EGFR and MET Amplifications Determine Response to HER2 Inhibition in ERBB2-Amplified Esophagogastric Cancer

Abstract

The anti-HER2 antibody trastuzumab is standard care for advanced esophagogastric (EG) cancer with ERBB2 (HER2) amplification or overexpression, but intrinsic and acquired resistance are common. We conducted a phase II study of afatinib, an irreversible pan-HER kinase inhibitor, in trastuzumab-resistant EG cancer. We analyzed pretreatment tumor biopsies and, in select cases, performed comprehensive characterization of postmortem metastatic specimens following acquisition of drug resistance. Afatinib response was associated with coamplification of EGFR and ERBB2. Heterogeneous 89Zr-trastuzumab PET uptake was associated with genomic heterogeneity and mixed clinical response to afatinib. Resistance to afatinib was associated with selection for tumor cells lacking EGFR amplification or with acquisition of MET amplification, which could be detected in plasma cell-free DNA. The combination of afatinib and a MET inhibitor induced complete tumor regression in ERBB2 and MET coamplified patient-derived xenograft models established from a metastatic lesion progressing on afatinib. Collectively, differential intrapatient and interpatient expression of HER2, EGFR, and MET may determine clinical response to HER kinase inhibitors in ERBB2-amplified EG cancer. SIGNIFICANCE: Analysis of patients with ERBB2-amplified, trastuzumab-resistant EG cancer who were treated with the HER kinase inhibitor afatinib revealed that sensitivity and resistance to therapy were associated with EGFR/ERBB2 coamplification and MET amplification, respectively. HER2-directed PET imaging and cell-free DNA sequencing could help guide strategies to overcome the emergence of resistant clones.See related commentary by Klempner and Catenacci, p. 166.This article is highlighted in the In This Issue feature, p. 151.

Trial registration: ClinicalTrials.gov NCT01522768.

©2018 American Association for Cancer Research.

Figures

Figure 1.. Individual treatment outcomes of 20 patients treated with afatinib and 12 patients treated with afatinib and trastuzumab.

A, Percentage best change from baseline in the target lesion assessed by RECIST 1.1. Relevant clinical features (time on therapy, type of sequenced sample, and collection time point of the sequenced sample) plus key genomic alterations in sequenced samples are shown for each patient. SLD, sum of longest diameter on CT scan. Pt1 to Pt32 are IDs for all the patients in the study. For patients with multiple sequenced samples, the sample included in the oncoprint is designed after a decimal. *Non-evaluable. **Sample uninformative due to low tumor content. B, dual probe EGFR and ERBB2 FISH from Patients 9, 30 and 32, all of which were collected prior to protocol treatment, demonstrated diffuse and uniform ERBB2 and EGFR co-amplification in virtually all tumor cells. There was no additional tissue for dual probe FISH for Patient 31. C, Percent change in 89Zr-trastuzumab PET standard uptake value (SUV) pre- vs post-therapy for each individual lesion on CT in 1 patient treated with afatinib monotherapy and 7 patients treated with afatinib/trastuzumab. White stars inside the bar plots denote lesions with resolution of uptake to baseline SUV.

Figure 2.. Intrapatient disease heterogeneity and selection for a non-EGFR amplified tumor clone as mechanisms of afatinib resistance in Patient 30.

A, Radiographic tumor assessment using 89Zr-trastuzumab PET and conventional CT scan, with corresponding bar graphs. 89Zr-trastuzumab PET values in SUV and CT measurements in mm. 89Zr-trastuzumab PET images include three panels: left panel (pre-afatinib) is a baseline pretreatment image showing uptake in segment 2/3 liver metastasis (SUV 16.4), GE junction (SUV 21.5) and retroesophageal lymph node (SUV 9.1); middle panel (on afatinib) corresponds to 3 weeks post-treatment initiation, showing resolution of retroesophageal lymph node (SUV 3.0) with decrease in size but persistent high uptake in segment 2/3 liver lesion (SUV 15.8), similar uptake in GE junction (SUV 23.7) and a new lesion in liver segment 7/8 which appeared on 89Zr-trastuzumab PET (SUV 8.6); right panel (progression) shows post-progression 89Zr-trastuzumab PET uptake in GE junction (SUV 28.5) and segment 7/8 liver lesion (SUV 9.0), decreased uptake in liver segment 2/3 (SUV 12.3) and ongoing resolution of retroesophageal lymph node. B, Genomic comparison of matched pre- and post-treatment primary and liver segment 2/3 and post-treatment progression liver segment 7/8 obtained at warm autopsy. EGFR amplification was unique to the segment 2/3 liver lesion with ongoing response. The enlarging GE junction tumor and segment 7/8 liver metastasis were not EGFR-amplified. In addition to a likely pathogenic mutation in PTEN (P95R, also present in a segment 2/3 liver lesion), the non-responding metastases acquired an amplification of CCND3. SUV, standardized uptake value on 89Zr-trastuzumab PET; SLD, sum of longest diameter on CT scan. C, Phylogenetic tree showing the inferred patterns of clonal evolution for the samples described in B.

Figure 3.. Acquired MET amplification as a mechanism of afatinib resistance in Patient 31 with EGFR- and ERBB2-amplified EG cancer.

A, Genomic comparison of matched pretreatment biopsy with post-treatment metastases obtained at warm autopsy demonstrating acquired MET amplification. B, SRM-MS analysis of liver metastasis sample obtained on afatinib with post-progression autopsy sample revealing increased MET and HER2 as well as decreased EGFR expression. PCTL, percentile. C, Efficacy of afatinib and afatinib/AMG 337 in a MET/EGFR/ERBB2-amplified PDX model established from Patient 31. The combination of afatinib/AMG 337 resulted in complete tumor response at 21 days in MET/EGFR/ERBB2-amplified PDX. Red arrow indicates when the mice were rechallenged with the afatinib/AMG 337 combination following discontinuation of drug treatment, with durable tumor control again achieved.

Figure 4.. Non-invasive detection of acquired MET amplification as a mechanism of afatinib/trastuzumab resistance using cfDNA.

A, Radiographic tumor assessment using 89Zr-trastuzumab PET and conventional CT scan, with corresponding bar graphs in Patient 28. 89Zr-trastuzumab PET values in SUV and CT measurements in mm. Left panel is a baseline pretreatment 89Zr-trastuzumab PET image showing uptake in a left ovary metastasis (SUV 5.1) not visible in this projection, perigastric peritoneal metastasis (SUV 7.2, lower arrow) and gastric mass (SUV 8.2, upper arrow). Right panel is 89Zr-trastuzumab PET 5 weeks post-treatment initiation, showing resolution of uptake in the perigastric peritoneal metastasis (SUV 1.8) and gastric mass (SUV 3.9) and persistently high uptake in the left ovarian metastasis (SUV 5.2) not seen on MIP images. B, cfDNA analysis at the time of disease progression on afatinib/trastuzumab, demonstrating ERBB2 and MET amplification (results for additional time points are provided in Supplementary Table S9). C, Genomic comparison of matched pretreatment biopsy with post-treatment tumor tissue collected from the left ovarian metastasis and subsequently at rapid autopsy, demonstrating acquired MET amplification unique to the progressing lesions. The perigastric metastasis and the primary tumor, both of which demonstrated ongoing response to afatinib/trastuzumab, did not harbor MET amplification. D, dual probe EGFR and ERBB2 FISH from a perirectal tumor biopsy collected after treatment demonstrated ERBB2 amplification and low-level gain of EGFR in a subset of tumor cells.