Anti-EGFR Treatment for EGFR-Amplified Gastroesophageal Adenocarcinoma

Published OnlineFirst February 15, 2018; DOI: 10.1158/2159-8290.CD-17-1260

RESEARCH ARTICLE

Targeted Therapies for Targeted Populations: Anti-EGFR Treatment for EGFR-Amplifi ed Gastroesophageal Adenocarcinoma

Steven B. Maron 1 , Lindsay Alpert 2 , Heewon A. Kwak 2 , Samantha Lomnicki 1 , Leah Chase 1 , David Xu 1 , Emily O’Day 1 , Rebecca J. Nagy 3 , Richard B. Lanman 3 , Fabiola Cecchi 4 , Todd Hembrough 4 , Alexa Schrock 5 , John Hart 2 , Shu-Yuan Xiao 2 , Namrata Setia 2 , and Daniel V.T. Catenacci 1

ABSTRACT Previous anti-EGFR trials in unselected patients with gastroesophageal adenocarcinoma (GEA) were resoundingly negative. We identifi ed EGFR amplifi cation in 5% (19/363) of patients at the University of Chicago, including 6% (8/140) who were prospectively screened with intention-to-treat using anti-EGFR therapy. Seven patients received ≥1 dose of treatment: three fi rst-line FOLFOX plus ABT-806, one second-line FOLFIRI plus cetuximab, and three third/ fourth-line cetuximab alone. Treatment achieved objective response in 58% (4/7) and disease control in 100% (7/7) with a median progression-free survival of 10 months. Pretreatment and posttreatment tumor next-generation sequencing (NGS), serial plasma circulating tumor DNA (ctDNA) NGS, and tumor IHC/FISH for EGFR revealed preexisting and/or acquired genomic events, including EGFR- negative clones, PTEN deletion, KRAS amplifi cation/mutation,NRAS, MYC , and HER2 amplifi cation, andGNAS mutations serving as mechanisms of resistance. Two evaluable patients demonstrated interval increase of CD3 + infi ltrate, including one who demonstrated increased NKp46+, and PD-L1 IHC expression from baseline, suggesting an immune therapeutic mechanism of action. EGFR amplifi cation predicted benefi t from anti-EGFR therapy, albeit until various resistance mechanisms emerged.

SIGnIFICAnCE: This paper highlights the role of EGFR inhibitors in EGFR-amplifi ed GEA—despite negative results in prior unselected phase III trials. Using serial ctDNA and tissue NGS, we identifi ed mechanisms of primary and acquired resistance in all patients, as well as potential contribution of antibody-dependent cell-mediated cytotoxicity to their clinical benefi t. Cancer Discov; 8(6); 696–713. ©2018 AACR.

See related commentary by Strickler, p. 679.

1 Department of Medicine, University of Chicago, Chicago, Illinois. 2 Depart- Corresponding Author: Daniel V.T. Catenacci , University of Chicago, ment of Pathology, University of Chicago, Chicago, Illinois. 3 Guardant Chicago, IL 60637. Phone: 773-702-1000; E-mail: dcatenac@bsd. Health, Inc., Redwood City, California. 4NantOmics, LLC, Rockville, uchicago.edu 5 Maryland. Foundation Medicine, Inc., Cambridge, Massachusetts. doi: 10.1158/2159-8290.CD-17-1260 note: Supplementary data for this article are available at Cancer Discovery © 2018 American Association for Cancer Research. Online (http://cancerdiscovery.aacrjournals.org/).

696 | CANCER DISCOVERY june 2018 www.aacrjournals.org

INTRODUCTION kinase inhibitors (erlotinib, gefitinib, afatinib, lapatinib, and osimertinib) have been approved for various cancers, includ- Gastric and esophagogastric junction (EGJ) adenocarci- ing lung, head and neck, and colon. Early-phase clinical trials noma, together gastroesophageal adenocarcinoma (GEA), had suggested potential benefit in unselected patients with has the third highest incidence and second highest cancer- GEA (14–26). These results supported further evaluation in related mortality, and it remains a significant global health larger phase III trials—EXPAND (cetuximab plus chemother- problem (1). When routine screening is not conducted, most apy, first line), REAL-3 (panitumumab plus chemotherapy, patients present with de novo metastatic disease or locally first line), and COG (gefitinib monotherapy, second–fourth advanced disease with high risk of recurrence. Approximately lines; refs. 6–8). Disappointingly, each of these phase III trials 55% to 60% of patients recur within 5 years after curative- was negative, and panitumumab actually resulted in worse intent resection despite perioperative therapy (2). The median survival compared with the control; evaluation of EGFR inhi- overall survival (OS) of stage IV GEA is 11 to 12 months with bition was abruptly abandoned for GEA. optimal palliative chemotherapy and increases to 16 months If HER2-targeted therapeutic development was devised in for patients with HER2-amplified tumors with the addi- the same biologically unselected manner, it likely would have tion of trastuzumab to first-line chemotherapy (3). To date, suffered the same fate as anti-EGFR therapy—to the detri- ramucirumab, an anti-VEGFR2 monoclonal antibody, is the ment of that subset of patients with HER2-amplified tumors only other approved second-line biologic therapy for GEA as who we now understand to derive significant benefit from monotherapy or in combination with paclitaxel, with modest this targeted approach. In the registration TOGA trial, nearly clinical benefit (4, 5). 4,000 patients were screened in order to identify the necessary Numerous other targeted therapies have been evaluated number of HER2-amplified patients n( = 584) to adequately in metastatic GEA in various lines and settings, but all of power the study. Despite this patient selection hurdle repre- these have been uniformly negative. Recent examples include senting only ∼15% of GEA, screening for HER2 amplification EGFR, MET, mTOR, and hedgehog pathway inhibitors— by FISH enriched for those most likely to benefit based on a generally in genomically unselected patients (6–13). strong preclinical rationale (3). Analogously, EGFR amplifica- EGFR is a well-recognized mediator of oncogenic phe- tion reportedly occurs in ∼4% of GEA in The Cancer Genome notype. EGFR inhibitors including monoclonal antibodies Atlas (TCGA; CbioPortal; refs. 27, 28), yet prospective EGFR (cetuximab, panitumumab, and necitumumab) and tyrosine amplification screening in stage IV patients and targeting

june 2018 CANCER DISCOVERY | 697

RESEARCH ARTICLE Maron et al. therapy in this biologically relevant population is currently an additional mechanism of action (48, 49). This has been lacking. reported with multiple IgG1 monoclonal antibodies, includ- TCGA as well as other sequencing efforts identified a high ing rituximab, trastuzumab, and cetuximab, and is believed degree of chromosomal instability in GEA (27–37). This to be mediated by natural killer (NK) cells with resultant instability generates additional oncogenic drivers, including dendritic cell and CD8+ T-cell priming (48–51). gene amplifications of well-known receptor tyrosine kinases, Due to these preclinical and clinical subset analyses sug- including HER2, MET, FGFR2, and EGFR. Preclinical and gesting potential benefit of EGFR inhibitors for patients clinical evidence suggests benefit of EGFR inhibitors forEGFR with EGFR-amplified GEA, we sought to first describe the genomically driven tumors. In lung cancer, patients with incidence of EGFR amplification in a large cohort of patients EGFR-mutated tumors derive a greater response and survival with GEA across stages and to evaluate for a direct correla- than EGFR wild-type patients (38, 39). More relevantly, analy- tion of gene copy number with protein expression levels. We ses were reported from two large phase III trials for squamous then prospectively screened 140 clinic patients with stage IV cell lung cancer evaluating the subset of patients with EGFR GEA (in any line of therapy) over 27 months at our center amplification or increased gene copy number. In this “EGFR- for EGFR amplification and, when present and otherwise positive” subgroup of patients, the addition of cetuximab to eligible, treated with EGFR monoclonal antibody therapy chemotherapy in the SWOG S0819 trial increased median under institutional review board (IRB)–approved protocols, OS from 6.4 to 11.8 months (P = 0.007; ref. 40), and in the when appropriate. We report their clinical responses and SQUIRE trial (41), this same molecular subgroup trended disease control to EGFR blockade as well as characterization toward a significant benefit with necitumumab [progression- of pretreatment and posttreatment tumor biopsies and serial free survival (PFS)/OS: HR, 0.71/0.70; refs. 41, 42). Simi- circulating tumor DNA (ctDNA) next-generation sequencing larly, phase II evaluation of second-line or greater icotinib (NGS) in an attempt to explain clinical outcomes and mecha- in patients with advanced squamous esophageal cancer with nisms of resistance, and to evaluate the contribution of NK either high expression by IHC or amplification by FISH dem- cell–dependent ADCC to antitumoral effect. onstrated a 16.7% objective response rate and 46.3% disease control rate (43). Most relevant, however, are studies focusing on EGFR gene copy and benefit from anti-EGFR therapy in RESULTS GEA samples and patients. Preclinical xenograft models of EGFR Gene Amplification and Protein GEA demonstrated that all responders possessed ≥4 EGFR Overexpression in GEA Cell Lines copies and suggested an even smaller population of those with “high” gene copy number having the highest probability EGFR/CEP7 FISH ratio and EGFR-selected reaction moni- of benefit (44). Apost hoc subset analysis of a phase II trial toring (SRM) mass spectrometry expression were assessed, of FOLFOX with cetuximab in GEA confirmed an association as previously described (52), in 24 GEA cancer lines, lympho between EGFR amplification and OS (P = 0.011; ref. 45). In blast and breast cancer–negative controls, as well as two the TRANS-COG correlative study of the prospective phase III positive control head and neck cancer cells lines (HN5 and COG trial of gefitinib monotherapy, 16.3% of patients pos- SQ20B) both known to harbor EGFR amplification (Fig. 1; sessed “EGFR-positive” tumors, and the smaller subset of Supplementary Table S1). EGFR was amplified (FISH ratio these patients with true gene amplification (6.1%, 18/294) EGFR/CEP7 ≥2) in only the two head and neck cell lines derived a statistically significant survival benefit with the addi- (Fig. 1A). EGFR-SRM ranged from <100 to 41,383 amol/μg tion of gefitinib in apost hoc analysis (HR, 0.19; P = 0.007; ref. (median 575 amol/μg; Fig. 1C). EGFR/CEP7 ratio ≥2 and 46). The EXPAND trial demonstrated survival benefit in the SRM values ≥ 4,000 amol/μg were strongly correlated (Fisher small subset with extremely high EGFR H-score expression (6, exact test P = 0.002) in the cell lines. No EGFR expression 47), which was possibly attributable to an underlying subset ≥4,000 amol/μg was observed in cell lines in the absence of of EGFR-amplified tumors, but this has yet to be confirmed. EGFR amplification by FISH (Supplementary Table S1). EGFR monoclonal antibodies reportedly act by prevent- ing activating ligands from binding the extracellular domain EGFR Amplification and Overexpression in and/or receptor internalization/degradation of the receptor, GEA FFPE Tissues but induction of antibody-dependent cell-mediated cytotox- Five hundred two samples from 363 patients in the University icity (ADCC) via the Fc portion of the antibody may serve as of Chicago GEA tumor bank underwent NGS by Foundation

Figure 1. EGFR amplification and expression assessment in cancer cell lines and tissues. A, FISH demonstrating EGFR amplification of HN5 cells (left) and lack of EGFR amplification in SNU-5 cells [right; polysomy with 4 copies of bothEGFR (green) and CEP7 (orange) control probes]. B, FISH demonstrating EGFR amplification (orange) within the gastric primary tumor of patient 2 (left) and absence ofEGFR amplification within a liver metastasis of patient 5 (right). C, EGFR SRM by amplification status in selected cell lines (red amplified by FISH ratio≥ 2; blue not amplified). D, EGFR SRM by amplification status across retrospective cohort and overlay including EGFR-amplified patients from the prospective cohort when adequate tissue was available for analysis (red amplified by FISH ratio≥ 2 and/or NGS copy ≥8; blue not amplified). E, Of 4,645 patients undergoing Foundation One testing at Foundation Medicine, 259 (5.6%) demonstrated EGFR amplification, displayed by copy-number ranges> 100, 50–90, 20–49, and 8–19. Although clinical reports do note “equivocal” amplification with 6–7 copies, these were considered negative for this study. F, Concurrent genomic alterations occurring in ≥5% of EGFR-amplified tumors (≥8 EGFR gene copies) within the Foundation Medicine cohort (N = 259). G, PD-L1 IHC testing (Ventana SP142; see Methods) of 632 GEA samples performed at Foundation Medicine in the entire cohort, by anatomic location (proximal EGJ versus distal gastric) and EGFR amplification status, reported as percent tumor positivity score, percent tumor-infiltrating lymphocyte (TIL) positivity, and combination of these for a combined positivity score (CPS).

