Background – Osteosarcoma (OS) is the most common primary bone malignancy. OS presents across all ages, though it is most prevalent among adolescents in the second decade of life. 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-normal matched (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 (either loss or amplification) were assessed to describe the somatic landscape. Incidental germline analyses were restricted to patients with T/N-matched sequencing and SNVs/indels, where small indels were included. For each T/N-matched sample, the rate of incidental germline findings detected in 65 reportable hereditary cancer genes was calculated. Note, the incidental germline panel is not a validated comprehensive germline panel and cannot detect larger deletions/duplications. Immune environment features (i.e., TMB, PD-L1, MSI, neoantigen tumor burden, immune cell composition) were also measured from DNA-seq, RNA seq, or IHC data.
Results – A total of 126 OS patient records were analyzed (median age=30 years old, range: 1-87, interquartile range: 16-51), including 55 tumor-only (44%) and 71 T/N-matched samples (56%) mostly from the metastatic disease (86%). Of the 126 samples, 117 (93%) had at least one P/LP mutation or 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%), UTG1A1 (n=19, 15%), CDK4 (n=18, 14%), and many others (Table). We detected a total of ten distinct fusions out of 125 patients who had fusion data available (8%), three of which were detected via RNA only (Table). To our knowledge 7 of the fusions have not been described before (Table). Among the 71 T/N-matched samples, we detected incidental P/LP germline variants in 5 OS patients (7%). The most prevalent P/LP germline variants were 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 out of 125 (0.8%), TMB was high in two out of 113 (2%), and PD-L1 positivity was observed in 6 out of 55 patients (11%). Immune cell infiltration assays by RNA-seq showed that macrophages (median=64% of immune cells) were the predominant immune infiltrating cell over B cells, CD4/8 T cells, and NK cells.
Conclusions – 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 that has been previously recognized and reported. Accordingly, 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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