Clonal lineage from normal endometrium to ovarian clear cell carcinoma through ovarian endometriosis

Abstract

Clear cell carcinoma of the ovary is thought to arise from endometriosis. In addition, retrograde menstruation of shed endometrium is considered the origin of endometriosis. However, little evidence supports cellular continuity from uterine endometrium to clear cell carcinoma through endometriosis at the genomic level. Here, we performed multiregional whole-exome sequencing to clarify clonal relationships among uterine endometrium, ovarian endometriosis and ovarian clear cell carcinoma in a 56-year-old patient. Many somatic mutations including cancer-associated gene mutations in ARID1A, ATM, CDH4, NRAS and PIK3CA were shared among epithelium samples from uterine endometrium, endometriotic lesions distant from and adjacent to the carcinoma, and the carcinoma. The mutant allele frequencies of shared mutations increased from uterine endometrium to distant endometriosis, adjacent endometriosis, and carcinoma. Although a splice site mutation of ARID1A was shared among the four epithelium samples, a frameshift insertion in ARID1A was shared by adjacent endometriosis and carcinoma samples, suggesting that the biallelic mutations triggered malignant transformation. Somatic copy number alterations, including loss of heterozygosity events at PIK3CA and ATM, were identified only in adjacent endometriosis and carcinoma, suggesting that mutant allele-specific imbalance is another key factor driving malignant transformation. By reconstructing a clonal evolution tree based on the somatic mutations, we showed that the epithelium samples were derived from a single ancestral clone. Although the study was limited to a single patient, the results from this illustrative case could suggest the possibility that epithelial cells of ovarian endometriosis and clear cell carcinoma were descendants of uterine endometrial epithelium.

Keywords: clonal evolution; endometriosis; endometrium; genome; ovarian neoplasms.

FIGURE 1

A macroscopic picture of a surgical specimen with sampling sites. A, An image of a surgical specimen from a 56‐year‐old woman with ovarian clear cell carcinoma, representing multiregional sampling sites of the following tissue components: cancer epithelial cells (C), cancer stromal cells (S), epithelial cells of adjacent endometriosis (AE) and distant endometriosis (DE), and epithelial cells of uterine endometrium (U). Each tick mark represents 1 cm

FIGURE 2

Histological images of multiregional samples. Frozen sections of 8‐µm thickness from each sample were stained with H&E to histologically confirm (A) the cancer site accompanied by adjacent endometriosis (boxed lesion), (B) adjacent endometriosis, (C) distant endometriosis and (D) uterine endometrium. The scale bars represent 100 µm

FIGURE 3

Sharing of somatic mutation profiles defined by multiregional sequencing. A, Sharing pattern of somatic single nucleotide variants (SNV) among five tissues. Somatic SNV fulfilling the following criteria were used: (a) SNV whose mutant allele frequency (MAF) were greater than or equal to 0.15 in at least one epithelium or stromal sample; (b) SNV located on coding exons; and (c) SNV whose MAF in blood did not exceed 0.15. B, Distribution of MAF of the SNV shared by epithelium samples. The statistical significance of the differences was assessed using the Wilcoxon‐Mann‐Whitney test

FIGURE 4

Profiles of mutant allele frequency (MAF) for cancer‐associated gene mutations in the four epithelium samples. Cancer‐associated genes were selected according to the COSMIC Cancer Gene Census. Nonsilent mutations shared among at least two epithelium samples are shown

FIGURE 5

Landscape of somatic copy number alterations (SCNA) in the four epithelium samples. A, Genome‐wide profiles of SCNA for four epithelium samples. The absolute values of the log odds ratios for the variant allele read counts at heterozygous single nucleotide variant (SNV) sites in each pair of epithelium and blood samples are plotted according to chromosome coordinates. The log odds ratios were estimated by FACETS. B, SCNA on chromosome 3 leading to the loss of heterozygosity (LOH) status of PIK3CA detected only in cancer epithelium. Regions with copy number alterations are highlighted in light green. The numbers separated by a colon are the major and minor copy numbers for the regions indicated by the arrows. C, SCNA on chromosome 11 leading to the LOH status of ATM detected both in cancer epithelium and adjacent endometriosis. Regions with copy number alterations are highlighted in light green. The numbers separated by a colon are the major and minor copy numbers for the regions indicated by the arrows

FIGURE 6

Clonal relationships among the four epithelium samples. A, Somatic single nucleotide variants (SNV) and indels were classified into six clusters based on their mutant allele frequency (MAF) profiles with PyClone. The prevalence of the six clones in each of the epithelium samples was evaluated, and clonal ordering was then implemented with ClonEvol to create fish plots showing clonal evolutions within the samples. B, A branch‐based clonal evolution tree was generated with ClonEvol. Mutations in cancer‐associated genes except for PIK3CA and ATM were assigned to branches based on the result of mutation clustering by PyClone. Mutations in PIK3CA and ATM were excluded from the analysis of mutation clustering because they were located in SCNA. The PIK3CA and ATM mutations were manually assigned to the initial branch because they were shared at a clonal state among all epithelium samples. The lengths of the branches are associated with the number of somatic mutations