Anti-FOXL2 monoclonal antibody, clone 373D2b

During mouse sex determination, transient expression of the Y-linked gene Sry up-regulates its direct target gene Sox9, via a 3.2 kb testis specific enhancer of Sox9 (TES), which includes a core 1.4 kb element, TESCO. SOX9 activity leads to differentiation of Sertoli cells, rather than granulosa cells from the bipotential supporting cell precursor lineage. Here, we present functional analysis of TES/TESCO, using CRISPR/Cas9 genome editing in mice. Deletion of TESCO or TES reduced Sox9 expression levels in XY fetal gonads to 60 or 45% respectively relative to wild type gonads, and reduced expression of the SOX9 target Amh. Although human patients heterozygous for null mutations in SOX9, which are assumed to have 50% of normal expression, often show XY female sex reversal, mice deleted for one copy of Sox9 do not. Consistent with this, we did not observe sex reversal in either TESCO-/- or TES-/- XY embryos or adult mice. However, embryos carrying both a conditional Sox9 null allele and the TES deletion developed ovotestes. Quantitative analysis of these revealed levels of 23% expression of Sox9 compared to wild type, and a significant increase in the expression of the granulosa cell marker Foxl2. This indicates that the threshold in mice where sex reversal begins to be seen is about half that of the similar to 50% levels predicted in humans. Our results demonstrate that TES/TESCO is a crucial enhancer regulating Sox9 expression in the gonad, but point to the existence of additional enhancers that act redundantly.

Adult granulosa cell tumor (AGCT) is a low-grade malignant neoplasm with a significant propensity for late recurrence and metastasis. Almost all AGCTs are composed of cells with bland nuclear features and even when these tumors recur or metastasize, the nuclear features are almost always low-grade. We report 5 cases of AGCT in patients aged 37 to 88 years composed of areas of typical AGCT with low-grade morphology admixed with areas of high-grade morphology, with marked nuclear atypia, often with bizarre multinucleate cells and high mitotic activity; this is the first reported series of high-grade transformation in AGCTs. The high-grade areas often morphologically closely resembled juvenile granulosa cell tumor with abundant eosinophilic cytoplasm, significant mitotic activity, and intermediate sized follicles. Four cases were FIGO stage IA at diagnosis and 1 was stage IIIC with omental involvement. FOXL2 mutation analysis of both the morphologically low-grade and high-grade areas in 4 of 5 cases confirmed the presence of missense point mutation, c.402C>G, p.(Cys134Trp), providing conclusive evidence that the high-grade component represents transformation of typical AGCT rather than the coexistence of another sex cord-stromal tumor, such as juvenile granulosa cell tumor, which has been suggested for such neoplasms. In 3 of 4 cases where immunohistochemistry was undertaken, there was a striking difference between the p53 staining in the low-grade and high-grade components with wild-type staining in the former and diffuse mutation-type immunoreactivity in the latter, suggesting that TP53 mutation is likely to play a role in high-grade transformation. TP53 mutation analysis covering exons 4 to 10 was undertaken in 4 cases and TP53 mutations were identified in the high-grade component of 2 of the cases. In 1 case, there was diffuse block-type p16 staining in the high-grade component. Follow-up in the 4 stage IA neoplasms revealed no evidence of tumor recurrence in 3 (6 to 9 mo follow-up) while the other patient developed mediastinal, peritoneal, and pulmonary metastasis 17 months after diagnosis. High-grade transformation is uncommon in AGCTs and given that one of our cases was advanced stage at diagnosis, another exhibited widespread metastasis within a short period and there have been occasional case reports of aggressive behavior in AGCTs with high-grade transformation, this event may herald an aggressive clinical course.