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Liver metastasis is one of the most important prognostic factors in lung cancer patients. However, current therapies are not sufficient. RNA interference provides us a powerful and promising approach for treating human diseases including cancers. Herein, we investigated the in vitro effects of PLK-1 small interfering RNA (siRNA) on human lung cancer cell lines and the in vivo usage of PLK-1 siRNA with atelocollagen as a drug delivery system in a murine liver metastasis model of lung cancer. PLK-1 was overexpressed in cell lines and in cancerous tissues from lung cancer patients. PLK-1 siRNA treatment inhibited growth and induced apoptosis in a concentration-dependent manner. To verify in vivo efficacy, we confirmed that atelocollagen was a useful drug delivery system in our model of implanted luciferase-labeled A549LUC cells by detecting reduced bioluminescence after an i.v. injection of luciferase GL3 siRNA/atelocollagen. PLK-1 siRNA/atelocollagen was also successfully transfected into cells and inhibited the progression of metastases. This study shows the efficacy of i.v. administration of PLK-1 siRNA/atelocollagen for liver metastases of lung cancer. We believe siRNA therapy will be a powerful and promising strategy against advanced lung cancer. [Mol Cancer Ther 2008;7(9):2904–12]

Supplementary Fig. S1 from Administration of PLK-1 small interfering RNA with atelocollagen prevents the growth of liver metastases of lung cancer

Supplementary Figure Legends from Administration of PLK-1 small interfering RNA with atelocollagen prevents the growth of liver metastases of lung cancer

Thiopurines are effective immunosuppressants and anticancer agents, but intracellular accumulation of their active metabolites (6-thioguanine nucleotides, 6-TGN) causes dose-limiting hematopoietic toxicity. Thiopurine S-methyltransferase deficiency is known to exacerbate thiopurine toxicity. However, many patients are highly sensitive to thiopurines for unknown reasons. We show that multidrug-resistance protein 4 (Mrp4) is abundant in myeloid progenitors and tested the role of the Mrp4, an ATP transporter of monophosphorylated nucleosides, in this unexplained thiopurine sensitivity. Mrp4-deficient mice experienced Mrp4 gene dosage-dependent toxicity caused by accumulation of 6-TGNs in their myelopoietic cells. Therefore, Mrp4 protects against thiopurine-induced hematopoietic toxicity by actively exporting thiopurine nucleotides. We then identified a single-nucleotide polymorphism (SNP) in human MRP4 (rs3765534) that dramatically reduces MRP4 function by impairing its cell membrane localization. This SNP is common (>18%) in the Japanese population and indicates that the increased sensitivity of some Japanese patients to thiopurines may reflect the greater frequency of this MRP4 SNP. [Cancer Res 2008;68(13):4983–9]

Supplementary Methods, Figure Legends 1-3 from Deoxycytidine Kinase Modulates the Impact of the ABC Transporter ABCG2 on Clofarabine Cytotoxicity

Supplementary Figure 1 from Substrate Overlap between Mrp4 and Abcg2/Bcrp Affects Purine Analogue Drug Cytotoxicity and Tissue Distribution

Supplementary Figure 2 from Deoxycytidine Kinase Modulates the Impact of the ABC Transporter ABCG2 on Clofarabine Cytotoxicity

Supplementary Figure 3 from Deoxycytidine Kinase Modulates the Impact of the ABC Transporter ABCG2 on Clofarabine Cytotoxicity

Supplementary Figure 1 from Deoxycytidine Kinase Modulates the Impact of the ABC Transporter ABCG2 on Clofarabine Cytotoxicity

The use of probe substrates and combinations of ATP-binding cassette (ABC) transporter knockout (KO) animals may facilitate the identification of common substrates between apparently unrelated ABC transporters. An unexpectedly low concentration of the purine nucleotide analogue, 9-(2-(phosphonomethoxy)ethyl)-adenine (PMEA), and up-regulation of Abcg2 in some tissues of the Mrp4 KO mouse prompted us to evaluate the possibility that Abcg2 might transport purine-derived drugs. Abcg2 transported and conferred resistance to PMEA. Moreover, a specific Abcg2 inhibitor, fumitremorgin C, both increased PMEA accumulation and reversed Abcg2-mediated PMEA resistance. We developed Mrp4 and Abcg2 double KO mice and used both single KOs of Abcg2 and Mrp4 mice to assess the role of these transporters in vivo. Abcg2 contributed to PMEA accumulation in a variety of tissues, but in some tissues, this contribution was only revealed by the concurrent absence of Mrp4. Abcg2 also transported and conferred resistance to additional purine analogues, such as the antineoplastic, 2-chloro-2′-deoxyadenosine (cladribine) and puromycin, a protein synthesis inhibitor that is often used as a dominant selectable marker. Purine analogues interact with ABCG2 by a site distinct from the prazosin binding site as shown by their inability to displace the substrate analogue and photoaffinity tag [125I]iodoarylazidoprazosin. These studies show that Abcg2, like Mrp4, transports and confers resistance to purine nucleoside analogues and suggest that these two transporters work in parallel to affect drug cytotoxicity and tissue distribution. This new knowledge will facilitate an understanding of how Abcg2 and Mrp4, separately and in combination, protect against purine analogue host toxicity as well as resistance to chemotherapy. [Cancer Res 2007;67(14):6965–72]

