Incidence of ERBB fusions (EGFR, ERBB2, ERBB4) across tumor types Laura Schubert1, Andrew Elliott2, Robert C. Doebele3, Emil Lou4, Hossein Borghaei5, Michael J. Demeure6, Razelle Kurzrock7, Anh T. Le1, Joshua E. Reuss8, Sai-Hong Ignatius Ou9, David R. Braxton6, Christian A. Thomas10, Sourat Darabi6, Wolfgang Michael Korn11, Wafik S. El-Deiry12, Stephen V. Liu13; 1University of Colorado School of Medicine, Denver, CO; 2CARIS Life Sciences, Irving, TX; 3Rain Therapeutics, Newark, CA; 4University of Minnesota School of Medicine, Minneapolis, MN; 5Fox Chase Cancer Center, Philadelphia, PA; 6Hoag Memorial Hospital Presbyterian, Newport Beach, CA; 7University of California San Diego, Moores Cancer Center, La Jolla, CA; 8Georgetown University, Washington, DC; 9University of California Irvine, Orange, CA; 10New England Cancer Specialists, Scarborough, ME; 11Caris Life Sciences, Phoenix, AZ; 12Cancer Center at Brown University, Providence, RI; 13Georgetown University, Department of Hematology and Oncology, School of Medicine, Washington, DC