698 | CANCER DISCOVERY june 2018 www.aacrjournals.org

Anti-EGFR Therapy for EGFR-Amplified Gastroesophageal Cancer RESEARCH ARTICLE

A B

60,000 Amplified 40,000 Nonamplified

20,000 EGFR amol/ µ g 0

HN5 CP-A OE33 CP-B Cat2 CP-D SNU-1 CP-C CAT-3 OE19CAT-4 SQ20B SNU-5 CAT-17CAT-12 CAT-14 MKN-1CAT-15CAT-16 Hs746t SNU-16KATOIII HGC-27 CAT-2R3 NCI-N87CAT2R2 AGS-KD MKN-45 CAT-2R3 AGS-SC AGS-WT GM14667 Cell line D E 90 25,000 89 14,000 70 Amplified 12,000 20,000 60 Nonamplified 10,000 8,000 50 15,000 6,000 40 4,000 GEA cases 30 2,000 10,000 . of EGFR -amplified 20 0 No EGFR amol/ µ g Pt Pt Pt Pt 10 5,000 2478 0 ≥100 50–99 20–49 8–19 6–7 0 EGFR copy number Patient FG EGFR amp PD-L1 tumor EGFR nonamp PD-L1 tumor

100 3% 12% 14% 90

80 88% 83%

60 EGFR amp PD-L1 TILs EGFR nonamp PD-L1 TILs

12% 18% 40 % Altered cases

30 88% 82%

10 EGFR amp CPS score EGFR nonamp CPS score 0 17% 27%

TP53 MYC MET EGFR KRAS FGF3FGF4 CDK6 CDH1 ERBB2 FGF19 GATA6 EMSY CCND1 ARID1A SMAD4CCND3 ZNF217 KDM6A CDKN2A CDKN2B RICTOR 83% 73%

Multiple alterations Copy-number change Short variant Rearrangement

june 2018 CANCER DISCOVERY | 699

RESEARCH ARTICLE Maron et al.

Table 1A. Demographics at diagnosis by EGFR Table 1B. Demographics by EGFR amplifi cation status amplifi cation status across the entire U of C cohort across all stage IV or recurrent esophagogastric cases in retrospective U of C cohort, excluding those prospectively screened between September 2014 and December 2016 EGFR EGFR Characteristic amplifi ed nonamplifi ed P Number of patients (%) 19 (5) 344 (95) <2.2 × EGFR EGFR 10 –16 Characteristic amplifi ed nonamplifi ed P Median age, y (range) 64 (37–77) 63 (16–91) 0.97 Number of 10 (7) 134 (93) < 2.2 × patients (%) 10 –16 Male sex, n (%) 16 (84) 238 (69) 0.20 Median age, y 64.5 (37–77) 62 (22–83) 0.83 Disease site, n (%) (range) Esophagus/GEJ 12 (63) 183 (53) 0.54 Gastric 7 (37) 161 (47) Male sex, n (%) 9 (90) 93 (69) 0.28 Tumor grade, n (%) Disease site, n (%) Well differentiated 0 (0) 13 (4) 0.557 Esophagus/GEJ 6 (60) 63 (47) 0.52 Moderately 3 (16) 89 (26) Gastric 4 (40) 71 (53) differentiated Tumor grade, n (%) Poorly 15 (79) 229 (67) Well 0 (0) 4 (3) 0.76 differentiated differentiated Unknown 1 (5) 13 (4) Moderately 1 (10) 32 (24) Stage at diagnosis, n (%) differentiated I 0 (0) 35 (10) 0.11 Poorly 8 (80) 97 (72) II 0 (0) 43 (13) differentiated III 4 (21) 93 (27) Unknown 1 (10) 1 (1) IV 15 (79) 172 (50) Stage at diagnosis, n (%) Unknown 0 (0) 1 (0) I 0 (0) 9 (7) 0.94 HER2, n (%) II 0 (0) 12 (9) Positive 5 (26) 81 (24) 0.91 III 2 (20) 30 (22) Negative 12 (63) 212 (62) IV 8 (80) 83 (62) Equivocal 0 (0) 17 (5) Unknown 0 (0) 0 (0) Unknown 2 (11) 34 (10) HER2, n (%) Race, n (%) Positive 3 (30) 30 (22) 0.74 Caucasian 15 (79) 250 (73) 0.68 Negative 6 (60) 83 (62) African American 2 (11) 58 (17) Equivocal 0 (0) 11 (8) Asian 1 (5) 27 (8) Unknown 1 (10) 10 (11) Hispanic 1 (5) 9 (3) Race, n (%) Caucasian 7 (70) 104 (78) 0.57 Abbreviation: U of C, University of Chicago. African 2 (20) 21 (16) American Asian 1 (10) 9 (7) One and/or SRM mass spectrometry by Nantomics and were Hispanic 0 (0) 0 (0) included in the overall cohort ( Table 1A ; Supplementary Table S2A). Among these cases, 18, 183, and 292 patients underwent EGFR FISH ( Fig. 1B ), EGFR-SRM ( Fig. 1D ), and NGS, respectively. One hundred twelve patients had both SRM and NGS, and 11 patients underwent testing by all three modalities. There was All cases with EGFR expression ≥1,200 amol/μg were EGFR a statistically signifi cant linear correlation between EGFR copy- amplifi ed. number and EGFR expression by SRM (Pearson correlation = 0.87, P < 2.2 × 10 −16), with a trend to signifi cance when evaluating Incidence of EGFR Amplifi cation and Concurrent binary “presence” or “absence” of amplifi cation versus expression Genomic Aberrations in Metastatic GEA (P = 0.08). EGFR amplifi cation was identifi ed in 19 of 363 (5%) To further defi ne the 6% to 7% EGFR amplifi cation overall patients across all disease stages in both the retrospective incidence observed in our stage IV cohorts as compared and prospective cohorts (Table 1A), 10 of 144 (7%) retrospec- with the incidence noted from the TCGA (4%) comprising tively evaluated stage IV patients only (Table 1B), and 8 of 140 earlier stage tumors, we queried the Foundation Medicine (6%) prospectively screened stage IV patients only ( Table 1C ). database for the incidence of EGFR amplifi cation among Only one EGFR- amplifi ed case was identifi ed in the absence of all unique patients with GEA (N = 4,645) sequenced with EGFR expression ≥1,200 amol/μg in formalin-fi xed, paraffi n- the Foundation One test between 2012 and 2017. These embedded (FFPE) samples (Supplementary Tables S2A–S2C). samples were considered, for the most part, to be from

700 | CANCER DISCOVERY june 2018 www.aacrjournals.org

Anti-EGFR Therapy for EGFR-Ampliﬁ ed Gastroesophageal Cancer RESEARCH ARTICLE

EGFR Amplifi cation Is Associated with Lower Table 1C. Demographics by EGFR amplifi cation status PD-L1 Expression by IHC across all stage IV or recurrent esophagogastric cases prospectively screened at U of C for anti-EGFR therapy Given the growing interest and importance of PD-1 and between September 2014 and December 2016 PD-L1 checkpoint inhibition in GEA, we also assessed the incidence of PD-L1 positivity by tumor positivity score (TPS), tumor-infi ltrating lymphocytes (TIL), and combined positiv- EGFR EGFR ity score (CPS) by EGFR amplifi cation status ( Table 2B ; Fig. Characteristic ampliﬁ ed nonampliﬁ ed P 1G). Of the 632 patients with GEA in the Foundation Medi- Number of 8 (6) 132 (94) < 2.2 × cine database for whom PD-L1 IHC was performed (N = 632), patients (%) 10 –16 26% of samples were CPS score positive (≥1%; see Materials and Methods). EGFR-amplifi ed tumors had lower incidence Median age, y 61.5 (48–74) 61.5 (19–91) 0.68 of CPS positivity (17%) compared with nonamplifi ed tumors (range) (27%), which was not statistically signifi cant. Male sex, n (%) 6 (75) 95 (72) 1 Disease site, n (%) Clinicopathologic Characteristics of Esophagus/GEJ 5 (63) 73 (55) 1 EGFR Amplifi cation Gastric 3 (38) 59 (45) In the overall University of Chicago cohort (N = 363), Tumor grade, n (%) which comprised 223 retrospectively accrued and 140 pro- Well 0 (0) 4 (3) 1 spectively accrued patients, there was no statistically sig- differentiated nifi cant difference in gender, race, age, stage, tumor grade, Moderately 2 (25) 27 (20) primary tumor location, or HER2 positivity among patients differentiated with EGFR amplifi cation versus those without amplifi cation Poorly 6 (75) 93 (70) (Table 1A). However, among the EGFR-amplifi ed patients, differentiated 63% had esophageal or junctional tumors whereas 37% had Unknown 0 (0) 8 (6) distal gastric tumors, as compared with 53% and 47% in the Stage at diagnosis, n (%) nonamplifi ed patients, respectively. When evaluating only I 0 (0) 3 (2) 0.79 patients having stage IV and recurrent disease, no statistical II 0 (0) 11 (8) differences in gender, race, age, tumor grade, tumor location, III 1 (13) 29 (22) or HER2 status were identifi ed in the retrospective ( Table 1B ) IV 7 (88) 88 (67) nor the prospective cohorts (Table 1C). Unknown 0 (0) 1 (1) HER2, n (%) Anti-EGFR Antibody Therapy for Positive 2 (25) 29 (22) 1 EGFR -Amplifi ed Patients Negative 6 (75) 90 (68) Eight of the 140 screened patients (6%) during the prospec- Equivocal 0 (0) 2 (2) tive screening period (September 2014–December 2016) dem- Unknown 0 (0) 11 (8) onstrated baseline tumor tissue EGFR amplifi cation (defi ned Race, n (%) as ≥8 copies by NGS) ranging from 54 to 167 EGFR gene Caucasian 7 (88) 91 (69) 0.43 copies by NGS ( Fig. 2A ). Evaluation of each patient’s samples African 0 (0) 18 (14) for plasma EGFR ctDNA ( Fig. 2B ), EGFR/CEP7 FISH ( Fig. American 2C), along with EGFR IHC and PD-L1 IHC (Fig. 2D), was Asian 0 (0) 14 (11) performed. Seven of these 8 patients ultimately underwent Hispanic 1 (13) 9 (7) at least one dose of EGFR-directed therapy (Supplementary Table S3)—three patients received fi rst-line FOLFOX plus ABT- 806 (investigational EGFR monoclonal antibody inhibitor as part of the PANGEA trial; ref. 53 ), one received second-line patients with advanced metastatic GEA; however, detailed therapy with FOLFIRI plus cetuximab, two received third-line staging information was unavailable. EGFR amplifi cation cetuximab monotherapy, and one received fourth-line cetuxi- was identifi ed in 5.6% of patients with GEA, with a higher mab monotherapy (for patient details, see Supplementary rate of 7.1% in proximal EGJ tumors as compared with 3.7% Data File S1). The eighth patient, who had concurrent MET in distal gastric tumors ( Table 2A ). The median EGFR gene and HER2 amplifi cation, was not eligible for EGFR-directed copy number was 40 copies with a range of 8 to 375 copies therapy due to poor clinical condition after failure of fi rst-line ( Table 2A ; Fig. 1E ). Forty-six percent of EGFR -amplifi ed FOLFOX therapy and enrollment in hospice. GEA samples (2.6% of all GEA samples) had ≥50 EGFR Objective best response, the primary endpoint, was gene copies. Concurrent genomic aberrations occurring in observed in 57% (4/7) of patients, including complete >5% of EGFR -amplifi ed samples in this dataset were mostly responses in 43% (3/7), partial responses in 14% (1/7), and short variant events in tumor suppressors, including TP53, disease control in the remaining 43% (3/7; Fig. 3A and B). CDKN2A, ARID1A, SMAD4, and CDH1 , and amplifi ca- Complete responses included one patient (patient 3) receiv- tions of various oncogenes, including MYC, ERBB2, KRAS, ing third-line cetuximab monotherapy who had a durable CCND1, and others (Fig. 1F). response of 14 months with resolution of his symptomatic

june 2018 CANCER DISCOVERY | 701

RESEARCH ARTICLE Maron et al.

