High-Throughput Mutation Profiling of Primary and Metastatic Endometrial Cancers Identifies KRAS, FGFR2 and PIK3CA to Be Frequently Mutated

Background Despite being the most common pelvic gynecologic malignancy in industrialized countries, no targeted therapies are available for patients with metastatic endometrial carcinoma. In order to improve treatment, underlying molecular characteristics of primary and metastatic disease must be explored. Methodology/Principal Findings We utilized the mass spectrometric-based mutation detection technology OncoMap to define the types and frequency of point somatic mutations in endometrial cancer. 67 primary tumors, 15 metastases corresponding to 7 of the included primary tumors and 11 endometrial cancer cell lines were screened for point mutations in 28 known oncogenes. We found that 27 (40.3%) of 67 primary tumors harbored one or more mutations with no increase in metastatic lesions. FGFR2, KRAS and PIK3CA were consistently the most frequently mutated genes in primary tumors, metastatic lesions and cell lines. Conclusions/Significance Our results emphasize the potential for targeting FGFR2, KRAS and PIK3CA mutations in endometrial cancer for development of novel therapeutic strategies.


Introduction
Despite being the most common pelvic gynecologic malignancy in industrialized countries, no targeted therapies are available for patients with metastatic endometrial carcinoma. Although 75% are treated at an early stage, 15% to 20% recur. For patients with advanced disease at diagnosis or recurrent disease, outcome is poor. In order to improve treatment, underlying molecular characteristics of primary and metastatic disease must be explored. Furthermore, improved tools for correct stratification of patients according to risk-groups and improved definitions of potential targets for novel therapeutics are of great importance and much work is undertaken to develop better criteria to select patients for individualized therapies [1].
To assess the risk of recurrent disease, traditionally endometrial cancer has been divided into two subgroups, type I and type II carcinomas [2]. Type I endometrial carcinoma is associated with good prognosis, low grade, endometrioid histology and rarely metastasize to regional and distant sites [3]. In addition, type I endometrial cancers are often hormone receptor positive with PTEN and KRAS mutations. Type II endometrial carcinomas are associated with poor prognosis, non-endometrioid histology, high grade, loss of hormone receptors and altered expression of p53 and p16. Still, the value of this classification to predict prognosis and for treatment stratification is limited as 20% of type I endometrial cancers recur and 50% of type II cancers do not [4].
Currently, conventional chemotherapy regimens and antihormonal treatment are basis for adjuvant and systemic treatment of recurrent or metastatic endometrial cancer as targeted therapies are not yet available in the clinic. However, mutational profiles are applied for selection of targeted therapeutics for several other cancers and also applied for clinical trials stratification. Our previous screening of a smaller number of endometrial cancer patients identified somatic mutations in FGFR2, KRAS, PIK3CA, PTEN, PT53 and CTNNB1 [5]. However, this study did not rule out possible mutations in other known oncogenes that could be potentially interesting for targeted treatment of endometrial cancer. Thus, the current study was undertaken to screen for a large panel of known oncogenic mutations in a series of primary and metastatic lesions from endometrial cancer patients using the high-throughput method OncoMap [6,7]. OncoMap provides a unique opportunity to simultaneously interrogate a large number of known mutations in a large number of genes, thus providing the opportunity to characterize the molecular subgroups of endometrial cancer with a potential relevance for targeting novel therapeutics.

Ethics statement
All parts of the study have been approved according to Norwegian legislation as well as international demands for ethical review. The study was approved by the Norwegian Data Inspectorate, Norwegian Social Sciences Data Services, and the Western Regional Committee for Medical and Health Research Ethics, REC West (NSD15501; REK 052.01). Patients were included in the study after written informed consent approved by the ethics committee (REK West).