Supplementary Figure 1 from Transporter-Mediated Protection against Thiopurine-Induced Hematopoietic Toxicity

Supplementary Table 1 from Transporter-Mediated Protection against Thiopurine-Induced Hematopoietic Toxicity

Supplementary Table 1 from Substrate Overlap between Mrp4 and Abcg2/Bcrp Affects Purine Analogue Drug Cytotoxicity and Tissue Distribution

The surface-polarity-dependent Raman spectra of ultrathin silicon carbide crystal are reported. The relative Raman intensity of the folded-transverse-acoustic phonon to the folded-transverse-optical phonon modes differs drastically between silicon-terminated face (Si-face) and carbon-terminated face (C-face) only for sample thickness below 150 nm. For samples thicker than 150 nm, the relative Raman intensity ratio takes an almost constant value for both Si- and C-faces. These results indicate that the phonon modes confined in the near-surface region are the possible origin of the observed surface-polarity-dependent Raman spectra.

Oncogenic gene mutations observed in lung adenocarcinomas, such as epidermal growth factor receptor (EGFR) and KRAS, have some predictive value for chemotherapeutic drugs or EGFR–tyrosine kinase inhibitors. However, the influence of these gene alterations on patients’ prognosis remains controversial. We retrospectively analyzed the tumors of 180 patients with completely resected pathological stage I–III lung adenocarcinoma which harbored either KRAS codon 12 mutation or EGFR gene mutations within exons 18–21 to investigate the impact of these gene mutations on the patients’ survival. Gene mutations were detected by established methods. Of 180 patients, 32 had KRAS codon 12 mutations (KRAS group), 148 had EGFR mutations within exon 18–21 (EGFR group). Pathological stage and operation mode were independent factors for disease-free survival. However, the EGFR group had better overall survival than the KRAS group (P = 0.0271). Cox proportional hazard model revealed pathological stage (P = 0.0001) and presence of EGFR gene mutations (P = 0.0408) were independent factors for overall survival. In survival after tumor recurrence, the EGFR group had a better median survival time (46.7 months) after recurrence than the KRAS group (26.0 months). In patients with completely resected lung adenocarcinomas, KRAS and EGFR gene mutation status of tumors was not associated with disease-free survival. However, the presence of an EGFR gene mutation boded well for the patient’s overall survival, and thus patients with EGFR mutations have a better prognosis than those with KRAS mutations.

Purine nucleoside antimetabolites, such as clofarabine, are effective antileukemic agents. However, their effectiveness depends on an initial activation step in which they are monophosphorylated by deoxycytidine kinase (dCK). Some purine nucleoside antimetabolites and their monophosphate derivatives are exported by the ABC transporter ABCG2. Because clofarabine is a dCK substrate, and we show substantial variation in dCK and ABCG2 in myeloid leukemia, we hypothesized that the activity of dCK may modulate ABCG2-mediated resistance to clofarabine by regulating the formation of clofarabine monophosphate. We show that ABCG2 influence on clofarabine cytotoxicity was markedly influenced by dCK activity. When dCK expression was reduced by siRNA, clofarabine cytotoxicity was strongly reduced by enhanced ABCG2-mediated efflux. Conversely, dCK overexpression blunted ABCG2-mediated efflux of clofarabine by increasing the formation of clofarabine nucleotides. The use of an ABCG2 inhibitor confirmed that ABCG2 export of clofarabine is maximal when dCK levels are minimal. Analysis of intracellular clofarabine metabolites suggested that ABCG2 exported clofarabine more readily than clofarabine monophosphate. That ABCG2 primarily effluxes clofarabine, but not chlorfarabine-monophosphate, was confirmed by HPLC analysis of drug exported from ABCG2-overexpressing cells. Because the level and function of dCK and ABCG2 vary substantially among other types of cancer, these findings have important implications not only for clofarabine therapy but for purine nucleoside therapy in general. Therefore, we propose that addition of ABCG2 inhibitors would effectively increase the antitumor efficacy of purine nucleosides by blocking drug efflux that may be a significant mode of resistance when dCK levels are low. Cancer Res; 71(5); 1781–91. ©2011 AACR.

