Seiji Yano

Multi-Institutional Randomized Phase II Trial of Gefitinib for Previously Treated Patients With Advanced Non–Small-Cell Lung Cancer

PURPOSE To evaluate the efficacy and tolerability of two doses of gefitinib (Iressa [ZD1839]; AstraZeneca, Wilmington, DE), a novel epidermal growth factor receptor tyrosine kinase inhibitor, in patients with pretreated advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS This was a randomized, double-blind, parallel-group, multicenter phase II trial. Two hundred ten patients with advanced NSCLC who were previously treated with one or two chemotherapy regimens (at least one containing platinum) were randomly assigned to receive either 250-mg or 500-mg oral doses of gefitinib once daily. RESULTS Efficacy was similar for the 250- and 500-mg/d groups. Objective tumor response rates were 18.4% (95% confidence interval [CI], 11.5 to 27.3) and 19.0% (95% CI, 12.1 to 27.9); among evaluable patients, symptom improvement rates were 40.3% (95% CI, 28.5 to 53.0) and 37.0% (95% CI, 26.0 to 49.1); median progression-free survival times were 2.7 and 2.8 months; and median overall survival times were 7.6 and 8.0 months, respectively. Symptom improvements were recorded for 69.2% (250 mg/d) and 85.7% (500 mg/d) of patients with a tumor response. Adverse events (AEs) at both dose levels were generally mild (grade 1 or 2) and consisted mainly of skin reactions and diarrhea. Drug-related toxicities were more frequent in the higher-dose group. Withdrawal due to drug-related AEs was 1.9% and 9.4% for patients receiving gefitinib 250 and 500 mg/d, respectively. CONCLUSION Gefitinib showed clinically meaningful antitumor activity and provided symptom relief as second- and third-line treatment in these patients. At 250 mg/d, gefitinib had a favorable AE profile. Gefitinib 250 mg/d is an important, novel treatment option for patients with pretreated advanced NSCLC [corrected]

The seed and soil hypothesis: vascularisation and brain metastases

The development of a relevant mouse model for the establishment and growth of brain metastases is essential for study of the biology and therapy of brain metastasis. Injection of human tumour cells into the internal carotid artery of syngeneic or nude mice produces experimental metastases in specific regions of the brain; these are not due to patterns of initial cell arrest, motility, or invasiveness, but rather to the ability of metastatic tumour cells to grow. Whether the progressive growth of brain metastases depends on neovascularisation is not clear. Immunohistochemical and morphometric analyses show that the density of blood vessels within experimental metastases in the brains of nude mice, or within brain metastases derived from human lung cancer, is lower than in the adjacent, tumour-free brain parenchyma. However, blood vessels associated with brain metastases are dilated and contain many dividing endothelial cells. Immunohistochemical analysis also reveals that tumour cells located less than 100 microm from a blood vessel are viable, whereas more distant tumour cells undergo apoptosis. The blood-brain barrier is intact in and around experimental brain metastases smaller than 0.25 mm in diameter, but is leaky in larger metastases. Nevertheless, the lesions are resistant to chemotherapeutic drugs. The way in which the brain microenvironment influences the biological behaviour of tumour cells is a subject of intense investigation.

Crosstalk to Stromal Fibroblasts Induces Resistance of Lung Cancer to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors

Purpose: Lung cancers with epidermal growth factor receptor (EGFR)–activating mutations show good clinical response to gefitinib and erlotinib, selective tyrosine kinase inhibitors (TKI) to EGFR, but these tumors invariably develop drug resistance. Host stromal cells have been found to have a considerable effect on the behavior of cancer cells. Little is known, however, about the role of host cells on the sensitivity of cancer cells to receptor TKIs. We have therefore assessed the effect of crosstalk between stromal cells and lung cancer cells harboring EGFR mutations on susceptibility to EGFR-TKIs. Experimental Design: We evaluated the gefitinib sensitivity of lung cancer cells with EGFR-activating mutations, PC-9 and HCC827, when cocultured with fibroblasts and coinjected into severe combined immunodeficient mice. We also examined the effect of lung cancer cells to fibroblast recruitment. Results: Both human fibroblast cell lines and primary cultured fibroblasts produced various levels of hepatocyte growth factor (HGF). Lung cancer cells markedly recruited fibroblasts. The lung cancer cells became resistant to EGFR-TKIs when cocultured in vitro with HGF-producing fibroblasts and coinjected into severe combined immunodeficient mice. Importantly, combined use of gefitinib plus anti-HGF antibody or the HGF antagonist, NK4, successfully overcame the fibroblast-induced EGFR-TKI resistance both in vitro and in vivo. Colocalization of fibroblasts and HGF was detected in both xenograft tumors in mouse model and lung cancer patient specimens. Conclusions: These findings indicate that crosstalk to stromal fibroblasts plays a critical role in lung cancer resistance to EGFR-TKIs and may be an ideal therapeutic target in lung cancer with EGFR-activating mutations. (Clin Cancer Res 2009;15(21):6630–8)

Serum levels of interleukin 6 in patients with lung cancer

Serum interleukin 6 (IL-6) levels were measured in 75 patients with lung cancer and in 20 patients with benign lung diseases. IL-6 was detectable in 29 patients with lung cancer (39%), but was not detectable in any of the patients with benign lung diseases. Serum C-reactive protein levels and plasma fibrinogen levels were significantly higher and serum albumin concentration was significantly lower in lung cancer patients with detectable serum IL-6 levels than in those without detectable serum IL-6 levels and in patients with benign lung diseases. On the other hand, no significant difference was observed in blood platelet counts in these three groups. Moreover, serum IL-6 levels were not significantly different in lung cancer patients with or without clinically demonstrated distant metastasis. These results suggest that IL-6 may be a mediator of various reactions including an inflammatory response in lung cancer patients.

Production of Experimental Malignant Pleural Effusions Is Dependent on Invasion of the Pleura and Expression of Vascular Endothelial Growth Factor/Vascular Permeability Factor by Human Lung Cancer Cells

We determined the molecular mechanisms that regulate the pathogenesis of malignant pleural effusion (PE) associated with advanced stage of human, non-small-cell lung cancer. Intravenous injection of human PC14 and PC14PE6 (adenocarcinoma) or H226 (squamous cell carcinoma) cells into nude mice yielded numerous lung lesions. PC14 and PC14PE6 lung lesions invaded the pleura and produced PE containing a high level of vascular endothelial growth factor (VEGF)-localized vascular hyperpermeability. Lung lesions produced by H226 cells were confined to the lung parenchyma with no PE. The level of expression of VEGF mRNA and protein by the cell lines directly correlated with extent of PE formation. Transfection of PC14PE6 cells with antisense VEGF165 gene did not inhibit invasion into the pleural space but reduced PE formation. H226 cells transfected with either sense VEGF 165 or sense VEGF 121 genes induced localized vascular hyperpermeability and produced PE only after direct implantation into the thoracic cavity. The production of PE was thus associated with the ability of tumor cells to invade the pleura, a property associated with expression of high levels of urokinase-type plasminogen activator and low levels of TIMP-2. Collectively, the data demonstrate that the production of malignant PE requires tumor cells to invade the pleura and express high levels of VEGF/VPF.

Colon Cancer Cell-Derived High Mobility Group 1/Amphoterin Induces Growth Inhibition and Apoptosis in Macrophages

