Alexa B. Schrock

Supplemental Table 1: Demographics and clinical characteristics of the ERBB2-amplified mBC cohort treated with first-line trastuzumab-based regimens Supplemental Table 2: Demographics and clinical characteristics of the ERBB2-amplified mBC cohort treated with T-DXd Supplemental Table 3: Demographics and clinical characteristics of the ERBB2-amplified aGE cohort treated with first-line trastuzumab-based regimens Supplemental Table 4: Demographics and clinical characteristics of the ERBB2-amplified CRC cohort treated with trastuzumab Supplemental Table 5: ERBB2 NGS and HER2 IHC/FISH concordance for 3,650 mBC patients Supplemental Table 6: Demographics and clinical characteristics of the HER2+ mBC cohort treated with 1L trastuzumab-based regimens Supplemental Table 7: Demographics and clinical characteristics of the HER2+ mBC cohort treated with T-DXd Supplemental Table 8: Demographics and clinical characteristics of the HER2+ mBC cohort treated with T-DM1 Supplemental Table 9: Demographics and clinical characteristics of the HER2-low/ultra-low mBC cohort treated with 2L + T-DXd Supplemental Table 10: ERBB2 NGS and HER2 IHC/FISH concordance for 1,168 aGE patients Supplemental Table 11: Demographics and clinical characteristics of the HER2+ aGE cohort treated with 1L trastuzumab-based regimens.

Purpose:Gene copy number (CN) amplifications and protein overexpression are common drug targets, and detection relies on various methodologies, including next-generation sequencing–based CN, immunohistochemistry (IHC), and in situ hybridization (ISH). We investigated the pan-tumor landscape of amplifications and developed AmpRatio, a novel method of CN quantitation.Experimental Design:Pan-tumor tissue (N = 486,340) and liquid (N = 85,635) samples underwent hybrid capture–based comprehensive genomic profiling. A genome-wide CN model for each sample was generated to estimate the purity, ploidy, and segment-level CN. AmpRatio was calculated by dividing gene CN/sample ploidy. A US-based deidentified clinicogenomic database was utilized to assess the relationship between ERBB2 AmpRatio and HER2 IHC/FISH and outcomes on anti-HER2 therapies.Results:Amplifications with varying degrees of gain were reported in 38.6% of pan-tumor tissue samples, most frequently MYC (5.6%), 11q13 (5.2%), ERBB2 (5.2%), and CCNE1 (3.2%). ERBB2 AmpRatio was associated with HER2 positivity by IHC/FISH in gastroesophageal [overall percent agreement (OPA) 90%] and breast (OPA 95%) cancers. Among patients treated with anti-HER2 therapies, ERBB2 AmpRatio significantly stratified outcomes within the ERBB2-amplified and IHC-defined HER2+ and HER2-low/ultralow populations. High concordance (sensitivity 88%) of amplification detection in liquid biopsy versus tissue was associated with higher AmpRatio and ctDNA tumor fraction ≥20%.Conclusions:CN amplifications are prevalent and diverse biomarkers, and AmpRatio is variable across genes and tumor types. ERBB2 AmpRatio is associated with outcomes to HER2-directed therapies and may have utility alongside IHC for clinical decision-making. With the increasing number of therapies targeting amplifications/overexpression, it will be important to define harmonized methods for CN quantification for optimal patient selection.

