Stefanie Schrauwen

Mismatch repair deficiency endows tumors with a unique mutation signature and sensitivity to DNA double-strand breaks

DNA replication errors that persist as mismatch mutations make up the molecular fingerprint of mismatch repair (MMR)-deficient tumors and convey them with resistance to standard therapy. Using whole-genome and whole-exome sequencing, we here confirm an MMR-deficient mutation signature that is distinct from other tumor genomes, but surprisingly similar to germ-line DNA, indicating that a substantial fraction of human genetic variation arises through mutations escaping MMR. Moreover, we identify a large set of recurrent indels that may serve to detect microsatellite instability (MSI). Indeed, using endometrial tumors with immunohistochemically proven MMR deficiency, we optimize a novel marker set capable of detecting MSI and show it to have greater specificity and selectivity than standard MSI tests. Additionally, we show that recurrent indels are enriched for the ‘DNA double-strand break repair by homologous recombination’ pathway. Consequently, DSB repair is reduced in MMR-deficient tumors, triggering a dose-dependent sensitivity of MMR-deficient tumor cultures to DSB inducers. DOI: http://dx.doi.org/10.7554/eLife.02725.001

Five endometrial cancer risk loci identified through genome-wide association analysis

We conducted a meta-analysis of three endometrial cancer genome-wide association studies (GWAS) and two follow-up phases totaling 7,737 endometrial cancer cases and 37,144 controls of European ancestry. Genome-wide imputation and meta-analysis identified five new risk loci of genome-wide significance at likely regulatory regions on chromosomes 13q22.1 (rs11841589, near KLF5), 6q22.31 (rs13328298, in LOC643623 and near HEY2 and NCOA7), 8q24.21 (rs4733613, telomeric to MYC), 15q15.1 (rs937213, in EIF2AK4, near BMF) and 14q32.33 (rs2498796, in AKT1, near SIVA1). We also found a second independent 8q24.21 signal (rs17232730). Functional studies of the 13q22.1 locus showed that rs9600103 (pairwise r2 = 0.98 with rs11841589) is located in a region of active chromatin that interacts with the KLF5 promoter region. The rs9600103[T] allele that is protective in endometrial cancer suppressed gene expression in vitro, suggesting that regulation of the expression of KLF5, a gene linked to uterine development, is implicated in tumorigenesis. These findings provide enhanced insight into the genetic and biological basis of endometrial cancer.

CYP19A1 fine-mapping and Mendelian randomization: estradiol is causal for endometrial cancer

Candidate gene studies have reported CYP19A1 variants to be associated with endometrial cancer and with estradiol (E2) concentrations. We analyzed 2937 single nucleotide polymorphisms (SNPs) in 6608 endometrial cancer cases and 37 925 controls and report the first genome wide-significant association between endometrial cancer and a CYP19A1 SNP (rs727479 in intron 2, P=4.8×10−11). SNP rs727479 was also among those most strongly associated with circulating E2 concentrations in 2767 post-menopausal controls (P=7.4×10−8). The observed endometrial cancer odds ratio per rs727479 A-allele (1.15, CI=1.11–1.21) is compatible with that predicted by the observed effect on E2 concentrations (1.09, CI=1.03–1.21), consistent with the hypothesis that endometrial cancer risk is driven by E2. From 28 candidate-causal SNPs, 12 co-located with three putative gene-regulatory elements and their risk alleles associated with higher CYP19A1 expression in bioinformatical analyses. For both phenotypes, the associations with rs727479 were stronger among women with a higher BMI (Pinteraction=0.034 and 0.066 respectively), suggesting a biologically plausible gene-environment interaction.