Thiopurines are effective immunosuppressants and anticancer agents, but intracellular accumulation of their active metabolites (6-thioguanine nucleotides, 6-TGN) causes dose-limiting hematopoietic toxicity. Thiopurine S-methyltransferase deficiency is known to exacerbate thiopurine toxicity. However, many patients are highly sensitive to thiopurines for unknown reasons. We show that multidrug-resistance protein 4 (Mrp4) is abundant in myeloid progenitors and tested the role of the Mrp4, an ATP transporter of monophosphorylated nucleosides, in this unexplained thiopurine sensitivity. Mrp4-deficient mice experienced Mrp4 gene dosage-dependent toxicity caused by accumulation of 6-TGNs in their myelopoietic cells. Therefore, Mrp4 protects against thiopurine-induced hematopoietic toxicity by actively exporting thiopurine nucleotides. We then identified a single-nucleotide polymorphism (SNP) in human MRP4 (rs3765534) that dramatically reduces MRP4 function by impairing its cell membrane localization. This SNP is common (>18%) in the Japanese population and indicates that the increased sensitivity of some Japanese patients to thiopurines may reflect the greater frequency of this MRP4 SNP. [Cancer Res 2008;68(13):4983–9]

Summary Maspin is a member of the serpin (serine protease inhibitor) superfamily, and some experimental studies revealed a potential tumor suppressor activity of maspin. To reveal clinical significance of maspin status in non-small cell lung cancer (NSCLC), we quantitatively evaluated maspin gene expression in lung primary tumors cut from a total of 55 resected NSCLC patients. Maspin expression in squamous cell carcinoma (Sq) was significantly higher than that in adenocarcinoma (Ad, p =0.011). Five-year overall survival rates of maspin-high and maspin-low patients were 67.7 and 41.4%, respectively, demonstrating a significant favorable prognosis of maspin-high patients (log-rank, p =0.042). A multivariate analysis confirmed that high maspin expression was an independent and significant factor to predict a favorable overall survival ( p =0.031). These results suggested that maspin expression was significantly increased in Sq than in Ad, and that increased maspin expression was a significant factor to predict a favorable prognosis in resected NSCLC.

Background: MUC1 is a transmembrane mucin that plays an important role in tumor progression. Many clinical studies have suggested that the expression pattern of MUC1 core protein can be a useful prognostic marker in various malignancies, but the prognostic significance in non–small cell lung cancer (NSCLC) remains uncertain. We performed a study to assess clinical significance, especially prognostic impact, of MUC1 expression in NSCLC. Methods: A total of 62 patients with completely resected pathologic stage I to IIIA NSCLC were retrospectively reviewed. Histologic sections cut from primary tumors were immunohistochemically stained with an anti-MUC1 monoclonal antibody (CA15-3, clone DF3), which recognizes unglycosylated epitope of MUC1 core protein. According to MUC1 expression pattern, each patient was classified into the high-grade polarized expression (HP), the low-grade polarized expression (LP), or the depolarized expression (D) group. Results: Twenty-four (38.7%), 21 (33.9%), and 17 (27.4%) patients were classified into the HP group, the LP group, and the D group, respectively. HP was exclusively seen in adenocarcinoma, mostly in well-differentiated adenocarcinoma. D was correlated with progressive stage and lymph node metastasis. Postoperative survival of the D group seemed to be poorer than that of the HP group for all NSCLC patients, and the difference was enhanced in adenocarcinoma patients. Conclusion: A novel classification of MUC1 expression pattern (HP, LP, and D) was correlated with tumor differentiation and postoperative survival in NSCLC, especially in lung adenocarcinoma.

Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a novel membrane-anchored matrix metalloproteinase inhibitor, and experimental studies have shown that RECK can suppress tumor progression through angiogenesis inhibition. We have already revealed that enhanced RECK expression is significantly correlated with a favorable prognosis in non-small-cell lung cancer (NSCLC). In this study, further analyses focused on pN2 disease were conducted to assess the clinical significance of RECK expression.A total of 118 patients with completely resected pathologic stage IIIA N2 NSCLC were retrospectively examined. RECK expression in the primary tumor, along with involved N2 nodes, was examined immunohistochemically.RECK expression in the primary tumor was strong in 53 patients (44.9%) and was weak in the other 65 patients. The 5-year survival rate of patients with RECK-strong tumor (42.9%) was significantly higher than that of patients with RECK-weak tumor (23.1%; P = .017). Reduced RECK expression significantly correlated with a poor prognosis for patients with a single N2 node involved (P = .019), but not for patients with multiple N2 nodes involved (P = .440). A multivariate analysis confirmed that reduced RECK expression was an independent and significant factor to predict a poor prognosis (P = .031). RECK expression in involved N2 nodes was significantly higher than in primary tumors (P.001).RECK status was a novel prognostic factor in pathologic stage IIIA N2 NSCLC.

