Stratification of Leiomyosarcoma molecular subtypes by 3' end RNA-sequencing: Toward precision medicine
Leiomyosarcoma (LMS) is a malignant neoplasm with smooth muscle differentiation.  Little is known about its molecular heterogeneity and no targeted therapy currently exists for LMS.  We demonstrate the existence of 3 molecular subtypes in a cohort of 99 cases and an independent cohort of 82 LMS.  Two new FFPE tissue-compatible diagnostic immunohistochemical markers are identified: LMOD1 for subtype I LMS and ARL4C for subtype II LMS. Subtype I and subtype II LMS are associated with good and poor prognosis, respectively.  The LMS subtypes show significant differences in expression levels for genes for which novel targeted therapies are being developed. Overall design: Gene expression profiling was performed by 3' End RNA Sequencing (3SEQ), a next generation sequencing approach that does not rely on frozen tissue but can be performed on archival FFPE tissue. Samples included 99 LMS, 6 Undifferentiated Pleomorphic Sarcomas (UPS), 3 leiomyomas, 4 normal myometrium samples, and 1 case of Lymphangioleiomyomatosis (LAM). This study only includes the 99 LMS Samples. After gene expression levels were quantified by 3SEQ analysis pipeline, Consensus Clustering with bootstrap method was used to determine that the dataset contained three robust subtypes, and Silhouette analysis was performed to validate the subtype assignments. Two class SAM analysis (Significance Analysis of Microarrays) was performed to identify genes expressed differentially between each subtype of LMS with FDR of 0.05. Immunohistochemical staining was used to validate the potential diagnostic and prognostic markers from 3SEQ data on a tissue microarray.