Uterine leiomyomas are benign smooth muscle tumors occurring in 70% of women of reproductive age. The majority of leiomyomas harbor one of three well-established genetic changes: a hotspot mutation in MED12, overexpression of HMGA2, or biallelic loss of FH. The majority of studies have classified leiomyomas by complex and costly methods, such as whole-genome sequencing, or by combining multiple traditional methods, such as immunohistochemistry and Sanger sequencing. The type of specimens and the amount of resources available often determine the choice. A more universal, cost-effective, and scalable method for classifying leiomyomas is needed. The aim of this study was to evaluate whether RNA sequencing can accurately classify formalin-fixed paraffin-embedded (FFPE) leiomyomas. We performed 3′RNA sequencing with 44 leiomyoma and 5 myometrium FFPE samples, revealing that the samples clustered according to the mutation status of MED12, HMGA2, and FH. Furthermore, we confirmed each subtype in a publicly available fresh frozen dataset. These results indicate that a targeted 3′RNA sequencing panel could serve as a cost-effective and robust tool for stratifying both fresh frozen and FFPE leiomyomas. This study also highlights 3′RNA sequencing as a promising method for studying the abundance of unexploited tissue material that is routinely stored in hospital archives.

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Uterine leiomyomas, or fibroids, are the most common tumors in women of reproductive age. Uterine leiomyomas can be classified into at least three main molecular subtypes according to mutations affecting MED12, HMGA2, or FH. FH-deficient leiomyomas are characterized by activation of the NRF2 pathway, including upregulation of the NRF2 target gene AKR1B10. Here, we have identified a novel leiomyoma subtype showing AKR1B10 expression but no alterations in FH or other known driver genes. Whole-exome and whole-genome sequencing revealed biallelic mutations in key genes involved in neddylation of the Cullin 3-RING E3 ligase, including UBE2M, NEDD8, CUL3, and NAE1. 3′RNA sequencing confirmed a distinct molecular subtype with activation of the NRF2 pathway. Most tumors displayed cellular histopathology, perivascular hypercellularity, and characteristics typically seen in FH-deficient leiomyomas. These results suggest a novel leiomyoma subtype that is characterized by distinct morphological features, genetic alterations disrupting neddylation of the Cullin 3-RING E3 ligase, and oncogenic NRF2 activation. They also present defective neddylation as a novel mechanism leading to aberrant NRF2 signaling. Molecular characterization of uterine leiomyomas provides novel opportunities for targeted treatment options.

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.

Uterine leiomyosarcomas are rare, aggressive cancers that represent the most common type of uterine sarcomas. They frequently harbor genetic alterations in TP53, RB1, PTEN, and ATRX. Their benign counterpart, uterine leiomyomas (or fibroids) are very common tumors that may significantly affect women’s quality of life. The majority of leiomyomas harbor genetic alterations affecting either MED12, HMGA2, or FH. A third of leiomyosarcomas have been reported to harbor leiomyoma-associated driver mutations, suggesting that some leiomyomas may have undergone malignant transformation. The leiomyoma/leiomyosarcoma diagnosis is largely based on histopathology. This can be specifically challenging in differentiating leiomyosarcomas from a subset of leiomyomas that display histopathological features resembling malignancy. The aim of this study was to identify molecular biomarkers that can accurately differentiate benign and malignant uterine smooth muscle tumors. 3’RNA sequencing of 48 leiomyosarcomas and 44 leiomyomas revealed that leiomyosarcomas are uniquely characterized by dysregulation of the retinoblastoma pathway. We confirmed upregulation of several members of this pathway, including TOP2A and CDK1, in a publicly available transcriptomic dataset of 49 uterine leiomyosarcomas. The second objective of this study was to explore the gene expression profile of leiomyosarcomas carrying leiomyoma-associated driver mutations. This revealed that unlike leiomyomas, leiomyosarcomas do not form distinct gene expression profiles based on the presence of leiomyoma-associated driver mutations such as MED12. However, leiomyosarcomas with a MED12 mutation showed upregulation of RAD51B and ADAM12, which we have previously highlighted as biomarkers of leiomyomas harboring a MED12 mutation. We identified upregulation of IRS4 and characteristic deletions affecting COL4A5 and COL4A6 in four in-house and three additional uterine leiomyosarcomas from The Cancer Genome Atlas (TCGA) collection. Similar driver alterations have previously been reported in a small subset of leiomyomas but not in leiomyosarcomas. In conclusion, leiomyosarcomas display a gene expression profile characterized by dysregulation of genes related to the retinoblastoma pathway. These include potential biomarkers that could be used in a clinical setting to differentiate leiomyosarcomas from leiomyomas. Citation Format: Sara Khamaiseh, Riitta Koivisto-Korander, Terhi Ahvenainen, Ralf Bützow, Miika Mehine, Pia Vahteristo. Identification of molecular biomarkers differentiating malignant uterine leiomyosarcoma from benign leiomyoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 996.