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Malignant pleural mesothelioma (MPM), a rare cancer a long latency period (up to 40 years) between asbestos exposure and disease presentation. The mechanisms coupling asbestos to recurrent somatic alterations are poorly defined. Gene-fusions arising through genomic instability may create novel drivers during early MPM evolution. We explored the gene fusions that occurred early in the evolutionary history. We conducted multiregional whole exome sequencing (WES) of 106 samples from 20 patients undergoing pleurectomy decortication and identified 24 clonal non-recurrent fusions, three of which were novel (FMO9P-OR2W5,GBA3andSP9). The number of early gene fusion events detected varied from zero to eight per tumour, and presence of gene fusions was associated with clonal SCNAs involving the Hippo pathway genes and homologous recombination DNA repair genes. Fusions involved known tumour suppressorsBAP1,MTAP, andLRP1B,and a clonal oncogenic fusion involvingCACNA1D-ERC2, PARD3B-NT5DC2andSTAB2-NT5DC2fusions were also identified as clonal fusions. Gene fusions events occur early during MPM evolution. Individual fusions are rare as no recurrent truncal fusions event were found. This suggests the importance of early disruption of these pathways in generating genomic rearrangements resulting in potentially oncogenic gene fusions.

MPM is a pleural tumour associated with asbestos exposure. There is a long latency period of more than 40 years between asbestos exposure and disease presentation, highlighting an unknown evolutionary trajectory. Throughout the latent years, different somatic structural alterations are acquired by the growing tumour. To elucidate this evolutionary path, we conducted multiregional whole exome sequencing (WES) of 106 samples (5 or 4 regions per tumour and a matched blood sample) for 20 patients as part of the MEDUSA study (Mesothelioma evolution: Drugging somatic alterations). Gene fusions have been shown to have a clinical, diagnostic, and therapeutic significance i.e., the driver fusion EM4-ALK expressed in a minority of peritoneal mesotheliomas. Previous descriptions of gene fusions in MPM have mostly been limited to isolated case reports, with the only genome-wide assessment study identifying fusion events involving known MPM genes including NF2 and BAP1. Therefore, our aim was to identify potential truncal inter- and intra-chromosomal rearrangements generating gene fusions that occurred early in the evolutionary history of the tumour. We used two software tools - Meerkat (v.0.189) and Delly (v0.8.7), to identify gene fusion events in the regional tumour sites for a given patient. We identified tumour-specific events by filtering against the calls made on exome sequence from the matched blood of each patient. Finally, we identified truncal gene fusion events as ones observed across all regional samples of a particular patient. We validated the gene fusions by PCR and Sanger sequencing of the breakpoint junctions. Further work was carried out to identify truncal fusion transcripts at the transcriptomic level using RNA sequencing data (Arriba v2.1.0) and compare the gene fusion predictions detected at the genomic level made by WES. We found a total of 24 truncal fusion events, 3 of which were novel (FMO9P-OR2W5, GBA3 and SP9). Tumour suppressor genes involved included (BAP1, MTAP, and LRP1B). Oncogenic fusions involved (CACNA1D-ERC2). In addition to this, our analysis revealed truncal copy number neutral events such as in-frame PARD3B-NT5DC2 and out of frame STAB2-NT5DC2. Moreover, with transcriptome sequencing we characterised the fusion transcripts events initially detected by WES (i.e. fusions involving AHRR and B3GAL2 genes). Our findings suggest that gene fusions events occur during early evolution with marked heterogeneity across the cohort (no recurrent truncal fusions event were observed in a subset). Some truncal fusion events are likely to be drivers of cancer progression, as they involve mesothelioma associated genes (e.g. MTAP and BAP1). The role of other gene fusions remains unclear, and larger studies are needed to clarify whether somatic rearrangements of these genes are involved in tumour progression in MPM. Citation Format: Maymun Jama, Edward J Hollox, Apostolos Nakas, Lee Brannan, Alan Dawson, frank dudbridge, Dean Fennell. Mesotheliomas harbor early clonal fusions involving both tumor suppressor drivers and novel oncogenic alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5693.

Malignant Pleural Mesothelioma (MPM) is typically diagnosed 20–50 years after exposure to asbestos and evolves along an unknown evolutionary trajectory. To elucidate this path, we conducted multi-regional exome sequencing of 90 tumour samples from 22 MPMs acquired at surgery. Here we show that exomic intratumour heterogeneity varies widely across the cohort. Phylogenetic tree topology ranges from linear to highly branched, reflecting a steep gradient of genomic instability. Using transfer learning, we detect repeated evolution, resolving 5 clusters that are prognostic, with temporally ordered clonal drivers. BAP1/−3p21 and FBXW7/-chr4 events are always early clonal. In contrast, NF2/−22q events, leading to Hippo pathway inactivation are predominantly late clonal, positively selected, and when subclonal, exhibit parallel evolution indicating an evolutionary constraint. Very late somatic alteration of NF2/22q occurred in one patient 12 years after surgery. Clonal architecture and evolutionary clusters dictate MPM inflammation and immune evasion. These results reveal potentially drugable evolutionary bottlenecking in MPM, and an impact of clonal architecture on shaping the immune landscape, with potential to dictate the clinical response to immune checkpoint inhibition., The impact of intratumour heterogeneity on immune surveillance and clinical outcomes has not been adequately explored in malignant pleural mesothelioma (MPM). Here the authors analyse the influence of evolution on the survival and immune landscape of MPM patients using multi-region sequencing data.

Malignant Pleural Mesothelioma (MPM) is typically diagnosed 20-50 years after exposure to asbestos and evolves along an unknown evolutionary trajectory. To elucidate this path, we conducted multi-regional exome sequencing of 90 tumour samples from 22 MPMs acquired at surgery. Here we show that exomic intratumour heterogeneity varies widely across the cohort. Phylogenetic tree topology ranges from linear to highly branched, reflecting a steep gradient of genomic instability. Using transfer learning, we detect repeated evolution, resolving 5 clusters that are prognostic, with temporally ordered clonal drivers. BAP1/-3p21 and FBXW7/-chr4 events are always early clonal. In contrast, NF2/-22q events, leading to Hippo pathway inactivation are predominantly late clonal, positively selected, and when subclonal, exhibit parallel evolution indicating an evolutionary constraint. Very late somatic alteration of NF2/22q occurred in one patient 12 years after surgery. Clonal architecture and evolutionary clusters dictate MPM inflammation and immune evasion. These results reveal potentially drugable evolutionary bottlenecking in MPM, and an impact of clonal architecture on shaping the immune landscape, with potential to dictate the clinical response to immune checkpoint inhibition.