Search

Uterine leiomyomas, or fibroids, are very common smooth muscle tumors that arise from the myometrium. They can be divided into distinct molecular subtypes. We have previously shown that 3'RNA-sequencing is highly effective in classifying archival formalin-fixed paraffin-embedded (FFPE) leiomyomas according to the underlying mutation. In this study, we performed 3'RNA-sequencing with 111 FFPE leiomyomas previously classified as negative for driver alterations in mediator complex subunit 12 (MED12), high mobility group AT-hook 2 (HMGA2), and fumarate hydratase (FH) by Sanger sequencing and immunohistochemistry. This revealed 43 tumors that displayed expression features typically seen in HMGA2-positive tumors, including overexpression of PLAG1. We explored 12 such leiomyomas by whole-genome sequencing to identify their underlying genomic drivers and to evaluate the feasibility of detecting chromosomal driver alterations from FFPE material. Four tumors with significant HMGA2 overexpression at the protein-level served as controls. We identified chromosomal rearrangements targeting either HMGA2, HMGA1, or PLAG1 in all 16 tumors, demonstrating that it is possible to detect chromosomal driver alterations in archival leiomyoma specimens as old as 18 years. Furthermore, two tumors displayed biallelic loss of DEPDC5 and one tumor harbored a COL4A5-COL4A6 deletion. These observations suggest that instead of only HMGA2-positive leiomyomas, a distinct leiomyoma subtype is characterized by rearrangements targeting either HMGA2, HMGA1, or PLAG1. The results indicate that the frequency of HMGA2-positive leiomyomas may be higher than estimated in previous studies where immunohistochemistry has been used. This study also demonstrates the feasibility of detecting chromosomal driver alterations from archival FFPE material.

Problem statementUterine leiomyomas (ULM) are benign smooth muscle tumors, occurring in up to 70% of women by the age of 50 years. Myomectomy is a surgical treatment option where the uterus and thereby reproductive potential are preserved. The most important known genetic changes in ULM are mutations in mediator complex subunit 12 (MED12), changes leading to high mobility group AT-hook (HMGA2) overexpression, and heterozygous germline mutations in fumarate hydratase (FH), resulting in hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. The earlier studies on molecular background of ULM are mostly based on hysterectomy samples from middle aged women. Therefore, we wanted to know whether the pathogenesis of ULM is similar in younger women.MethodsThe research material comprises of women up to 45 years old, who have undergone myomectomy at Helsinki University Central Hospital. We have collected formalin fixed paraffin embedded samples from patients operated during 2009u20142014. MED12 mutation frequency was determined by direct sequencing of exons 1 and 2, and HMGA2 expression levels were assessed by immunohistochemistry. Biallelic FH inactivation was detected with 2SC staining. Patientsu00b4 medical records were reviewed. ResultsWe analyzed 234 patients with 361 leiomyomas. The median age of patients at operation was 34 years. 6.4% of the patients had had a prior myomectomy. The majority of patients (58%) were operated for a single leiomyoma. The frequency of MED12 mutation was 71%, as reported in tumors obtained through hysterectomy. 9% of leiomyomas showed HMGA2 overexpression, 3% were FH deficient, and 17% were wildtype for all of the studied alterations. Interestingly, in solitary leiomyomas, the MED12 frequency was only 49%, and 27% of the solitary tumors were wildtype. Leiomyomas with MED12 mutation were smaller in size compared to tumors with HMGA2 overexpression, and more often subserosal compared to tumors with other drivers. ConclusionThis study is a comprehensive sample series of young ULM patients. Our findings emphasize the meaning of solitary leiomyomas in clinical practice. Molecular changes behind the notable proportion of wildtype leiomyomas need more research. The authors declare that they have no conflict of interest.