Abstract The BostonGene Tumor PortraitTM Test (TPT) is a comprehensive molecular profiling assay with both integrated genomic and transcriptomic analysis. The combined analysis offers additional information that is potentially critical for personalized clinical decision making. We retrospectively reviewed 2 sarcoma cohorts that received the TPT, MG (n=21) and MD (n=40) (Table 1), and evaluated reported events that were not previously identified by institute respective targeted panels. A total of 19 (MG) and 23 (MD) additional clinically-relevant SNV/indels, CNAs, and fusions were identified by the TPT, including an amplification of EZH2, 2 novel fusions with MDM2 as a binding partner, and a known diagnostic fusion, NAB2-STAT6. A high TMB was observed in 5 patients. MSI was stable for all patients. HLA class I loss of heterozygosity was found in 7 patients. In both cohorts, we observed significant changes in the expression of potentially targetable genes, with 81% of patients presenting with increased expression of such targets. Transcriptomic-based tumor microenvironment (TME) classification found that 51% and 49% of patients had an Immune-Enriched (IE) subtype (predicted favorable IO treatment outcomes) and Fibrotic/Desert (F/D) subtypes (predicted poor response to IO treatment), respectively. Predicted response to IO therapy through TME subtyping agreed with previous reports, as angiosarcomas were enriched in the IE subtype (83%), and bone sarcomas were enriched in the F (30%) compared to the IE (6%) subtype. Further, these results supported matching patients with biomarker-driven clinical trials at their respective centers. In summary, the TPT identified more clinically-relevant biomarkers compared to respective institute targeted gene panels, highlighting the potential value of comprehensive molecular profiling for patients with highly heterogeneous diseases, such as sarcoma, to support personalized treatment decisions. Table 1. Genetic and expression alterations identified by the BostonGene Tumor PortraitTM Test Event type analyzed in targeted panels Event type Cohort (N patients)* % cohort with event or # biomarkers Events, status, or subtype Yes SNV/Indel MG (21) 10% FAT4 Y558_V590delinsCAfs*5 LOF mutation COL2A1 W1348Nfs*13 LOF mutation Yes SNV/Indel MD (40) 6% KMT2B R1779* LOF mutation KMT2C S143Vfs*3 LOF mutation PRKDC Y4046* LOF mutation Yes Germline MG (21) 5% MUTYH rs36053993, germline, pathogenic Yes Germline MD (40) 3% BTD rs13078881, germline, pathogenic Yes CNA MG (21) 43% IGF1R amplification +8 copies MGMT loss FANCA loss MCL1 amplification +10 copies MYOCD amplification +3 copies AKT2 amplification +8 copies AMACR amplification +6 copies YAP1 amplification +7 copies TRAF2 loss EZH2 amplification +2 copies HMGA2 amplification +100 copies FRS2 amplification +61 copies DDIT3 amplification +39 copies Yes CNA MD (40) 27.5% ERK1 amplification +2 copies FOXO1 amplification +5 copies HMGA2 amplification +2, +8 copies KMT2C amplification +3 copies NCOR1 amplification +7 copies TERT amplification +6 copies TSPAN31 amplification +30 copies HSF1 loss Yes Fusions MG (20) 10% MDM2-CR1 MDM2-TXNDC12 AC090825.1-IGF1R Yes Fusions MD (39) 15% FUS-KIAA1549 KIAA1549-CREB3L2 HMGA2-LRRC37A3 NAB2-STAT6 PPP1R12A-PAWR RB1-ZAR1L No TMB** MG+MD (61) 8% 35.67 mut/Mb (Desmoid fibromatosis) 8.48 mut/Mb (Cutaneous angiosarcoma) 8.9 mut/Mb (Sarcoma, NOS) 16.1 mut/Mb (Skin Angiosarcoma) 18.8 mut/Mb (Undifferentiated pleomorphic sarcoma) No MSI status MG+MD (61) 100% Stable No HLA loss of heterozygosity MG+MD (61) 11% HLA-I LOH (Leiomyosarcoma (N=1), Ewing Sarcoma (N=1)) HLA-A (Dedifferentiated liposarcoma (N=1)) HLA-I (Chondrosarcoma (N=3), High-grade sarcoma (N=1)) No Targetable surface molecule overexpression MG+MD (59) 81% CTLA4, EGFR, ERBB2, MAGEA3, PD1, PDL1, TIM3, TROP, ERBB2, FOLR1, NECTIN4, ROR1, TROP2 No Molecular Functional PortraitTM type MG+MD (59) 32% Fibrotic No Molecular Functional PortraitTM type MG+MD (59) 17% Immune Desert No Molecular Functional PortraitTM type MG+MD (59) 24% Immune-Enriched, Fibrotic No Molecular Functional PortraitTM type MG+MD (59) 27% Immune-Enriched, Non-fibrotic No MHC deficiency MG+MD (59) 3% MHC class I/II deficiency No MHC deficiency MG+MD (59) 3% MHC class II deficiency No FDA label biomarkers MG+MD (59) 7 biomarkers - Desmoid fibromatosis, TMB 35.67 mut/Mb (Pembrolizumab) - Angiosarcoma, TMB 16.1 mut/Mb (Pembrolizumab); - Undifferentiated pleomorphic sarcoma, MSI, TMB 18.8 mut/Mb (Pembrolizumab); - Cutaneous angiosarcoma, PALB2 pathogenic germline variant (Olaparib); - Chondrosarcoma IDH1 R132L (Ivosidenib) No Transcriptomic biomarkers MG+MD (59) 29 biomarkers CD8+ T cell number; PDL1 expression level; SLFN11 expression level; SMARCB1 expression; CD8+ T cell number No Diagnostic biomarkers MG+MD (59) 9 biomarkers NAB2-STAT6; MXI1-NUTM1; EWSR1-NR4A3; EWSR1-FLI1; EWSR1-CREB3L1; NAB2-STAT6 - - - - *RNA analysis failed for 2 patients. Therefore, any expression based analysis was performed on n=20 and n=39 for the MG and MD cohort, respectively. **Tumor mutational burden is considered high for FFPE samples when TMB>8mut/Mb. Citation Format: Gregory M. Cote, Anna Novokreshchenova, Irina Zhuk, Aida Aukhadieva, Sandrine Degryse, Aleksei Shevkoplias, Anna Belozerova, Krystle Nomie, Nikita Kotlov, Georgy Sagardze, Lev Bedniagin, Jessica Brown, Ekaterina Postovalova, Alexander Bagaev, Nathan Fowler, J. Andrew Livingston, Vinod Ravi. Use of comprehensive molecular profiling to identify additional clinically-relevant alterations compared to targeted gene panels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 244.