Is the FOXL2 gene mutation involved in the development of granulosa cell tumours in equines?

Introduction and aim: Granulosa-theca cell tumour (GTCT) is a significant cause of infertility in mares, representing for 85% of ovarian tumours and approximately 2.5% of all neoplasms in horses [1]. In women, granulosa cell tumours (GCT) comprise about 5% of ovarian neoplasms and 70% of ovarian sex cord-stromal tumours. Human GCTs are categorized into adult and juvenile types, distinguished by clinicopathologic features. The adult type, the most common, occurs in peri- or post-menopausal women, while the juvenile type, affects prepubertal girls and young women [2]. In the adult type, a somatic "missense" mutation in the FOXL2 gene (402C→G; C134W) is present in 97% of cases but is absent in the juvenile type [2]. Adult GCTs exhibit a variety of histological patterns from well-differentiated to poorly differentiated. Well-differentiated patterns include microfollicular, macrofollicular, trabecular, insular, solid tubular, and empty tubular. "Call-Exner bodies” are typical. Adult GCTs contain round to oval cells with characteristic "coffee-bean" nuclei, few mitotic figures, mild nuclear atypia, scant cytoplasm and often luteinized cells. Juvenile GCTs share the macroscopic appearance with the adult type but differ significantly histologically, exhibiting a follicular or solid diffuse pattern with larger luteinized granulosa cells containing hyperchromatic or markedly bizarre nuclei and high mitotic activity [3]. This study aimed to explore if this categorization is possible in equines. Methods: 12 cases of equine GTCT were obtained by unilateral ovariectomy, using a flank-assisted laparoscopic technique. Ovaries were classified based on their macroscopic and microscopic appearance. For histopathology specimens were fixed in formalin and embedded in paraffin blocks. Slides were evaluated by experienced veterinary and human pathologists. GTCTs were classified according to the most prevalent pattern [4]. Additionally, a molecular test for the FOXL2 gene mutation was performed. The DNA was extracted using a commercially available kit, from 12 samples of formalin-fixed paraffin-embedded (FFPE) tissue. PCR was used to amplify the DNA region of interest before Sanger sequencing. Results: The median age of the mares was 11.6 years (3-25 years). All mares presented infertility problems; 8/12 exhibited stallion-like behaviour, while the remaining mares showed anoestrous or abnormal oestrous behaviour. All cases showed unilateral ovarian enlargement and a hypotrophic contralateral ovary. Macroscopically, the neoplastic ovary had an ovoid or spherical shape with a smooth surface, enclosed by a fibrous capsule. The cut surface revealed a multicystic structure in 9 cases and a solid structure in 3 cases. Cystic structures contained serous or serohaematic material, surrounded by solid connective septa of varying thickness and a greyish-white or yellowish colour. Histological patterns included follicular (macrofollicular, microfollicular, or macro-microfollicular) in 6/12 cases, solid (insular, tubular, and diffuse) in 2/12 cases, and mixed in 4/12 cases. The cell population was characterized by neoplastic granulosa cells in all cases and luteinized cells in 12/12 cases. Additionally, 6/12 cases presented "Sertoli-like" elements and 12/12 cases exhibited a fibro-thecal component. Call-Exner bodies were present in only 1/12 cases. Mitotic activity was low in all cases. The FOXL2 amplification and sequencing demonstrated a missense point mutation at position C135W, with a cytosine for guanine, in only one case (8.3%). Discussion and conclusions: Comparing the morphological features observed in these cases with the two subtypes of the tumour in women, pathologists agreed that mare cases exhibit greater homology with the adult type of the human tumour regarding growth patterns and low mitotic activity. Even the age of onset of equine GTCTs (median of 11.6 years) is similar to the human adult type. The typical features of the juvenile form in women were not observed in any equine cases, including the case in a 3-year-old mare, which could be age-equivalent to the juvenile form in women. Only 1/12 equine cases presented the classic Call-Exner bodies, suggesting these structures are not so typical in equine tumours. In contrast to the adult type, equine GTCT seems to not carry constantly the FOXL2 mutation (1 case, 8.3%). In horses, the FOXL2 gene consists of 378 amino acids, compared to 376 amino acids in women, explaining why the amino acid change occurs at position C134W in women and C135W in mares. The lack of mutation of FOXL2 in most cases would suggest a juvenile-type similarity. Likely, equine GTCTs cannot be separated into juvenile or adult types. FOXL2 mutation cannot be used as a diagnostic marker [5] lacking in most of the tumours and it has probably a limited role in the genesis of these tumours in this species. Keywords: Mare, ovary, neoplasm, histopathology, gene mutation References: 1. McCue PM, Roser JF, Munro CJ, Liu IK, Lasley BL. Granulosa cell tumors of the equine ovary. Vet Clin North Am Equine Pract (2006) 22:799-817. doi: 10.1016/j.cveq.2006.08.008 2. Li X, Tian B, Liu M, Miao C, Wang D. Adult-type granulosa cell tumor of the ovary. Am J Cancer Res (2022) 12:3495-511. 3. Jamieson S, Fuller PJ. Molecular pathogenesis of granulosa cell tumors of the ovary. Endocr Rev (2012) 33:109-44. doi: 10.1210/er.2011-0014 4. Edwards JF, Cullen JM, Kennedy PC. Histological Classification of Tumors of the Genital System of Domestic Animals. 2nd series, Volume IV. Washington: Armed Forces Institute of Pathology (1998). 5. Kim JH, Kim YH, Kim HM, Park HO, Ha NC, Kim TH, Park M, Lee K, Bae J. FOXL2 posttranslational modifications mediated by GSK3β determine the growth of granulosa cell tumours. Nat Commun (2014) 5:2936. doi: 10.1038/ncomms3936