(P 039) THE GENOMIC AND IMMUNE LANDSCAPE OF OSTEOSARCOMA

Objective: Osteosarcoma (OS) is the most common primary bone malignancy. Previous genetic sequencing studies have shown that among pediatric cancers, OS has one of the highest rates of structural variation with numerous somatic mutations and copy number alterations (CNAs). Additionally, several heritable genetic syndromes can predispose to the development of OS such as Li-Fraumeni syndrome (germline TP53 mutation) and hereditary retinoblastoma (germline RB1 mutation). Although germline alterations are frequently found in OS, genetic testing is not routinely offered. Here, we evaluate tumor genomic alterations, incidental germline findings, and immune profiles of OS patients from one of the largest paired tumor to normal (T/N) tissue datasets.

Methods: De-identified records of patients diagnosed with OS were identified from the Tempus database and filtered to those who underwent Tempus xT next-generation sequencing (NGS). For patients with multiple samples sequenced, the most recent sample was utilized for analyses. The prevalence of pathogenic/likely pathogenic (P/LP) single-nucleotide variants (SNVs), insertions/deletions (indels), and copy number alterations (CNAs, either loss or amplification) were assessed to describe the somatic landscape. Incidental germline analyses were restricted to patients with T/N-matched sequencing and included SNVs and small indels. For each T/N-matched sample, the rate of incidental germline findings detected in 65 reportable hereditary cancer genes was calculated. Of note, the incidental germline panel is not a validated comprehensive germline panel and cannot detect larger deletions/duplications. Immune environmental features including tumor mutational burden (TMB); Programmed Death Ligand 1 (PD-L1); micro-satellite instability (MSI); neoantigen burden; and immune cell composition were also measured from DNA sequencing, RNA Sequencing and immunohistochemistry data.

Results: A total of 126 OS patient records were analyzed (median age = 30 years old (yo), range 1-87 yo, interquartile range: 16-51 yo), including 55 tumor only (44%), and 71 T/N matched-samples (56%) mostly from metastatic disease (86%). Of the 126 samples, 117 (93%) had at least one somatic P/LP mutation, CNA or fusion. We identified frequent somatic mutations in TP53 (n = 44, 35%), RB1 (n=22, 17%), CDKN2A (n=21, 17%), CDKN2B (n=19, 15%), CDK4 (n=18, 14%) and many others (see Table). We detected a total of ten distinct fusions out of 125 patients who had fusion data available, three of which were detected via RNA only (Table). To our knowledge, 6 of the fusions have not been described before. Among the 71 T/N-matched samples, we detected P/LP germline variants in 5(7%) of OS patients, which included MUTYH (n=2, 3%), PMS2 (n=2, 3%) and RB1 (n=1, 1.4%). In the immune environment of OS, MSI was high in one patient, TMB was high in 2 patients (2%) and PD-L1 positivity was observed in 6 out of 55 patients (11%). Immune cell infiltration assays by RNA-sequencing showed that macrophages (median=64% of immune cells) were the predominant immune infiltrating cell over B cells, CD4/8 T cells, and NK cells.

Conclusion: Here we provide an analysis of T/N-matched NGS of a real world OS cohort. Overall, the paired germline samples revealed a greater understanding of both somatic and germline findings than has been previously recognized and reported as well as the immune landscape. Grasping a better understanding of genetic variability in this highly heterogeneous disease, may allow better classification and stratification of osteosarcoma patients. Finally, T/N-matched sequencing in OS patients should be considered as results may have clinical implications for the patient, as well as at-risk family members, thereby providing the opportunity for genetic counseling and risk-stratified interventions.

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