Histopathological and Molecular Profiling of Clear Cell Sarcoma and Correlation with Response to Crizotinib: An Exploratory Study Related to EORTC 90101 'CREATE' Trial

Simple Summary Clear cell sarcoma (CCSA) is a rare subtype of soft tissue sarcoma characterized by EWSR1 rearrangement and subsequent MET upregulation. The European Organisation for Research and Treatment of Cancer 90101 phase II trial evaluated the MET inhibitor crizotinib in CCSA but resulted in only sporadic responses. The aim of this exploratory study was to identify the molecular alterations potentially relevant for the treatment outcome by using archival CCSA samples and trial-related clinical data. We characterized MET signaling and revealed an infrequent activation of MET, which may explain the lack of response to crizotinib in the disease cohort. Based on sequencing analyses, we discovered copy number alterations, mutations and dysregulated pathways with potentially predictive or prognostic values for patients’ outcomes. This work describes the molecular heterogeneity in CCSA and provides deep insight into the biology of this ultra-rare malignancy, which may potentially lead to better therapeutic approaches. Abstract Clear cell sarcoma (CCSA) is characterized by a chromosomal translocation leading to EWSR1 rearrangement, resulting in aberrant transcription of multiple genes, including MET. The EORTC 90101 phase II trial evaluated the MET inhibitor crizotinib in CCSA but resulted in only sporadic responses. We performed an in-depth histopathological and molecular analysis of archival CCSA samples to identify alterations potentially relevant for the treatment outcome. Immunohistochemical characterization of MET signaling was performed using a tissue microarray constructed from 32 CCSA cases. The DNA from 24 available tumor specimens was analyzed by low-coverage whole-genome sequencing and whole-exome sequencing for the detection of recurrent copy number alterations (CNAs) and mutations. A pathway enrichment analysis was performed to identify the pathways relevant for CCSA tumorigenesis. Kaplan–Meier estimates and Fisher’s exact test were used to correlate the molecular findings with the clinical features related to crizotinib treatment, aiming to assess a potential association with the outcomes. The histopathological analysis showed the absence of a MET ligand and MET activation, with the presence of MET itself in most of cases. However, the expression/activation of MET downstream molecules was frequently observed, suggesting the role of other receptors in CCSA signal transduction. Using sequencing, we detected a number of CNAs at the chromosomal arm and region levels. The most common alteration was a gain of 8q24.21, observed in 83% of the cases. The loss of chromosomes 9q and 12q24 was associated with shorter survival. Based on exome sequencing, 40 cancer-associated genes were found to be mutated in more than one sample, with SRGAP3 and KMT2D as the most common alterations (each in four cases). The mutated genes encoded proteins were mainly involved in receptor tyrosine kinase signaling, polymerase-II transcription, DNA damage repair, SUMOylation and chromatin organization. Disruption in chromatin organization was correlated with longer progression-free survival in patients receiving crizotinib. Conclusions: The infrequent activation of MET may explain the lack of response to crizotinib observed in the majority of cases in the clinical trial. Our work describes the molecular heterogeneity in CCSA and provides further insight into the biology of this ultra-rare malignancy, which may potentially lead to better therapeutic approaches for CCSA.