treatment benefi t, yet resolved by the time of disease pro- Table 2A. EGFR amplifi cation incidence and gression), whereas the three patients receiving ABT-806 did characteristics in the Foundation Medicine GEA not; this was consistent with low rash frequency in phase I database (August 2012–november 2017) evaluation of ABT-806 ( 54 ). There were no new safety signals with the addition of EGFR monoclonal antibody therapy as All GEA cases EGJ Gastric monotherapy or in combination with chemotherapy. Patients, n 4,645 2,534 2,111 Mechanisms of Resistance to EGFR Blockade EGFR amp (%) 259 (5.6) 181 (7.1) 78 (3.7) Underlying baseline and acquired mechanisms of resist- Median EGFR 40 (8–375) 39 (8–375) 44 (8–281) ance to therapy were evaluated using baseline and serial CN (range) tumor tissue NGS in parallel with baseline and serial plasma EGFR ≥ 50 119 (46%; 82 37 ctDNA NGS in all treated patients, with confi rmation by copies 2.6% overall) IHC/FISH, when applicable ( Table 3 ; Fig. 4A ). Likely mechanisms of resistance existing prior to treatment initiation were Median age, y 63 (27–90) 63 (32–84) 62.5 (27–90) identifi ed in seven of eight patients, and included intra- and/ (range) for or intertumoral EGFR amplifi cation heterogeneity in fi ve of EGFR amp seven patients (n = 5, patients 1, 2, 5, 7, and 8), as observed Gender for 47 F:212 M 19 F:162 M 28 F:50 M by areas with and without EGFR amplifi cation within the EGFR amp primary tumor itself and/or across different tumor sites

Abbreviation: CN, copy number. anatomically. Additional baseline mechanisms of resistance included coamplifi cation ofHER2 (n = 3, patients 2, 4, 8), NRAS (n = 1, patient 4), KRAS (n = 1, patient 6), MYC (n = 4, (cough) pulmonary metastases ( Fig. 3C ). Median PFS was 10 patients 1, 2, 4, and 6), or CCNE1 (n = 2, patients 4 and 6), as months (range, 0.5+ to 14; Fig. 3B). Among the seven patients well as mutation in KRAS (n = 1, patient 5) or mutation of treated, all four radiographic responses were seen in patients another stimulatory G-protein alpha subunit, GNAS (n = 1, with baseline plasma-detected EGFR amplifi cation over the patient 6; Table 3 and Fig. 4A). 50th percentile (2.4 copies in plasma), and the degree of There were two observed groups of patients upon disease plasma copy-number amplifi cation correlated with objective progression—those with retained and those without retained RECIST response, with the mean ctDNA copy number being tissue EGFR amplifi cation/overexpression ( Fig. 4B–E ). Serial 2.5 in nonresponders and 33.9 in responders, and mean dif- ctDNA demonstrated a steep decline in EGFR copy number ference 31.4 copies between responders and nonresponders with EGFR-directed therapy in all evaluable patients including (P = 0.049, 95% CI, 0.25–62.5; Fig. 3A ). Notably, however, those receiving anti-EGFR antibody monotherapy, but even- patients 5 and 7, both having EGFR amplifi cation observed in tual recovery and increase was seen in some patients (patients only their primary tumors and not metastases, had clinically 1 and 3) upon disease progression and development of resist- signifi cant improvements in their dysphagia/dyspepsia only ance mechanisms, which correlated with rise in serum CA19-9 once ABT-806 was added to their chemotherapy (Supplemen- (Fig. 4B ; Supplementary Fig. S2A and S2C). In a patient with tary Data File S1; Supplementary Fig. S1). retained EGFR amplifi cation in tissue, acquired PTEN deletion All four patients receiving cetuximab developed a stereo- contributed to resistance, along with a de novo PIK3CA muta- typical acneiform rash (which interestingly continued during tion identifi ed in ctDNA (patient 1). In contrast, loss ofEGFR

Table 2B. PD-L1 expression in the GEA Foundation Medicine cohort, by EGFR amplifi cation status

All GEA EGJ Gastric GEA EGFR WT GEA EGFR amp Cases with PD-L1 IHC, n 632 377 255 591 41 PD-L1 tumor (%) High positive 20 (3) 12 (3) 8 (3) 20 (3) 0 Low positive 89 (14) 56 (15) 33 (13) 84 (14) 5 (12) Negative 523 (83) 309 (82) 214 (84) 487 (82) 36 (88) PD-L1 TILs (%) High positive 1 (0.2) 0 1 (0.4) 1 (0.2) 0 Low positive 110 (17) 72 (19) 38 (15) 105 (18) 5 (12) Negative 521 (82) 305 (81) 216 (85) 485 (82) 36 (88) PD-L1 CPS (%) Positive (≥1%) 166 (26) 106 (28) 60 (24) 159 (27) 7 (17) Negative 466 (74) 271 (72) 195 (76) 432 (73) 34 (83)

Abbreviation: WT, wild-type.

702 | CANCER DISCOVERY june 2018 www.aacrjournals.org

Anti-EGFR Therapy for EGFR-Amplified Gastroesophageal Cancer RESEARCH ARTICLE

A 167

150 145

111 103 100 77

tumor 54 54

EGFR GCN 50

0 1234567 Patient number B 60 59

41 40

ctDNA 21

EGFR G CN 20 15

5.1 2.4 0 0 1234567 Patient number C

FISH ratio: 5.00 11.50 13.80 3.50 10 8.80 10

EGFR:CEP7 14.2:2.8 19.5:1.7 18:1.3 20:5.6 20:2 20:2.3 20:2

EGFR IHC

3+ 3+ 3+ 3+ 3+ 3+ 3+

Baseline PD-L1 IHC 01+ 00 01+ 0

Figure 2. Assessment and correlation of baseline tumor tissue EGFR amplification and expression as well asEGFR plasma ctDNA amplification in seven patients treated with anti-EGFR therapy. EGFR status assessment by (A) tumor NGS, (B) ctDNA NGS, (C) EGFR/CEP7 FISH, and (D) EGFR IHC, as well as PD-L1 IHC (Abcam antibody; see Methods). All samples demonstrated concordant tumor/ctDNA (n = 5 > 90th percentile, n = 1 > 50th percentile in patient 5, where the metastatic biopsy was not EGFR amplified) except patient 7, whose metastatic biopsy was also notEGFR amplified (see Tables 2A and B). amplification/expression (likely a selection of preexisting but never harbored systemic EGFR amplification in his metastatic not previously identifiedEGFR nonamplified clones) was seen biopsy nor ctDNA. Patient 3 demonstrated persistent EGFR in three cases (patients 2, 3, and 5), as well as patient 1 in a amplification by ctDNA, but his posttreatment biopsies (new separate large region of the primary tumor, whereas patient 7 lung, residual primary tumor) were not EGFR amplified and

june 2018 CANCER DISCOVERY | 703

RESEARCH ARTICLE Maron et al.

A B 100 3 75 Therapy status Continued 50 Discontinued 7

25 1 0

2 −25

50 number Patient

Change from baseline (%) − 5 Drug Cetuximab −75 FOLFIRI + Cetuximab 4 FOLFOX + ABT806 −100 Complete Patient # 6 574123 6 Partial

Therapy 0816 24 32 40 48 56 ABT-806 ABT-806 ABT-806 FOLFIRI + Cetuximab Cetuximab Cetuximab Cetuximab FOLFOX + FOLFOX + FOLFOX + FOLFOX PFS (Weeks) Tissue 98 54 77 54 145 111 167 EGFR CN ctDNA 5.14 2.41 0 58.5 15.2 41 20.8 EGFR CN Therapy 3 1 1 4 2 1 3 line

C Baseline 6 weeks 31 weeks 62 weeks

Lung metastases Complete response Recurrence

Figure 3. Clinical outcomes of EGFR-amplified patients treated with anti-EGFR therapy. A, Waterfall plot of seven evaluable patients. CN, copy number. B, Swimmer plot of seven treated patients. Black arrow, patient continued on therapy at time of data analysis. C, Computed tomography demonstrating radiographic resolution of lung metastasis (arrows) in patient 3. he developed BRAF, MET, and MYC coamplification in ctDNA At baseline, six of seven patients also had NK cells present (Fig. 4C). He also developed new brain metastases after disease in the stroma, but only one had baseline tumoral NKp46 progression that were not biopsied. cell staining (patient 2). CD3 stromal staining in patient 1 increased from 2+ to 4+ with concomitant increased NKp46 Immune IHC Evaluation Pre– and and PD-L1 staining in a biopsy taken during therapeu- Post–Anti-EGFR Therapy tic response (obtained prior to receiving anti–PD-L1 and Pretreatment tissue IHC revealed weak tumoral and stro- anti-CTLA4 combination therapy previously, because a post– mal CD3 staining in all patients with available tissue (Fig. immuno-oncologic and pre–anti-EGFR biopsy could not be 4F; Supplementary Table S4). Both tumoral and/or stromal obtained; Supplementary Table S4). Increased intratumoral CD3 baseline staining persisted in all five available pre/post- CD3 staining was also observed in patient 2. Conversely, therapy biopsy pairs (patients 1–5). Four of the five posttreat- patient 2 demonstrated decreased NKp46 stromal cell stain- ment biopsies were performed on treatment while clinically ing and no tumoral staining in a biopsy obtained after disease stable, and one at the time of clinical progression. progression on anti-EGFR ABT-806 plus FOLFOX therapy.

704 | CANCER DISCOVERY june 2018 www.aacrjournals.org

Anti-EGFR Therapy for EGFR-Ampliﬁ ed Gastroesophageal Cancer RESEARCH ARTICLE

Table 3. Observed mechanisms of resistance by tissue and ctDnA nGS in pretherapy, on-therapy, and posttherapy samples

Patient number Baseline Acquired 1EGFR amp heterogeneity (only in metastasis, not in MYC amp, PTEN deletion tissue, PIK3CA ctDNA primary tumor), MYC amp 2EGFR/HER2 coamp primary tumor: 50% HER2 amp, Residual HER2 amp and now-absent EGFR amp in residual other 50% EGFR amp; EGFR/MYC amp in ctDNA primary tumor; KRAS amp clone in ctDNA 3 None identifi ed Absent EGFR amp in residual primary tumor and recurrent lung metastases. New BRAF/MET/MYC amp in ctDNA 4K-NRAS/HER2/MYC/CCNE1/CCND1 coamp in primary Increase in NRAS / HER2/MYC amp and de novo GNAS- tumor and ctDNA mutated clone in ctDNA 5MET/EGFR coamp in tumor and ctDNA, but Increasing KRAS -mutated ctDNA, absent EGFR amp in KRAS- mutated liver biopsy but no EGFR amp tissue and ctDNA 6KRAS/MYC/CCNE1 amp and GNAS -mutated clone in KRAS/MYC/CCNE1 amp and GNAS -mutated clone in ctDNA ctDNA 7EGFR -amp heterogeneity (only in 10% of primary None yet identifi ed tumor, not metastasis or ctDNA) 8EGFR/MET/HER2 coamp in primary tumor (different Never treated clones by FISH)

