Targeting EGFR and HER-2 with cetuximab- and trastuzumab-mediated immunotherapy in oesophageal squamous cell carcinoma

Abstract

We previously reported that oesophageal squamous cell carcinoma (SCC) had a relatively high incidence of EGFR and HER-2 overexpression. Thus, anti-HER family targeting may become a promising approach to treat oesophageal SCC. In the present study, we investigated (a) the distribution of EGFR and HER-2 expression in oesophageal SCC (n=66) detected by immunohistochemistry and (b) cetuximab- and/or trastuzumab-mediated biological activity (antiproliferative effect by the MTT assay, apoptosis-inducing activity by the annexin V/propidium iodide assay, and antibody-dependent cellular cytotoxicity (ADCC) by the (51)Cr-release assay) against oesophageal SCC cell lines with various levels of EGFR and HER-2. Twelve of the 66 patients (18%) showed both EGFR- and HER-2 expression. Out of both EGFR- and HER-2-positive cases, nine cases (75%) showed EGFR and HER-2 expression in individually distinct regions. Furthermore, the combination of cetuximab and trastuzumab could induce synergistic antiproliferative effects and additional ADCC activities against not all, but several oesophageal SCC cell lines with EGFR and HER-2 expression. The combination of cetuximab and trastuzumab may be useful in the treatment of oesophageal SCC with EGFR and HER-2 expression.

Figure 1

Immunohistochemical staining of EGFR and HER-2 in oesophageal SCC. In oesophageal SCC, EGFR and HER-2 expressions were evaluated by IHC in the serial sections. Representative stainings for EGFR (A) and HER-2 (B) are shown, and both expressions were seen in the same regions of the oesophageal SCC tumour ( × 100).

Figure 2

Immunohistochemical staining of EGFR and HER-2 in oesophageal SCC. In oesophageal SCC, EGFR and HER-2 expressions were evaluated by IHC in the serial sections ( × 100). The region with EGFR-positive expression (A) was negative for HER-2 (B) in the same tumour. On the contrary, the region with HER-2-positive expression (D) was negative for EGFR (C) in the same tumour.

Figure 3

Survival curves of the patients with oesophageal SCC in relation to EGFR and HER-2 expression. Actuarial overall survival rates were analysed by the Kaplan–Meier method, and survival was measured in months from operation to death or the last review.

Figure 4

The degree of EGFR and HER-2 expression on oesophageal SCC cell lines and freshly isolated SCC tumours. EGFR and HER-2 expression was evaluated by flow cytometric analysis on oesophageal SCC cell lines and freshly isolated SCC tumours derived from two different patients.

Figure 5

Synergistic antiproliferative effects of cetuximab and trastuzumab for oesophageal SCC cell lines. The oesophageal SCC lines, TE3 (HER-2 low and EGFR high) and TE5 (HER-2 moderate and EGFR moderate), were analysed by the MTT assay in various dose combinations of cetuximab and trastuzumab. The inhibition of proliferation was shown as % cell growth inhibition induced by cetuximab and/or trastuzumab in comparison with that induced by control mAb.

Figure 6

Cetuximab- and/or trastuzumab-mediated ADCC for oesophageal SCC cell lines. Oesophageal SCC cell lines with various levels of EGFR and HER-2 expression (TE3, KYSE50, and TE4) were analysed for ADCC derived from healthy donor’s PBMC in various dose combinations of cetuximab, trastuzumab, and control mAb by the 6-h ⁵¹Cr release assay. E : T, effector/target ratios.

Figure 7

Cetuximab- and/or trastuzumab-mediated ADCC for oesophageal SCC in healthy donors and oesophageal SCC patients. Oesophageal SCC cell lines with various levels of EGFR and HER-2 expression were analysed for ADCC by healthy donors’ PBMC and patients’ PBMC in the presence of cetuximab (0.5 μg ml⁻¹) and/or trastuzumab (10 μg ml⁻¹) or control mAb (1 μg ml⁻¹) by the 6-h ⁵¹Cr release assay. Summarised data from healthy donors’ PBMC (n=5) and oesophageal cancer patients’ PBMC (n=5) are shown. E : T, effector/target ratios.