MED12 and HMGA2 mutations: two independent genetic events in uterine leiomyoma and leiomyosarcoma

Abstract

Recent identification of somatic MED12 mutations in most uterine leiomyomas brings a new venue for the study of the tumorigenesis of leiomyomas. We are particularly interested in the correlation of MED12 and HMGA2 gene products in leiomyomas and leiomyosarcomas with and without MED12 mutations. To address these issues, in this study we examined MED12 mutations in a large cohort of usual type leiomyomas (178 cases) and uterine leiomyosarcomas (32 cases). We found that 74.7% (133/178) of leiomyomas had MED12 mutations, which was consistent with several independent studies. In contrast, only 9.7% (3/32) of leiomyosarcomas harbored MED12 mutations. Expression analysis by western blot and immunohistochemistry revealed that those leiomyomas with complex MED12 mutations had significantly lower protein products than the matched myometrium. Interestingly, most leiomyosarcomas without MED12 mutations also had very low levels of MED12 expression in comparison to the matched myometrium. These findings suggest a potential functional role of MED12 in both benign and malignant uterine smooth muscle tumors. When we further examined HMGA2 expression in all leiomyomas and leiomyosarcomas, we found that HMGA2 overexpression was exclusively present in those leiomyomas with no MED12 mutation, accounting for 10.1% (18/178) of total leiomyomas and 40% (18/45) of non-MED12 mutant leiomyomas. Twenty-five percent (8/32) of leiomyosarcomas had HMGA2 overexpression, and no MED12 mutations were found in HMGA2 positive leiomyosarcoma. These findings strongly suggest that MED12 mutations and HMGA2 overexpression are independent genetic events that occur in leiomyomas, and they may act differently in the tumorigenesis of uterine leiomyomas.

Figure 1

MED12 mutation analysis in uterine leiomyomas. A. The photomicrograph illustrates the histology of usual type leiomyoma. B. Sequencing chromatographs of 12 types of MED12 mutations identified in usual type leiomyomas. Red arrows indicate the mutation sites. C. Genomic analysis of the types and distribution of MED12 mutations in exon2 and intron1. The point mutations were listed in the above sequences, and each dot represents a case with mutation. The complex mutations were listed below the sequence with thin bars illustrating the actual length of deletions.

Figure 2

Three leiomyosarcomas with MED12 mutations. A–C illustrate the high power histology of three leiomyosarcomas (upper) and their corresponding sequencing chromatographs for MED12 mutations (lower). Red arrows indicate the mutation sites. Case two (B) is a leiomyosarcoma of stage IV disease.

Figure 3

Reduced MED12 protein levels in uterine leiomyomas with complex MED12 mutations. A. Confocal microscopic images illustrate an example of immunofluorescent staining for MED12 (red), smooth muscle actin (green) and DAPI (blue) in leiomyoma with a complex MED12 mutation (with a partial MED12 Exon 2 deletion) and its matched myometrium. In myometrium, the expression pattern and distribution are punctuated with high intensity staining enriched in perinuclear localization. In contrast, the expression pattern for leiomyoma is diffused in the cytoplasm and has lesser staining intensity. Scale bars = 50 µm. Higher power image of MED12 staining in leiomyoma and myometrium are shown in the insert (Scale bar = 25 µm). B. The expression of MED12 protein was detected by Western blot analysis in leiomyomas with complex MED12 mutations (Pt81LM and Pt151LM for Exon 2 deletion on right) and leiomyomas with simple MED12 point mutations (Pt137LM and Pt173LM for c.131G>A on left). MED12 expression in the matched myometrium (MM) was shown next to tumors. Anti-α-smooth muscle actin, Anti-Actin, and anti-GAPDH were used as protein loading controls.

Figure 4

Immunohistochemical analysis of MED12 expression in formalin-fixed and paraffin-embedded leiomyomas and leiomyosarcomas. A. Photomicrographs illustrate examples of immunointensity of MED12 in tumor myometrium (MM), leiomyomas (ULM) and leiomyosarcoma (LMS). wt: wild type MED12; point: point/single nucleotide mutation of MED12; complex: complex mutations of MED12. B. Statistical analysis of immunointensity for MED12 in tumor myometrium (MM), leiomyomas (ULM) and leiomyosarcoma (LMS). MED12 mutation types and number of cases were listed below the histobars. **: p<0.01.

Figure 5

HMGA2 overexpression in uterine leiomyomas. A. Photomicrographs illustrate examples of strong and diffuse immunoreactivity for HMGA2 in uterine leiomyomas. High grade serous ovarian carcinoma was used as the positive control (left upper). B and C. Dot plot analysis of distribution of MED12 mutations and HMGA2 overexpression in 178 cases of uterine leiomyomas. 74% of leiomyomas have MED12 mutations and lack of HMGA2 overexpression; 10% of leiomyomas have wild type MED12 and HMGA2 overexpression; and remainder 16% of leiomyomas have wild type MED12 and lack of HMGA2 overexpression.