Objective: To reveal the incidence and clinical significance of mediastinal nodal metastases without N1-station nodal metastases ('skip-N2 metastases') in non-small cell lung cancer (NSCLC). Methods: A total of 450 NSCLC patients who underwent tumor resection with a systemic mediastinal nodal dissection were retrospectively reviewed. p53 status and proliferative activity represented as proliferative index (PI) were also examined immunohistochemically. Results: Skip-N2 metastases were documented in 49 (13%) patients of all 450 patients; among 334 patients without N1-nodal involvement, 18% patients had skip-N2 metastases. The postoperative survival of skip-N2 patients was almost same as that for patients with metastases to both N 1 and N2 nodes. Skip-N2 metastases were significantly more frequent in male patients and squamous cell carcinoma patients. In addition, the mean PI for tumor with skip-N2 metastases was significantly higher than that for any other pathologic nodal (pN)-status diseases. Combined with histologic type and PI, the incidences of skip-N2 metastases for adenocarcinoma showing lower PI were only 5% (7/137) of all patients and 7% (7/94) of patients without Nl-nodal involvement. Conclusions: N1 nodal status is not a useful predictor of N2 nodal status in NSCLC, because skip-N2 metastases were documented in 18% patients showing no Nl-nodal involvement. However, N1 node-guided dissection might be performed in patients with adenocarcinoma showing lower PI, because the incidence of skip-N2 metastases was extremely low.

Controlling the activation of platelets is a key strategy to mitigate cardiovascular disease. Previous studies have suggested that the ATP-binding cassette (ABC) transporter, ABCC4, functions in platelet-dense granules. Using plasma membrane biotinylation and super-resolution microscopy, we demonstrate that ABCC4 is primarily expressed on the plasma membrane of both mouse and human platelets. Platelets lacking ABCC4 have unchanged dense-granule function, number, and volume, but harbor a selective impairment in collagen-induced aggregation. Accordingly, Abcc4 knockout (KO) platelet attachment to a collagen substratum was also faulty and associated with elevated intracellular cyclic AMP (cAMP) and reduced plasma membrane localization of the major collagen receptor, GPVI. In the ferric-chloride vasculature injury model, Abcc4 KO mice exhibited markedly impaired thrombus formation. The attenuation of platelet aggregation by the phosphodiesterase inhibitor EHNA (a non-ABCC4 substrate), when combined with Abcc4 deficiency, illustrated a crucial functional interaction between phosphodiesterases and ABCC4. This was extended in vivo where EHNA dramatically prolonged the bleeding time, but only in Abcc4 KO mice. Further, we demonstrated in human platelets that ABCC4 inhibition, when coupled with phosphodiesterase inhibition, strongly impaired platelet aggregation. These findings have important clinical implications because they directly highlight an important relationship between ABCC4 transporter function and phosphodiesterases in accounting for the cAMP-directed activity of antithrombotic agents.

気胸手術で得られた切除ブラ壁にアスペルギルスを認めた2症例を経験したので報告する. (症例1) 48歳男性.左気胸で入院.持続ドレナージを行ったが, 気胸の改善がないため, 手術を行った.切除した肺尖のブラ壁にアスペルギルスを認めた. (症例2) 46歳男性.右胸痛にて受診.胸部X線写真上, 右気胸と両側に肺嚢胞を認め, 右肺嚢胞切除術を行った.病理標本にて胸膜下にアスペルギルスを認めた.自験例のように基礎疾患がなく, 気胸手術で発見された, ブラ内に限局したアスペルギルス感染はまれである.気胸手術例においても肺組織の肥厚があれば肺癌, 肺結核と同様に真菌感染も考慮した病理組織の検討が必要で, 組織浸潤を認めた場合は, 抗真菌剤の投与も考慮すべきである.

背部痛にて発症した胸壁浸潤を伴う肋骨原発の好酸球性肉芽腫を経験したので報告する.症例は24歳女性.左背部痛にて1998年1月近医を受診.胸部X線で左肋骨融解像を指摘され, 当院を紹介.胸部CT, MRIで, 左第5肋骨を中心に胸壁に浸潤した腫瘤影を認めた.病変の広がりの検索のため胸腔鏡検査を行い, 広範に胸壁へ進展した病変を認めた.左第5肋骨に沿う皮切を行い, 肋骨の部分切除を施行した.病理組織学的検索で多数の好酸球と, S-100蛋白陽性の褐色に染まる組織球を多数認め, 好酸球性肉芽腫と診断した.病変が広範囲に及ぶことから, 局所に放射線治療と, ステロイドと抗癌剤の全身投与を行い, 軽快した.治療1年半経過, 再発を認めていない.