L1CAM expression in endometrial carcinomas: an ENITEC collaboration study

Background:Identification of aggressive endometrioid endometrial carcinomas (EECs) and non-endometrioid carcinomas (NEECs) is essential to improve outcome. L1 cell adhesion molecule (L1CAM) expression is a strong prognostic marker in stage I EECs, but less is known about L1CAM expression in advanced-stage EECs and NEECs. This study analyses L1CAM expression in a clinically representative cohort of endometrial carcinomas.Methods:The expression of L1CAM was immunohistochemically determined in 1199 endometrial carcinomas, treated at one of the European Network for Individualized Treatment of Endometrial Cancer (ENITEC) centres. Staining was considered positive when >10% of the tumour cells expressed L1CAM. The association between L1CAM expression and several clincopathological characteristics and disease outcome was calculated.Results:In all, L1CAM was expressed in 10% of the 935 stage I EECs, 18% of the 160 advanced stage EECs, and 75% of the 104 NEECs. The expression of L1CAM was associated with advanced stage, nodal involvement, high tumour grade, non-endometrioid histology, lymphovascular space invasion, and distant recurrences in all cases, and with reduced survival in the EECs, but not in the NEECs.Conclusions:The expression of L1CAM is a strong predictor of poor outcome in EECs, but not NEECs. It is strongly associated with non-endometrioid histology and distant spread, and could improve the postoperative selection of high-risk endometrial carcinomas. The value of L1CAM expression in the preoperative selection of high-risk endometrial carcinomas should be studied.

Characterization of patient-derived tumor xenograft models of endometrial cancer for preclinical evaluation of targeted therapies.

OBJECTIVE Endometrial carcinoma (EC) is the sixth most common cancer in women and therapies are limited for advanced and recurrent disease. Patient-derived tumor xenograft (PDTX) models are becoming popular tools in translational research because of their histological and genetic similarity to the original tumors and the ability to predict therapeutic response to treatments. Here, we established and characterized a panel of 24 EC PDTX models which includes the major histological and genetic subtypes observed in patients. METHODS Fresh tumor tissues collected from primary, metastatic and recurrent type I and type II EC patients were engrafted in immunocompromised mice. Histology, vimentin, and cytokeratin expression were evaluated, together with Microsatellite instability (MSI), mutation profiling by Whole Exome Sequencing and copy number profiling by Whole Genome Low Coverage Sequencing. The efficacy of both PI3K and MEK inhibitors was evaluated in a model of endometrioid carcinoma harboring PTEN, PIK3CA and KRAS mutations. RESULTS We observed good similarity between primary tumors and the corresponding xenografts, at histological and genetic level. Among the engrafted endometrioid models, we found a significant enrichment of MSI and POLE mutated tumors, compared to non-engrafted samples. Combination treatment with NVP-BEZ235 and AZD6244 showed the possibility to stabilize the tumor growth in one model originated from a patient who already received several lines of chemotherapy. CONCLUSION The established EC PDTX models, resembling the original human tumors, promise to be useful for preclinical evaluation of novel combination and targeted therapies in specific EC subgroups.

Dual blockade of PI3K/AKT/mTOR (NVP-BEZ235) and Ras/Raf/MEK (AZD6244) pathways synergistically inhibit growth of primary endometrioid endometrial carcinoma cultures, whereas NVP-BEZ235 reduces tumor growth in the corresponding xenograft models

OBJECTIVES Endometrial carcinoma (EC) is the most common gynecological cancer in the Western World. Treatment options are limited for advanced and recurrent disease. Therefore, new treatment options are necessary. Inhibition of the PI3K/AKT/mTOR and/or the Ras/Raf/MEK pathways is suggested to be clinically relevant. However, the knowledge about the effect of combination targeted therapy in EC is limited. The aim of this study was to investigate the effect of these therapies on primary endometrioid EC cell cultures in vitro and in vivo. METHODS Primary endometrioid EC cell cultures were incubated with Temsirolimus (mTORC1 inhibitor), NVP-BKM120 (pan-PI3K inhibitor), NVP-BEZ235 (pan-PI3K/mTOR inhibitor), or AZD6244 (MEK1/2 inhibitor) as single treatment. In vitro, the effect of NVP-BEZ235 with or without AZD6244 was determined for cell viability, cell cycle arrest, apoptosis induction, and cell signaling. In vivo, the effect of NVP-BEZ35 was investigated for 2 subcutaneous xenograft models of the corresponding primary cultures. RESULTS NVP-BEZ235 was the most potent PI3K/AKT/mTOR pathway inhibitor. NVP-BEZ235 and AZD6244 reduced cell viability and induced cell cycle arrest and apoptosis, by reduction of p-AKT, p-S6, and p-ERK levels. Combination treatment showed a synergistic effect. In vivo, NVP-BEZ235 reduced tumor growth and inhibited p-S6 expression. The effects of the compounds were independent of the mutation profile of the cell cultures used. CONCLUSIONS A synergistic antitumor effect was shown for NVP-BEZ235 and AZD6244 in primary endometrioid EC cells in vitro. In addition, NVP-BEZ235 induced reduction of tumor growth in vivo. Therefore, targeted therapies seem an interesting strategy to further evaluate in clinical trials.