Supplemental Figure 1: Modeled CN vs. AmpRatio Supplemental Figure 2: Amplification ratio purity invariance Supplemental Figure 3: Modeled gene CN vs. AmpRatio Supplemental Figure 4: CONSORT diagram Supplemental Figure 5: Pan-tumor MDM2 amplification landscape Supplemental Figure 6: MDM2 co-alteration landscape Supplemental Figure 7: Tissue ploidy distribution by tumor type Supplemental Figure 8: CN segment length vs. AmpRatio Supplemental Figure 9: Amplicon focality Supplemental Figure 10: Pan-tumor AmpRatio distribution of emerging targets Supplemental Figure 11: Breast ERBB2 co-alteration landscape Supplemental Figure 12: GE ERBB2 co-alteration landscape Supplemental Figure 13: CRC ERBB2 co-alteration landscape Supplemental Figure 14: Breast CGDB CONSORT diagram Supplemental Figure 15: GE CGDB CONSORT diagram Supplemental Figure 16: CRC CGDB CONSORT diagram Supplemental Figure 17: rwPFS for ERBB2-amplified mBC, aGE, and mCRC patients treated with trastuzumab-based regimens Supplemental Figure 18: Exploratory AmpRatio cutoffs for ERBB2-amplified mBC, aGE, and mCRC patients treated with trastuzumab-based regimens Supplemental Figure 19: Outcomes for ERBB2-amplified mBC patients treated with T-DXd Supplemental Figure 20: Exploratory AmpRatio cutoffs for ERBB2-amplified mBC patients treated with T-DXd Supplemental Figure 21: Performance of ERBB2 AmpRatio for predicting HER2-positivity Supplemental Figure 22: Association between ERBB2 AmpRatio and HER2 IHC/FISH Supplemental Figure 23: rwPFS on anti-HER2 regimens within HER2 IHC-defined subgroups Supplemental Figure 24: Exploratory AmpRatio cutoffs for anti-HER2 regimens within HER2 IHC-defined subgroups Supplemental Figure 25: Outcomes for HER2+ mBC patients treated with T-DXd Supplemental Figure 26: Exploratory AmpRatio cutoffs for HER2+ mBC patients treated with T-DXd Supplemental Figure 27: Outcomes for HER2+ mBC patients treated with T-DM1 Supplemental Figure 28: Exploratory AmpRatio cutoffs for HER2+ mBC patients treated with T-DM1 Supplemental Figure 29: Sensitivity analysis for outcomes of HER2+ mBC treated with 1L trastuzumab-based regimens Supplemental Figure 30: Sensitivity analysis for outcomes of HER2+ mBC treated with T-DXd Supplemental Figure 31: Sensitivity analysis for outcomes of HER2+ mBC treated with T-DM1 Supplemental Figure 32: Outcomes for HER2+ aGE patients treated with 1L trastuzumab-based regimens Supplemental Figure 33: Sensitivity analysis for outcomes of HER2+ aGE treated with 1L trastuzumab-based regimens Supplemental Figure 34: NSCLC MET co-alteration landscape Supplemental Figure 35: NSCLC EGFR co-alteration landscape Supplemental Figure 36: Ovarian CCNE1 amplification landscape Supplemental Figure 37: Serous Ovarian CCNE1 co-alteration landscape Supplemental Figure 38: ctDNA TF distribution by alteration type Supplemental Figure 39: ctDNA TF distribution by tumor type Supplemental Figure 40: All-gene liquid amplification concordance Supplemental Figure 41: Tissue and Liquid AmpRatio Correlation Supplemental Figure 42: Distribution of ctDNA TF and AmpRatio for discordant vs. concordant liquid samples.

SUPPLEMENTARY MATERIALS & METHODS Foundation Medicine Comprehensive Genomic Profiling

Overview of MET TKD mutations detected in pan-cancer cohorts. (A) Type (missense, truncating, splice site, in-frame) of MET TKD mutations detected in the cohort #1 (711 MET TKD mutations among 664 unique patients with a tumor harboring MET TKD mutations without concurrent METex14 alterations). (B) Type of MET TKD mutations according to OncoKB annotation (biological function: oncogenic / likely oncogenic, unknown) detected in cohort #1. (C) Distribution of MET TKD mutations across MET kinase domain for cohort #1. (D) Type (missense, truncating, splice site, in-frame) of MET TKD mutations detected in cohort #2 (2,559 MET TKD mutations among 2,477 unique patients with a tumor harboring MET TKD mutations without concurrent METex14 alterations). (E) Type of MET TKD mutations according to OncoKB annotation (biological function: oncogenic / likely oncogenic, unknown) detected in cohort #2. (F) Distribution of MET TKD mutation across MET kinase domain for cohort #2.

Frequency of MET tyrosine kinase domain (TKD) mutations in cohort #2 according to amino acid residue, out of a total of 2,559 MET TKD mutations.

Sensitivity pattern of MET TKD mutations in 293T cells. (A) ERK-mediated transcriptional activation assessed by luciferase assay in 293T cells co-transfected with MET WT or TKD mutant constructs, an ERK activation reporter vector (pGL4.33), and a control co-reporter vector (pRL-TK). The lower panel shows western blot analysis of transfected samples. (B) Western blot analysis of MET and ERK1/2 phosphorylation in response to 1 μM crizotinib. MET TKD mutants showing sensitivity and resistance to crizotinib are colored in green and orange, respectively.

Concurrent driver mutations according to oncogenic / likely oncogenic MET TKD mutations detected in cohort #2.

Western blot analysis of MET and ERK1/2 phosphorylation in MET R1170Q cell models. MET and ERK1/2 phosphorylation in (A) Ba/F3 cells stably transduced with MET R1170Q and (B) 293T cells transiently overexpressing MET R1170Q. Cells were treated with MET TKIs at the indicated dose for Ba/F3 cells or at 1 μM for 293T cells.

TCGA PanCancer Atlas Studies included in cohort #1 downloaded from cBioPortal.