell line and compare it with the well-characterized Etx-sensitive Madin-Darby canine kidney epithelial cell line. Several experimental approaches, including morphological and cytotoxic assays, clearly demonstrate that the 1G11 cell line is highly sensitive to Etx and show the specific binding, oligomerization, and pore-forming activity of the toxin in these cells. Recently, the myelin and lymphocyte (MAL) protein has been postulated as a putative receptor for Etx. Here, we show the presence of Mal mRNA in the 1G11 cell line and the presence of the MAL protein in the endothelium of some mouse lung vessels, supporting the hypothesis that this protein is a key element in the Etx intoxication pathway. The existence of an Etx-sensitive cell line of endothelial origin would help shed light on the cellular and molecular mechanisms underlying Etx-induced edema and its consequences.

Targeted sequencing with a customized panel to assess histological typing in endometrial carcinoma.

The two most frequent types of endometrial cancer (EC) are endometrioid (EEC) and serous carcinomas (SC). Differential diagnosis between them is not always easy. A subset of endometrial cancers shows misleading microscopical features, which cause problems in differential diagnosis, and may be a good scenario for next-generation sequencing. Previous studies have assessed the usefulness of targeted sequencing with panels of generic cancer-associated genes in EC histological typing. Based on the analysis of TCGA (The Cancer Genome Atlas), EEC and SC have different mutational profiles. In this proof of principle study, we have performed targeted sequencing analysis with a customized panel, based on the TCGA mutational profile of EEC and SC, in a series of 24 tumors (16 EEC and 8 SC). Our panel comprised coding and non-coding sequences of the following genes: ABCC9, ARID1A, ARID5B, ATR, BCOR, CCND1, CDH19, CHD4, COL11A1, CSDE1, CSMD3, CTCF, CTNNB1, EP300, ERBB2, FBXW7, FGFR2, FOXA2, KLLN, KMT2B, KRAS, MAP3K4, MKI67, NRAS, PGAP3, PIK3CA, PIK3R1, PPP2R1A, PRPF18, PTEN, RPL22, SCARNA11, SIN3A, SMARCA4, SPOP, TAF1, TP53, TSPYL2, USP36, and WRAP53. Targeted sequencing validation by Sanger sequencing and immunohistochemistry was performed in a group of genes. POLE mutation status was assessed by Sanger sequencing. The most mutated genes were PTEN (93.7%), ARID1A (68.7%), PIK3CA (50%), and KMT2B (43.7%) for EEC, and TP53 (87.5%), PIK3CA (50%), and PPP2R1A (25%) for SC. Our panel allowed correct classification of all tumors in the two categories (EEC, SC). Coexistence of mutations in PTEN, ARID1A, and KMT2B was diagnostic of EEC. On the other hand, absence of PTEN, ARID1A, and KMT2B mutations in the presence of TP53 mutation was diagnostic of SC. This proof of concept study demonstrates the suitability of targeted sequencing with a customized endometrial cancer gene panel as an additional tool for confirming histological typing.