A 100% B 50% Caris Life Sciences 90% 45% Introduction 80% 40% 70% 35% Gene fusions involving receptor tyrosine kinases are 5 NSCLC ERBB2 FUSIONS ERBB4 FUSIONS 60% 30% EGFR FUSIONS 0.04% 20 Esophageal 16 Breast 0.26% established in multiple cancer types. Gene 50% 25% 4 Esophageal 1 Small intestinal 28 40% 20% 1 Salivary 0.28% fusions can be successfully targeted with small 3 Breast Breast 40 14 Bladder, 30% 15% 0.05% 0.45% 10 Colorectal Ovarian urothelial 0.72% Percent of Fusions Percent Percent of Fusions Percent 35 0.68% molecule inhibitors. ALK, ROS1, RET, and NTRK fusions 20% 10% High 3 Colorectal 1 NOTA 0.30% Grade 10% 5% 2 Pancreatic 4 Cholangio 9 Uterine 0.21% all have FDA approved targeted inhibitors. HER family Glioma 0% 0% 2 Soft tissue 4 Cervical 0.42% 12 Uterine 0.28% 2 Uterine 8 Gastric 0.72% fusions (EGFR, ERBB2, ERBB4) have been previously EGFR ERBB2 ERBB4 EGFR ERBB2 ERBB4 4 CUP 0.23% 2 Soft tissue 0.17% 8 NSCLC 0.07% 7 Ovarian 0.12% In-frame Kinase domain retained Deletion Duplication Inversion Translocation 1 Cervical 5 Bladder, urothelial described, however, there has not been a 2 Prostatic 0.10% 2 CUP 0.11% 1 Gastric 0.10% 6 Pancreatic 0.19% 6 NSCLC 0.05% 2 Cholangio 1 Small Intestinal 0.09% 2 Esophageal comprehensive study of their frequency. HER family Figure 2 – Characteristics of fusions detected in the Caris Life Sciences cohort. (A) Proportion of ERBB fusions that 0.11% 2 Colorectal 0.02% were in-frame or retained the ERBB kinase domain. (B) Distribution of fusion topologies determined from analysis of fusions are important potential candidates for [N fusion-positive samples detected, Cancer Type-specific Prevalence] targeted therapies. In this study, we sought to fusion breakpoints. comprehensively analyze the frequency and Public Data Sets EGFR Fusions ERBB2 Fusions molecular features of EGFR, ERBB2 and ERBB4 EGFR TCGA ERBB2 TCGA ERBB4 TCGA Public data sets Public data sets 3.81% Non-Small Cell Lung Cancer Figure 6 – Proposed mechanisms of activation of HER 4.00% Ovarian Epithelial Tumor fusions. We assessed HER family fusion partners, MSK AACR MSK AACR 5.43% Hepatocellular Carcinoma 4.00% Head and Neck 14.29% Non-Small Fusion Partner Caris LS TCGA IMPACT GENIE Total Fusion Partner Caris LS TCGA IMPACT GENIE Total 30.00% Cell Lung Cancer family fusions and proposed classification 7.80% Esophagogastric Squamous Cell Carcinoma genomic features, cancer types and co-occurring EGFR-SEPT14 20 13 2 12 27 ERBB2-PPP1R1B 3 10 1 11 Invasive Adenocarcinoma Breast 6.00% Endometrial Carcinoma 42.86% nomenclature. SEPT14-EGFR 17 17 ERBB2-IKZF3 10 6 4 10 37.26% 14.29% Endometrial 11.50% Head and Carcinoma 12.00% Non-Small Cell Lung Invasive Breast Carcinoma mutations. EGFR-PSPH 4 ERBB2-CTTN 8 8 Neck Squamous Cancer Carcinoma EGFR-VSTM2A 3 1 2 3 GRB7-ERBB2 2 2 4 6 Cell Carcinoma 4.00% Pancreatic EGFR-LAMA2 3 3 ERBB2-GRB7 2 14.64% Bladder Adenocarcinoma 14.29% Ovarian Conclusions Urothelial Carcinoma 14.00% Esophagogastric 6.00% Cervical Squamous Cell Epithelial Tumor VOPP1-EGFR 1 3 3 C17orf37-ERBB2 2 2 4 Adenocarcinoma Carcinoma Methods 19.55% Diffuse Glioma 10.00% Bladder Urothelial Carcinoma 14.29% Head and Neck • HER fusions are rare, recurrent genomic SEC61G-EGFR 2 2 2 GP2-ERBB2 5 Squamous Cell Carcinoma Tumor samples (n = 64,354; representing > 40 tumors EGFR-GBAS 2 2 ERBB2-PSMB3 3 3 alterations across multiple cancer types. EGFR-SEC61G 2 2 JUP-ERBB2 3 EGFR MSK IMPACT ERBB2 MSK IMPACT ERBB4 AACR GENIE types) submitted to Caris Life Sciences (Phoenix, AZ) EGFR-TNS3 2 2 ERBB2-GSDMA 3 • We identified 811 EGFR fusions, 287 ERBB2 EGFR-VOPP1 2 2 ERBB2-GSDMB 2 8.05% Non- 10% Non-Small Cell Lung Cancer 10% Cancer of Unknown Primary 5.88% Colorectal Cancer 5.88% Soft Tissue Sarcoma VSTM2A-EGFR 1 2 2 ERBB2-TCAP 1 1 1 2 Small Cell Lung were molecularly profiled by next-generation Cancer 5.88% Mixed Cancer Types fusions and 153 ERBB4 fusions across 182,590 ELDR-EGFR 2 2 ERBB2-WIPF2 1 1 2 85.06% Glioma 10% Colorectal 10% Esophagogastric Cancer Cancer 5.88% Cancer of sequencing of DNA (NextSeq, 592-gene panel; or ZNF713-EGFR 2 2 RARA-ERBB2 1 1 2 Unknown Primary 11.76% tumor samples. EGFR-GRB2 2 ERBB2-SHC1 2 10% Prostate 10% Melanoma NovaSeq, whole exome) and RNA (whole LANCL2-EGFR 2 SHC1-ERBB2 1 1 2 Cancer 17.65% Non- • Small Cell Lung Collectively, EGFR, ERBB2, and ERBB4 fusions ERBB2-CDK12 10 2 2 5.88% Endometrial Cancer 10% Bladder Cancer Cancer ERBB2-PGAP3 10 2 2 30% Breast transcriptome). Gene fusion partners, in/out-of-frame Cancer represent up to 1% of cancer patients. PGAP3-ERBB2 8 ERBB4 Fusions 17.65% Breast Cancer status, retention of ERBB kinase domain, topology of ERBB2-STARD3 5 2 2 EGFR AACR GENIE ERBB2 AACR GENIE ERBB4 MSK IMPACT • We identified an increased frequency of TP53 Public data sets ERBB2-IGFBP4 2 fusion breakpoints, and co-alterations were MSK AACR ERBB2-MED1 2 2.40% Colorectal Cancer mutations co-occurring with HER family Fusion Partner Caris LS TCGA IMPACT GENIE Total ERBB2-MED24 2 3.19% Breast 4.55% Endometrial 10.00% Esophagogastric Cancer Cancer characterized for each ERBB fusion transcript IKZF2-ERBB4 20 ERBB2-PLXDC1 2 Cancer 25.00% Colorectal 25.00% Glioma fusions in >70% of Caris Life Sciences data and 65.18% Glioma Cancer ERBB4-IKZF2 13 ERBB2-RAPGEFL1 2 11.82% Non-Small 36.36% Breast Cancer Cell Lung Cancer detected. Fusion prevalence was further examined in LANCL1-ERBB4 3 IKZF3-ERBB2 1 2 2 21.25% Non- >60% across public datasets. Small Cell Lung public data sets (TCGA, MSK-IMPACT and AACR KANSL1L-ERBB4 3 Cancer 3.64% Prostate • HER family fusions are excellent candidates for ERBB4-TRIM33 3 3 Cancer GENIE). ERBB4-PARD3B 1 1 2 Figure 3 – Recurrent fusion isoforms detected in 5.45% Colorectal Cancer targeted therapies. ERBB4-PXMP2 1 1 2 6.36% Ovarian Cancer 4.55% Bladder Cancer 50.00% Non-Small Cell AGAP1-ERBB4 2 Caris Life Sciences (Caris LS) and public data sets. Lung Cancer KLF7-ERBB4 2 ERBB4-FN1 2 Public data sets Figure 4 – HER family fusion frequency by cancer type References Fusion Caris Life TCGA MSK AACR Overall 1. The results shown here are in part based upon data generated by the Sciences PanCancer IMPACT GENIE Frequency Caris Life Sciences Public Data Sets TCGA Research Network: https://www.cancer.gov/tcga. (N= 64,354) (N= 10,967) (N= 10,945) (N=96,324) 2. Cerami et al. The cBio Cancer Genomics Portal: An Open Platform for EGFR ERBB2 ERBB4 EGFR Exploring Multidimensional Cancer Genomics Data. Cancer Discovery. ERBB2 ERBB4 60% 80% 90% May 2012 2; 401. EGFR 0.1% (59) 0.3% (27) 0.8% (88) 0.6% (637) 0.6% (752) 50 80 80 50% 70% 80% 3. Gao et al. Integrative analysis of complex cancer genomics and clinical 60 60 60% 70% 40 ERBB2 0.2% (114) 0.5% (50) <0.1% (10) 0.1% (113) 0.1% (173) 40% 40 40 profiles using the cBioPortal. Sci. Signal. 6, pl1 (2013). 50% 60% 20 30 30 4. The AACR Project GENIE Consortium. AACR Project GENIE: Powering 30% 40% 50% 15 Precision Medicine Through An International Consortium, Cancer ERBB4 0.2% (108) <0.1% (7) <0.1% (4) <0.1% (34) <0.1% (45) 40% 30% 20 20% 20