Human immunodeficiency virus (HIV) pharmacotherapy, by combining different drug classes such as nucleoside analogs and HIV protease inhibitors (PIs), has increased HIV-patient life expectancy. Consequently, among these patients, an increase in non-HIV–associated cancers has produced a patient cohort requiring both HIV and cancer chemotherapy. We hypothesized that multidrug resistance protein 4/ATP binding cassette transporter 4 (MRP4/ABCC4), a widely expressed transporter of nucleoside-based antiviral medications as well as cancer therapeutics might interact with PIs. Among the PIs evaluated (nelfinavir, ritonavir, amprenavir, saquinavir, and indinavir), only nelfinavir both effectively stimulated MRP4 ATPase activity and inhibited substrate-stimulated ATPase activity. Saos2 and human embryonic kidney 293 cells engineered to overexpress MRP4 were then used to assess transport and cytotoxicity. MRP4 expression reduced intracellular accumulation of nelfinavir and consequently conferred survival advantage to nelfinavir cytotoxicity. Nelfinavir blocked Mrp4-mediated export, which is consistent with its ability to increase the sensitivity of MRP4-expressing cells to methotrexate. In contrast, targeted inactivation of Abcc4/Mrp4 in mouse cells specifically enhanced nelfinavir and 9-(2-phosphonylmethoxyethyl) adenine cytotoxicity. These results suggest that nelfinavir is both an inhibitor and substrate of MRP4. Because nelfinavir is a new MRP4/ABCC4 substrate, we developed a MRP4/ABCC4 pharmacophore model, which showed that the nelfinavir binding site is shared with chemotherapeutic substrates such as adefovir and methotrexate. Our studies reveal, for the first time, that nelfinavir, a potent and cytotoxic PI, is both a substrate and inhibitor of MRP4. These findings suggest that HIV-infected cancer patients receiving nelfinavir might experience both enhanced antitumor efficacy and unexpected adverse toxicity given the role of MRP4/ABCC4 in exporting nucleoside-based antiretroviral medications and cancer chemotherapeutics.

著明なfoamy macrophage浸潤を示した肺のinHammatory pseudotumorの手術症例を経験した.症例は33歳の男性.前胸部痛が出現し近医にて右上肺野の腫瘤状陰影を発見され当科に紹介入院した.術前検査で確定診断が得られず, 右上葉切除術を施行しinflammatory pseudotumorと診断された.腫瘍は4×4×3cm大, 黄色調で非常に軟らかく境界明瞭であった.組織学的には腫瘍の中心部分に著明なfoamy macrophageの浸潤を, 周辺部分にはPlasmace11の浸潤を認めた.Foarny macrophageの寿命は短いことから本症例はinHamrnatory pseudotumorの初期像を示す1例と考えられた.

Aminopeptidase-N (APN) is a membranebound protein that acts as a zinc-binding protease and participates in extracellular proteolysis. APN plays important roles in tumor progression through promoting invasion and metastasis, prolonging survival of tumor cells, and tumor angiogenesis.We evaluated APN expression in non-small-cell lung cancer patients by immunohistochemistry.Of the 95 patients reviewed in the present study, 9 (9.5%), all with adenocarcinoma (Ad), showed positive APN expression on the tumors' cells. In all, 31 (32.6%) and 19 (20.0%) patients showed positive APN expression on the tumors' stromal cells (fibroblasts) and microvessels, respectively. APN expression on the tumors' stromal cells was more frequently observed in squamous cell carcinoma patients than in adenocarcinoma patients (P = 0.005). The mean microvessel density (MVD) for APNpositive tumor stromal cells was 59.9, which was significantly higher than that for APN-negative tumor stromal cells (mean MVD 27.4; P = 0.001). The 5-year survival rates for APN-positive and APN-negative tumor stromal cells were 66.0% and 69.8%, respectively, showing no difference in patient survival according to APN status on the tumors' stromal cells.That APN expression on stromal cells was observed predominantly in squamous cell carcinoma may account for the efficacy of ubenimex in the postoperative adjuvant setting for squamous cell carcinoma.

Experimental studies have revealed that D2-40 is useful in identifying the presence of lymphatic invasion in various malignant neoplasms, but the clinical significance remains unclear. The purpose of this study is to assess the clinical significance of D2-40 status in completely resected non-small cell lung cancer.A total of 215 consecutive patients with resected pathological stage I-IIIA non-small cell lung cancer were reviewed. Expression of D2-40 in tumor cells and in endothelial cells was examined immunohistochemically, and D2-40-positive lymphatic vessel density (LVD) at the tumor periphery were quantitatively evaluated.D2-40 expression on tumor cells was positive in 55 (25.6%) of 215 patients, and the incidence was significantly higher in squamous cell carcinoma (SCC) patients than in adenocarcinoma patients (48.8% vs. 8.6%, P.001). D2-40 was also seen on lymphatic vessels in tumor tissues, and the mean number of D2-40-LVD was significantly decreased along with differentiation of tumor cells (P = .038). For all histologic types of tumors, there was no difference in the postoperative survival between higher D2-40-LVD patients and lower D2-40-LVD patients. For SCC, however, lower D2-40-LVD patients showed a significantly better survival than higher D2-40-LVD patients (5-year survival rates, 52.9% vs. 78.9%, P = .040), which was confirmed by a multivariate analyses (P = .048).D2-40 expression on tumor cells was more frequently seen in SCC than in adenocarcinoma of the lung. In addition, D2-40 expression on lymphatic vessels in tumor tissues was a statistically significant prognostic factor in SCC.

