Tatsu Shimoyama

Antibody-dependent cellular cytotoxicity of cetuximab against tumor cells with wild-type or mutant epidermal growth factor receptor

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)

A Phase I Dose-Escalation Study of ZD6474 in Japanese Patients with Solid, Malignant Tumors

Introduction: ZD6474 (vandetanib) is an orally available inhibitor of vascular endothelial growth factor receptor, epidermal growth factor receptor, and RET receptor tyrosine kinase activity. This study assessed the safety and tolerability of escalating doses of ZD6474 in Japanese patients with solid, malignant tumors. Methods: Adult patients with solid tumors refractory to standard therapy received a once-daily oral dose of ZD6474 (100–400 mg) in 28-day cycles, until disease progression or unacceptable toxicity was observed. Results: Eighteen patients were treated at doses of 100 mg (n = 3), 200 mg (n = 6), 300 mg (n = 6), and 400 mg (n = 3). Dose-limiting toxicities at the completion of cycle 2 were hypertension (n = 3), diarrhea (n = 1), headache (n = 1), toxic skin eruption (n = 1), and alanine aminotransferase increase (n = 1). A dose of 400 mg/day was considered to exceed the maximum tolerated dose (MTD). Toxicities were manageable with dose interruption and/or reduction. Objective tumor response was observed in four of nine patients with non-small cell lung cancer (NSCLC) at doses of either 200 or 300 mg. Terminal half-life was about 90–115 hours. Plasma trough concentrations achieved steady-state conditions after approximately 1 month of daily dosing. Conclusions: It was concluded that a dose of 400 mg/day was considered to exceed the MTD, and doses for phase II study were thought to be not more than 300 mg/day. The objective response observed in some NSCLC patients is encouraging for further studies in this tumor type.

Establishment of a human non‐small cell lung cancer cell line resistant to gefitinib

The epidermal growth factor receptor (EGFR) tyrosine‐kinase inhibitor gefitinib (Iressa®, ZD1839) has shown promising activity preclinically and clinically. Because comparative investigations of drug‐resistant sublines with their parental cells are useful approaches to identifying the mechanism of gefitinib resistance and select factors that determine sensitivity to gefitinib, we established a human non‐small cell lung carcinoma subline (PC‐9/ZD) that is resistant to gefitinib. PC‐9/ZD cells are ∼180‐fold more resistant to gefitinib than their parental PC‐9 cells and PC‐9/ZD cells do not exhibit cross‐resistance to conventional anticancer agents or other tyrosine kinase inhibitors, except AG‐1478, a specific inhibitor of EGFR. PC‐9/ZD cells also display significant resistance to gefitinib in a tumor‐bearing animal model. To elucidate the mechanism of resistance, we characterized PC‐9/ZD cells. The basal level of EGFR in PC‐9 and PC‐9/ZD cells was comparable. A deletion mutation was identified within the kinase domain of EGFR in both PC‐9 and PC‐9/ZD, but no difference in the sequence of EGFR cDNA was detected in either cell line. Increased EGFR/HER2 (and EGFR/HER3) heterodimer formations were demonstrated in PC‐9/ZD cells by chemical cross‐linking and immunoprecipitation analysis in cells unexposed to gefitinib. Exposure to gefitinib increased heterodimer formation in PC‐9 cells, but not in PC‐9/ZD cells. Gefitinib inhibits EGFR autophosphorylation in a dose‐dependent manner in PC‐9 cells but not in PC‐9/ZD cells. A marked difference in inhibition of site‐specific phosphorylation of EGFR was observed at Tyr1068 compared to other tyrosine residues (Tyr845, 992 and 1045). To elucidate the downstream signaling in the PC9/ZD cellular machinery, complex formation between EGFR and its adaptor proteins GRB2, SOS, and Shc was examined. A marked reduction in the GRB2‐EGFR complex and absence of SOS‐EGFR were observed in PC‐9/ZD cells, even though the protein levels of GRB2 and SOS in PC‐9 and PC‐9/ZD cells were comparable. Expression of phosphorylated AKT was increased in PC‐9 cells and inhibited by 0.02 μM gefitinib. But the inhibition was not significant in PC‐9/ZD cells. These results suggest that alterations of adaptor‐protein‐mediated signal transduction from EGFR to AKT is a possible mechanism of the resistance to gefitinib in PC‐9/ZD cells. These phenotypes including EGFR–SOS complex and heterodimer formation of HER family members are potential biomarkers for predicting resistance to gefitinib.

Gene expression profiling of ATP-binding cassette (ABC) transporters as a predictor of the pathologic response to neoadjuvant chemotherapy in breast cancer patients.