Of the four biopsies performed on treatment during disease than a higher proportion in proximal EGJ tumors compared stability, only two had persistent EGFR expression and EGFR with distal gastric tumors, consistent with the known higher amplifi cation. Both of these persistent EGFR -amplifi ed tumors incidence of chromosomal instability (CIN) tumors proxi- (patients 1 and 4) also expressed PD-L1, whereas patients now mally in the TCGA cohort. We then prospectively screened lacking EGFR amplifi cation (patient 3) or postprogression patients for EGFR amplifi cation and treated them with EGFR- and no longer EGFR amplifi ed (patient 5) did not demonstrate targeting agents when possible. As expected, in this relatively PD-L1 expression. Patient 2 also exhibited PD-L1 expression, large cohort of 140 stage IV patients screened, only 6% of but only in the moderately differentiated HER2 + region of his patients were found to be EGFR amplifi ed, slightly higher than primary tumor both pre- and posttreatment, yet again not in the TCGA 4% incidence. Notwithstanding, a demonstrable the poorly differentiated residual HER2 − / EGFR− nonamplifi ed and robust treatment response and disease control to EGFR component posttreatment, where EGFR -amplifi ed clones were antagonists was observed in this select population. Notably, no longer detected. A biopsy of progressing peritoneal carci- with monotherapy in heavily pretreated patients, two signifi - nomatosis, which was EGFR -nonamplifi ed, harbored stromal cant responses (one of which was a complete response) were CD3+ staining, but absent PD-L1 and NKp46 expression (Sup- observed, and a third patient had disease control. Moreover, plementary Table S4). using tumor NGS in parallel with ctDNA NGS allowed iden- These observations suggest that while deriving clinical ben- tifi cation and understanding of multiple likely baseline and efi t from therapy, anti-EGFR ADCC may have elicited a refl ex- acquired resistance mechanisms, often concurrently within ive upregulation of PD-L1 expression in tumor cells, a so-called the same patient across and within tumor sites. interferon alpha/gamma T cell–induced immune response None of the 24 available GEA cell lines demonstrated ( 55), yet over time as tumor response occurred (i.e., EGFR- EGFR amplifi cation or extremely high expression by mass amplifi ed clones eradicated in patients 2, 3, 5 at posttreatment spectrometry, as compared with the EGFR -amplifi ed head biopsies) the immune response appeared to have dissipated, and neck cell line controls. An effort to establish more and therefore PD-L1 expression subsequently downregulated. EGFR -amplifi ed cell lines and xenografts is needed in order to enhance understanding of this molecular subset of the disease. Notably, none of the patients identifi ed for treatment DISCUSSION in this report had peritoneal ascites or pleural effusions— Herein we quantifi ed the incidence ofEGFR amplifi cation both recognized metastatic sites that are easily accessible and and consequent signifi cant EGFR overexpression in 24 GEA conducive for establishing tumor cell lines ( 56 ). Finally, we cell lines and 502 samples from 363 patients with GEA within demonstrated that tissue EGFR amplifi cation correlated well the University of Chicago Gastrointestinal Tumor Bank, as with protein expression by IHC and mass spectrometry in well as from a large commercial NGS database of 4,645 cell lines and tissues analyzed from the same time point and patients with GEA. We observed no statistically signifi cant dif- anatomic location, with the caveat of stromal cellular and ferences in clinicopathologic characteristics in patients with tumoral molecular heterogeneity affecting this relationship, EGFR amplifi cation versus those without amplifi cation, other as previously described with MET and HER2 (57–59 ).

june 2018 CANCER DISCOVERY | 705

RESEARCH ARTICLE Maron et al.

A B C Pt 1 Pt 2 Pt 3 Pt 4 Pt 5 Pt 6 Pt 7 Pt 8 MET copy number ACVR1B 12 AR BRAF copy number ARID1A 60 EGFR copy number BRAF 10 60 BRCA1 CCND1 8 CCND2 CCNE1 40 40 CD274 6 CDK6 CDKN2A/B 4 EGFR number Copy 20 20 EMSY 2 ERBB2 number EGFR copy EZH2 FGF19 0 FGF3 0 0 FGF4 copy number in cfDNA MET/BRAF copy FGFR1 010203040 50 60 70 0 100 200 300 400 GNAS JAK2 Weeks on therapy KIT Days on therapy KRAS MAP3K1 MET D E MYC KRAS copy number 14 ERBB2 copy number 60 NOTCH1 EGFR copy number 40 EGFR copy number NOTCH1 8 NRAS 12 50 PDCD1LG2 PDGFRA 30 PIK3CA 6 10 40 PIK3CB PIK3R1 PRKCI 8 30 PTCH1 4 20 PTEN RBM10 6 20 SMAD4 SPTA1 2 10 copy number EGFR copy TERC 4 10 number EGFR copy TP53

TSC2 number in cfDNA KRAS copy copy number in cfDNA ERBB2 copy 2 0 0 0 Primary Pretreatment Amplification Metastatic Posttreatment Deletion 050 100 150 200 250 300 020406080 100 ctDNA Mutation Days on therapy Days on therapy

FGPre Post Low concurrent molecular Combination events (vs. high) therapy (chemotherapy and/ CD3 or targeted) Immune infiltration

NKp46

Monoclonal antibody therapy (vs. TKI) High copy number (vs. low copy PD-L1 number) Homogeneity of EGFR amp within patient (vs. heterogeneity)

Figure 4. Molecular and immunologic correlatives of EGFR-amplified samples treated with anti-EGFR therapy. A, Comparison of intrapatient and interpatient significant genomic alterations in primary tumor DNA, metastatic tumor DNA, and ctDNA pre/post–anti-EGFR therapy. B, Serial ctDNA EGFR copy number for all treated patients. C, Serial ctDNA for patient 3 demonstrating decline of EGFR copy number when cetuximab was begun followed by rise in EGFR copy number when MET and BRAF amplification arose at the time of radiographic progression. D, Serial ctDNA for patient 2 revealing steep decline in EGFR copy number after FOLFOX and later ABT-806 initiation with cycle 3 followed by rise in KRAS copy number at the time of occult peritoneal disease growth leading to hydronephrosis. E, Serial ctDNA for patient 4 demonstrating sharp decline in EGFR copy number after cetuximab administration with concomitant rise in ERBB2 copy number at the time of clinical progression and progressive liver failure. F, IHC assessment for CD3+, NKp46+, and PD-L1+ cells pre-/post–anti-EGFR therapy, demonstrating increased inflammation within primary tumor from patient 1. G, “Genogram” figure of patient 3. A framework detailing predictive factors favoring response to genomic targeted therapy is toward the periphery including intrapatient homogeneity of EGFR amplification therapy; higherEGFR copy number; combination of chemotherapy + EGFR antagonist; fewer concurrent molecular events; monoclonal EGFR antibody use, and increased CD3 infiltration of tumor and stroma. Patient 3 demonstrated homogeneous and highEGFR amplification at baseline, with moderate immune infiltrate, and without concurrent resistance mechanisms. He therefore would be predicted to derive significant benefit, despite being in the third-line setting and with monotherapy; he had a complete response lasting 61 weeks.

706 | CANCER DISCOVERY june 2018 www.aacrjournals.org

Anti-EGFR Therapy for EGFR-Amplified Gastroesophageal Cancer RESEARCH ARTICLE

Among University of Chicago tissue samples analyzed, line ToGA trial combined trastuzumab with chemotherapy EGFR amplification incidence ranged from 5% to 7% across in treatment-naïve HER2-positive patients and achieved a all stages and cohorts, which is consistent with previous 6.7-month mPFS, and the second-line RAINBOW study of reports (28, 46, 60). Regarding incidence of EGFR amplifica- ramicirumab plus paclitaxel achieved a 4.4-month mPFS (3). tion specifically in metastatic patients, this was similar in the In our treated cohort, tumor reduction with clinical benefit retrospective and prospective stage IV patients (6%), suggest- was observed in all cases, and the best disease control rate ing a reflective prospective cohort. In the overall population, was 100%. Furthermore, as highlighted in the patient therapy EGFR amplification trended to be more commonly observed summaries (Supplementary Data File 1), subjective quality of in stage IV patients compared with earlier-stage patients life improvements were seen—even in those with the shortest (79% vs. 50% P = 0.11, Table 1A). As such, EGFR amplifica- duration of benefit and those with “primary-tumor-only” tion incidence was slightly higher in our study than the 4% EGFR amplification. seen in the TCGA GEA cohort, which was based entirely Baseline and serial ctDNA along with DNA from primary, upon early-stage resected specimens. This disparity may also metastatic, and serial tumor biopsies highlighted significant reflect the difference in incidence between esophageal/esoph- tumor heterogeneity and widespread potential of therapeutic agogastric junction and gastric primary tumor location, as resistance mechanisms in these patients. Resistance mecha- 14% of TCGA esophageal adenocarcinoma cases were EGFR nisms included regions of tumors at baseline not harboring amplified (28). Accordingly, our study was comprised of a EGFR amplification and regions withoutEGFR amplification majority of proximal tumors (54%), and EGFR amplification at the time of clinical progression. Resistance mechanisms incidence was consistent with the TRANS-COG analysis com- also included concomitant amplifications and mutations in prised exclusively of esophageal cancers (46). Our findings are genes putatively involved in circumventing EGFR signaling also consistent with previous work demonstrating that CIN in the setting of anti-EGFR therapy (65–70), which we also GEAs, which are more likely to harbor amplifications, tend observed in the larger Foundation Medicine GEA cohort (Fig. to have proximal locations (27, 28). The higher incidence in 1F). Patient 2 was observed to harbor both EGFR and ERBB2 EGJ versus distal gastric adenocarcinoma and the generally amplification with high expression of both, each within two higher incidence compared with TCGA was corroborated independent regions (50:50 ratio) of his primary tumor, in the larger Foundation Medicine database of 4,645 GEA whereas the retroperitoneal lymph node and bone marrow samples undergoing Foundation One testing (Table 2A). All biopsies, as well as ctDNA, at initial diagnosis harbored other pertinent positive/negative clinicopathologic findings EGFR amplification and lackedERBB2 amplification. For this (age, grade, HER2 status, gender, site, and race) were similar patient, from a standard-of-care perspective, chemotherapy regardless of EGFR status. combined with trastuzumab would be indicated (3), but this Despite amplification ofEGFR being found in only ∼5% would not likely have addressed his primarily HER2-negative to 7% of GEAs, with the high global incidence of distal gas- metastatic burden. In this patient, clinical resistance and pro- tric cancer alone, this may represent nearly 50,000 patients gression of peritoneal carcinomatosis after 10 months of anti- diagnosed each year with EGFR-amplified GEA. ALK-positive EGFR–based therapy corresponded with the rise of a ctDNA non–small cell lung cancer represents a similar paradigm, KRAS-amplified clone andKRAS amplification confirmed in with a 3% to 5% ALK-translocation frequency that has led the progressing peritoneal biopsy; the primary tumor at this to the approval of crizotinib, ceritinib, alectinib, and brig- time point was no longer EGFR amplified, norKRAS ampli- atinib (61–64). In this report, from 140 patients prospectively fied, but remainedHER2 amplified in∼ 75% of the biopsy screened at one treatment center, we identified and treated (Fig. 4D; Supplementary Fig. S2B). Patient 1, who had both seven patients with GEA with extreme amplification ofEGFR baseline EGFR-amplified and nonamplified regions of tumor, (54–167 gene copies) in tissue biopsies. By chance, we did not derived benefit from treatment with FOLFIRI and cetuximab, encounter any patients with EGFR amplification with tissue which together effectively controlled all disease including the gene copies between 8 and 53, but these patients are not EGFR-amplified clone for a period of time. However, as dem- uncommon, as demonstrated in the Foundation Medicine onstrated on repeat biopsy, this therapy eventually selected GEA cohort (Fig. 1E). As such, clinical benefit, or differential for an EGFR-amplified clone with concurrent downstream clinical benefit, in this “lower level tissue copy number sub- PTEN exon 6 deletion, along with persistence of the previ- set” cannot be determined from our study. However, we did ously identifiedEGFR -nonamplified region (Supplementary observe that higher plasma ctDNA copy number did correlate Fig. S2A), as well as a de novo PIK3CA mutation in the ctDNA. with response within our treated cohort. Cetuximab resistance via loss of PTEN has been previously We demonstrated clinical benefit with anti-EGFR tar- demonstrated in colorectal cancer (71). Of note, this dele- geted therapy in clinical scenarios that historically have poor tion was not detected by ctDNA, but rather by endoscopic response rates to conventional therapies. In particular, three biopsy and tumor NGS, and this highlights a potential patients were treated with third/fourth-line monotherapy challenge of detecting larger segment deletions in ctDNA. after exhausting all standard therapies. The observed best Patient 3 initially had homogenously EGFR-amplified disease, objective response rate by RECIST was 4 of 7 (57%) patients but after 14 months developed a combination of EGFR- across multiple lines of therapy, including a complete and nonamplified regions along with presumablyEGFR -amplified durable response lasting 14 months with monotherapy in and concurrent BRAF-, MET-, and MYC-amplified clones that the third-line setting (patient 3). We noted a median PFS circumvented EGFR inhibition (Fig. 4C; Supplementary Fig. (mPFS) of 10 months overall (nearly double the mPFS of S2C). His persistent EGFR amplification by ctDNA despite standard first-line chemotherapy). In comparison, the first- absence of amplification identified within postprogression

