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.

Current management of liver metastases from gastric cancer: what is common practice? New challenge of EORTC and JCOG.

The role of multidisciplinary treatment including surgery for liver metastases from gastric cancer (LMGC) is controversial. Studies to answer this clinical question are increasing in number, but all published data thus far are based on retrospective analyses with limited sample sizes. Thus, the European Organisation for Research and Treatment of Cancer (EORTC) Gastrointestinal Tract Cancer Group (GITCG) and the Japan Clinical Oncology Group (JCOG) Stomach Cancer Study Group (SCSG) initiated a collaboration to develop an optimal treatment strategy for LMGC. Before planning a prospective study, a questionnaire was sent out to the network members of both groups in June 2016 to clarify current common practice in each region. Sixty-seven sites from 17 countries in the EORTC network and 55 sites from Japan responded. According to the survey, for patients with resectable LMGC without extrahepatic metastases, preoperative chemotherapy followed by resection of both primary (if still in place) and liver lesions was the preferred option for both the synchronous and the metachronous setting. For patients with unresectable LMGC, most of the sites recommended chemotherapy only. In this article, the detailed results of this survey are reported, shedding light on current community practice, and a joint EORTC-JCOG strategy of investigation is delineated.

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.

Candidate locus analysis of the TERT-CLPTM1L cancer risk region on chromosome 5p15 identifies multiple independent variants associated with endometrial cancer risk.

Several studies have reported associations between multiple cancer types and single-nucleotide polymorphisms (SNPs) on chromosome 5p15, which harbours TERT and CLPTM1L, but no such association has been reported with endometrial cancer. To evaluate the role of genetic variants at the TERT-CLPTM1L region in endometrial cancer risk, we carried out comprehensive fine-mapping analyses of genotyped and imputed SNPs using a custom Illumina iSelect array which includes dense SNP coverage of this region. We examined 396 SNPs (113 genotyped, 283 imputed) in 4,401 endometrial cancer cases and 28,758 controls. Single-SNP and forward/backward logistic regression models suggested evidence for three variants independently associated with endometrial cancer risk (P = 4.9 × 10(-6) to P = 7.7 × 10(-5)). Only one falls into a haplotype previously associated with other cancer types (rs7705526, in TERT intron 1), and this SNP has been shown to alter TERT promoter activity. One of the novel associations (rs13174814) maps to a second region in the TERT promoter and the other (rs62329728) is in the promoter region of CLPTM1L; neither are correlated with previously reported cancer-associated SNPs. Using TCGA RNASeq data, we found significantly increased expression of both TERT and CLPTM1L in endometrial cancer tissue compared with normal tissue (TERT P = 1.5 × 10(-18), CLPTM1L P = 1.5 × 10(-19)). Our study thus reports a novel endometrial cancer risk locus and expands the spectrum of cancer types associated with genetic variation at 5p15, further highlighting the importance of this region for cancer susceptibility.

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.

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.

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.

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.

Addition of IMP3 to L1CAM for discrimination between low- and high-grade endometrial carcinomas: a European Network for Individualised Treatment of Endometrial Cancer collaboration study.

Discrimination between low- and high-grade endometrial carcinomas (ECs) is clinically relevant but can be challenging for pathologists, with moderate interobserver agreement. Insulin-like growth factor-II mRNA-binding protein 3 (IMP3) is an oncofoetal protein that is associated with nonendometrioid endometrial carcinomas but has been limited studied in endometrioid carcinomas. The aim of this study is to investigate the diagnostic and prognostic value of IMP3 in the discrimination between low- and high-grade ECs and its added value to L1CAM. IMP3 and L1CAM expression was assessed in tumors from 378 patients treated for EC at 1 of 9 participating European Network for Individualised Treatment of Endometrial Cancer centers. IMP3 was expressed in 24.6% of the tumors. In general, IMP3 was more homogeneously expressed than L1CAM. IMP3 expression was significantly associated with advanced stage, nonendometrioid histology, grade 3 tumors, deep myometrial invasion, lymphovascular space invasion, distant recurrences, overall mortality, and disease-related mortality. Simultaneous absence of IMP3 and L1CAM expression showed the highest accuracy for identifying low-grade carcinomas (area under the curve 0.766), whereas simultaneous expression of IMP3 and L1CAM was strongly associated with high-grade carcinomas (odds ratio 19.7; 95% confidence interval 9.2-42.2). Even within endometrioid carcinomas, this combination remained superior to IMP3 and L1CAM alone (odds ratio 8.6; 95% confidence interval 3.4-21.9). In conclusion, IMP3 has good diagnostic value and together with L1CAM represents the optimal combination of diagnostic markers for discrimination between low- and high-grade ECs compared to IMP3 and L1CAM alone. Because of the homogenous expression of IMP3, this marker might be valuable in preoperative biopsies when compared to the more patchy L1CAM expression.

Recurrent PPP2R1A Mutations in Uterine Cancer Act through a Dominant-Negative Mechanism to Promote Malignant Cell Growth.

Somatic missense mutations in the Ser/Thr protein phosphatase 2A (PP2A) Aα scaffold subunit gene PPP2R1A are among the few genomic alterations that occur frequently in serous endometrial carcinoma (EC) and carcinosarcoma, two clinically aggressive subtypes of uterine cancer with few therapeutic options. Previous studies reported that cancer-associated Aα mutants exhibit defects in binding to other PP2A subunits and contribute to cancer development by a mechanism of haploinsufficiency. Here we report on the functional significance of the most recurrent PPP2R1A mutations in human EC, which cluster in Aα HEAT repeats 5 and 7. Beyond predicted loss-of-function effects on the formation of a subset of PP2A holoenzymes, we discovered that Aα mutants behave in a dominant-negative manner due to gain-of-function interactions with the PP2A inhibitor TIPRL1. Dominant-negative Aα mutants retain binding to specific subunits of the B56/B' family and form substrate trapping complexes with impaired phosphatase activity via increased recruitment of TIPRL1. Accordingly, overexpression of the Aα mutants in EC cells harboring wild-type PPP2R1A increased anchorage-independent growth and tumor formation, and triggered hyperphosphorylation of oncogenic PP2A-B56/B' substrates in the GSK3ß, Akt, and mTOR/p70S6K signaling pathways. TIPRL1 silencing restored GSK3ß phosphorylation and rescued the EC cell growth advantage. Our results reveal how PPP2R1A mutations affect PP2A function and oncogenic signaling, illuminating the genetic basis for serous EC development and its potential control by rationally targeted therapies. Cancer Res; 76(19); 5719-31. ©2016 AACR.

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.

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 r(2) = 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.

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.

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.