Specimens
We have studied a total of 69 patients for mutations in 28 known oncogenes ( Table 1). 23 of the included patients had previously been screened for fewer oncogene mutations by another method [5]. The patients were recruited from a population based patient series of 701 patients with endometrial cancer prospectively collected at Haukeland University Hospital, Norway. Age at diagnosis, FIGO stage, histological subtype and grade, treatment and follow-up was registered as previously reported [8]. Distribution of clinico-pathologic variable for the 69 investigated cases did not differ significantly from the larger (n = 701) unselected patient cohort ( Table 2). Tissue was available from 67 primary tumors and 15 metastatic lesions from 9 patients of which 7 had corresponding tissues from primary lesions available for comparison. The majority of selected lesions were verified by frozen sections to contain .80% malignant epithelial component with a minimum cut off for inclusion of 50% purity.
OncoMap and DNA sequencing DNA from primary and metastatic lesions was extracted from fresh frozen biopsies. DNA was isolated by digestion over night at 65uC in lysis buffer containing proteinase K, followed by a standard ethanol precipitation. DNA from 11 endometrial cancer cell lines was extracted using Qiagen Tissue DNA kit according to manufacturers protocol. DNA quantity was measured using the Quant-iT TM PicogreenH Assay (Invitrogen) and high quality of the DNA assured on a 0.7% agarose gel before genomic DNA was amplified using the Repli-g Midi Kit (Qiagen, Germany) according to manufacturers' instructions. Amplified DNA was diluted 1:10 in 1xTE buffer (pH 8.0) and after hydration for 24 h at room temperature further diluted to a working concentration of 5 ng/ml in water. Mutations were detected in genome-amplified DNA using a mass spectrometry-based single base extension technique (Sequenom, Inc.) as previously described [7]. Primers for additional assays to detect mutations described in several cancer studies since 2008 [9,10,11,12,13] were designed using the Sequenom Assay Design Software. Following amplification and mutation site specific probe elongation analytes were spotted on SpectroCHIPs I and masses detected using a Bruker matrixassisted laser desorption/ionization-time of flight mass spectrometer (Sequenom). Spectra were manually reviewed using the Typer 4.0 Software (Sequenom). A list of the mutations included in OncoMap and the corresponding amino acid changes is given in Table S1.
To validate the proportion of the most frequently mutated oncogenes detected by OncoMap, genomic DNA was extracted from freshly frozen primary tumor tissue from 199 additional patients. In total 264 patients were screened for point mutations in KRAS (exon 2) and PIK3CA (exon 9 and 20) as described [14]. Details regarding primers and conditions are available upon request. Sequencing reactions were analyzed on an ABI Prism 3100 genetic analyzer using the Sequencing Analysis software, version 3.7. Oligonucleotide DNA microarray analyses A microarray dataset corresponding to the 69 primary tumor samples included in the OncoMap screen was generated. RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and hybridized to Agilent Whole Human Genome Microarrays 44k (Cat.no. G4112F), according to the manufacturers instructions. Arrays were scanned using the Agilent Microarray Scanner Bundle and data were imported and analyzed in J-Express software (Molmine, Norway). Median spot signal was used as intensity measure. Expression data were normalized using quantile normalization. Microarraydata have been deposited in the ArrayExpress Archive database, http://www.ebi.ac.uk/ arrayexpress/ (ArrayExpress accession: E-MTAB-1358).
A SAM (Significance Analysis of Microarray) analysis between grade I-II and grade III was performed to identify significantly differentially expressed genes according to histologic grade. 306 genes were significantly differentially expressed (FDR,0.01) between the two groups. Hierarchical clustering was performed on this list of genes using weighted average linkage and Pearson correlation as similarity measures. Clinico-pathological data and mutational status were mapped manually to the cluster-tree to visualize the distribution of mutation across the patient population.

Results
The OncoMap screen for 387 oncogenic mutations in 28 commonly mutated genes in cancer (Table S1) (Table 3). FGFR2 mutation frequency had been validated previously [5]. The most common single mutation found by OncoMap screening was FGFR2 aaS252W (9.0%), however the most frequently mutated gene was KRAS (17.4%) ( Table 3). The OncoMap screen of the 11 established endometrial cancer cell lines identified as expected KRAS G12D and PIK3CA G1049R mutations in both Hec1A and Hec1B, while FGFR2 mutation S252W was found in MFE280 and MFE319. Additionally, two PIK3CA mutations were identified in MFE280 and MFE296 (E545K and P539R, respectively). We did not find any of the cell lines to have mutations in any of the other genes included in the OncoMap panel.
To explore a possible link between type of mutations and gene expression patterns in primary tumors, a hierarchical cluster analysis of 306 genes significantly differentially expressed (SAM analysis, FDR,0.01) according to histologic grade was performed. We found that there was no significant association between specific oncogene mutations and patient clusters based on transcriptional signatures (Figure 1). This finding appears to be in line with our previous report on a smaller data set applying an earlier generation of mRNA genearrays, with no enrichment for PIK3CA mutations in the patient cluster capturing aggressive phenotype [15].
To further investigate if mutation pattern changed during disease progression, 15 metastatic lesions from 9 patients from which seven had primary tumors available for comparison, were analyzed for mutations. KRAS, PIK3CA and FGFR2 were found to be the most frequently mutated genes also in metastatic lesions, with no significant increase in mutation frequency (Table 4). In two cases, mutations were detected in the metastatic lesions but not in the primary lesion, while one case with mutation in the primary lesion had no detectable mutation in the metastatic lesion. The small sample set available for this analysis, tumor heterogeneity and differences in stromal contamination should call for caution in the conclusions.