june 2018 CANCER DISCOVERY | 707

RESEARCH ARTICLE Maron et al. biopsies (of both the residual primary tumor and new lung double the median OS in this cancer (Supplementary Table metastasis) suggests additional nonbiopsied sites harboring S3). This may suggest that EGFR amplification portends a EGFR amplification, potentially within new brain metasta- relatively favorable prognosis, but further larger studies will ses that were not biopsied. Similarly, selective pressure with need to sort this out. It should be noted that patient 3 refused cetuximab led to expansion of preexisting HER2-, NRAS-, surgery while locally advanced, and therefore the duration of and MYC-amplified subclones and emergence ofde novo “first-line” therapy in this case is distorted (he was stage IV to GNAS mutation in patient 4, which all likely conferred thera- the lung by the time of initiating “third-line” monotherapy peutic resistance in various sites within the patient (Fig. cetuximab). Despite this, these late-line patients 3, 4, and 6 4E; Supplementary Fig. S2D). Patient 5 had baseline MET all demonstrated clinical benefit from cetuximab monother- coamplification as well asKRAS mutation in different ana- apy, suggesting that this is indeed an actionable alteration. tomic locations. In this patient, significant clinical benefit Notably, each of these three patients had identifiedEGFR of anti-EGFR therapy was reported despite lack of RECIST amplification in their original stage IV diagnostic samples as response, with improved local esophagogastric symptoms of well as their profiling just prior to anti-EGFR therapy in later pain/dyspepsia, which was potentially explained by the EGFR lines, suggesting stability (and dependence) over time of this amplification identified only in the primary tumor. Notably aberration with standard therapies. In contrast, and interest- this patient’s posttreatment residual primary tumor biopsy ingly, most patients treated in early lines in combination with no longer identified anEGFR -amplified region. Again, as in chemotherapy in our cohort had evidence of loss of the aber- patient 1 above, this heterogeneity highlights the benefit of ration in all or at least some of their tissue/plasma samples concurrent combination chemotherapy in suppressing other and/or acquisition of likely concurrent resistance mechanisms preexisting or acquired resistant clones that would otherwise (e.g., PTEN deletion) after experiencing disease progression on progress at an accelerated rate if treated with anti-EGFR anti-EGFR therapy. This again confirms the specific targeting monotherapy. Patient 6 was unable to obtain drug after cycle of anti-EGFR therapy toward EGFR-amplified clones, with 1 due to insurance denial, but presumably would have a more consequent EGFR-amplified clonal eradication and/or pres- limited benefit in the face of preexistingKRAS/MYC/CCNE1 sure to select for concurrent circumventing alterations. amplifications andGNAS mutation in the ctDNA at baseline. Previous phase II and III trials (including COG with gefi- Her best response was short-lived stable disease after one tinib and EXPAND using cetuximab) demonstrated an OS dose of cetuximab, yet somewhat impressively after disease benefit in the small subset of patients withEGFR- amplified progression on first-line FOLFOX and second-line paclitaxel/ (TRANS-COG) or overexpressed (EXPAND) GEA. This was ramucirumab. Finally, patient 7 further highlights the intra- despite an unimpressive response rate (15, 46), particularly tumoral and intertumoral heterogeneity of EGFR amplifica- in the TRANS-COG analysis. Of 13 patients in TRANS-COG tion, as only a fraction of his primary tumor exhibited EGFR with EGFR amplification who received gefitinib, none had an amplification, but not in the liver metastasis nor ctDNA. objective response. Our results from seven EGFR-amplified The patient did however derive significant benefit, similar to patients treated with anti-EGFR monoclonal antibodies sug- patient 5 above, as demonstrated with improved dysphagia gest a similar benefit, with four patients having durable PFS only after anti-EGFR therapy was added after 4 cycles of inef- of over 6 months, but also a high response rate, even in those fective standard FOLFOX chemotherapy (see Supplementary treated with monotherapy (2/3, 66%), with minimal adverse File S1). These seven cases highlight the utility of a compos- drug reactions. The difference in response rates between TKI ite of tumor and ctDNA sequencing in tailoring therapy for and antibodies is intriguing and although it has a number of patients with GEA and using anti-EGFR, cytotoxic therapy, possible explanations, it could be explained by ADCC and/or and other targeted and immuno-oncologic agents combined receptor internalization/downregulation, which is not seen for optimal tumor control. with TKIs. Antibody therapy was intentionally chosen for A limitation of this study in terms of defining benefit from treatment in our study due to potential for ADCC via NK anti-EGFR therapy is the combination of anti-EGFR therapy cells seen with cetuximab and other IgG1 monoclonal anti- with chemotherapy in 4 of the 7 patients. The individual con- bodies, such as ABT-806, as well as less toxicity in combina- tribution of cetuximab/ABT-806 in combination with chemo tion with chemotherapy relative to TKIs. On the other hand, therapy is therefore difficult to discern in this small cohort. panitumumab, an IgG2 antibody, may act via myeloid cell However, the median PFS with FOLFIRI in second line is lineage ADCC (75, 76), and differences/similarities between only 4 to 5 months (72). Also, clinical benefit in patient 7 these two IgG classes with respect to ADCC are not well delin- was not experienced with four cycles of FOLFOX, and only eated, certainly so for EGFR-amplified GEA. after the addition of anti-EGFR ABT806 antibody at cycle Regardless, in head and neck cancer, cetuximab stimulates 5 (when biological grouping was determined on PANGEA NK cell recruitment and IFNγ secretion, which mediates den- study; see Supplementary Fig. S1) was dysphagia dramatically dritic cell maturation and cross-presentation to cytotoxic T improved, which avoided further intervention including stent lymphocytes against EGFR (77). IFNγ and its associated genes and/or palliative radiation. Data regarding the prognostic are currently under evaluation as a predictive biomarker of significance and natural progression ofEGFR amplification response to PD-1 and PD-L1 antagonists due to their asso- remain unknown, but EGFR amplification and EGFR over- ciation with a T cell–inflamed tumor environment (78–82). expression have been associated with shortened survival in Interestingly, from a large Foundation Medicine cohort of some reports (73, 74). Strikingly, however, all three patients GEA samples undergoing PD-L1 IHC testing, we observed a who received late-line cetuximab monotherapy began their slightly lower rate of positivity by TPS, TILs, and CPS scoring therapy approximately 2 years after initial stage IV diagnosis— in EGFR-amplified tumors as compared with nonamplified

708 | CANCER DISCOVERY june 2018 www.aacrjournals.org

Anti-EGFR Therapy for EGFR-Amplified Gastroesophageal Cancer RESEARCH ARTICLE tumors (Table 2B; Fig. 1G). A limitation of this analysis is rent chemotherapy for synergy on EGFR-amplified clones as the use of the Ventana SP142 PD-L1 antibody as opposed to well as simultaneous suppression of EGFR-negative clones the 22C3 pharmDx companion antibody, which was recently versus anti-EGFR monotherapy, (iv) lack of baseline genomic approved for pembrolizumab in PD-L1–expressing patients. resistance mechanisms versus “molecular chaos,” (v) ADCC SP142 has lower sensitivity and therefore possibly underesti- mechanism of monoclonal antibodies as compared with TKI mates PD-L1 expression (83–85). Regardless, relatively lower lacking this mechanism of action, with the patient’s general frequency of PD-L1 expression by EGFR-amplified tumors immune status playing an important role, and (vi) addition compared with nonamplified tumors as we observed in the of concurrent PD-1/PD-L1 checkpoint blockade to increase large Foundation Medicine cohort, if confirmed, may cor- immune response. This has been conceptualized in a “geno- respond to lower responses to anti–PD-1/anti–PD-L1 check- gram,” or “EGFR ampligram,” akin to the recently suggested point inhibitor monotherapy in EGFR-amplified patients. “immunogram” (55), to serve as a framework to predict This requires further investigation. clinical benefit from anti-EGFR therapy inEGFR -amplified To evaluate the effect of anti-EGFR antibodies on the tumor tumors (Fig. 4G; Supplementary Fig. S3). The degree to which immune environment, including evidence of ADCC, we eval- each of these variables contributes to predicted response and uated pretreatment and posttreatment tumor biopsies for response duration will require further investigation. Further EGFR, NKp46, CD8, and PD-L1 expression when possible. In assessment of anti-EGFR treatment for EGFR amplification is this study, results from “during therapy” biopsies imply that warranted. Given the relatively low frequency of EGFR ampli- treatment with EGFR-directed monoclonal antibodies led to fication, not to mention the issues with intrapatient hetero- increased tumoral infiltration by CD3+ T cells and NKp46+ NK geneity, evaluation in a traditional phase III study has been cells as well as increased PD-L1 expression, which suggested that elusive and remains difficult, as demonstrated in all phase III consequent ADCC may create, or “trigger,” a reflexive immuno- EGFR-directed GEA trials to date having only small subsets suppressed tumor environment. On-treatment PD-L1 expres- to evaluate this event, without definitive practice-changing sion appeared more common in cases with persistent EGFR results. Novel trial designs, such as PANGEA, a type II expan- amplification, which also supports this proposed mechanism. sion platform design trial in GEA, test a treatment strategy Furthermore, patient 1 had previously received, though not of cytotoxic therapy plus matched targeted therapies across responded to, CTLA4 and PD-L1 combination inhibition, and a number of biological subgroups, including EGFR ampli- so we cannot detail if his increased post-therapy CD3/PD-L1 fication. This design may optimally identify and treat these staining represents a delayed effect from prior immunotherapy low incidence aberrations as well as addressing the various alone, anti-EGFR ADCC alone, sequential immunotherapy mechanisms of resistance at baseline and progression over and targeted monoclonal antibodies, or spatial heterogeneity. time (53, 59, 88, 89). Therefore, although suggestive, these results are limited due to the low sample size and temporospatial biopsy variability. These METHODS hypothesis-generating findings merit further prospective investigation to tease out the individual contributions of canonical GEA Clinical Samples and Cell Lines EGFR ligand–binding inhibition and receptor internalization/ Retrospective and prospective GEA patient samples, with linked degradation versus ADCC/immune phenomena. Should fur- clinical and pathological correlates, were obtained from the University ther studies confirm an upregulation of PD-L1 in anti-EGFR of Chicago (Chicago, IL) under IRB-approved tissue banking proto- antibody–treated EGFR-amplified tumors, combination with cols. This work was conducted in full concordance with the principles PD-1 checkpoint blockade would be an appealing combina- of the Declaration of Helsinki. All patients provided written informed tion strategy. consent, where applicable. The human GEA lines and lymphoblast/ breast cancer–negative controls were obtained and cultured as previ- In summary, we report EGFR amplification with over- ously described (57, 90). The genetic identity of parental cell lines expression in 5% (19/363) of a large GEA patient cohort. was authenticated by short-tandem repeat profiling (Cell ID System; Prospectively, 6% (8/140) of stage IV advanced patients dem- Promega) at 10 different loci not fewer than 2 months before profiling onstrated EGFR amplification, of which seven patients were and experiments. Cell lines tested negative for Mycoplasma contamina- successfully treated with at least one dose of anti-EGFR tion with the VenorGeM Classic Kit (Minerva Biolabs). These included monoclonal antibody therapy. A 57% objective response rate AGS, CAT-2, CAT-3, CAT-4, CAT11B, CAT12, CAT13, CAT14A, and 100% disease control rate was observed. Within our CAT15pl, CP-A, CP-B, CP-C, CP-D, GM14667, HGC-27, Hs746T, cohort, elevated plasma-based ctDNA NGS copy-number KATO III, MKN-1, MKN-45, NCI-N87, OE19, OE33, SNU-1, SNU-16, estimation correlated with objective response by RECIST SNU-5, and ZR-75-30 obtained between 2008 and 2012. The head and criteria. This is consistent with a similar-sized prospective neck cancer lines (HN5 and SQ20B) were graciously provided by Dr. Ezra Cohen (UCSD) in 2012. CAT lines were established between 2009 study of HER2 amplification in plasma associated with an and 2016 from malignant ascites or pleural effusion aspirates from 80% response rate to targeted therapy in GEA (86, 87). It is patients at the University of Chicago under preapproved guidelines likely that response to anti-EGFR therapy will be optimized and IRB protocols. for depth and duration with the following contributing factors (quite analogous to anti-HER2 for HER2 amplification EGFR FISH and likely also to anti-MET for MET amplification and anti- FISH results for cell lines and retrospective samples included mean FGFR2 for FGFR2 amplification): (i) homogeneity ofEGFR EGFR and CEP7 copies/nucleus and EGFR/CEP7 ratio as previously gene amplification within and across all sites of a patient’s described (90–92) and prospectively screened patients using Clarient tumor burden versus heterogeneous “EGFR-negative” sites, Diagnostics Services Inc. FISH amplification was defined asEGFR/ (ii) higher EGFR gene copy number versus lower, (iii) concur- CEP7 ratio ≥2 in all settings.

june 2018 CANCER DISCOVERY | 709

RESEARCH ARTICLE Maron et al.

