Whole Transcriptome Sequencing Reveals Oncogenic Fusions in Melanoma

Sourat Darabi1, Andrew Elliott2, David R. Braxton1, Jia Zeng2, Kelsey Poorman2, Geoffrey T. Gibney3, Justin Moser4, Thuy Phung5, Michael B. Atkins3, Gino K. In6, W. Michael Korn2, Burton L. Eisenberg1, and Michael J. Demeure1 1Hoag Family Cancer Institute, Newport Beach, CA; 2Caris Life Sciences, Phoenix, AZ; 3MedStar Georgetown University Hospital, Washington, DC; 4HonorHealth Scottsdale Shea Medical Center, Scottsdale, AZ; 5University of South Alabama, Mobile, AL; 6USC Keck School of Medicine, Los Angeles, CA

RAF Kinase Fusions Other Oncogenic Fusions Introduction Results Breakpoint Breakpoint • Somatic genomic alterations occur frequently in melanoma Partner Oncogene Partner Oncogene • BRAF mutations are the most clinically relevant as they predict for response to AGK-Exon 2 Tyr Kinase Exon 8 (n=4) SEPT9-Exon 1 Tyr Kinase Exon 3 targeted therapies * AKAP9-Exon 18 Exon 9 ZNF814-Exon 1 Exon 4 • Oncogenic gene fusions are frequently identified in different cancers with an * AKAP9-Exon 10 Exon 11 NF1-Exon 1 Exon 9 PRKCA unknown incidence in melanoma CEP68-Exon 5 Exon 10 * PPM1D-Exon 5 PP2C Exon 9 * CAP- CAP- • Targeted therapies are approved for specific gene fusions in other tumor types and CLIP2-Exon 8 Gly Gly Exon 9 * FAM172A-Exon 5 Arb2 Telo-RBD RVT Exon 2 are now standard of care CUX1-Exon 7 Exon 8 * TERT 1 LPCAT1-Exon 1 Telo-RBD RVT Exon 2 • The aim of this retrospective study was to determine the prevalence of oncogenic ERC1-Exon 15 CAST Exon 8 Oncogene Partner fusions in metastatic or locally advanced melanoma * FLOT1-Exon 11 SPFH Exon 9 • Gene expression analysis across a broad group of melanomas with and without GTF2I-Exon 8 GTF2I Exon 10 AXL-Exon 19 V-set Ig2 Fn3 Fn3 Tyr Kinase MEF2B-Exon 3 BRAF * fusions was performed in order to better elucidate the functional consequences of ITSN1-Exon 14 EF Hand EF Hand Exon 11 FGFR3-Exon 17 Ig I-set TACC3-Exon 3 gene fusions in this aggressive malignancy KIAA1549-Exon 10 DUF3827 Exon 9 NUDCD3-Exon 5 NUDC CS Exon 9 ↑Figure 1b. Other Oncogenic Fusions * PDE4D-Exon 6 Exon 9 (n=2) Identified 8 non-RAF Kinase fusion involving * PLGRKT-Exon 3 Exon 11 PRKCA (4), TERT (2), AXL (1), and FGFR3 (1), I-setStudy Highlights SND1-Exon 10 SNase SNase Exon 11 including 6 novel (*) fusions. Methods TRIM24-Exon 6 B-box B-box Exon 9 • 33 patients with an in-frame oncogenic fusions identified • Retrospective analysis of FFPE patient tumors that were profiled as part of routine YWHAZ-Exon 2 14-3-3 Exon 9 • Including 15 novel fusions clinical testing (Caris Life Sciences, Phoenix, AZ) * ZSCAN25-Exon 7 SCAN ZF Exon 10 ←Figure 1a. RAS Kinase Fusions • 10 (32.3%) exhibited PD-L1 expression by IHC (SP142 or 28-8) • Samples were profiled by next-generation sequencing of DNA/RNA (592-gene ANO10-Exon 13 Anoctamin Tyr Kinase Exon 10 We identified 25 patients with fusions • 11 (33.3%) were TMB-High panel/whole transcriptome [WTS]) and immunohistochemistry IMPDH2-Exon 13 CBS CBS IMPDH Exon 7 • Fusion transcripts with unknown pathogenicity were detected in 668 (53.2%) of * RAF1 involving BRAF (21) or RAF1 (4) genes, • In addition to detection of fusions by WTS, samples were exampled for co- * NAP1L4-Exon 14 NAP Exon 8 including 9 novel (*) fusions. melanoma samples alterations, including tumor mutational burden (TMB), deficient mismatch * PITPNA-Exon 10 IP-trans Exon 8 • 2,404 total unclassified fusion isoforms detected repair/high microsatellite instability (dMMR/MSI-High), and PD-L1 protein • 10 recurrent fusions (≥ 3 occurrences) expression. • RNA expression analysis of Wnt/β-catenin, PI3K/AKT/MTOR, DNA repair, INFG, and JAK/STAT pathways showed a high degree of variability among fusion-positive Figure 2. Fusion prevalence compared to other biomarkers in melanoma. tumors Oncogenic fusions were rare (2.6%, n=33 samples) compared to other Immune cell populations T cells Expression Max • DNA damage repair pathway mutation frequency similar in patients with fusion- RAS/RAF pathway alterations (63.4%), including BRAF-V600X (30.0%) and CD8 T cells Results Cytotoxic lymphocytes positive and fusion-negative tumors NF1 mutations (62.3%). TMB-High and/or PD-L1 expression were observed Median • 1,255 melanoma specimens were screened for fusions (Table) NK cells in nearly half (47.5% and 43.4%, respectively) of all melanoma samples Min • We identified 33 (2.6%) tumors with in-frame oncogenic fusions (Figures 1a and 1b) B lineage examined. TMB • 25 Raf kinase fusion: 21 BRAF fusions and 4 RAF1 fusions Monocytic lineage Max Myeloid dendritic cells • 796 (63.4%) cases with RAS/RAF pathogenic or likely pathogenic mutations 10 mut/Mb 70% 63.4% 62.3% Neutrophils • 373 (30.0%) BRAF p.V600X mutations 60% Stromal cell populations Endothelial cells Min 47.5% Conclusions • Tumors harboring PRKCA and TERT fusions were each detected with at least one MAPK 50% 43.4% Fibroblasts Alteration/IHC 40% Markers of potential TMB Positive • Oncogenic gene fusions are rare in melanoma pathway co-alteration (NRAS, NF1, or BRAF p.V600E mutation) 30.0% 30% response to IO-therapy dMMR/MSI-High Negative • Potentially actionable fusions, in addition to co-alterations in fusion-positive 17.7% Indeterminate

