The FOXA2 transcription factor is frequently somatically mutated in uterine carcinosarcomas and carcinomas

Abstract

Background: Uterine carcinosarcomas (UCSs) are a rare but clinically aggressive form of cancer. They are biphasic tumors consisting of both epithelial and sarcomatous components. The majority of uterine carcinosarcomas are clonal, with the carcinomatous cells undergoing metaplasia to give rise to the sarcomatous component. The objective of the current study was to identify novel somatically mutated genes in UCSs.

Methods: We whole exome sequenced paired tumor and nontumor DNAs from 14 UCSs and orthogonally validated 464 somatic variants using Sanger sequencing. Fifteen genes that were somatically mutated in at least 2 tumor exomes were Sanger sequenced in another 39 primary UCSs.

Results: Overall, among 53 UCSs in the current study, the most frequently mutated of these 15 genes were tumor protein p53 (TP53) (75.5%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) (34.0%), protein phosphatase 2, regulatory subunit A, alpha (PPP2R1A) (18.9%), F-box and WD repeat domain containing 7 (FBXW7) (18.9%), chromodomain helicase DNA binding protein 4 (CHD4) (17.0%), and forkhead box A2 (FOXA2) (15.1%). FOXA2 has not previously been implicated in UCSs and was predominated by frameshift and nonsense mutations. One UCS with a FOXA2 frameshift mutation expressed truncated FOXA2 protein by immunoblotting. Sequencing of FOXA2 in 160 primary endometrial carcinomas revealed somatic mutations in 5.7% of serous, 22.7% of clear cell, 9% of endometrioid, and 11.1% of mixed endometrial carcinomas, the majority of which were frameshift mutations.

Conclusions: Collectively, the findings of the current study provide compelling genetic evidence that FOXA2 is a pathogenic driver gene in the etiology of primary uterine cancers, including UCSs. Cancer 2018;124:65-73. © 2017 American Cancer Society.

Keywords: carcinosarcoma; endometrial cancer; endometrial carcinoma; exome; mutation; uterine cancer.

Figure 1. Oncoprint displaying the occurrence of somatic mutations in 15 prevalence screen genes and the MSI, MSH6 and POLE status, of 53 primary UCSs forming the discovery and prevalence screens

The frequency (% of altered cases) of each aberration is shown (left). Coded tumor numbers are shown at top. Somatically mutated tumors are shown by the green bars; dark blue bars indicate that no somatic mutation was detected. Discovery screen tumors are indicated in bold. (*) Mutations in TP53, PPP2R1A and PIK3CA among 36 of 39 prevalence screen tumors were detected here by Sanger sequencing and previously detected in these tumors by targeted next generation sequencing (Supplementary Table 7) .

Figure 2. Localization of somatic FOXA2 mutations uncovered in uterine carcinosarcomas and in endometrial carcinomas relative to functional domains of the encoded protein

(a) Location of somatic FOXA2 mutations in endometrial carcinomas (green squares) and in uterine carcinosarcomas (black squares), relative to mapped functional domains of the protein; the A346Gfs*11 variant found in UCS-5, which did not have paired normal DNA available for analysis, is indicated (yellow square) (b) Overlapping nucleotide deletions result in a cluster of somatic frameshift mutations in the C-terminal region of FOXA2. Deleted nucleotides are indicated by dashes; inserted nucleotides are shown in lower case.

Figure 3. Variable expression of the FOXA2 protein in uterine cancer cell lines and expression of truncated FOXA2 in a UCS harboring a FOXA2 frameshift mutation

Immunoblots showing variable FOXA2 protein expression levels among (a) endometrial carcinoma (EC) cell lines and (b) primary UCS tumors; a truncated FOXA2 protein (~30KDa) was detected in one tumor (UCS-5), (c) Partial FOXA2 nucleotide sequence traces for tumor UCS-5 showing heterozygosity for a frameshift mutation (c.1036_1049delGCGGCCCACCTGCT; p.A346Gfs*11) in the genomic DNA (gDNA) (middle panels) and homozygosity for this mutation in the cDNA (lower panel); an unmatched normal FOXA2 sequence is shown for reference, the nucleotides boxed in green (upper panel) are deleted in UCS-5.