High mobility group (HMGB)1/amphoterin is a multifunctional cytokine involved in invasion and metastasis of cancer and in inflammation. To investigate HMGB1/amphoterin effects on macrophages, U937 human monocytic leukemia cells and rat peritoneal and human alveolar macrophages were examined. U937 cells expressed low levels of an HMGB1/amphoterin receptor, receptor for advanced glycation end-products (RAGE), whereas RAGE production was induced in differentiated phorbol 12-myristate 13-acetate (PMA)-U937 cells. Treatment with cultured medium of HMGB1/amphoterin-secreting WiDr human colon cancer cells showed growth inhibition of both U937 and PMA-U937 cells and apoptosis in PMA-U937 cells. The number of PMA-U937 cells was markedly decreased by co-culture with WiDr cells exposed to HMGB1/amphoterin sense S-oligodeoxynucleotide (ODN) in spheroids or monolayers. In contrast, PMA-U937 cells co-cultured with WiDr cells exposed to HMGB1/amphoterin anti-sense S-ODN were preserved in number. PMA-U937 cells exposed to RAGE anti-sense S-ODN were insensitive to WiDr-cultured medium. Recombinant human HMGB1/amphoterin induced growth inhibition in thioglycollate-induced rat peritoneal macrophages, PMA-U937 cells, and human alveolar macrophages, an effect that was abrogated by absorption with anti-HMGB1 antibody. Phosphorylation of JNK and Rac1 was induced in PMA-U937 cells treated with HMGB1/amphoterin. These results suggest that HMGB1/amphoterin induces growth inhibition and apoptosis in macrophages through RAGE intracellular signaling pathway.

Interleukin-10 is a potent inhibitor of tumor cytotoxicity by human monocytes and alveolar macrophages

The effects of purified human interleukin‐10 (IL‐10) on the expression of antitumor activity of human monocytes and alveolar macrophages (AMs) obtained by centrifugal elutriation and bronchoalveolar lavage, respectively, from the same healthy donors were examined. Monocytes and AMs were incubated for 16 h in medium with lipopolysaccharide (LPS) in the presence or absence of IL‐10 or IL‐4, and then their tumoricidal activity was assayed by measuring 125I‐IUdR release from human melanoma (A375) cells. Addition of IL‐10 to cultures of monocytes or AMs with LPS resulted in dose‐dependent suppression of their cytotoxicity against A375 cells, the suppression of the activity of monocytes being the higher. IL‐10 also suppressed the synergistic effects of interferon‐γ and desmethyl muramyldipeptide in activation of monocytes. IL‐10 inhibited the early induction phase of monocyte activation but not the effector phase (monocyte‐mediated cytotoxicity). IL‐10 plus IL‐4 inhibited the antitumor activities of AMs and monocytes much more than either IL‐10 or IL‐4 alone. IL‐10 and IL‐4 at sub‐optimal concentrations also showed synergistic inhibitory effects. These findings suggest that IL‐10 may be important in vivo in down‐regulating the antitumor activities of monocytes and AMs in the lung by inhibiting their productions of antitumor effector molecules. J. Leukoc. Biol. 55: 437–442; 1994.

Novel metastasis model of human lung cancer in SCID mice depleted of NK cells.

Metastasis is a critical problem in the treatment of human lung cancer. Thus, a suitable animal model of metastasis of human lung cancer is required for in vivo biological and preclinical studies. In this study, we tried to establish a suitable model for this, using SCID mice. Neither human SCLC H69/VP cells (5 × 106) nor squamous‐cell carcinoma RERF‐LC‐AI cells (1 × 106), injected through a tail vein, formed metastases in untreated SCID mice. Pre‐treatment of SCID mice with anti‐asialo GM1 serum resulted in only a few metastases of H69/VP cells, but pre‐treatment with anti‐mouse IL‐2 receptor β chain Ab (TM‐β1) resulted in numerous lymph‐node metastases 56 days after tumor inoculation. H69/VP‐M cells, an in vivo‐selected variant line, formed significant numbers of lymph‐node metastases even in SCID mice pre‐treated with anti‐asialo GM1 serum. SCID mice depleted of NK cells by treatment with TM‐β1 showed different patterns of metastasis when inoculated intravenously with the 2 different human lung cancer cell lines (H69/VP and RERF‐LC‐AI cells): H69/VP cells formed metastases mainly in systemic lymph nodes and the liver, whereas RERF‐LC‐AI cells formed metastases mainly in the liver and kidneys, with only a few in lymph nodes. A histopathological study showed that the metastatic colonies consisted of cancer cells. The numbers of metastatic colonies formed by the 2 cell lines increased with the number of cells inoculated. TM‐β1 treatment of SCID mice efficiently removed NK cells from peripheral blood for at least 6 weeks, whereas, after treatment of the mice with anti‐asialo GM1 serum, NK cells were recovered within 9 days. These findings suggest that NK‐cell‐depleted SCID mice may be useful as a model in biological and pre‐clinical studies on metastasis of human lung cancer.