Liver metastasis is one of the most important prognostic factors in lung cancer patients. However, current therapies are not sufficient. RNA interference provides us a powerful and promising approach for treating human diseases including cancers. Herein, we investigated the in vitro effects of PLK-1 small interfering RNA (siRNA) on human lung cancer cell lines and the in vivo usage of PLK-1 siRNA with atelocollagen as a drug delivery system in a murine liver metastasis model of lung cancer. PLK-1 was overexpressed in cell lines and in cancerous tissues from lung cancer patients. PLK-1 siRNA treatment inhibited growth and induced apoptosis in a concentration-dependent manner. To verify in vivo efficacy, we confirmed that atelocollagen was a useful drug delivery system in our model of implanted luciferase-labeled A549LUC cells by detecting reduced bioluminescence after an i.v. injection of luciferase GL3 siRNA/atelocollagen. PLK-1 siRNA/atelocollagen was also successfully transfected into cells and inhibited the progression of metastases. This study shows the efficacy of i.v. administration of PLK-1 siRNA/atelocollagen for liver metastases of lung cancer. We believe siRNA therapy will be a powerful and promising strategy against advanced lung cancer. [Mol Cancer Ther 2008;7(9):2904–12]

Double ion implanted 4H-SiC bipolar junction transistors (BJTs) are fabricated by Al and N ion implantation to the base and emitter. The current gain of 3 is obtained at the base Al concentration of 1 × ∼ 1017 /cm3. The collector current as a function of the base Gummel number suggests that double ion implanted 4H-SiC BJT operates in the intrinsic region below the emitter in the low injection level. The high base resistance restricts the base current at VBE as low as 3 V.

Background: Pemetrexed, a multi-targeted antifolate (MTA), is a promising agent in the treatment of malignant pleural mesothelioma (MPM) and non-small cell lung cancer (NSCLC). With the aim of finding an optimal schedule for the combination therapy of MTA and gemcitabine (GEM), we investigated their interaction against an MPM cell line, 211H, and the NSCLC cell lines A549 and H1299. Methods: Combination index analysis was used in 3 different schedules. Cell cycle analysis by flow cytometry and real-time RT-PCR analysis of thymidylate synthase (TS), folylpolyglutamate synthetase (FPGS) and reduced folate carrier 1 (RFC1) genes were performed to understand the biological consequences of their interaction. Results: MTA showed potent cytotoxicity against 211H cells (IC50, 67 nM for 48 h exposure), compared to NSCLC cell lines. Significantly higher expression of FPGS and RFC1 mRNAs in 211H cells were associated with MTA sensitivity. Simultaneous exposure of MTA and GEM was antagonistic in all cell lines tested. Strong synergism was observed in 211H cells when MTA preceded GEM, but the inverted sequence showed antagonism. Similar results were exhibited in H1299 cells, whereas a moderately synergistic effect was observed in A549 cells when GEM preceded MTA. S phase accumulation by MTA treatment partly supported these results. Conclusion: Sequential administration of MTA and GEM is active, and the schedule of MTA followed by GEM is recommended for treating MPM.

The use of probe substrates and combinations of ATP-binding cassette (ABC) transporter knockout (KO) animals may facilitate the identification of common substrates between apparently unrelated ABC transporters. An unexpectedly low concentration of the purine nucleotide analogue, 9-(2-(phosphonomethoxy)ethyl)-adenine (PMEA), and up-regulation of Abcg2 in some tissues of the Mrp4 KO mouse prompted us to evaluate the possibility that Abcg2 might transport purine-derived drugs. Abcg2 transported and conferred resistance to PMEA. Moreover, a specific Abcg2 inhibitor, fumitremorgin C, both increased PMEA accumulation and reversed Abcg2-mediated PMEA resistance. We developed Mrp4 and Abcg2 double KO mice and used both single KOs of Abcg2 and Mrp4 mice to assess the role of these transporters in vivo. Abcg2 contributed to PMEA accumulation in a variety of tissues, but in some tissues, this contribution was only revealed by the concurrent absence of Mrp4. Abcg2 also transported and conferred resistance to additional purine analogues, such as the antineoplastic, 2-chloro-2′-deoxyadenosine (cladribine) and puromycin, a protein synthesis inhibitor that is often used as a dominant selectable marker. Purine analogues interact with ABCG2 by a site distinct from the prazosin binding site as shown by their inability to displace the substrate analogue and photoaffinity tag [125I]iodoarylazidoprazosin. These studies show that Abcg2, like Mrp4, transports and confers resistance to purine nucleoside analogues and suggest that these two transporters work in parallel to affect drug cytotoxicity and tissue distribution. This new knowledge will facilitate an understanding of how Abcg2 and Mrp4, separately and in combination, protect against purine analogue host toxicity as well as resistance to chemotherapy. [Cancer Res 2007;67(14):6965–72]