Sample Preparation and EGFR-SRM Assay in order to correlate cfDNA levels and genomic findings with initial Laser microdissection isolated tumor cells were obtained from response outcomes and for potential mechanisms of resistance over FFPE tumor sections as previously described (52, 57, 58, 92, 93). time (98–100). Absolute plasma copy number was determined utiliz- Total protein content for lysates was measured using the Micro-BCA ing the mode of the normalized number of cfDNA fragments covering assay (Thermo Fisher Scientific Inc.). The EGFR-SRM assay followed each gene to estimate the fragment number corresponding to two cop- previously described methods and quantified expression in attomols/ ies to derive a baseline diploid value. All values of unique fragments for each gene were then normalized by this baseline value. The baseline microgram (amol/μg; ref. 93). derivation was informed by molecule counts data from a large set of EGFR Copy Number by Tissue NGS normal samples from healthy donors’ plasma. Note that the plasma copy number was related to two variables—the copy number in the tis- All NGS results were obtained through routine clinical testing sue and the amount of shedding of tumor DNA into the blood where using Foundation One (94); EGFR amplification was defined asEGFR the tumor DNA—and thus the copy number was expected to be diluted copy ≥8. Equivocal amplification as noted in the clinical report (copy by abundant leukocyte-derived EGFR fragments, the latter having a number 6–7) was considered EGFR nonamplified in this study. copy number of 2.0. Centiles of EGFR copy number reported in the clinical G360 results were denoted by a “+” for absolute plasma copy PD-L1 IHC from Foundation Medicine Cohort number greater than 2.14 (<50th percentile), “++” for copy number GEA samples having PD-L1 testing through Foundation Medicine greater than 2.4 but less than 4 (<90th percentile), or “+++” for copy were identified for analysis N( = 632). PD-L1 testing was performed number greater than 4 (≥90th percentile). In this study, we reported using the Ventana antibody (SP142) as previously described (95) and absolute plasma copy number, not these percentiles. was scored three ways: % TPS, % TILs, and combination of these two for a CPS given recent approval for third-line therapy with pembrolizumab ADCC for PD-L1–positive GEA tumors as defined by CPS score≥ 1% (83–85). The contribution of ADCC was assessed in the prospectively identified anti-EGFR–treatedEGFR -amplified cohort by IHC Quantitative Analysis and Validation of EGFR in Clinical using pretreatment and posttreatment IHC for CD3 (Agilent GEA Tissues and Cell lines A0452), NKp46 (R&D Systems, Clone 195314), and PD-L1 (Abcam EGFR-SRM for 225 retrospective GEA FFPE samples and 28 cell ab205921) in order to evaluate for treatment-related tumor– lines was performed by Nantomics and expression was calculated stroma modulation. from the ratio of AUC for the endogenous and isotopically labeled standard peptide multiplied by the known amount of isotopically Statistical Analysis labeled standard peptide spiked into the sample before analysis, as Comparisons between EGFR-amplified and nonamplified cases previously described (93). were performed using χ2 testing or a two-sided Fisher exact test. The relationship between EGFR amplification and expression by SRM or Identification and Treatment of EGFR-Amplified GEA RECIST response was evaluated using the Student t test and by linear Patients with Anti-EGFR Therapy regression. PFS was estimated by the Kaplan–Meier method. Patients at the University of Chicago with metastatic GEA (any line of therapy) were prospectively screened for EGFR amplification Disclosure of Potential Conflicts of Interest between September 2014 and December 2016 with NGS using the R.B. Lanman has ownership interest (including patents) in Guardant Foundation One test (Foundation Medicine). When remaining tissue Health, Inc. T. Hembrough has ownership interest (including patents) was available, EGFR amplification identified by NGS was confirmed in NantOmics, LLC. A. Schrock has ownership interest (including by FISH (Clarient Diagnostics Services Inc.), EGFR overexpression patents) in Foundation Medicine, Inc. D.V.T. Catenacci reports receiv- was confirmed with IHC (Invitrogen, Clone 31G7, Ventana Ultra ing commercial research support from Genentech/Roche, has received View Detection Kit), and EGFR-SRM through Nantomics (93). All honoraria from the speakers bureaus of Genentech/Roche, Merck, assays used were Clinical Laboratory Improvement Amendments and Lilly, and is a consultant/advisory board member for Genentech/ (CLIA) certified. Roche, Amgen, Lilly, Merck, BMS, and Gritstone. No potential conflicts of interest were disclosed by the other authors. Treatment Assignment Newly diagnosed metastatic, or recurrent after previous curative Authors’ Contributions intent surgery, first-line patients were treated on the PANGEA pro- Conception and design: S.B. Maron, F. Cecchi, T. Hembrough, tocol (NCT02213289; ref. 53) with anti-EGFR antibody ABT-806 D.V.T. Catenacci 24 mg/kg q2 weeks i.v., in combination with FOLFOX per proto- Development of methodology: S.B. Maron, R.B. Lanman, col (Supplementary Fig. S1). Otherwise, patients were treated with T. Hembrough, N. Setia, D.V.T. Catenacci off-label cetuximab 500 mg q2 weeks i.v. (96), in combination with Acquisition of data (provided animals, acquired and man- FOLFIRI in the second line, or as monotherapy in the third- and aged patients, provided facilities, etc.): S.B. Maron, H.A. Kwak, fourth-line settings. S. Lomnicki, D. Xu, E. O’Day, R.J. Nagy, R.B. Lanman, T. Hembrough, Clinical Outcome Assessment A. Schrock, J. Hart, S.-Y. Xiao, N. Setia, D.V.T. Catenacci Analysis and interpretation of data (e.g., statistical analysis, bio- The primary objective of this study was clinical response as assessed statistics, computational analysis): S.B. Maron, R.J. Nagy, R.B. Lanman, by CT using RECIST 1.1 (97). Measurements were performed inde- F. Cecchi, T. Hembrough, A. Schrock, N. Setia, D.V.T. Catenacci pendently by clinical interpreting radiologists. Secondary endpoints Writing, review, and/or revision of the manuscript: S.B. Maron, included PFS and toxicity. D. Xu, R.J. Nagy, T. Hembrough, A. Schrock, N. Setia, D.V.T. Catenacci Administrative, technical, or material support (i.e., reporting or Circulating Cell-Free DNA NGS organizing data, constructing databases): S.B. Maron, S. Lomnicki, Circulating free DNA (cfDNA) sequencing was obtained at baseline L. Chase, E. O’Day, S.-Y. Xiao, D.V.T. Catenacci prior to anti-EGFR therapy and serially monitored by Guardant 360 Study supervision: D.V.T. Catenacci

710 | CANCER DISCOVERY june 2018 www.aacrjournals.org

Anti-EGFR Therapy for EGFR-Amplified Gastroesophageal Cancer RESEARCH ARTICLE

Other (analysis of pathology specimens and interpretation of the randomized, double-blind, phase III GRANITE-1 study. J Clin immunohistochemical studies): L. Alpert Oncol 2013;31:3935–43. Other (pathology): S.-Y. Xiao 12. Cohen DJ, Christos PJ, Kindler HL, Catenacci DVT, Bekaii-Saab TB, Tahiri S, et al. Vismodegib (V), a hedgehog (HH) pathway inhibitor, combined Acknowledgments with FOLFOX for first-line therapy of patients (pts) with advanced gas- The authors would like to acknowledge Kyle Holen, Brian Panzl, tric and gastroesophageal junction (GEJ) carcinoma: a New York cancer James Ward, Vincent Blot, and Earl Bain from AbbVie Pharmaceuti- consortium led phase II randomized study. J Clin Oncol 2013;31:4011. cals. This work was supported by the NIH (K23 CA178203 and P30 13. Maron SB, Catenacci DV. Novel targeted therapies for esophagogastric cancer. Surg Oncol Clin N Am 2017;26:293–312. CA014599), University of Chicago Comprehensive Cancer Center 14. Kim C, Lee JL, Ryu MH, Chang HM, Kim TW, Lim HY, et al. A pro- (UCCCC) Award in Precision Oncology, Live Like Katie (LLK) Foun- spective phase II study of cetuximab in combination with XELOX dation Award, Castle Foundation Award, and the Sal Ferrara II Fund (capecitabine and oxaliplatin) in patients with metastatic and/or for PANGEA (to D.V.T. Catenacci). recurrent advanced gastric cancer. Invest New Drugs 2011;29:366–73. 15. Lordick F, Luber B, Lorenzen S, Hegewisch-Becker S, Folprecht G, Woll Received November 9, 2017; revised January 11, 2018; accepted E, et al. Cetuximab plus oxaliplatin/leucovorin/5-fluorouracil in first- February 9, 2018; published first February 15, 2018. line metastatic gastric cancer: a phase II study of the Arbeitsgemein- schaft Internistische Onkologie (AIO). Br J Cancer 2010;102:500–5. 16. Pinto C, Di Fabio F, Barone C, Siena S, Falcone A, Cascinu S, et al. References Phase II study of cetuximab in combination with cisplatin and doc- . 1 Sehdev A, Catenacci DV. Gastroesophageal cancer: focus on epide- etaxel in patients with untreated advanced gastric or gastro-oesophageal miology, classification, and staging. Discov Med 2013;16:103–11. junction adenocarcinoma (DOCETUX study). Br J Cancer 2009;101: 2. Al-Batran S-E, Homann N, Schmalenberg H, Kopp H-G, Haag 1261–8. GM, Luley KB, et al. Perioperative chemotherapy with docetaxel, 17. Pinto C, Di Fabio F, Siena S, Cascinu S, Rojas Llimpe FL, Ceccarelli C, oxaliplatin, and fluorouracil/leucovorin (FLOT) versus epirubicin, et al. Phase II study of cetuximab in combination with FOLFIRI in cisplatin, and fluorouracil or capecitabine (ECF/ECX) for resect- patients with untreated advanced gastric or gastroesophageal junction able gastric or gastroesophageal junction (GEJ) adenocarcinoma adenocarcinoma (FOLCETUX study). Ann Oncol 2007;18:510–7. (FLOT4-AIO): A multicenter, randomized phase 3 trial. J Clin Oncol 18. Woell E, Greil R, Eisterer W, Fridrik M, Grunberger B, Gattringer 2017;35:4004. K, et al. Oxaliplatin, irinotecan and cetuximab in advanced gastric 3. Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L, Sawaki cancer: first results of a multicenter phase II trial (AGMT Gastric-2). A, et al. Trastuzumab in combination with chemotherapy versus J Clin Oncol 2008;26:15587. chemotherapy alone for treatment of HER2-positive advanced gas- 19. Yeh K, Hsu C, Hsu C, Lin C, Shen Y, Wu S, et al. Phase II study tric or gastro-oesophageal junction cancer (ToGA): a phase 3, open- of cetuximab plus weekly cisplatin and 24-hour infusion of high- label, randomised controlled trial. Lancet 2010;376:687–97. dose 5-fluorouracil and leucovorin for the first-line treatment of 4. Fuchs CS, Tomasek J, Yong CJ, Dumitru F, Passalacqua R, Gos- advanced gastric cancer. J Clin Oncol 2009;27:4567. wami C, et al. Ramucirumab monotherapy for previously treated 20. Enzinger PC, Burtness BA, Niedzwiecki D, Ye X, Douglas K, Ilson DH, advanced gastric or gastro-oesophageal junction adenocarcinoma et al. CALGB 80403 (Alliance)/E1206: a randomized phase II study of (REGARD): an international, randomised, multicentre, placebo- three chemotherapy regimens plus cetuximab in metastatic esopha- controlled, phase 3 trial. Lancet 2014;383:31–9. geal and gastroesophageal junction cancers. J Clin Oncol 2016;34: 5. Wilke H, Muro K, Van Cutsem E, Oh SC, Bodoky G, Shimada Y, 2736–42. et al. Ramucirumab plus paclitaxel versus placebo plus paclitaxel in 21. Dragovich T, McCoy S, Fenoglio-Preiser CM, Wang J, Benedetti JK, patients with previously treated advanced gastric or gastro-oesoph- Baker AF, et al. Phase II trial of erlotinib in gastroesophageal junc- ageal junction adenocarcinoma (RAINBOW): a double-blind, ran- tion and gastric adenocarcinomas: SWOG 0127. J Clin Oncol 2006; domised phase 3 trial. Lancet Oncol 2014;15:1224–35. 24:4922–7. 6. Lordick F, Kang YK, Chung HC, Salman P, Oh SC, Bodoky G, et al. 22. Wainberg ZA, Lin LS, DiCarlo B, Dao KM, Patel R, Park DJ, et al. Capecitabine and cisplatin with or without cetuximab for patients Phase II trial of modified FOLFOX6 and erlotinib in patients with with previously untreated advanced gastric cancer (EXPAND): a metastatic or advanced adenocarcinoma of the oesophagus and randomised, open-label phase 3 trial. Lancet Oncol 2013;14:490–9. gastro-oesophageal junction. Br J Cancer 2011;105:760–5. 7. Waddell T, Chau I, Cunningham D, Gonzalez D, Okines AF, Okines 23. Tomblyn MB, Goldman BH, Thomas CR Jr, Benedetti JK, Lenz HJ, C, et al. Epirubicin, oxaliplatin, and capecitabine with or without Mehta V, et al. Cetuximab plus cisplatin, irinotecan, and thoracic panitumumab for patients with previously untreated advanced radiotherapy as definitive treatment for locally advanced, unresect- oesophagogastric cancer (REAL3): a randomised, open-label phase able esophageal cancer: a phase-II study of the SWOG (S0414). J 3 trial. Lancet Oncol 2013;14:481–9. Thorac Oncol 2012;7:906–12. 8. Dutton SJ, Ferry DR, Blazeby JM, Abbas H, Dahle-Smith A, Man- 24. Chan JA, Blaszkowsky LS, Enzinger PC, Ryan DP, Abrams TA, Zhu soor W, et al. Gefitinib for oesophageal cancer progressing after AX, et al. A multicenter phase II trial of single-agent cetuximab in chemotherapy (COG): a phase 3, multicentre, double-blind, placebo- advanced esophageal and gastric adenocarcinoma. Ann Oncol 2011; controlled randomised trial. Lancet Oncol 2014;15:894–904. 22:1367–73. 9. Catenacci DVT, Tebbutt NC, Davidenko I, Murad AM, Al-Batran 25. Adelstein DJ, Rodriguez CP, Rybicki LA, Ives DI, Rice TW. A phase SE, Ilson DH, et al. Rilotumumab plus epirubicin, cisplatin, and II trial of gefitinib for recurrent or metastatic cancer of the esoph- capecitabine as first-line therapy in advanced MET-positive gastric agus or gastroesophageal junction. Invest New Drugs 2012;30: or gastro-oesophageal junction cancer (RILOMET-1): a randomised, 1684–9. double-blind, placebo-controlled, phase 3 trial. Lancet Oncol 2017;18: 26. Gold PJ, Goldman B, Iqbal S, Leichman LP, Zhang W, Lenz HJ, 1467–82. et al. Cetuximab as second-line therapy in patients with metastatic 10. Shah MA, Bang YJ, Lordick F, Alsina M, Chen M, Hack SP, et al. esophageal adenocarcinoma: a phase II Southwest Oncology Group Effect of fluorouracil, leucovorin, and oxaliplatin with or without Study (S0415). J Thorac Oncol 2010;5:1472–6. onartuzumab in HER2-negative, MET-positive gastroesophageal 27. Cancer Genome Atlas Research Network. Comprehensive molecu- adenocarcinoma: the METGastric randomized clinical trial. JAMA lar characterization of gastric adenocarcinoma. Nature 2014;513: Oncol 2017;3:620–7. 202–9. 11. Ohtsu A, Ajani JA, Bai YX, Bang YJ, Chung HC, Pan HM, et al. 28. Cancer Genome Atlas Research Network. Integrated genomic charac- Everolimus for previously treated advanced gastric cancer: results of terization of oesophageal carcinoma. Nature 2017;541:169–75.

