HER family receptor abnormalities in lung cancer brain metastases and corresponding primary tumors

Abstract

Purpose: To compare the characteristics of deregulation of HER receptors and their ligands between primary tumor and corresponding brain metastases of non-small cell lung carcinoma (NSCLC).

Experimental design: Fifty-five NSCLC primary tumors and corresponding brain metastases specimens were examined for the immunohistochemical expression of epidermal growth factor receptor (EGFR), phosphorylated EGFR, Her2, Her3, and phosphorylated Her3, and their ligands EGF, transforming growth factor-alpha, amphiregulin, epiregulin, betacellulin, heparin-binding EGFR-like growth factor, neuregulin (NRG) 1, and NRG2. Analysis of EGFR copy number using fluorescence in situ hybridization and mutation by PCR-based sequencing was also done.

Results: Metastases showed significantly higher immunohistochemical expression of EGF (membrane: brain metastases 66.0 versus primary tumors 48.5; P = 0.027; nucleus: brain metastases 92.2 versus 67.4; P = 0.008), amphiregulin (nucleus: brain metastases 53.7 versus primary tumors 33.7; P = 0.019), phosphorylated EGFR (membrane: brain metastases 161.5 versus primary tumors 76.0; P < 0.0001; cytoplasm: brain metastases 101.5 versus primary tumors 55.9; P = 0.014), and phosphorylated Her3 (membrane: brain metastases 25.0 versus primary tumors 3.7; P = 0.001) than primary tumors did. Primary tumors showed significantly higher expression of cytoplasmic transforming growth factor-alpha(primary tumors 149.8 versus brain metastases 111.3; P = 0.008) and NRG1 (primary tumors 158.5 versus brain metastases 122.8; P = 0.006). In adenocarcinomas, a similar high frequency of EGFR copy number gain (high polysomy and amplification) was detected in primary (65%) and brain metastasis (63%) sites. However, adenocarcinoma metastases (30%) showed higher frequency of EGFR amplification than corresponding primary tumors (10%). Patients whose primary tumors showed EGFR amplification tended to develop brain metastases at an earlier time point.

Conclusions: Our findings suggest that NSCLC brain metastases have some significant differences in HER family receptor-related abnormalities from primary lung tumors.

Figure 1

Representative microphotographs of immunohistochemical expression of EGFR and p-EGFR, and the ligands amphiregulin, TFG-α, and NRG1 in primary tumors and corresponding brain metastases (magnification, ×400). All markers showed protein expression (brown staining) in tumor cells from primary and/or metastasis sites at the membrane and cytoplasm levels. Amphiregulin showed also nuclear expression in malignant cells.

Figure 2

Box plots showing scores of immunohistochemical expression of p-EGFR, TGF-α, p-Her3 and NRG1 markers comparing primary tumors with corresponding brain metastases. P values comparing primary tumors and and brain metastasis are shown for all comparisons. Bars indicate median, and x mean score.

Figure 3

Representative microphotographs of FISH showing EGFR copy number in primary tumors (PT) and corresponding brain metastases (BM) (magnification, ×1000). Red signals (red arrows) represent EGFR gene copies and green signals (white arrows) represent the chromosome 7 centromere probe. Cell nuclei stained blue with DAPI. High polysomy is defined by ≥4 copies in ≥40% of cells, and gene amplification by the presence of loose or tight EGFR gene clusters and a ratio of EGFR gene to chromosome of 2 or 15 copies of EGFR per cell in 10% of the analyzed cells.