Identification of molecular pathway aberrations in uterine serous carcinoma by genome-wide analyses

Abstract

Background: Uterine cancer is the fourth most common malignancy in women, and uterine serous carcinoma is the most aggressive subtype. However, the molecular pathogenesis of uterine serous carcinoma is largely unknown. We analyzed the genomes of uterine serous carcinoma samples to better understand the molecular genetic characteristics of this cancer.

Methods: Whole-exome sequencing was performed on 10 uterine serous carcinomas and the matched normal blood or tissue samples. Somatically acquired sequence mutations were further verified by Sanger sequencing. The most frequent molecular genetic changes were further validated by Sanger sequencing in 66 additional uterine serous carcinomas and in nine serous endometrial intraepithelial carcinomas (the preinvasive precursor of uterine serous carcinoma) that were isolated by laser capture microdissection. In addition, gene copy number was characterized by single-nucleotide polymorphism (SNP) arrays in 23 uterine serous carcinomas, including 10 that were subjected to whole-exome sequencing.

Results: We found frequent somatic mutations in TP53 (81.6%), PIK3CA (23.7%), FBXW7 (19.7%), and PPP2R1A (18.4%) among the 76 uterine serous carcinomas examined. All nine serous carcinomas that had an associated serous endometrial intraepithelial carcinoma had concordant PIK3CA, PPP2R1A, and TP53 mutation status between uterine serous carcinoma and the concurrent serous endometrial intraepithelial carcinoma component. DNA copy number analysis revealed frequent genomic amplification of the CCNE1 locus (which encodes cyclin E, a known substrate of FBXW7) and deletion of the FBXW7 locus. Among 23 uterine serous carcinomas that were subjected to SNP array analysis, seven tumors with FBXW7 mutations (four tumors with point mutations, three tumors with hemizygous deletions) did not have CCNE1 amplification, and 13 (57%) tumors had either a molecular genetic alteration in FBXW7 or CCNE1 amplification. Nearly half of these uterine serous carcinomas (48%) harbored PIK3CA mutation and/or PIK3CA amplification.

Conclusion: Molecular genetic aberrations involving the p53, cyclin E-FBXW7, and PI3K pathways represent major mechanisms in the development of uterine serous carcinoma.

Figure 1.

Single-nucleotide polymorphism array analysis of DNA copy number alteration. A) Copy number alteration in uterine serous carcinoma and other types of gynecological neoplasia. Individual chromosomes (vertical axis) of each tumor (horizontal axis) are plotted using a pseudocolor gradient indicating the overall level of copy number alteration (low [black] to high [red]). Normal = normal tissue (n = 25); UEMC = uterine endometrioid carcinoma (n = 13); USC = uterine serous carcinoma (n = 23); SBT = ovarian serous borderline tumor (n = 12); LGSC = ovarian low-grade serous carcinoma (n = 12); OEM = ovarian endometrioid carcinoma (n = 7); CCC = ovarian clear cell carcinoma (n = 12); HGSC = ovarian high-grade serous carcinoma (n = 33). B) Scatter plot of genome-wide chromosome instability index for individual tumors (open circles). Vertical blue lines indicate mean values; horizontal red lines indicate plus or minus one SD. C) Overall view of DNA copy number gain and loss in uterine serous carcinoma. To generate an overview of DNA copy number alterations, we performed circular binary segmentation analysis and combined the segmentation results for all 23 uterine serous carcinomas. Based on the Segments-of-Gain-Or-Loss (SGOL) scores (horizontal axis), DNA copy number gains (green) and losses (red) are plotted as a function of distance along the normal genome (vertical axis). Dashed vertical lines are generated based on the values of 3 SDs of the SGOL scores of gains or losses. Tumor-associated genes located in representative amplified and deleted regions are annotated. D) Heat map of DNA copy number ratio along the chromosome 19. Arrow shows amplification in the CCNE1 locus in 23 uterine serous carcinoma samples. A = CCNE1 amplification; red circles = FBXW7 mutation; gray circle = FBXW7 hemizygous deletion. Suffix of the tumor numbers: TS and TCS = fresh tumor with affinity-purified tumor cells; T = tumor samples without affinity purification; N = normal control.

Figure 2.

Molecular genetic alterations involving the FBXW7–cyclin E and phosphatidylinositol 3-kinase (PI3K) pathways in uterine serous carcinoma. A) Summary of sequence mutations and DNA copy number changes in FBXW7, CCNE1, and PIK3CA. B) Schematic presentation of possible mechanisms involving the FBXW7–cyclin E and PI3K pathways in the development of uterine serous carcinoma. FBXW7 gene deletion or mutation results in an increased level of cyclin E because of the suppression of FBXW7-mediated ubiqutination and subsequent proteasome-mediated degradation of cyclin E proteins. Alternatively, activation of PI3K can be a result of increased DNA copy number of PIK3CA and activating mutation of the gene. Increased E2F-1 and PI3K activity leads to activation of their downstream targets that promote cell cycle progression and other tumor-promoting phenotypes. Ub = ubiquitin; P = phosphate.

Figure 3.

Mutation profiles of the most commonly mutated genes and cyclin E expression in uterine serous carcinoma (USC) and serous endometrial intraepithelial carcinoma (SEIC). A) Mutation profiles of FBXW7, PIK3CA, PPP2R1A, and TP53 in nine USCs with a concurrent SEIC component. Manual microdissection and laser-capture microdissection were used to isolate USC and SEIC, respectively, for DNA analysis. Mutations are highlighted in different colors for each gene. ND = not done; wt = wildtype; NA = not available for analysis; *tumor (5FFPE) has two additional mutations in PIK3CA: S553Ifs, H1047R; #tumor (10FFPE) has one additional mutation in PIK3CA: G1007R. B) A uterine serous carcinoma with a serous endometrial intraepithelial carcinoma component. Top panel, SEIC; middle panel, USC. The tumor cells in both lesions show highly atypical and enlarged nuclei. The arrow indicates the junction between normal-appearing uterine surface epithelium (left) and serous endometrial intraepithelial carcinoma (right). Cyclin E immunoreactivity (brown) was detected in both uterine serous carcinoma and serous endometrial intraepithelial carcinoma cells but not in adjacent normal epithelium or stromal cells (scale bar = 100 µm). Bottom panels: Mutational analysis showing an identical somatic mutation of FBXW7, from CGT to CAT (arrows), leading to the amino acid change R465H in both the serous endometrial intraepithelial carcinoma and the uterine serous carcinoma. C) Locations of FBXW7 mutations identified among the 76 uterine serous carcinomas. Each circle represents an individual mutation.