Genetic alterations in endometrial cancer by targeted next-generation sequencing

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Abstract

Many genetic factors play important roles in the development of endometrial cancer. The aim of this study was to investigate genetic alterations in the Taiwanese population with endometrial cancer. DNA was extracted from 10 cases of fresh-frozen endometrial cancer tissue. The exomes of cancer-related genes were captured using the NimbleGen Comprehensive Cancer Panel (578 cancer-related genes) and sequenced using the Illumina Genomic Sequencing Platform. Our results revealed 120 variants in 99 genes, 21 of which were included in the Oncomine Cancer Research Panel used in the National Cancer Institute Match Trial. The 21 genes comprised 8 tumor suppressor candidates (ATM, MSH2, PIK3R1, PTCH1, PTEN, TET2, TP53, and TSC1) and 13 oncogene candidates (ALK, BCL9, CTNNB1, ERBB2, FGFR2, FLT3, HNF1A, KIT, MTOR, PDGFRA, PPP2R1A, PTPN11, and SF3B1). We identified a high frequency of mutations in PTEN (50%) and genes involved in the endometrial cancer-related molecular pathway, which involves the IL-7 signaling pathway (PIK3R1, n = 1; AKT2, n = 1; FOXO1, n = 1). We report the mutational landscape of endometrial cancer in the Taiwanese population. We believe that this study will shed new light on fundamental aspects for understanding the molecular pathogenesis of endometrial cancer and may aid in the development of new targeted therapies.

Introduction

Endometrial cancer is the fourth most common gynecological malignancy in Europe and the United States. More than 280,000 women are diagnosed each year worldwide and 74,000 women die from endometrial cancer annually. It is also the second most common gynecological cancer in Taiwan.

Endometrial cancer is broadly divided into two groups: endometrioid carcinoma and uterine serous carcinoma (Bokhman, 1983). Close to 80–90% of endometrial cancers are endometrioid carcinomas and 2–10% are uterine serous carcinomas (Dedes et al., 2011). Although uterine serous carcinoma is in the minority, it is much more aggressive than endometrioid carcinoma and has a poor outcome (del Carmen et al., 2012, Hamilton et al., 2006). Therefore, understanding the genomic alterations associated with this disease may provide opportunities for genome-guided clinical trials and drug development.

Next-generation sequencing (NGS) technology has emerged as a powerful tool to investigate the genetic etiology of diseases. NGS has been applied in a variety of ways, such as whole genome sequencing, targeted capture, high-throughput RNA sequencing, and chromatin immunoprecipitation followed by sequencing. The ability to generate a huge quantity of sequencing data also presents the challenge of deciding which variants to validate. Several organizations, such as the College of American Pathologists, Centers for Disease Control and Prevention, and U.S. Food and Drug Administration, have published guidelines for clinical NGS analysis.

Over the past two years (2012–2014), a number of studies have reported the genomic landscapes of endometrial cancer. The first study to characterize the genomic landscape of uterine serous carcinoma was described by Kuhn et al. (2012). Among the most frequently mutated genes in 10 uterine serous carcinomas were TP53, PIK3CA, FBXW7, and PPP2R1A. Subsequently, Gallo et al. found frequent somatic mutations not only in FBXW7, but also in ubiquitin ligase complex and chromatin remodeling genes (Le Gallo et al., 2012). Zhao et al. then decoded the exomes of a uterine serous carcinoma cohort five times larger than those reported earlier. They identified a significantly increased burden of mutation in 14 genes, including the previously reported and well-known cancer genes TP53, PIK3CA, PPP2R1A, KRAS, PTEN, FBXW7, and CDKN1A (Zhao et al., 2013). The Cancer Genome Atlas (TCGA) project revealed 14 pathogenic driver genes of uterine serous carcinoma (TP53, PIK3CA, FBXW7, PPP2R1A, CHD4, CSMD3, SLC9A11, PTEN, COL11A1, PRPF18, SPOP, CDH19, HIST1H2AM, and CELP) (Kandoth et al., 2013).

The first whole exome sequencing study of endometrioid carcinoma investigated 13 cases. Ten tumor suppressor genes (ARID1A, INHBA, KMO, TTLL5, GRM8, IGFBP3, AKTIP, PHKA2, TRPS1, and WNT11) and two oncogenes (ERBB3 and RPS6KC1) were identified as potential candidate driver genes (Liang et al., 2012). In addition, the frequent occurrence of mutations in PTEN (64%), PIK3CA (59%), ARID1A (55%), CTNNB1 (32%), MLL2 (32%), FBXW7 (27%), RNF43 (27%), APC (23%), FGFR2 (18%), and EGFR (14%) among endometrioid carcinomas was confirmed by Kinde et al. based on the exome sequencing results of 22 cases (Kinde et al., 2013).

The aim of the current study was to identify genetic alterations in endometrial cancer in the Taiwanese population. We performed deep sequencing (> 500 ×) to detect the mutational status in 578 cancer-related genes (NimbleGen Comprehensive Cancer Panel) using fresh-frozen tissues from 10 Taiwanese patients with endometrial cancer.

Section snippets

Sample preparation and DNA extraction

The specimens consisted of 10 fresh-frozen endometrial cancer tissues that were submitted for targeted sequencing. DNA was isolated using proteinase K and a QIAamp® DNA Micro Extraction Kit (QIAGEN) according to the manufacturer's protocol. This study was approved by the Institutional Review Board (KMUH-IRB-970488).

Library preparation and amplification, targeted capture, and Illumina-based sequencing

Genomic DNA (1 μg) was fragmented using a Covaris S2 Focused-ultrasonicator (Covaris, Woburn, MA), and quality control (QC) was performed using an Agilent Bioanalyzer 2100 (Agilent

Pathway analysis of mutated genes in endometrial cancer

We sequenced each sample on the Illumina HiSeq Platform to an average sequencing depth of 535.79. We generated a mean of 16 M raw reads per sample, of which 98.45% to 99.71% were aligned to the human reference genome (Grch38, Table 1). Table 2 shows an overview of our approach to identify variants.

After data filtering, we identified 120 variants in 99 genes, including 107 missense variants, 7 nonsense variants and 6 frame shift variants (Supplemental 2). Functional annotation of the 99 mutated

Discussion

In this study, we screened for mutations in 578 genes in endometrial cancer using the NimbleGen Comprehensive Cancer Panel on the Illumina HiSeq. We identified several cancer driver genes and defined the pathway involved in the development of endometrial cancer. Overall, 50% of endometrial cancers harbored mutations in the PTEN gene. We also detected molecular aberrations that led to putative activation of the IL-7 signaling pathway (n = 2 [PIK3R1, AKT2, FOXO1]), which has not previously been

Acknowledgments

We thank the National Center for Genome Medicine of the National Core Facility Program for Biotechnology, Ministry of Science and Technology, for the technical support. We also thank Wei-Chi Wang from Health GeneTech Corporation, Taoyuan, Taiwan for his assistance with bioinformatics analysis. This work was supported by grants from China Medical University Hospital (DMR-103-121) and the Ministry of Science and Technology, Taiwan, R.O.C. (98-2320-B-037-010-MY3).

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