Yasuhiro Fujiwara

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.

Prognostic significance of p53 and ras gene abnormalities in lung adenocarcinoma patients with stage I disease after curative resection.

We investigated the prognostic significance of p53 gene abnormalities and ras gene mutations in patients with curatively resected stage I lung adenocarciiioma. Formalin‐fixed and paraffin‐embedded tissues were obtained from 30 patients who had undergone curative resection for stage I lung adenocarciiioma. Abnormalities of the p53 gene were detected using polymcrasc chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) analysis and immunohistochemistry and ras mutations were detected using PCR‐restriction fragment length polymorphism (RFLP) analysis. Both univariate and multivariate analyses were performed to assess the relationship between the presence of abnormalities of these genes and the patients’disease‐free survival. Eleven tumors (37%) had mutated p53 sequences and 11 (37%) showed p53 overexpression. A total of 15 tumors (50%) had p53 gene abnormalities and the concordance rate was 73%. Seven tumors (23%) showed mutated ras sequences. The univariate analysis revealed that the disease‐free survival of patients with any p53 abnormality was shorter than that of those without abnormalities (P=0.02, generalized Wilcoxon test), and survival of those with p53 protein overexpression was more significantly shorter (P=0.003, generalized Wilcoxon test). Multivariate analysis using the Cox proportional hazards model indicated that the presence of p53 abnormalities was a significantly (P=0.01) unfavorable prognostic factor. There was no significant correlation between the presence of ras mutation and survival. These results suggest that analysis of the p53 gene may be helpful for the selection of high‐risk patients for clinical trials of adjuvant therapy for stage I lung adenocarcinoma.

The Role of Glucuronidation in 7-Ethyl-10-hydroxycamptothecin Resistance in vitro

Although glucuronidation catalyzed by uridine 5′‐diphosphoglucuronosyltransferase (UGT) is a major pathway of drug inactivation in humans, glucuronidation in malignant cells has received little attention as a cause of anti‐cancer drug resistance. In this study, we tried to elucidate the role of SN‐38 glucuronidation in the CPT‐11‐resistant human lung cancer cell line PC‐7/CPT. PC‐7/CPT cells possessed an increased activity to glucuronidate SN‐38 compared to the parent cells, PC‐7, Furthermore, sensitivity of PC‐7/CPT cells to SN‐38 was improved by inhibiting UGT activity. Western and northern blot analyses demonstrated that this increased activity was due to increased levels of UGT protein and mRNA. These results not only imply that npregulation of UGT activity in PC‐7/CPT cells may contribute in part to SN‐38 resistance, hut also illustrate the importance of drug metabolism within malignant cells themselves, as a cause of drug resistance

Increased expression of the MRP5 gene is associated with exposure to platinum drugs in lung cancer.

To investigate the role of the multidrug resistance‐associated protein (MRP1) homologue MRP5 in relation to platinum drug resistance, we examined the steady‐state levels of the mRNAs for MRP5 in both lung cancer cell lines and peripheral mononuclear cells (PMN) after exposure to platinum drug and in normal lung and lung cancer tissue specimens. Firstly, we examined MRP5 gene expression levels in 80 autopsy samples (40 primary tumors and 40 corresponding normal lung tissues) from 40 patients who had died from lung cancer. Next, we monitored MRP5 gene expression levels within 24 hr in both lung cancer cell lines incubated with cisplatin and in PMN from 10 previously untreated lung cancer patients after carboplatin administration alone. The MRP5 gene expression levels were assessed by quantitative reverse transcription polymerase chain reaction or RNase protection assay. The MRP5 expression levels in normal lung tissues and in tumors from patients exposed to platinum drugs during their lifetime were significantly higher than those in tissues from non‐exposed patients. On the other hand, the MRP5 expression levels were not rapidly induced by platinum drugs either in lung cancer cell lines or in PMN within 24 hr. Our results suggest that increased expression levels of the MRP5 gene are associated with exposure to platinum drugs in lung cancer in vivo and/or the chronic stress response to xenobiotics. Int. J. Cancer 86:95–100, 2000.

High-performance liquid chromatographic determination of irinotecan (CPT-11) and its active metabolite (SN-38) in human plasma

A simplified method for the simultaneous determination of irinotecan (CPT-11, I) and its active metabolite (SN-38, II) in human plasma by high-performance liquid chromatography (HPLC) with fluorescence detection has been developed. Following the addition of the internal standard (I.S.) camptothecin, the drugs were extracted from plasma using methanol. The average extraction efficiencies were 87% for I, 90% for II and 90% for the I.S. Chromatography was performed using a TSK gel ODS-80Ts column, monitored at 556 nm (excitation wavelength, 380 nm) and the mobile phase was acetonitrile-50 mM disodium hydrogen phosphate (28:72) containing 5 mM heptanesulphonate (pH 3.0). The linear quantitation ranges for I and II were 30-2000 and 1-30 ng/ml, respectively.

