Dottorato in MEDICINA MOLECOLARE

Titolo della tesi: Comprehensive analysis of gene expression profiles in paediatric myofibroblastic neoplasms

ABSTRACT OBJECTIVES. Myofibroblastic lesions in children are a group of soft tissue proliferations that range from reactive to hamartomatous to neoplastic including a full spectrum of benign, intermediate, and malignant neoplasms. The aim of this study was to characterize the gene expression profiles of paediatric myofibroblastic neoplasms carrying TRK fusions and classified as Inflammatory Myofibroblastic Tumors (IMT), Infantile Fibrosarcoma (IFS) or TRK-driven mesenchymal tumors according to Paediatric tumors WHO classification 2022 in order to define their differences and the potential pathways responsible for their clinical diversity. MATERIALS AND METHODS. We performed unbiased transcriptome-wide RNA-sequencing analysis on all the cases of myofibroblastic lesions arrived at Bambino Gesù Hospital of Rome from January 2000 to December 2023 as part of diagnostic work up or for central review in the context of AIEOP protocols. RNA was extracted from paraffin embedded tissues. NGS sequencing was performed on an Illumina platform NextSeq analyzed through the software Archer FusionPlex OPBG custom V2, (Invitae, San Francisco, CA) and SureSelectXT HS2 RNA system with Human All Exon V6 + COSMIC Probe (Agilent Technologies, Santa Clara, CA, USA). Analysis results were validated using bulk transcriptomic datasets. RESULTS. Our integrative analysis was performed on 46 samples and included 15 IF (7 with prominent vascular pattern, all with ETV6::NTRK3 fusion), 4 vascular lesions from 2 patients, arising in the site of previous IF or at distance, after 10 and 2 years respectively, all carrying the ETV6::NTRK3 fusion, 9 IF-like lesions and 6 TRK rearranged paediatric mesenchymal tumors. In addition there were 12 IMT. The RNA seq analysis demonstrated three main groups of myofibroblastic neoplasms with distinct expression profiles: Classic IFS (cluster 1), IFS with prominent vascular pattern (IFSv)/(cluster 2) and IMT (cluster 3). Hemangiomas which were separated from the myofibroblastic lesions and formed an additional small cluster (cluster 4). IFS-like tumors are IFS showing TRK rearrangement different from ETV6::NTRK3. These IFS-like and the small subset of TRK-rearranged mesenchymal tumors arising in older children formed 2 heterogeneous clusters, one including lesions harboring RAF and BRAF fusions closely related to IMT group (cluster 5) and the other one very close to IFS (cluster 6). The latter did not form a homogeneous cluster. DISCUSSION. The classification of paediatric myofibroblastic lesions has been deeply modified with the advent of molecular characterization. The initial studies demonstrated that IFs carry an ETV6::NTRK3 rearrangement, while IMT are mostly characterized by ALK, ROS or, less frequently NTRK, RET rearrangements. The routinely use of molecular characterization has highlighted the heterogeneity of molecular alterations in these lesions with identification of ALK rearrangement in IF and NTRK3 fusions in IMT, raising the need for a redefinition of diagnostic criteria of these entities, and their classification according to morphology, molecular alterations or clinical features. Our study demonstrates that ETV6::NTRK3 rearranged IMT cluster with ALK rearranged IMT, and IFS-like lesions with ALK rearrangement cluster with other IFS-like lesions. Thus the molecular profile reflects and confirms their classification based on morphology. The different pathways involved in IMT, IFS, TRK are being investigated by bioinformatic analyses. The preliminary results highlight also a different molecular profile in IFSv and classic IFS. The occurrence in two patients of hemangiomas with ETV6::NTRK fusion in the site of previous IFS or at distance after years is an unusual event, so far never reported and may be related to an evolution of the tumoral stem cell, probably under the influence of environmental and epigenetic factors towards a vascular rather than a myofibroblastic lineage. This study may pave the way to an evolution towards an integrated classification of myofibroblastic tumors, therapy oriented and prognostically significant.