SummaryDrug resistance is a major obstacle to the successful chemotherapy. Several ATP-binding cassette (ABC) transporters including ABCB1, ABCC1 and ABCG2 have been known to be important mediators of chemoresistance. Using oligonucleotide microarrays (HG-U133 Plus 2.0; Affymetrix), we analyzed the ABC transporter gene expression profiles in breast cancer patients who underwent sequential weekly paclitaxel/FEC (5-fluorouracil, epirubicin and cyclophosphamide) neoadjuvant chemotherapy. We compared the ABC transporter expression profile between two classes of pretreatment tumor samples divided by the patients’ pathological response to neoadjuvant chemotherapy (residual disease [RD] versus pathologic complete response [pCR]) ABCB3, ABCC7 and ABCF2 showed significantly high expression in the pCR. Several ABC transporters including ABCC5, ABCA12, ABCA1 ABCC13, ABCB6 and ABCC11 showed significantly increased expression in the RD (p<0.05). We evaluated the feasibility of developing a multigene predictor model of pathologic response to neoadjuvant chemotherapy using gene expression profiles of ABC transporters. The prediction error was evaluated by leave-one-out cross-validation (LOOCV). A multigene predictor model with the ABC transporters differentially expressed between the two classes (p≤0.003) showed an average 92.8% of predictive accuracy (95% CI, 88.0–97.4%) with a 93.2% (95% CI, 85.2–100%) positive predictive value for pCR, a 93.6% (95% CI, 87.8–99.4%) negative predictive value, a sensitivity of 88.1%(95% CI, 76.8–99.4%), and a specificity of 95.9% (91.1% CI, 87.8–100%). Our results suggest that several ABC transporters in human breast cancer cells may affect the clinical response to neoadjuvant chemotherapy, and transcriptional profiling of these genes may be useful to predict the pathologic response to sequential weekly paclitaxel/FEC in breast cancer patients.

Akt-dependent nuclear localization of Y-box-binding protein 1 in acquisition of malignant characteristics by human ovarian cancer cells

Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.

Randomized Pharmacokinetic and Pharmacodynamic Study of Docetaxel: Dosing Based on Body-Surface Area Compared With Individualized Dosing Based on Cytochrome P450 Activity Estimated Using a Urinary Metabolite of Exogenous Cortisol

PURPOSE Docetaxel is metabolized by cytochrome P450 (CYP3A4) enzyme, and the area under the concentration-time curve (AUC) is correlated with neutropenia. We developed a novel method for estimating the interpatient variability of CYP3A4 activity by the urinary metabolite of exogenous cortisol (6-beta-hydroxycortisol [6-beta-OHF]). This study was designed to assess whether the application of our method to individualized dosing could decrease pharmacokinetic (PK) and pharmacodynamic (PD) variability compared with body-surface area (BSA) -based dosing. PATIENTS AND METHODS Fifty-nine patients with advanced non-small-cell lung cancer were randomly assigned to either the BSA-based arm or individualized arm. In the BSA-based arm, 60 mg/m(2) of docetaxel was administered. In the individualized arm, individualized doses of docetaxel were calculated from the estimated clearance (estimated clearance = 31.177 + [7.655 x 10(-4) x total 6-beta-OHF] - [4.02 x alpha-1 acid glycoprotein] - [0.172 x AST] - [0.125 x age]) and the target AUC of 2.66 mg/L . h. RESULTS In the individualized arm, individualized doses of docetaxel ranged from 37.4 to 76.4 mg/m(2) (mean, 58.1 mg/m(2)). The mean AUC and standard deviation (SD) were 2.71 (range, 2.02 to 3.40 mg/L . h) and 0.40 mg/L . h in the BSA-based arm, and 2.64 (range, 2.15 to 3.07 mg/L . h) and 0.22 mg/L . h in the individualized arm, respectively. The SD of the AUC was significantly smaller in the individualized arm than in the BSA-based arm (P < .01). The percentage decrease in absolute neutrophil count (ANC) averaged 87.1% (range, 59.0 to 97.7%; SD, 8.7) in the BSA-based arm, and 87.4% (range, 78.0 to 97.2%; SD, 6.1) in the individualized arm, suggesting that the interpatient variability in percent decrease in ANC was slightly smaller in the individualized arm. CONCLUSION The individualized dosing method based on the total amount of urinary 6-beta-OHF after cortisol administration can decrease PK variability of docetaxel.

Dimerization and the signal transduction pathway of a small in-frame deletion in the epidermal growth factor receptor