20% PD-L1 IHC (SP142/28-8) cases, can be identified through comprehensive tumor molecular profiling Percent of cohort of Percent 10% Key signaling and regulatory IL6/JAK/STAT3 Signaling Gender Patient/Tumor Characteristics 2.6% Male • Clinical trials or targeted treatments would be an options for patients with fusion- 0% pathway gene expression Interferon Gamma Response DNA Repair Female positive advanced melanoma Total, N cases 1,255 Apoptosis Tumor site MYC Targets Metastatic Median Age, years (SD) 67 (13.5) WNT/Beta Catenin Signaling Primary - Age Range, years 3-90+ PI3K/AKT/MTOR Signaling Age Max MAPK Pathway Activation Score Female/Male, N cases 478/777 RAS/RAF pathway mutations BRAF-V600E Median Selected References NRAS-MT Min - (% Female/% Male) (38.1%/61.9%) Birkeland, E., Zhang, S., Poduval, D., Geisler, J., Nakken, S., Vodak, D., ... & Lønning, P. E. (2018). Patterns of genomic Fusions BRAF Fusion evolution in advanced melanoma. Nature communications, 9(1), 2665. 780/456 RAF1 Fusion Joseph, E. W., Pratilas, C. A., Poulikakos, P. I., Tadi, M., Wang, W., Taylor, B. S., ... & Rosen, N. (2010). The RAF inhibitor Metastatic/Primary, N cases Figure 3. Expression analysis. PRKCA Fusion PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF-selective manner. Proceedings of the National (63.1%/36.9%) TERT Fusion - (% Metastatic/% Primary) The heatmap shows BRAF fusions have a trend toward Academy of Sciences, 107(33), 14903-14908. - [N unclear] [19] FGFR3 Fusion Kumar-Sinha, C., Kalyana-Sundaram, S., & Chinnaiyan, A. M. (2015). Landscape of gene fusions in epithelial cancers: seq and low immune cell abundance and increased stromal AXL Fusion ye shall find. Genome medicine, 7(1), 129. population. Hierarchical clustering based on immune Patient/Tumor characteristicsGender (Male) Quan, V. L., Panah, E., Zhang, B., Shi, K., Mohan, L. S., & Gerami, P. (2019). The role of gene fusions in melanocytic Table. Patient/Tumor Characteristics Metastatic neoplasms. Journal of cutaneous pathology. and stromal cell population abundance. Age AACR, April 2021