june 2018 CANCER DISCOVERY | 711

RESEARCH ARTICLE Maron et al.

29. Beroukhim R, Mermel CH, Porter D, Wei G, Raychaudhuri S, Dono- 47. Lordick F, Kang Y-K, Salman P, Oh SC, Bodoky G, Kurteva GP, van J, et al. The landscape of somatic copy-number alteration across et al. Clinical outcome according to tumor HER2 status and EGFR human cancers. Nature 2010;463:899–905. expression in advanced gastric cancer patients from the EXPAND 30. Murugaesu N, Wilson GA, Birkbak NJ, Watkins TB, McGranahan study. J Clin Oncol 2013;31:4021. N, Kumar S, et al. Tracking the genomic evolution of esophageal 48. Kurai J, Chikumi H, Hashimoto K, Yamaguchi K, Yamasaki A, Sako adenocarcinoma through neoadjuvant chemotherapy. Cancer Discov T, et al. Antibody-dependent cellular cytotoxicity mediated by cetuxi- 2015;5:821–31. mab against lung cancer cell lines. Clin Cancer Res 2007;13:1552–61. 31. Dulak AM, Stojanov P, Peng S, Lawrence MS, Fox C, Stewart C, et al. 49. Kimura H, Sakai K, Arao T, Shimoyama T, Tamura T, Nishio K. Anti- Exome and whole-genome sequencing of esophageal adenocarci- body-dependent cellular cytotoxicity of cetuximab against tumor noma identifies recurrent driver events and mutational complexity. cells with wild-type or mutant epidermal growth factor receptor. Nat Genet 2013;45:478–86. Cancer Sci 2007;98:1275–80. 32. Nones K, Waddell N, Wayte N, Patch AM, Bailey P, Newell F, et al. 50. Hatjiharissi E, Xu L, Santos DD, Hunter ZR, Ciccarelli BT, Verselis Genomic catastrophes frequently arise in esophageal adenocarci- S, et al. Increased natural killer cell expression of CD16, augmented noma and drive tumorigenesis. Nat Commun 2014;5:5224. binding and ADCC activity to rituximab among individuals express- 33. Ross-Innes CS, Becq J, Warren A, Cheetham RK, Northen H, ing the Fc{gamma}RIIIa-158 V/V and V/F polymorphism. Blood O’Donovan M, et al. Whole-genome sequencing provides new 2007;110:2561–4. insights into the clonal architecture of Barrett’s esophagus and 51. Arnould L, Gelly M, Penault-Llorca F, Benoit L, Bonnetain F, Migeon esophageal adenocarcinoma. Nat Genet 2015;47:1038–46. C, et al. Trastuzumab-based treatment of HER2-positive breast can- 34. Stachler MD, Taylor-Weiner A, Peng S, McKenna A, Agoston AT, cer: an antibody-dependent cellular cytotoxicity mechanism? Br J Odze RD, et al. Paired exome analysis of Barrett’s esophagus and Cancer 2006;94:259–67. adenocarcinoma. Nat Genet 2015;47:1047–55. 52. Hembrough T, Thyparambil S, Liao WL, Darfler MM, Abdo J, Bengali 35. Dahle-Smith A, Stevenson D, Massie D, Murray GI, Dutton SJ, Rob- KM, et al. Application of selected reaction monitoring for multiplex erts C, et al. Epidermal Growth Factor (EGFR) copy number aber- quantification of clinically validated biomarkers in formalin-fixed, rations in esophageal and gastro-esophageal junctional carcinoma. paraffin-embedded tumor tissue. J Mol Diagn 2013;15:454–65. Mol Cytogenet 2015;8:78. 53. Catenacci DV. Next-generation clinical trials: Novel strategies to 36. Ali SM, Sanford EM, Klempner SJ, Rubinson DA, Wang K, Palma NA, address the challenge of tumor molecular heterogeneity. Mol Oncol et al. Prospective comprehensive genomic profiling of advanced gastric 2015;9:967–96. carcinoma cases reveals frequent clinically relevant genomic alterations 54. Cleary JM, Reardon DA, Azad N, Gandhi L, Shapiro GI, Chaves J, and new routes for targeted therapies. Oncologist 2015;20:499–507. et al. A phase 1 study of ABT-806 in subjects with advanced solid 37. Zhou J, Wu Z, Wong G, Pectasides E, Nagaraja A, Stachler M, et al. tumors. Invest New Drugs 2015;33:671–8. CDK4/6 or MAPK blockade enhances efficacy of EGFR inhibition in 55. Blank CU, Haanen JB, Ribas A, Schumacher TN. Cancer Immuno oesophageal squamous cell carcinoma. Nat Commun 2017;8:13897. logy. The “cancer immunogram.” Science 2016;352:658–60. 38. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, 56. Shepherd TG, Theriault BL, Campbell EJ, Nachtigal MW. Primary Brannigan BW, et al. Activating mutations in the epidermal growth culture of ovarian surface epithelial cells and ascites-derived ovarian factor receptor underlying responsiveness of non-small-cell lung cancer cells from patients. Nat Protoc 2006;1:2643–9. cancer to gefitinib. N Engl J Med 2004;350:2129–39. 57. Catenacci DV, Liao WL, Thyparambil S, Henderson L, Xu P, Zhao 39. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR L, et al. Absolute quantitation of Met using mass spectrometry for mutations in lung cancer: correlation with clinical response to gefi- clinical application: assay precision, stability, and correlation with tinib therapy. Science 2004;304:1497–500. MET gene amplification in FFPE tumor tissue. PLoS One 2014;9: 40. Herbst RS, Redman MW, Kim ES, Semrad TJ, Bazhenova L, Masters e100586. G, et al. Cetuximab plus carboplatin and paclitaxel with or without 58. Catenacci DV, Liao WL, Zhao L, Whitcomb E, Henderson L, O’Day bevacizumab versus carboplatin and paclitaxel with or without E, et al. Mass-spectrometry-based quantitation of Her2 in gastroe- bevacizumab in advanced NSCLC (SWOG S0819): a randomised, sophageal tumor tissue: comparison to IHC and FISH. Gastric phase 3 study. Lancet Oncol 2018;19:101–14. Cancer 2016;19:1066–79. 41. Thatcher N, Hirsch FR, Luft AV, Szczesna A, Ciuleanu TE, Dediu M, 59. Pectasides E, Stachler MD, Derks S, Liu Y, Maron S, Islam M, et al. et al. Necitumumab plus gemcitabine and cisplatin versus gemcit- Genomic heterogeneity as a barrier to precision medicine in gastroe- abine and cisplatin alone as first-line therapy in patients with stage sophageal adenocarcinoma. Cancer Discov 2018;8:37–48. IV squamous non-small-cell lung cancer (SQUIRE): an open-label, 60. Lennerz JK, Kwak EL, Ackerman A, Michael M, Fox SB, Bergethon K, randomised, controlled phase 3 trial. Lancet Oncol 2015;16:763–74. et al. MET amplification identifies a small and aggressive subgroup 42. Hirsch F, Boyle T, Thatcher N, Paz-Ares L, Varella-Garcia M, Kow- of esophagogastric adenocarcinoma with evidence of responsiveness alewski A, et al. EGFR IHC and FISH correlative analyses (SQUIRE to crizotinib. J Clin Oncol 2011;29:4803–10. Trial): necitumumab plus gemcitabine-cisplatin vs gemcitabine- 61. Shaw AT, Ou SH, Bang YJ, Camidge DR, Solomon BJ, Salgia R, et al. cisplatin in 1st-line squamous NSCLC. J Thoracic Oncol 2015; Crizotinib in ROS1-rearranged non-small-cell lung cancer. N Engl J 10(Suppl 2):S797. Med 2014;371:1963–71. 43. Huang J, Fan Q, Lu P, Ying J, Ma C, Liu W, et al. Icotinib in patients 62. Shaw AT, Engelman JA. Ceritinib in ALK-rearranged non-small-cell with pretreated advanced esophageal squamous cell carcinoma with lung cancer. N Engl J Med 2014;370:2537–9. EGFR overexpression or EGFR gene amplification: a single-arm, 63. Ou SH, Ahn JS, De Petris L, Govindan R, Yang JC, Hughes B, et al. multicenter phase 2 study. J Thorac Oncol 2016;11:910–7. Alectinib in crizotinib-refractory ALK-rearranged non-small-cell 44. Zhang L, Yang J, Cai J, Song X, Deng J, Huang X, et al. A subset lung cancer: a phase II global study. J Clin Oncol 2016;34:661–8. of gastric cancers with EGFR amplification and overexpression 64. Kim DW, Tiseo M, Ahn MJ, Reckamp KL, Hansen KH, Kim SW, et al. respond to cetuximab therapy. Sci Rep 2013;3:2992. Brigatinib in patients with crizotinib-refractory anaplastic lym- 45. Luber B, Deplazes J, Keller G, Walch A, Rauser S, Eichmann M, et al. phoma kinase-positive non-small-cell lung cancer: a randomized, Biomarker analysis of cetuximab plus oxaliplatin/leucovorin/5- multicenter phase II trial. J Clin Oncol 2017;35:2490–8. fluorouracil in first-line metastatic gastric and oesophago-gastric 65. Misale S, Yaeger R, Hobor S, Scala E, Janakiraman M, Liska D, et al. junction cancer: results from a phase II trial of the Arbeitsgemein- Emergence of KRAS mutations and acquired resistance to anti- schaft Internistische Onkologie (AIO). BMC Cancer 2011;11:509. EGFR therapy in colorectal cancer. Nature 2012;486:532–6. 46. Petty RD, Dahle-Smith A, Stevenson DAJ, Osborne A, Massie D, 66. De Roock W, Claes B, Bernasconi D, De Schutter J, Biesmans B, Clark C, et al. Gefitinib and EGFR gene copy number aberrations in Fountzilas G, et al. Effects of KRAS, BRAF, NRAS, and PIK3CA esophageal cancer. J Clin Oncol 2017;35:2279–87. mutations on the efficacy of cetuximab plus chemotherapy in