A short, in‐frame deletional mutant (E746‐A750del) is one of the major mutant forms of epidermal growth factor receptor (EGFR) and has been reported to be a determinant of response to EGFR tyrosine kinase inhibitors such as gefitinib and erlotinib. However, the biological and pharmacological functions of mutational EGFR remain unclear. To clarify these biological functions of deletional EGFR, we examined the cellular response to EGF ligand stimulation. Dimerization and phosphorylation of EGFR were observed without any ligand stimulation in the 293(D) cells transfected with deletional EGFR as compared with those transfected with wild‐type EGFR (293(W) cells). When the 293(D) cells were exposed to gefitinib, an immunoblotting analysis revealed remarkable inhibition of AKT phosphorylation but not phospho‐p44/42 MAPK. To examine the cellular response in a lung cancer cell line intrinsically expressing deletional EGFR, phospho‐EGFR, and downstream reactions were monitored under EGF stimulation with a beads‐based mulitiplex assay. EGFR and its downstream proteins were constitutively phosphorylated in the PC‐9 cells without any ligand stimulation as compared with A549 lung cancer cells expressing wild‐type EGFR. In conclusion, deletional EGFR is constitutively active and phosphorylates p44/42 MAPK and AKT in the cells, although the fact that the EGFR phosphorylation in the PC‐9 cells is still modulated by EGF stimulation cannot be ignored. Gefitinib‐inhibited phospho‐AKT predominantly in deletional EGFR expressing cells.

Phase I pharmacokinetic and pharmacogenomic study of E7070 administered once every 21 days

E7070 is a novel sulfonamide anticancer agent that disrupts the G1/S phase of the cell cycle. The objectives of this phase I study of E7070 were to estismate the maximal tolerated dose (MTD), to determine the recommended dose for phase II, and to clarify the pharmacokinetic profile of E7070 and its relation to polymorphisms of CYP2C9 (*2, *3) and CYP2C19 (*2, *3) in Japanese patients. Patients received 1–2‐h i.v. infusions of E7070 (400, 600, 700, 800 or 900 mg/m2) on day 1 of a 21‐day cycle. Twenty‐one patients received between one and eight cycles of E7070. The dose‐limiting toxicities (DLT) comprised leukopenia, neutropenia, thrombocytopenia, elevation of aspartate aminotransferase, colitis, and ileus. The mean area under the plasma concentration–time curve (AUC) for successive dose levels increased in a non‐dose‐proportional manner. Two patients were heterozygous for the CYP2C9 mutation. For CYP2C19, eight patients were wild type and the remainder had heterozygous (n = 8) or homozygous mutations (n = 5). Regarding the CYP2C19 genotype, the AUC of patients with mutant alleles were higher than those of patients with wild type at a dose of 600 mg/m2 or more. The severity of toxic effects, such as myelosuppression, seemed to depend on the AUC. No partial responses were observed. One patient treated at a dose of 700 mg/m2 experienced a maximum tumor volume reduction of 22.5%. The MTD was estimated to be 900 mg/m2. A dose of 800 mg/m2 is recommended for further phase II studies. The pharmacokinetic/pharmacodynamic properties of E7070 seemed to be influenced by CYP2C19 genotype. The observed safety profile and preliminary evidence of antitumor activity warrant further investigation of this drug in monotherapy or in combination chemotherapy. (Cancer Sci 2005; 96: 721 –728)

FOLFIRI plus bevacizumab as second-line therapy in patients with metastatic colorectal cancer after first-line bevacizumab plus oxaliplatin-based therapy: the randomized phase III EAGLE study

EAGLE was a randomized, multicenter phase III study which evaluated the superiority of bevacizumab 10 mg/kg plus FOLFIRI compared with bevacizumab 5 mg/kg plus FOLFIRI in patients with mCRC previously treated with first-line bevacizumab plus an oxaliplatin-based regimen. The results suggest that the higher 10 mg/kg dose offers no clear clinical benefit compared with bevacizumab 5 mg/kg in this setting.

Translational studies for target-based drugs.

The biological background for the clinical and prognostic heterogeneity among tumors within the same histological subgroup is due to individual variations in the biology of tumors. The number of investigations looking at the application of novel technologies within the setting of clinical trials is increasing. The most promising way to improve cancer treatment is to build clinical research strategies on intricate biological evidence. New genomic technologies have been developed over recent years. These techniques are able to analyze thousands of genes and their expression profiles simultaneously. The purpose of this approach is to discover new cancer biomarkers, to improve diagnosis, predict clinical outcomes of disease and response to treatment, and to select new targets for novel agents with innovative mechanisms of action. Gene expression profiles are also used to assist in selecting biomarkers of pharmacodynamic effects of drugs in the clinical setting. Biomarker monitoring in surrogate tissues may allow researchers to assess “proof of principle” of new treatments. Clinical studies of biomarkers monitoring toxicity profiles have also been done. Such pharmacodynamic markers usually respond to treatment earlier than clinical response, and as such may be useful predictors of efficacy. Epidermal growth factor receptor (EGFR) mutation in lung cancer tissues is a strong predictive biomarker for EGFR-targeted protein tyrosine kinase inhibitors. Monitoring of EGFR mutation has been broadly performed in retrospective and prospective clinical studies. However, global standardization for the assay system is essential for such molecular correlative studies. A more sensitive assay for EGFR mutation is now under evaluation for small biopsy samples. Microdissection for tumor samples is also useful for the sensitive detection of EGFR mutation. Novel approaches for the detection of EGFR mutation in other clinical samples such as cytology, pleural effusion and circulating tumor cells are ongoing.