Cetuximab (Erbitux, IMC‐C225) is a monoclonal antibody targeted to the epidermal growth factor receptor (EGFR). To clarify the mode of antitumor action of cetuximab, we examined antibody‐dependent cellular cytotoxicity (ADCC) activity against several tumor cell lines expressing wild‐type or mutant EGFR. ADCC activity and complement‐dependent cytolysis activity were analyzed using the CytoTox 96 assay. ADCC activities correlated with the EGFR expression value (R = 0.924). ADCC activities were detected against all tumor cell lines, except K562 cells in a manner dependent on the cellular EGFR expression level, whereas complement‐dependent cytolysis activity was not detected in any of the cell lines. The ADCC activity mediated by cetuximab was examined in HEK293 cells transfected with wild‐type EGFR (293W) and a deletional mutant of EGFR (293D) in comparison with the mock transfectant (293M). ADCC activity was detected in 293W and 293D cells, in a cetuximab dose‐dependent manner, but not in 293M cells (<10%). These results indicate that ADCC‐dependent antitumor activity results from the degree of affinity of cetuximab for the extracellular domain of EGFR, independent of EGFR mutation status. These results suggest ADCC activity to be one of the modes of therapeutic action of cetuximab and to depend on EGFR expression on the tumor cell surface. (Cancer Sci 2007; 98: 1275–1280)

Frequency of FGFR2 amplification, its clinicopathological features, and the results of high-throughput screening assays in a large cohort of gastric clinical samples remain largely unclear. Drug sensitivity to a fibroblast growth factor receptor (FGFR) inhibitor was evaluated in vitro. The gene amplification of the FGFRs in formalin-fixed, paraffin-embedded (FFPE) gastric cancer tissues was determined by a real-time PCR-based copy number assay and fluorescence in situ hybridisation (FISH). FGFR2 amplification confers hypersensitivity to FGFR inhibitor in gastric cancer cell lines. The copy number assay revealed that 4.1% (11 out of 267) of the gastric cancers harboured FGFR2 amplification. No amplification of the three other family members (FGFR1, 3 and 4) was detected. A FISH analysis was performed on 7 cases among 11 FGFR2-amplified cases and showed that 6 of these 7 cases were highly amplified, while the remaining 1 had a relatively low grade of amplification. Although the difference was not significant, patients with FGFR2 amplification tended to exhibit a shorter overall survival period. FGFR2 amplification was observed in 4.1% of gastric cancers and our established PCR-based copy number assay could be a powerful tool for detecting FGFR2 amplification using FFPE samples. Our results strongly encourage the development of FGFR-targeted therapy for gastric cancers with FGFR2 amplification.

The response rate to sorafenib in hepatocellular carcinoma (HCC) is relatively low (0.7%-3%), however, rapid and drastic tumor regression is occasionally observed. The molecular backgrounds and clinico-pathological features of these responders remain largely unclear. We analyzed the clinical and molecular backgrounds of 13 responders to sorafenib with significant tumor shrinkage in a retrospective study. A comparative genomic hybridization analysis using one frozen HCC sample from a responder demonstrated that the 11q13 region, a rare amplicon in HCC including the loci for FGF3 and FGF4 , was highly amplified. A real-time polymerase chain reaction–based copy number assay revealed that FGF3 / FGF4 amplification was observed in three of the 10 HCC samples from responders in which DNA was evaluable, whereas amplification was not observed in 38 patients with stable or progressive disease ( P = 0.006). Fluorescence in situ hybridization analysis confirmed FGF3 amplification. In addition, the clinico-pathological features showed that multiple lung metastases (5/13, P = 0.006) and a poorly differentiated histological type (5/13, P = 0.13) were frequently observed in responders. A growth inhibitory assay showed that only one FGF3 / FGF4 -amplified and three FGFR2 -amplified cancer cell lines exhibited hypersensitivity to sorafenib in vitro . Finally, an in vivo study revealed that treatment with a low dose of sorafenib was partially effective for stably and exogenously expressed FGF4 tumors, while being less effective in tumors expressing EGFP or FGF3 . Conclusion: FGF3 / FGF4 amplification was observed in around 2% of HCCs. Although the sample size was relatively small, FGF3/FGF4 amplification, a poorly differentiated histological type, and multiple lung metastases were frequently observed in responders to sorafenib. Our findings may provide a novel insight into the molecular background of HCC and sorafenib responders, warranting further prospective biomarker studies.

