Research Article: The Specificity of the FOXL2 c.402C>G Somatic Mutation: A Survey of Solid Tumors

Date Published: November 24, 2009

Publisher: Public Library of Science

Author(s): Kasmintan A. Schrader, Bella Gorbatcheva, Janine Senz, Alireza Heravi-Moussavi, Nataliya Melnyk, Clara Salamanca, Sarah Maines-Bandiera, Susanna L. Cooke, Peter Leung, James D. Brenton, C. Blake Gilks, John Monahan, David G. Huntsman, Anita Brandstaetter.

Abstract: A somatic mutation in the FOXL2 gene is reported to be present in almost all (97%; 86/89) morphologically defined, adult-type, granulosa-cell tumors (A-GCTs). This FOXL2 c.402C>G mutation changes a highly conserved cysteine residue to a tryptophan (p.C134W). It was also found in a minority of other ovarian malignant stromal tumors, but not in benign ovarian stromal tumors or unrelated ovarian tumors or breast cancers.

Partial Text: Malignant adult ovarian granulosa-cell tumors (A-GCTs) are malignant sex cord-stromal tumors known for their genomic stability and varied prognosis [1]. Until recently, there has been little insight into the molecular characteristics of A-GCTs. Using whole-transcriptome paired-end RNA sequencing, we identified a somatic missense mutation (c.402C>G, p. Cys134Trp) in the Forkhead transcription factor gene, FOXL2[2]. This mutation was present in 97% of 89 morphologically identified A-GCTs [2]. Foxl2 has been shown to be crucial for granulosa-cell differentiation [3]. This was the first association of a somatic mutation in FOXL2 associated with cancer, however aberrant expression of Foxl2 has been reported in juvenile granulosa-cell tumor of the testis [4]. The mutation was also found at a lower frequency in two other related ovarian stromal tumors; 21% (3/14) thecomas and 10% (1/10) juvenile-type GCTs were mutation positive [2]. This single, recurrent mutation suggests that it is characteristic of granulosa-cell tumors, and its high frequency implies that it is potentially a driver in disease initiation.

Samples for the high resolution melt assay were purchased as either DNA or tissue blocks from vendors who provided unlinked anonymized specimens collected in accordance with applicable review boards approval, regulations and laws. Novartis does not require an ethical review committee for samples collected in this manner. Control DNA, used to validate the high resolution melt assay, was extracted from anonymized tumor specimens compiled by the frozen tumor bank, OvCaRe (Ovarian Cancer Research), under written informed consent. Approval for analysis of these samples for the FOXL2 mutation was obtained through the British Columbia Cancer Agency’s research ethics board.

All 11 previously reported FOXL2 c.402C>G mutation-positive A-GCT specimens as well as an unreported A-GCT case and the A-GCT cell line, KGN, validated the HRM assay by demonstrating a variant melt curve distinct from the common (wild-type) pattern. None of the 14 FOXL2 c.402C>G mutation negative samples exhibited this variant melt profile. However, three of the 10 high grade serous ovarian cancers showed an alternative variant profile; sequencing confirmed them to be false positives.

Loss-of-function germline mutations in FOXL2 are associated with blepharophimosis–ptosis–epicanthus–inversus syndrome [BPES;OMIM#110100]; an autosomal dominant developmental disorder characterized by eyelid malformations and premature ovarian failure due to a dysfunction of granulosa-cells [10]. The FOXL2 c.402C>G mutation is seen in the heterozygous state in most A-GCTs. Unlike in BPES, where germline FOXL2 mutations are spread across the gene [11], the somatic FOXL2 mutation in A-GCTs involves the same base pair in all cases. This favors a specific functional consequence such as a dominant negative effect or a change or gain of function as opposed to a generic loss of function and the ultimate impact of this mutation is oncogenic. Additionally, immunohistochemical data indicating that Foxl2 expression is maintained in the nuclei in A-GCTs, that were heterozygous or appeared to be hemizygous or homozygous for the mutation, implies that this mutation does not affect protein localization [2].



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