Heterogeneity of amplification of HER2, EGFR, CCND1 and MYC in gastric cancer

Abstract

Background: Intra-tumor heterogeneity is a potential cause for failure of targeted therapy in gastric cancer, but the extent of heterogeneity of established (HER2) or potential (EGFR, CCND1) target genes and prognostic gene alterations (MYC) had not been systematically studied.

Methods: To study heterogeneity of these genes in a large patient cohort, a heterogeneity tissue microarray was constructed containing 0.6 mm tissue cores from 9 different areas of the primary gastric cancers of 113 patients and matched lymph node metastases from 61 of these patients. Dual color fluorescence in-situ hybridization was performed to assess amplification of HER2, EGFR, CCND1 and MYC using established thresholds (ratio ≥ 2.0). Her2 immunohistochemistry (IHC) was performed in addition.

Results: Amplification was found in 17.4% of 109 interpretable cases for HER2, 6.4% for EGFR, 17.4% for CCND1, and 24.8% for MYC. HER2 amplification was strongly linked to protein overexpression by IHC in a spot-by-spot analysis (p < 0.0001). Intra-tumor heterogeneity was found in the primary tumors of 9 of 19 (47.3%) cancers with HER2, 8 of 17 (47.0%) cancers with CCND1, 5 of 7 (71.4%) cancers with EGFR, and 23 of 27 (85.2%) cancers with MYC amplification. Amplification heterogeneity was particularly frequent in case of low-level amplification (<10 gene copies). While the amplification status was often different between metastases, unequivocal intra-tumor heterogeneity was not found in individual metastases.

Conclusion: The data of our study demonstrate that heterogeneity is common for biomarkers in gastric cancer. Given that both TMA tissue cores and clinical tumor biopsies analyze only a small fraction of the tumor bulk, it can be concluded that such heterogeneity may potentially limit treatment decisions based on the analysis of a single clinical cancer biopsy.

Figure 1

Representative images of FISH- and IHC findings in gastric cancer. a-h) Examples of amplified and non-amplified cancers: a, b) HER2, c, d) EGFR, e, f) CCND1 and g, h) MYC. i-l): Examples of HER2-immunohistochemistry negative (score 0–1, i, j) and positive (score 2–3, k, l) cancers.

Figure 2

HER2 FISH and IHC findings in primary gastric cancers and their metastases in 19 cancers with HER2 amplification.^a“Tumor” indicates the cases number referred to in the text. ^bThe FISH and IHC results of individual tissue spots are indicated by integers reflecting the copy numbers of centromere 17/HER2 (i.e. 2/20, black face) and the HercepTest IHC score (i.e. 3, white face) in the primary and metastases spots. Green color in the primary/metastases spots area indicates high-level HER2 amplification or positive IHC findings, orange color indicates low-level amplification, and blue color indicates negative IHC findings. ^cBold black outline in the “metastases spots” highlights spots that were derived from the same metastasis. ^d“Heterogeneity” indicates whether FISH or IHC findings were homogeneous (Homo.) or heterogeneous (Hetero). ^e“Validation” shown the large section FISH results in 3 cases obtained from the primary cancer (case #11 and #19) and from the amplified metastasis (#12).

Figure 3

EGFR FISH findings in primary gastric cancers and their metastases in 7 cancers with EGFR amplification.^a“Tumor” indicates the cases number referred to in the text. ^bThe FISH results of individual tissue spots are indicated by integers reflecting the copy numbers of centromere 7/EGFR (i.e. 2/20, black face) in the primary and metastases spots. Green color in the primary/metastases spots area indicates high-level EGFR amplification, and blue color indicates negative IHC findings. ^cBold black outline in the “metastases spots” highlights spots that were derived from the same metastasis.

Figure 4

CCND1 FISH findings in primary gastric cancers and their metastases in 19 cancers with CCND1 amplification.^a“Tumor” indicates the cases number referred to in the text. ^bThe FISH results of individual tissue spots are indicated by integers reflecting the copy numbers of centromere 11/CCND1 (i.e. 2/20, black face) in the primary and metastases spots. Green color in the primary/metastases spots area indicates high-level CCND1 amplification, and blue color indicates negative IHC findings. ^cBold black outline in the “metastases spots” highlights spots that were derived from the same metastasis.

Figure 5

MYC FISH findings in primary gastric cancers and their metastases in 27 cancers with MYC amplification.^a“Tumor” indicates the cases number referred to in the text. ^bThe FISH results of individual tissue spots are indicated by integers reflecting the copy numbers of centromere 8/MYC (i.e. 2/20, black face) in the primary and metastases spots. Green color in the primary/metastases spots area indicates high-level MYC amplification, and blue color indicates negative IHC findings. ^cBold black outline in the “metastases spots” highlights spots that were derived from the same metastasis.

Figure 6

Comparison of the FISH finding in 8 tumors showing high-level co-amplification of at least 2 of the 4 analyzed genes.^a“Tumor” indicates the cases number referred to in the text. ^bGreen color in the primary/metastases spots area indicates high-level amplification, orange color indicates low-level amplification, and blue color indicates negative findings. ^cBold black outline in the “metastases spots” highlights spots that were derived from the same metastasis.