Ryan Lacinski ^1,2 Sebastian Dziadowicz ^3,4 Clark Roth ⁵ Li Ma ^3,4 Vincent Melemai ² Brody Fitzpatrick ² Edwin Chaharbakhshi ² Tanya Heim ⁵ Ines Lohse ⁵ Karen Schoedel ⁵ Gangqing Hu ^3,4 Nicolas Llosa ⁶ Kurt Weiss ⁵ Brock Lindsey ^6*

• ¹ Cancer Institute, West Virginia University, Morgantown, West Virginia, United States
• ² Department of Orthopaedics, West Virginia University School of Medicine, Morgantown, United States
• ³ Department of Microbiology, Immunology and Cell Biology, School of Medicine, West Virginia University, Morgantown, West Virginia, United States
• ⁴ Bioinformatics Core, West Virginia University School of Medicine, Morgantown, United States
• ⁵ Department of Orthopaedic Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
• ⁶ Department of Orthopaedic Surgery, School of Medicine, Johns Hopkins Medicine, Baltimore, Maryland, United States

Background: The large-scale proteomic platform known as the SomaScan® assay is capable of simultaneously measuring thousands of proteins in patient specimens through next-generation aptamer-based multiplexed technology. While previous studies have utilized patient peripheral blood to suggest serum biomarkers of prognostic or diagnostic value in osteosarcoma (OSA), the most common primary pediatric bone cancer, they have ultimately been limited in the robustness of their analyses. We propose utilizing this aptamer-based technology to describe the systemic proteomic milieu in patients diagnosed with this disease. Methods: To determine novel biomarkers associated with overall survival in OSA, we deployed the SomaLogic SomaScan® 7k assay to investigate the plasma proteomic profile of naive primary, recurrent, and metastatic OSA patients. Following identification of differentially expressed proteins (DEPs) between 2-year deceased and survivor cohorts, publicly available databases including Survival Genie, TIGER, and KM Plotter Immunotherapy, among others, were utilized to investigate the significance of our proteomic findings. Results: Apo-transcobalamin-II (APO-TCN2) was identified as the most DEP between 2-year deceased and survivor cohorts (Log2 fold change = 6.8, P-value = 0.0017). Survival analysis using the Survival Genie web-based platform indicated that increased intratumoral TCN2 expression was associated with better overall survival in both OSA (TARGET-OS) and sarcoma (TCGA-SARC) datasets. Cell-cell communication analysis using the TIGER database suggested that TCN2+ Myeloid cells likely interact with marginal zone and immunoglobin-producing B lymphocytes expressing the TCN2 receptor (CD320) to promote their proliferation and survival in both non-small cell lung cancer and melanoma tumors. Analysis of publicly available OSA scRNA-sequencing datasets identified similar populations in naive primary tumors. Furthermore, circulating APO-TCN2 levels in OSA were then associated with a plasma proteomic profile likely necessary for robust B lymphocyte proliferation, infiltration, and formation of intratumoral tertiary lymphoid structures for improved anti-tumor immunity. Conclusions: Overall, APO-TCN2, a circulatory protein previously described in various lymphoproliferative disorders, was associated with 2-year survival status in patients diagnosed with OSA. The relevance of this protein and apparent immunological function (anti-tumor B lymphocyte responses) was suggested using publicly available solid tumor RNAsequencing datasets. Further studies characterizing the biological function of APO-TCN2 and its relevance in these diseases is warranted.