Genomic Heterogeneity as a Barrier to Precision Medicine in Gastroesophageal Adenocarcinoma

Abstract

Gastroesophageal adenocarcinoma (GEA) is a lethal disease where targeted therapies, even when guided by genomic biomarkers, have had limited efficacy. A potential reason for the failure of such therapies is that genomic profiling results could commonly differ between the primary and metastatic tumors. To evaluate genomic heterogeneity, we sequenced paired primary GEA and synchronous metastatic lesions across multiple cohorts, finding extensive differences in genomic alterations, including discrepancies in potentially clinically relevant alterations. Multiregion sequencing showed significant discrepancy within the primary tumor (PT) and between the PT and disseminated disease, with oncogene amplification profiles commonly discordant. In addition, a pilot analysis of cell-free DNA (cfDNA) sequencing demonstrated the feasibility of detecting genomic amplifications not detected in PT sampling. Lastly, we profiled paired primary tumors, metastatic tumors, and cfDNA from patients enrolled in the personalized antibodies for GEA (PANGEA) trial of targeted therapies in GEA and found that genomic biomarkers were recurrently discrepant between the PT and untreated metastases. Divergent primary and metastatic tissue profiling led to treatment reassignment in 32% (9/28) of patients. In discordant primary and metastatic lesions, we found 87.5% concordance for targetable alterations in metastatic tissue and cfDNA, suggesting the potential for cfDNA profiling to enhance selection of therapy.Significance: We demonstrate frequent baseline heterogeneity in targetable genomic alterations in GEA, indicating that current tissue sampling practices for biomarker testing do not effectively guide precision medicine in this disease and that routine profiling of metastatic lesions and/or cfDNA should be systematically evaluated. Cancer Discov; 8(1); 37-48. ©2017 AACR.See related commentary by Sundar and Tan, p. 14See related article by Janjigian et al., p. 49This article is highlighted in the In This Issue feature, p. 1.

Figure 1. Paired whole exome sequencing of primary tumor and synchronous distant metastasis reveals discrepancies in key oncogenes between paired samples

A) Schematic depicting analyses of collected paired synchronous primary and metastatic samples in 11 gastric adenocarcinoma patients, including one patient with 2 primary and 2 metastatic samples (cohort 1). B) Comparison of the percentage of mutations (left) or amplifications (right) that were either identified in the primary only, metastasis only, or shared between both samples. Patient C1-11, which had 4 samples, was excluded from the comparison. C) Phylogenetic trees showing the genomic relationship of clones and subclones within the paired primary (green and blue) and metastatic (purple and red) samples. Trees go from germline on far left with events shared by all samples depicted on the grey line. Branches off this grey line represent events discrepant between primary and metastatic lesions. Branches noted with filled circles represent clonal mutations, present in all sampled cancer cells within a given sample, and open circles represent subclonal events, present in only a subset of sampled cancer cells. The thickness of the subclonal branches correlates with the estimated percent representation of that subclone in the sample. Key alterations and the number of shared mutations are annotated. D) Results of whole exome sequencing depicting the genomic status of key pathogenic oncogenes and tumor suppressors in paired samples with each patient represented by a column and each box a gene. The bottom triangle of the box represents the primary sample and the top triangle represents metastasis

Figure 2. Multi-region targeted sequencing of matched primary tumor sites, lymph nodes and distant metastases reveals significant heterogeneity of key clinical biomarkers

A) Schematic depicting the analyses of geographically distinct areas of primary/metastases in 26 GEA patients (cohort 2). B) Results of massively parallel sequencing of distinct tumor regions using a 243-GEA gene panel. Genes being evaluated are at the left and each patient’s samples are arrayed vertically with the type of sample (primary or metastases) marked by color. The status of each sample for each given gene is noted with colors as in the legend. Those cases where all the samples from a given patient share the same alteration are marked with a diagonal line. Proportions at right indicate the concordance by gene among patients with a genomic alteration in one of their samples for the given gene. C) Fluorescent in situ hybridization validation of heterogeneous sequencing results noting distinct gene amplification profiles of KRAS, EGFR and MET distinct tumor areas from one patient.

Figure 3. Sequencing of paired primary tumor and circulating cell-free DNA (cfDNA) reveals shared and discrepant results

A) Schematic of sampling of paired tumor and circulating plasma DNA in 11 GEA patients where the tumor was subjected to a clinical targeted sequencing panel and paired cfDNA was subjected to low pass whole genome sequencing. B) A chart representing amplifications identified in key GEA oncogenes from tissue and cfDNA sequencing is presented, where each column represents a patient and each box a gene. The bottom triangle of the box represents the primary sample and the top triangle representing the cfDNA. Amplifications are shown in red. When a sample with a low level gain in the copy-number for a given gene has a paired sample that gene amplified, the low level gain is shown in orange. C) A depiction of the copy number profile of chromosome 12 from patient C3-10 showing a high-level amplification of KRAS detected in both tissue and cfDNA. D) A depiction of the copy-number profiles on chromosome 17 in patient C3-7 showing a high level amplification of ERBB2 in the primary tumor and no copy number gain in the paired cfDNA.

Figure 4. Discrepant biomarker profiling between paired primary tumor, metastasis, and circulating free DNA in a clinical trial for untreated metastatic gastroesophageal adenocarcinoma

A) Schematic of sampling of paired primary tumor, metastasis and cfDNA in 28 patients enrolled in the PANGEA clinical trial. Treatment assignment was altered in 32% of cases, based on discrepant biomarker profiles between the primary tumor and metastasis. B) PANGEA cases where discordances between the biomarker profiles of the primary tumor and metastases led to treatment reassignment. The details of the biomarker states in the primary, metastases, cfDNA are shown. C) Patient 3 profiling showed ERRB2 and EGFR both to be amplified in distinct regions of the primary tumor biopsy. The metastases and cfDNA both showed EGFR amplification but no abnormality in ERBB2. D) Patient 5 showed no evidence for ERBB2 amplification in the primary tumor, but the metastasis and cfDNA were both ERBB2-positive. CCNE1 amplification was also detected in cfDNA, and was also present in PT and metastases (not shown). E) In Patient 7, ERRB2 amplification was detected in the primary tumor by sequencing, FISH and immunohistochemistry. Additionally, the primary tumor showed no evidence of FGFR2 overexpression or amplification (by FISH). By contrast, the metastasis and cfDNA were ERBB2-negative but positive for FGFR2 amplification, as confirmed by FISH.