Increased expression of placental growth factor in high-grade endometrial carcinoma

Placental growth factor (PlGF), a homolog of vascular endothelial growth factor (VEGF), exerts pleiotropic functions in cancer by affecting tumor cells as well as endothelial and inflammatory cells. Moreover, PlGF expression correlates with tumor stage, recurrence, metastasis and patient outcome in different types of cancer. Recently, administration of anti-PlGF therapy reduced tumor growth and metastasis in preclinical tumor models. In the present study, we evaluated the diagnostic and prognostic value of systemic and local expression of PlGF in primary endometrial carcinomas. PlGF levels in tumor lysates (n=128) and serum (n=88) of patients with primary endometrial cancer were determined using ELISA. PlGF mRNA expression in endometrial carcinoma tissues was quantified by quantitative qRT-PCR. Results were compared to endometrial cancer stage and grade. Systemic PlGF levels were not altered in patients with endometrial cancer (FIGO stage I-II-III) as compared to healthy controls. Only in FIGO stage IV patients, serum PlGF levels were slightly increased. Local PlGF mRNA and protein expression in endometrial tumors progressively increased with tumor grade. In endometrioid carcinomas, PlGF mRNA expression was significantly increased in endometrioid grade 3 tumors as compared to normal endometrial tissue. PlGF protein expression was significantly increased in endometrioid grade 2 and 3 carcinomas and in serous carcinomas as compared to normal endometrial tissue. Our study showed that systemic/serum PlGF levels cannot be used as a diagnostic or prognostic marker in endometrial cancer. However, the increased local expression of PlGF, primarily in high-grade carcinomas, underscores the possibility for preclinical assessment of anti-PlGF therapy in endometrial cancer.

Microsatellite instable and microsatellite stable primary endometrial carcinoma cells and their subcutaneous and orthotopic xenografts recapitulate the characteristics of the corresponding primary tumor

Objective Well-characterized, low-passage, primary cell cultures established directly from patient tumors are an important tool for drug screening because these cultures faithfully recapitulate the genomic features of primary tumors. Here, we aimed to establish these cell cultures from primary endometrial carcinomas (ECs) and to develop subcutaneous and orthotopic xenograft models as a model to validate promising treatment options for EC in the in vivo setting. Methods Primary cell cultures of EC tumors were established and validated by analysing histologic and genetic characteristics, telomerase activity, and in vitro and in vivo growth characteristics. Using these primary cell cultures, subcutaneous and orthotopic mouse models were subsequently established. Results We established and characterized 7 primary EC cell cultures and corresponding xenograft models of different types of endometrioid tumors. Interestingly, we observed that the chance to successfully establish a primary cell culture seems higher for microsatellite instable than microsatellite stable tumors. For the first time, we also established an orthotopic murine model for EC derived from a primary cell culture. In contrast to EC cell lines, grafted tumor cultures preserved the original tumor structure and mimicked all histologic features. They also established abdominal and distant metastases, reflecting the tumorigenic behavior in the clinical setting. Remarkably, the established cell cultures and xenograft tumors also preserved the genetic characteristics of the primary tumor. Conclusions The established EC cultures reflect the epithelial genetic characteristics of the primary tumor. Therefore, they provide an appropriate model to investigate EC biology and apply high-throughput drug screening experiments. In addition, the established murine xenograft models, in particular the orthotopic model, will be useful to validate promising therapeutic strategies in vivo, as the grafted tumors closely resemble the primary tumors from which they were derived. Microsatellite instable status seems to determine the success rate of establishing primary cell cultures.