Frequency (%) Discov. 2017 Aug;7(8):818-831 Frequency (%) 30% 20% Frequency (%) 10 Frequency (%) Frequency (%) Frequency (%) 5. Zehir et al. Mutational landscape of metastatic cancer revealed from 10% 20% 10 10 10% 5 10% prospective clinical sequencing of 10,000 patients. Nat Med. 2017 0% 0% 0% 0 0 0 Jun;23(6):703-713. doi: 10.1038/nm.4333. Epub 2017 May 8. Erratum NF1 APC RB1 APC TP53 TERT SPEN RB1 KRAS Figure 1 – Overall ERBB family fusion incidence in the Caris Life NF1 TERT EGFR ATRX PTEN B2M ASXL1 TP53 TERT

RHOA NF1 KRAS ATRX ERBB2 ERBB3 CHEK2 PTEN RB1 in: Nat Med. 2017 Aug 4;23 (8):1004. PMID: 28481359; PMCID: NSD1 NF1 NF1 RUNX1

NTHL1 RB1 RB1 PIK3CA FBXW7 TP53 KMT2D ASXL1 PIK3R2 PIK3R1 KDM6A PIK3CA KMT2C ARID1A FGFR3 PTEN TP53 TP53 BRCA1 KDM5C EGFR ARID1A GATA3 MUTYH PTEN PTEN RUNX1 CDKN2A

PIK3CA EGFR EGFR FBXW7 KMT2D CTNNB1 CDKN2A ARID1A KDM6A BRCA1BRCA2 ERBB2ERBB3ERBB4 BRCA1BRCA2 ERBB2ERBB3ERBB4 BRCA1BRCA2 ERBB2ERBB3ERBB4

CDKN2A PIK3CA

MAP2K4 PIK3CA PIK3CA SMARCA4 CDKN2A CDKN2A CDKN2A Sciences and public data sets. SMARCA4 PMC5461196. ASCO June 2021 [email protected] Figure 5 – Co-occuring alterations in selected cancer-related TCGA MSK IMPACT AACR GENIE