Discussion
Activating mutations in specific proto-oncogenes may confer oncogene-addiction. Such mutations have been identified in several genes and may drive malignant disease progression. This principal for oncogene-addiction can be exploited to develop new targeted therapies [16]. Currently, mutational profiles are applied for selection of targeted therapeutics for e.g. BRAF inhibitors in malignant melanoma [17] and BRAF and EGFR targeting in lung-and colorectal cancers [18,19]. For endometrial cancer, none of the novel targeted therapeutics is available in the clinic at present. However, several ongoing clinical trials aim at exploiting   targets supported by recent comprehensive molecular profiling of primary endometrial carcinoma lesion [1], dominated by trials targeting the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) or FGFR2. However, to our knowledge, no previous study has reported as comprehensive mutational data for a large panel of oncogenes in endometrial cancers including metastatic lesions. A large number of oncogene mutations has been identified to be important in cancer development. Recently, several papers have reported the usefulness of the high-throughput genotyping platform OncoMap to screen for mutations in a large panel of known cancer oncogenes [6,20,21,22]. The high degree of concordance between our findings using OncoMap for the investigated genes and the validated frequency in the present study as well as previously published mutation frequencies in endometrial cancer samples based on traditional sequencing, is assuring. Using OncoMap we found that 40.3% of the analyzed endometrial cancer samples harbored at least one mutation. Of the 28 oncogenes included, mutations were only found at high frequency (.10%) in KRAS, PIK3CA and FGFR2. These genes have been linked to endometrial cancer previously, both by us [5,15] and others [23,24].
In the present study, the S252W mutation in FGFR2 was identified as the most frequent single mutation (9%) in endometrial cancer. The somatic FGFR2 mutations include the S252W and P253R alleles, where autosomal dominant mutations are associated with the congenital developmental disorder Apert syndrome [25]. We, and others [26], have previously linked these mutations to endometrial cancer, through increased tumor cell survival and anchoring independent growth in endometrial cancer cell lines, and indicated the potential for FGFR2 inhibitors in mutated cell lines [5]. It has also been reported that FGFR2 inhibitors induce cell death in endometrial cancer cells despite PTEN inactivating mutations [27]. The frequency of FGFR2 mutations detected in the present OncoMap screen of 10.4% is in concordance with our previous findings from 122 endometrial cancer patients from the same region, finding FGFR2 to be mutated in 12.3% [5]. Recently, a frequency in this range of 10.3% was also published by others [23].
Several of the PIK3CA mutations were detected at relatively low frequencies (,3%), however the total frequency of any detected PIK3CA mutations was 13.4%. We have validated this frequency of point mutations in PIK3CA (exon 9 and 20) in 14.6% in a cohort of 264 endometrial cancer patients. This is consistent with the reported mutational frequency of PIK3CA in endometrial carcinoma in the COSMIC database for PIK3CA mutations tested for in OncoMap [28]. A potential relevance for targeting therapy in patients harboring PIK3CA mutations was recently supported in a study demonstrating higher response rate to PI3K/AKT/mTOR inhibitors for patients with mutated compared to wild type PIK3CA in breast and gynecologic malignancies [29].
KRAS mutations were found in 17.9% of the cases, with high frequency of point mutations in exon 2 (G12A, G12C, G12D and G13D), validated in 264 endometrial cancer patients (14,7%; [14]) and also in line with previous studies (18%; [30]). KRAS mutations have been associated with low grade, and endometrioid histologic subtype, although not with prognosis [31,32]. Interestingly, KRAS and FGFR2 mutations were found to be mutually exclusive, in line with a previous report [23]. In terms of therapy KRAS mutational status has been linked to EGFR inhibitor resistance in colorectal cancer [33], but further studies are needed in endometrial carcinoma to explore such potential link.
In line with the present study, we previously reported a low frequency (2%) of mutations in BRAF in endometrial cancer [30]. Interestingly, with the exception of a few mutations in NRAS, HRAS, EGFR and BRAF (1.5%), no other hot-spot mutations were identified in the remaining 21 oncogenes screened for, neither in primary tumors nor in metastatic lesions.
The present work used a version of OncoMap covering 387 mutations in a total of 28 different oncogenes. In endometrial carcinomas, the oncogenes CTNNB1 and tumor suppressor genes PTEN and P53 have also been reported to be frequently mutated [34,35] but were not included in the present sceen and can therefore not be accounted for. Among the included genes and mutations, we have identified and validated KRAS, PIK3CA and FGFR2 to be the most frequently mutated oncogenes in endometrial cancer. Although transcriptional signature pattern according to histologic grade did not identify any distinct subgroups linking any of the mutations to phenotype, PIK3CA, KRAS and FGFR2 mutations may still be of relevance for targeting novel therapeutics in endometrial cancer. Nevertheless, more knowledge regarding functional aspects of the different mutations and their implications for response to drugs will be important to guide further selection of patients for molecularly based clinical trials.