Background and Objectives Tissue inhibitor of metalloproteinase-3 (TIMP-3) inhibits the activity of metalloproteinases that play important roles in development and progression of malignant tumors. We conducted a retrospective study of TIMP-3 expression in resected non-small cell lung cancer (NSCLC). Methods TIMP-3 expression was examined immunohistochemically in primary tumor specimens from 143 patients who underwent complete resection for NSCLC. Correlations between TIMP-3 expression grade and tumor histology, TNM classification, MMP-2 and MMP-9 expression grade, VEGF expression grade, intra-tumoral microvessel density, proliferative index, apoptosis index, and prognosis were analyzed. Results TIMP-3 expression was low in 40, moderate in 71, and high in 32 patients. Higher TIMP-3 expression was seen in squamous cell carcinoma than in adenocarcinoma (P = 0.001), and reduced TIMP-3 expression was significantly associated with nodal involvement (P = 0.016) and advanced pathologic stage (P = 0.036). MMP-2 expression was reduced along with enhanced TIMP-3 expression (P = 0.010). The 5-year overall survival rates of low, moderate, and high TIMP-3 patients were 53, 64, and 84%, respectively (P = 0.037). Multivariate analysis confirmed that enhanced TIMP-3 expression was an independent factor for a favorable prognosis (P = 0.037). Conclusions TIMP-3 expression status was significantly correlated with pathologic stage and nodal involvement, and was an independent prognostic factor in resected NSCLC. J. Surg. Oncol. 2007;95:250–257. © 2007 Wiley-Liss, Inc.

Fms-like tyrosine kinase 1 (Flt-1), a receptor for vascular endothelial growth factor (VEGF), have two isoforms: membrane-bound form (mFlt-1) and soluble form. In the present study, we quantitatively evaluated expression level of mFlt-1 mRNA and VEGF mRNA in non-small cell lung cancer, and demonstrated the clinical significance of the ratio of mFlt-1 mRNA to VEGF mRNA (mFlt-1/VEGF). High mFlt-1/VEGF tumor showed a significantly lower microvessel density (P=0.004), and patients with high mFlt-1/VEGF tumor had a significantly favorable survival (P=0.037). Thus, the ratio of mFlt-1 mRNA to VEGF mRNA was inversely correlated with tumor angiogenesis, and was a significant prognostic factor.

Carbonyl reductase (CBR) is a cytosolic NADPH-dependent oxidoreductase metabolizing prostaglandins, steroids, quinines, and anthracycline antibiotics. Many experimental studies have shown that CBR plays important roles in the regulation of tumor progression, but clinical significance of CBR status remains unclear. Thus, we conducted a retrospective study on CBR mRNA expression in lung cancer. Tumor tissues obtained from 59 non–small-cell lung cancer patients were analyzed by quantitative real-time reverse transcription-PCR assay to reveal clinical significance of CBR expression. Angiogenesis was measured immunohistochemically as intratumoral microvessel density (IMVD) using anti-CD34 monoclonal antibody CD34-IMVD) and anti-CD105 monoclonal antibody (CD105-IMVD). CBR mRNA expression was significantly reduced along with progression of primary tumors (the mean CBR mRNA/GAPDH mRNA, 3.288 × 10−2 for pT1, 1.628 × 10−2 for pT2, and 1.175 × 10−2 for pT3-4 disease, respectively; P = 0.02). Moreover, CBR mRNA expression in tumor with nodal involvement seemed to be reduced as compared with that in tumor without nodal involvement (the mean CBR mRNA/GAPDH mRNA, 1.446 × 10−2 and 2.531 × 10−2, respectively), whereas the difference did not reach a statistical significance (P = 0.09). The mean CD105-IMVD for CBR-high tumor was 59.2, which was significantly lower than that for CBR-low tumor (130.6, P = 0.02), whereas no significant difference between the mean CD34-IMVDs for CBR-high tumor and CBR-low tumor was found. The 5-year survival rate of CBR-high patients was 68.3%, significantly higher than that of CBR-low patients (36.5%; P = 0.03). A multivariate analysis confirmed that CBR-high expression was a significant factor to predict a favorable prognosis (P = 0.04; relative risk, 0.39; 95% confidence interval, 0.16-0.98). Expression of CBR mRNA was a significant prognostic factor in non–small-cell lung cancer and was inversely associated with tumor progression and angiogenesis.