No Alteration in DNA Topoisomerase I Gene Related to CPT‐11 Resistance in Human Lung Cancer

Mutations and reduced expression of DNA topoisomerase I (topo I) gene have been shown to be important in the acquisition of resistance to camptothecin and its analogues in vitro, but their significance has not been verified in vivo. We investigated possible topo I gene mutations and topo I mRNA expression levels in 127 samples obtained from 56 patients with lung tumors, including patients who had developed clinical resistance to topo I inhibitors. No mutations were detected in any of the samples examined and expression levels did not differ significantly between clinically resistant cases and others. However, the topo I mRNA expression level was significantly higher in small cell lung carcinomas than in non‐small cell lung carcinomas (P<0.05). These results suggest that topo I mRNA levels may affect CPT‐11 sensitivity in human lung cancer. However, topo I gene mutations and reduced topo I mRNA expression may not be the main mechanism of clinically acquired resistance to camptothecin analogues in vivo.

Development and validation of diagnostic prediction model for solitary pulmonary nodules

Background and objective:u2003 The aim of this study was to develop a simple prediction model for the underlying diagnosis of solitary pulmonary nodules (SPN) based on clinical characteristics and thin‐section CT findings.

Efficacy of weekly paclitaxel in patients with docetaxel-resistant metastatic breast cancer.

SummaryBackground. Partial cross-resistance to paclitaxel and docetaxel has been demonstrated in pre-clinical studies.n Patients and methods. We retrospectively evaluated the efficacy of weekly paclitaxel 80 mg/m2 in 82 patients with docetaxel-resisitant metastatic breast cancer. Docetaxel resistance was classified into primary resistance, defined as progressive disease while receiving docetaxel, and secondary resistance, defined as progression after achievement of a documented clinical response to docetaxel. Secondary resistance was subclassified according to the interval between the final infusion of docetaxel and the start of weekly paclitaxel into: (1) short interval, ≦120 days, and (2) long interval, >120 days.nn Results. The response rate of the 82 patients was 19.5% (95% confidence interval, 10.8–27.9%). The response rate according to the docetaxel resistance category was: primary resistance (n=24), 8.3%; secondary resistance (n=58), 24.1% (short interval [n=39], 17.9%, and long interval, [n=19], 36.8%). The differences in response rates among the three categories were statistically significant (p=0.0247, Cochran–Mantel–Haenszel test). The interval between from the final docetaxel infusion and disease progression were predictors for response of weekly paclitaxel.nn Conclusion. Weekly paclitaxel is modestly effective and safe in docetaxel-resistant metastatic breast cancer patients. However, weekly paclitaxel should not be recommended for primary resistance patients with docetaxel.n

Salivary drug monitoring of irinotecan and its active metabolite in cancer patients

Abstractu2003To assess the clinical usefulness of salivary monitoring of irinotecan (CPT-11) and its active metabolite (SN-38), we examined the clinical pharmacological profile of both drugs in 9 patients with thoracic malignancies who received 60 mg/m2 CPT-11 (21 courses). Plasma and unstimulated whole saliva were collected over a 24-h period, and concentrations of CPT-11 and SN-38 were measured by high-performance liquid chromatography. Both CPT-11 and SN-38 were detectable in saliva, and the concentration-time curves in plasma and saliva showed a very similar pattern. A good correlation was observed between the saliva concentration (Cs) and the plasma concentration (Cp) for both CPT-11 and SN-38 (r=0.732, P<0.0001 and r = 0.611, P<0.0001, respectively). The area under the concentration-time curve calculated for saliva (AUCs) correlated with that generated for plasma (AUCp) for both CPT-11 and SN-38 (r = 0.531, P = 0.012 and r = 0.611, P = 0.0025, respectively). These results suggest that it may be feasible to use saliva instead of plasma for pharmacokinetics/pharmacodynamics studies of CPT-11.

Induction of cytochrome P450 3A4 by docetaxel in peripheral mononuclear cells and its expression in lung cancer.

Abstract. Several recent studies have demonstrated that the cytochrome p450 (CYP) family plays an important role in the metabolism of taxanes. However, the role of CYP gene expression in tumors and peripheral mononuclear cells (PMN) is unknown. We therefore investigated the levels of CYP3A4 and CYP2C gene expression using reverse transcription polymerase chain reaction (RT-PCR) in PMN from 16 previously untreated lung cancer patients to determine whether the expression of the two genes is induced by docetaxel (TXT). Neither the CYP3A4 nor the CYP2C gene was induced after administration of carboplatin (CBDCA) alone. Expression of the CYP3A4 gene was induced by the administration of TXT alone or TXT and CBDCA, but expression of the CYP2C gene was unaffected. We also measured the expression of both genes using RT-PCR in 20 autopsy samples (ten non-small-cell lung cancers and their corresponding normal lung tissues) obtained from patients who had not received any chemotherapy during life. The level of CYP2C gene expression in samples of lung cancer was significantly higher than in normal lung tissue, but the level of CYP3A4 gene expression was not. These results suggest that the CYP3A4 gene is induced by TXT, and that it plays an important role in intracellular TXT metabolism.