The antitumor effect of metformin with and without carboplatin on primary endometrioid endometrial carcinoma in vivo

OBJECTIVES New treatment options for advanced and recurrent endometrial carcinoma (EC) are necessary. Epidemiological studies showed that diabetic patients using metformin have reduced risks of endometrial cancer (EC) incidence. Moreover, pre- and clinical studies demonstrated an antitumor effect by metformin, with and without additional treatments, for different solid malignancies. However, cancer cell-autonomous effects of metformin on EC have not been fully characterized yet. The aim of this study was to investigate the effect of metformin, with or without carboplatin, on patient-derived primary endometrioid EC cells xenografted in nude mice, to assess its ability to reduce or impair growth in already established tumors. METHODS Two xenograft models were established by subcutaneous inoculation of primary endometrioid EC cell suspensions. Tumors were allowed to grow and then mice were treated with metformin (250 mg/kg, daily, p.o.), carboplatin (50 mg/kg, 1×/week, i.p.), or the combination of both compounds at the same concentration as single treatment, for three weeks. Effects of metformin treatment on the tumor mass were determined by tumor growth follow-up. Metformin influences on AMPK/mTOR cell signaling were evaluated by investigating AKT, AMPK and S6 phosphorylation levels. RESULTS In vivo, metformin did not affect the growth of EC tumors established from patient-derived primary cultures and the phosphorylation of AKT, AMPK and S6. In addition, no enhanced antitumor effect was determined by combining metformin and carboplatin treatments. CONCLUSIONS Metformin, at clinically relevant concentrations, did not show effects on the growth of already established tumors. Adding metformin to carboplatin did not have synergistic effects.

Added Value of Estrogen Receptor, Progesterone Receptor, and L1 Cell Adhesion Molecule Expression to Histology-Based Endometrial Carcinoma Recurrence Prediction Models: An ENITEC Collaboration Study

Objectives Endometrial carcinoma mortality is mainly caused by recurrent disease, and various immunohistochemical markers to predict recurrences have been studied. Loss of the estrogen receptor (ER) and progesterone receptor (PR) and the presence of the L1 cell adhesion molecule (L1CAM) are promising markers, but their combined value has not been studied. Materials and Methods Expression of ER, PR, and L1CAM was immunohistochemically determined in 293 endometrial carcinomas from 11 collaborating European Network for Individualized Treatment of Endometrial Cancer centers. Estrogen receptor, PR, or L1CAM staining was considered positive or negative when expressed by greater than or equal to 10% or less than 10% of the tumor cells, respectively. The association between these markers and clinicopathological markers, and their combined value in predicting survival were calculated, both in the entire cohort and in a selected groups of stage I endometrioid and low-risk stage I endometrioid carcinomas. Results Estrogen receptor and PR were negative in 19% and 28% of the cases, respectively, and L1CAM was positive in 18%. All 3 were associated with advanced stage, high-grade, nonendometrioid histology, lymphovascular space invasion (LVSI), and reduced disease-free survival. Only advanced stage, loss of PR, and LVSI were associated with reduced disease-free survival in multivariate analysis. A prognostic model including these 3 markers was superior to 1 including only the 3 immunohistochemical markers, which was superior to the traditional model. In both the stage I endometrioid and the low-risk stage I endometrioid groups, only loss of PR was associated with reduced disease-free survival. Conclusions Loss of ER and PR, and the presence of L1CAM are associated with high risk characteristics, and loss of PR is the strongest predictor of recurrent disease. Although a combination of these 3 markers is slightly superior to the traditional histological markers, a prognostic model including stage, PR expression, and LVSI is the most promising model in the identification of high risk carcinomas. In the stage I endometrioid carcinomas, PR immunohistochemistry appears to be of additional value in predicting recurrences.