Massively Parallel Sequencing-Based Clonality Analysis of Synchronous Endometrioid Endometrial and Ovarian Carcinomas

Abstract

Synchronous early-stage endometrioid endometrial carcinomas (EECs) and endometrioid ovarian carcinomas (EOCs) are associated with a favorable prognosis and have been suggested to represent independent primary tumors rather than metastatic disease. We subjected sporadic synchronous EECs/EOCs from five patients to whole-exome massively parallel sequencing, which revealed that the EEC and EOC of each case displayed strikingly similar repertoires of somatic mutations and gene copy number alterations. Despite the presence of mutations restricted to the EEC or EOC in each case, we observed that the mutational processes that shaped their respective genomes were consistent. High-depth targeted massively parallel sequencing of sporadic synchronous EECs/EOCs from 17 additional patients confirmed that these lesions are clonally related. In an additional Lynch Syndrome case, however, the EEC and EOC were found to constitute independent cancers lacking somatic mutations in common. Taken together, sporadic synchronous EECs/EOCs are clonally related and likely constitute dissemination from one site to the other.

Figure 1.

Repertoires of somatic mutations, gene copy number alterations, and mutational signatures in sporadic synchronous endometrioid endometrial carcinomas (EECs) and endometrioid ovarian carcinomas (EOCs). Somatic mutations (left), gene copy number alterations (middle), and mutational signatures (right) identified in the five cases of synchronous EECs and EOCs subjected to whole-exome sequencing. The Venn diagrams represent the total number of somatic mutations (silent single-nucleotide variants (SNVs), nonsynonymous SNVs, and insertions/deletions) that are unique to the EEC and EOC of a given case and that are shared between the EEC and EOC. Driver mutations were defined as mutations classified as likely pathogenic by mutation effect prediction algorithms and/or associated with loss of heterozygosity of the wild-type allele (Supplementary Methods, available online) and that affected known cancer genes included in Kandoth et al. (39), the Cancer Gene Census (40), and/or Lawrence et al. (41), or genes statistically significantly mutated in nonultramutated EECs by The Cancer Genome Atlas (34). For case SYN4, three anatomically distinct areas were subjected to whole-exome sequencing, and the phylogenetic tree depicts the evolution of these regions, where the colored branches represent each of the subclones identified and selected somatic mutations that define a given clone are illustrated along the branches. The length of the branches is representative of the number of mutations that distinguishes a given clone from its ancestral clone (42). In the chromosome plots, the Log₂ ratios are plotted on the y-axis and the genomic positions are plotted on the x-axis. Gains and amplifications are highlighted in blue, and losses in red. Mutational signatures of all somatic SNVs in the EECs and EOCs of a given case are displayed according to the 96 substitution classification defined by the substitution classes (C>A, C>G, C>T, T>A, T>C, and T>G bins) and the 5’ and 3’ sequence context. All mutational signatures are normalized to the trinucleotide frequency of the human genome. The number in brackets following “EEC” and “EOC” is the mutational signature assigned according to Alexandrov et al. (35), where signature 1A relates to aging and signature 6 to defective DNA mismatch repair. Driv = driver mutation; EEC = endometrioid endometrial carcinoma; EOC = endometrioid ovarian carcinoma; I = >20% of mutations in case SYN4 were small insertions/deletions; ns = nonsynonymous SNVs and insertions/deletions; SNV = single-nucleotide variant.

Figure 2.

Clonal relatedness analysis of synchronous endometrioid endometrial carcinomas (EECs) and endometrioid ovarian carcinomas (EOCs) occurring in a sporadic or Lynch Syndrome context. A) Nonsynonymous and synonymous somatic single-nucleotide variants and small insertions/deletions identified by targeted MPS analysis in 18 cases of synchronous EECs/EOCs. Note that in all cases but SV2 the EEC and the EOC shared somatic mutations. B) Clonality Index (CI2) for the 23 cases of synchronous EECs/EOCs analyzed in this study, defined as the likelihood of two lesions sharing mutations not expected to have co-occurred by chance (Supplementary Methods, available online). Red dotted lines indicate the threshold to define clonal relatedness for the respective sequencing platform (whole-exome sequencing left, targeted capture massively parallel sequencing right). Blue dotted line indicates the CI2 at which two samples from a given patient did not share any mutation on the respective sequencing platform. With the exception of case SV2, a Lynch Syndrome case, all tumors from a given patient were found to be clonally related. C) In SV2, MPS analysis demonstrated that none of the somatic mutations were shared between the synchronous EEC and EOC. The mutational signatures of the nonsynonymous and synonymous somatic single-nucleotide variants in the EEC and EOC of SV2 are displayed according to the 96 substitution classification defined by the substitution classes (C>A, C>G, C>T, T>A, T>C, and T>G bins) and the 5’ and 3’ sequence context. All mutational signatures are normalized to the trinucleotide frequency of the human genome. The EEC displayed a mutational signature consistent with that of a hereditary ultra-hypermutated carcinoma (ie, a tumor with a germline mismatch repair gene mutation and a somatic POLE hotspot mutation), whereas the EOC displayed a mutational signature related to aging (35,37,38). A POLE S459F hotspot mutation was identified in the EEC but not in the synchronous EOC as shown in the sequence electropherograms. Both the EEC and the synchronous EOC displayed loss of MSH6 expression as assessed by immunohistochemistry (scale bar = 100 µm). The patient SV2 harbored a germline MSH6 (R1076C) mutation as shown in the sequence electropherograms. EEC = endometrioid endometrial carcinoma; EOC = endometrioid ovarian carcinoma; m = mutations; SNV = single-nucleotide variant.