Activin A is a multi-functional cytokine belonging to the transforming growth factor-β (TGF-β) superfamily; however, the effect of activin A on angiogenesis remains largely unclear. We found that inhibin β A subunit (INHBA) mRNA is overexpressed in gastric cancer (GC) specimens and investigated the effect of activin A, a homodimer of INHBA, on angiogenesis in GC. Anti-angiogenic effects of activin A via p21 induction were evaluated using human umbilical vein endothelial cells (HUVECs) in vitro and a stable INHBA-introduced GC cell line in vivo. Compared with TGF-β, activin A potently inhibited the cellular proliferation and tube formation of HUVECs with induction of p21. A promoter assay and a chromatin immunoprecipitation assay revealed that activin A directly regulates p21 transcriptional activity through Smads. Stable p21-knockdown significantly enhanced the cellular proliferation of HUVECs. Notably, stable p21-knockdown exhibited a resistance to activin-mediated growth inhibition in HUVECs, indicating that p21 induction has a key role on activin A-mediated growth inhibition in vascular endothelial cells. Finally, a stable INHBA-introduced GC cell line exhibited a decrease in tumour growth and angiogenesis in vivo. Our findings highlight the suppressive role of activin A, unlike TGF-β, on tumour growth and angiogenesis in GC.

Non–small cell lung cancer (NSCLC) patients with an activating mutation within the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) respond dramatically to EGFR tyrosine kinase inhibitors (TKIs).¹ x 1 Mitsudomi, T, Morita, S, Yatabe, Y..., and West Japan Oncology Group. Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol. 2010; 11: 121–128 Abstract | Full Text | Full Text PDF | PubMed | Scopus (2511) | Google Scholar See all References ,² x 2 Maemondo, M, Inoue, A, Kobayashi, K..., and North-East Japan Study Group. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med. 2010; 362: 2380–2388 Crossref | PubMed | Scopus (3114) | Google Scholar See all References The common activating mutations include in-frame deletions in exon 19 (dels) and a point mutation in exon 21 (L858R), all of which lead to the sustained activity of EGFR tyrosine kinase. Unfortunately, almost all patients with these activating mutations eventually develop acquired resistance while receiving EGFR-TKI treatment.³ x 3 Rosell, R, Moran, T, Queralt, C..., and Spanish Lung Cancer Group. Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med. 2009; 361: 958–967 Crossref | PubMed | Scopus (1583) | Google Scholar See all References ,⁴ x 4 Mok, TS, Wu, YL, Thongprasert, S et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 2009; 361: 947–957 Crossref | PubMed | Scopus (5086) | Google Scholar See all References A secondary mutation in exon 20 (T790M) is observed in approximately 50% of such cases.⁵ x 5 Kobayashi, S, Boggon, TJ, Dayaram, T et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med. 2005; 352: 786–792 Crossref | PubMed | Scopus (2631) | Google Scholar See all References ,⁶ x 6 Chmielecki, J, Foo, J, Oxnard, GR et al. Optimization of dosing for EGFR-mutant non-small cell lung cancer with evolutionary cancer modeling. Sci Transl Med. 2011; 3: 90ra59 Crossref | PubMed | Scopus (309) | Google Scholar See all References The T790M mutation alters the relative affinity of the mutant receptor in favor of adenosine triphosphate (ATP), abrogating the effect of EGFR-TKIs.⁷ x 7 Yun, CH, Mengwasser, KE, Toms, AV et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci USA. 2008; 105: 2070–2075 Crossref | PubMed | Scopus (1041) | Google Scholar See all References

Purpose: EGFR-tyrosine kinase inhibitor (TKI) is a standard first-line therapy for activated EGFR-mutated non–small cell lung cancer (NSCLC). Treatment options for patients with acquired EGFR-TKI resistance are limited. HER3 mediates EGFR-TKI resistance. Clinical trials of the HER3-targeting antibody–drug conjugate patritumab deruxtecan (HER3-DXd) demonstrated its anticancer activity in EGFR-mutated NSCLC; however, the mechanisms that regulate HER3 expression are unknown. This study was conducted with the aim to clarify the mechanisms underlying HER3 regulation in EGFR-mutated NSCLC tumors and explored the strategy for enhancing the anticancer activity of HER3-DXd in EGFR-mutated NSCLC. Experimental Design: Paired tumor samples were obtained from 48 patients with EGFR-mutated NSCLC treated with EGFR-TKI(s). HER3 expression was immunohistochemically quantified with H-score, and genomic alteration and transcriptomic signature were tested in tumors from pretreatment to post-EGFR-TKI resistance acquisition. The anticancer efficacy of HER3-DXd and osimertinib was evaluated in EGFR-mutated NSCLC cells. Results: We showed augmented HER3 expression in EGFR-mutated tumors with acquired EGFR-TKI resistance compared with paired pretreatment samples. RNA sequencing revealed that repressed PI3K/AKT/mTOR signaling was associated with HER3 augmentation, especially in tumors from patients who received continuous EGFR-TKI therapy. An in vitro study also showed that EGFR-TKI increased HER3 expression, repressed AKT phosphorylation in multiple EGFR-mutated cancers, and enhanced the anticancer activity of HER3-DXd. Conclusions: Our findings help clarify the mechanisms of HER3 regulation in EGFR-mutated NSCLC tumors and highlight a rationale for combination therapy with HER3-DXd and EGFR-TKI in EGFR-mutated NSCLC.