Phosphorylation, but not overexpression, of epidermal growth factor receptor is associated with poor prognosis of non-small cell lung cancer patients.

Epidermal growth factor receptor (EGFR) is commonly overexpressed in non-small cell lung cancer (NSCLC) and its tyrosine kinase phosphorylation is thought to be an ideal target in the treatment of patients with NSCLC. In the present study, we examined surgically obtained specimens from a series of 36 NSCLC patients for expression of EGFR, phosphorylated EGFR (p-EGFR), and HER2 by immunohistochemistry, and also examined the correlation with clinical characteristics. The positive rate of EGFR, p-EGFR, and HER2 was 97.2%, 44.4%, and 88.6%, respectively, and the overexpression rate was 80.6%, 0.0%, and 27.8%, respectively. EGFR overexpression and phosphorylation were seen at almost the same rate in each histological type of squamous and nonsquamous cell carcinoma (squamous vs. nonsquamous; 78.6% vs. 81.8% for EGFR, 35.7% vs. 50.0% for p-EGFR), while HER2 overexpression was seen less frequently in squamous cell carcinoma than in nonsquamous cell carcinoma (0.0% vs. 45.5%, P = 0.003). Univariate analysis revealed that EGFR overexpression was related to good performance status (P = 0.038) but not related to EGFR phosphorylation. EGFR phosphorylation was correlated to short time to progression (TTP) (P = 0.002) and poor prognosis (P = 0.002), although EGFR overexpression, HER2 overexpression, or EGFR-HER2 coexpression were not correlated to TTP or survival. Bivariate analysis showed EGFR phosphorylation was related to short TTP and poor prognosis both in early and advanced stages. Multivariate analyses confirmed that clinical stage, performance status, and p-EGFR expression were independently associated with increasing risk of short TTP and poor prognosis. These results suggest that phosphorylation, but not overexpression, of EGFR may be an important predictor for clinical outcome of NSCLCs.

Combined Therapy with Mutant-Selective EGFR Inhibitor and Met Kinase Inhibitor for Overcoming Erlotinib Resistance in EGFR-Mutant Lung Cancer

Although the EGF receptor tyrosine kinase inhibitors (EGFR-TKI) erlotinib and gefitinib have shown dramatic effects against EGFR mutant lung cancer, patients become resistant by various mechanisms, including gatekeeper EGFR-T790M mutation, Met amplification, and HGF overexpression, thereafter relapsing. Thus, it is urgent to develop novel agents to overcome EGFR-TKI resistance. We have tested the effects of the mutant-selective EGFR-TKI WZ4002 and the mutant-selective Met-TKI E7050 on 3 EGFR mutant lung cancer cell lines resistant to erlotinib by different mechanisms: PC-9/HGF cells with an exon 19 deletion, H1975 with an L858R mutation, and HCC827ER with an exon 19 deletion, with acquired resistance to erlotinib because of HGF gene transfection, gatekeeper T790M mutation, and Met amplification, respectively. WZ4002 inhibited the growth of H1975 cells with a gatekeeper T790M mutation, but did not inhibit the growth of HCC827ER and PC-9/HGF cells. HGF triggered the resistance of H1975 cells to WZ4002, whereas E7050 sensitized HCC827ER, PC-9/HGF, and HGF-treated H1975 cells to WZ4002, inhibiting EGFR and Met phosphorylation and their downstream molecules. Combined treatment potently inhibited the growth of tumors induced in severe-combined immunodeficient mice by H1975, HCC827ER, and PC-9/HGF cells, without any marked adverse events. These therapeutic effects were associated with the inhibition of EGFR and Met phosphorylation in vivo. The combination of a mutant-selective EGFR-TKI and a Met-TKI was effective in suppressing the growth of erlotinib-resistant tumors caused by gatekeeper T790M mutation, Met amplification, and HGF overexpression. Further evaluations in clinical trials are warranted. Mol Cancer Ther; 11(10); 2149–57. ©2012 AACR.