712 | CANCER DISCOVERY june 2018 www.aacrjournals.org

Anti-EGFR Therapy for EGFR-Amplified Gastroesophageal Cancer RESEARCH ARTICLE

chemotherapy-refractory metastatic colorectal cancer: a retrospec- 84. Joshi SS, Maron SB, Catenacci DV. Pembrolizumab for treatment of tive consortium analysis. Lancet Oncol 2010;11:753–62. advanced gastric and gastroesophageal junction adenocarcinoma. 67. Douillard JY, Oliner KS, Siena S, Tabernero J, Burkes R, Barugel M, Future Oncol 2018;14:417–30. et al. Panitumumab-FOLFOX4 treatment and RAS mutations in 85. Kang YK, Boku N, Satoh T, Ryu MH, Chao Y, Kato K, et al. colorectal cancer. N Engl J Med 2013;369:1023–34. Nivolumab in patients with advanced gastric or gastro-oesophageal 68. Frattini M, Saletti P, Romagnani E, Martin V, Molinari F, Ghisletta junction cancer refractory to, or intolerant of, at least two previ- M, et al. PTEN loss of expression predicts cetuximab efficacy in ous chemotherapy regimens (ONO-4538–12, ATTRACTION-2): a metastatic colorectal cancer patients. Br J Cancer 2007;97:1139–45. randomised, double-blind, placebo-controlled, phase 3 trial. Lancet 69. Sartore-Bianchi A, Martini M, Molinari F, Veronese S, Nichelatti M, 2017;390:2461–71. Artale S, et al. PIK3CA mutations in colorectal cancer are associated 86. Kim ST, Banks KC, Lee S-H, Kim K, Park JO, Park SH, et al. Prospec- with clinical resistance to EGFR-targeted monoclonal antibodies. tive Feasibility study for using cell-free circulating tumor DNA– Cancer Res 2009;69:1851–7. guided therapy in refractory metastatic solid cancers: an interim 70. Bardelli A, Corso S, Bertotti A, Hobor S, Valtorta E, Siravegna G, analysis. JCO Precision Oncol 2017;1:1–15. et al. Amplification of the MET receptor drives resistance to anti- 87. Liang DH, Ensor JE, Liu ZB, Patel A, Patel TA, Chang JC, et al. Cell- EGFR therapies in colorectal cancer. Cancer Discov 2013;3:658–73. free DNA as a molecular tool for monitoring disease progression 71. Loupakis F, Pollina L, Stasi I, Ruzzo A, Scartozzi M, Santini D, et al. PTEN and response to therapy in breast cancer patients. Breast Cancer Res expression and KRAS mutations on primary tumors and metastases in Treat 2016;155:139–49. the prediction of benefit from cetuximab plus irinotecan for patients 88. Catenacci DV. Expansion platform type II: testing a treatment strat- with metastatic colorectal cancer. J Clin Oncol 2009;27:2622–9. egy. Lancet Oncol 2015;16:1276–8. 72. Assersohn L, Brown G, Cunningham D, Ward C, Oates J, Waters JS, 89. Joshi SS, Maron SB, Lomnicki S, Polite BN, Sharma M, Ibe J, Allen K, et al. Phase II study of irinotecan and 5-fluorouracil/leucovorin in et al. Personalized antibodies for gastroesophageal adenocarcinoma patients with primary refractory or relapsed advanced oesophageal (PANGEA): a phase II precision medicine trial (NCT02213289). and gastric carcinoma. Ann Oncol 2004;15:64–9. J Clin Oncol 2017;36:abstr TPS198. 73. Nagatsuma AK, Aizawa M, Kuwata T, Doi T, Ohtsu A, Fujii H, et al. 90. Catenacci DV, Cervantes G, Yala S, Nelson EA, El-Hashani E, Kanteti Expression profiles of HER2, EGFR, MET and FGFR2 in a large R, et al. RON (MST1R) is a novel prognostic marker and therapeu- cohort of patients with gastric adenocarcinoma. Gastric Cancer tic target for gastroesophageal adenocarcinoma. Cancer Biol Ther 2015;18:227–38. 2011;12:9–46. 74. Terashima M, Kitada K, Ochiai A, Ichikawa W, Kurahashi I, Sakura- 91. Catenacci DV, Henderson L, Xiao SY, Patel P, Yauch RL, Hegde P, moto S, et al. Impact of expression of human epidermal growth et al. Durable complete response of metastatic gastric cancer with factor receptors EGFR and ERBB2 on survival in stage II/III gastric anti-Met therapy followed by resistance at recurrence. Cancer Discov cancer. Clin Cancer Res 2012;18:5992–6000. 2011;1:573–9. 75. Mellor JD, Brown MP, Irving HR, Zalcberg JR, Dobrovic A. A critical 92. Sellappan S, Blackler A, Liao WL, O’Day E, Xu P, Thyparambil S, review of the role of Fc gamma receptor polymorphisms in the response et al. Therapeutically induced changes in HER2, HER3, and EGFR to monoclonal antibodies in cancer. J Hematol Oncol 2013;6:1. protein expression for treatment guidance. J Natl Compr Canc Netw 76. Schneider-Merck T, Lammerts van Bueren JJ, Berger S, Rossen K, van 2016;14:503–7. Berkel PH, Derer S, et al. Human IgG2 antibodies against epidermal 93. Hembrough T, Thyparambil S, Liao WL, Darfler MM, Abdo J, growth factor receptor effectively trigger antibody-dependent cel- Bengali KM, et al. Selected reaction monitoring (SRM) analysis of lular cytotoxicity but, in contrast to IgG1, only by cells of myeloid epidermal growth factor receptor (EGFR) in formalin fixed tumor lineage. J Immunol 2010;184:512–20. tissue. Clin Proteomics 2012;9:5. 77. Srivastava RM, Lee SC, Andrade Filho PA, Lord CA, Jie HB, David- 94. Frampton GM, Fichtenholtz A, Otto GA, Wang K, Downing SR, son HC, et al. Cetuximab-activated natural killer and dendritic cells He J, et al. Development and validation of a clinical cancer genomic collaborate to trigger tumor antigen-specific T-cell immunity in profiling test based on massively parallel DNA sequencing. Nat head and neck cancer patients. Clin Cancer Res 2013;19:1858–72. Biotechnol 2013;31:1023–31. 78. Muro K, Chung HC, Shankaran V, Geva R, Catenacci D, Gupta S, 95. Rimm DL, Han G, Taube JM, Yi ES, Bridge JA, Flieder DB, et al. A et al. Pembrolizumab for patients with PD-L1-positive advanced Prospective, multi-institutional, pathologist-based assessment of 4 gastric cancer (KEYNOTE-012): a multicentre, open-label, phase 1b immunohistochemistry assays for PD-L1 expression in non-small trial. Lancet Oncol 2016;17:717–26. cell lung cancer. JAMA Oncol 2017;3:1051–8. 79. Gajewski TF, Louahed J, Brichard VG. Gene signature in melanoma 96. Tabernero J, Pfeiffer P, Cervantes A. Administration of cetuximab associated with clinical activity: a potential clue to unlock cancer every 2 weeks in the treatment of metastatic colorectal cancer: an immunotherapy. Cancer J 2010;16:399–403. effective, more convenient alternative to weekly administration? 80. Spranger S, Spaapen RM, Zha Y, Williams J, Meng Y, Ha TT, et al. Up-reg- Oncologist 2008;13:113–9. ulation of PD-L1, IDO, and T(regs) in the melanoma tumor microenvi- 97. Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, ronment is driven by CD8(+) T cells. Sci Transl Med 2013;5:200ra116. Ford R, et al. New response evaluation criteria in solid tumours: 81. Doi T, Piha-Paul SA, Jalal SI, Mai-Dang H, Saraf S, Koshiji M. revised RECIST guideline (version 1.1). Eur J Cancer 2009;45: Updated results for the advanced esophageal carcinoma cohort of 228–47. the phase Ib KEYNOTE-028 study of pembrolizumab (MK-3475). 98. Zill O, Banks K, Fairclough S, Mortimer S, Vowles J, Mokhtari R, J Clin Oncol 2016;34. et al. The landscape of actionable genomic alterations in cell-free cir- 82. Seiwert TY, Burtness B, Mehra R, Weiss J, Berger R, Eder JP, et al. culating tumor DNA from 21,807 advanced cancer patients. bioRxiv Safety and clinical activity of pembrolizumab for treatment of 2017:233205. recurrent or metastatic squamous cell carcinoma of the head and 99. Maron SB JS, Lomnicki S, Oliwa T, Landron S, Johnson J, Kiedrowski neck (KEYNOTE-012): an open-label, multicentre, phase 1b trial. LA, et al. Circulating tumorDNA (ctDNA) landscape and prognostic Lancet Oncol 2016;17:956–65. implications in gastroesophageal adenocarcinoma (GEA). J Clin 83. Fuchs CS, Doi T, Jang RW, Muro K, Satoh T, Machado M, et al. Oncol 2018;36:Abstract 45. Safety and efficacy of pembrolizumab monotherapy in patients with 100. Catenacci DVT, Nagi RJ, Braiteh FS, Kim S, Kwak EL, Maron previously treated advanced gastric and gastroesophageal junction SB, et al. Cell free circulating tumor DNA (ctDNA) landscape in cancer: phase 2 KEYNOTE-059 trial. JAMA Oncol 2018 Mar 15 patients with advanced gastroesophageal adenocarcinoma (GEC). [Epub ahead of print]. J Clin Oncol 2017;35:Abstract 47.

june 2018 CANCER DISCOVERY | 713

Targeted Therapies for Targeted Populations: Anti-EGFR Treatment for EGFR-Amplified Gastroesophageal Adenocarcinoma

Steven B. Maron, Lindsay Alpert, Heewon A. Kwak, et al.

Cancer Discov 2018;8:696-713. Published OnlineFirst February 15, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/2159-8290.CD-17-1260

Supplementary Access the most recent supplemental material at: Material http://cancerdiscovery.aacrjournals.org/content/suppl/2018/02/15/2159-8290.CD-17-1260.DC1

Cited articles This article cites 97 articles, 22 of which you can access for free at: http://cancerdiscovery.aacrjournals.org/content/8/6/696.full#ref-list-1

Citing articles This article has been cited by 16 HighWire-hosted articles. Access the articles at: http://cancerdiscovery.aacrjournals.org/content/8/6/696.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerdiscovery.aacrjournals.org/content/8/6/696. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.