Maspin is a member of the serpin (serine protease inhibitor) superfamily, and some experimental studies revealed a potential tumor suppressor activity of maspin. To reveal clinical significance of maspin status in non-small cell lung cancer (NSCLC), we quantitatively evaluated maspin gene expression in lung primary tumors cut from a total of 55 resected NSCLC patients. Maspin expression in squamous cell carcinoma (Sq) was significantly higher than that in adenocarcinoma (Ad, p=0.011). Five-year overall survival rates of maspin-high and maspin-low patients were 67.7 and 41.4%, respectively, demonstrating a significant favorable prognosis of maspin-high patients (log-rank, p=0.042). A multivariate analysis confirmed that high maspin expression was an independent and significant factor to predict a favorable overall survival (p=0.031). These results suggested that maspin expression was significantly increased in Sq than in Ad, and that increased maspin expression was a significant factor to predict a favorable prognosis in resected NSCLC.

MAGE-D4, originally termed MAGE-E1, is a novel MAGE family gene that is expressed at high levels in malignant tumors as compared to normal tissue. The present study was conducted to assess the clinical significance of MAGE-D4 expression in non-small cell lung cancer (NSCLC).Expression of MAGE-D4 protein was estimated by immunohistochemistry and MAGE-D4 mRNA expression was investigated using quantitative reverse transcription-PCR (RT-PCR).We assessed MAGE-D4 expression in NSCLC tissues and was found to be up-regulated in tumor tissues compared with normal lung tissues (mean MAGE-D4/GAPDH values, 0.035 for tumor tissues and 0.009 for normal lung tissues; p=0.002). However, there was no significant difference in MAGE-D4 expression among different pathological stages. The proliferative activity of tumor cells was significantly higher in high MAGE-D4 tumors (mean proliferative indices, 58.3 for high MAGE-D4 tumor levels and 34.0 for low MAGE-D4 tumor levels; p0.001). In addition, a high MAGE-D4 expression was more frequently seen in squamous cell carcinoma than in adenocarcinoma (p=0.008), and less frequently in well-differentiated tumors than in moderately to poorly differentiated tumors (p=0.036). There was no difference in the postoperative survival between low and high MAGE-D4 patients (5-year survival rates, 65% and 69%, respectively; p=0.742).MAGE-D4 plays some roles in tumor cells proliferation in NSCLC, but MAGE-D4 expression status did not provided a prognostic significance.

The melanoma differentiation-associated gene-7 (MDA-7) protein, also known as interleukin (IL)-24, is a novel candidate of tumor suppressor that can induce apoptosis experimentally in a variety of human malignant cells including lung cancer cells. However, only one clinical study has documented that MDA-7/IL-24 expression is down-regulated with progression of melanoma. Thus, the present study was conducted to assess the clinical significance of MDA-7/IL-24 expression in non-small cell lung cancer.A total of 183 consecutive patients with resected pathologic stage I-IIIA, non-small cell lung cancer were retrospectively reviewed, and immunohistochemical staining was used to detect MDA-7/IL-24 expression.MDA-7/IL-24 expression was high in 97 (53.0%) patients and low in the other patients. There was no significant correlation between MDA-7/IL-24 status and any patients' characteristic including pathologic stage. There was no significant difference in tumor angiogenesis or proliferative activity according to MDA-7/IL-24 status, but MDA-7/IL-24-high adenocarcinoma showed a significantly higher incidence of apoptotic tumor cell death than MDA-7/IL-24-low adenocarcinoma. MDA-7/IL-24-high patients seemed to show a favorable postoperative prognosis as compared with MDA-7/IL-24-low patients (5-year survival rates, 75.9% and 62.0%, respectively), although the difference did not reach a statistical significance (P = 0.061). Subset analyses showed that positive MDA-7/IL-24 expression was a significant factor to predict a favorable prognosis in adenocarcinoma (P = 0.033), which was confirmed by a multivariate analysis; there was no difference in the prognosis according to MDA-7/IL-24 status in squamous cell carcinoma.MDA-7/IL-24 status was a significant prognostic factor in lung adenocarcinoma, not in lung squamous cell carcinoma.

Purpose: Experimental studies have revealed that fms-like tyrosine kinase (Flt)-4 plays important roles in lymphangiogenesis in malignant tumors, but the clinical significance remains unclear. We assessed Flt-4 expression in tumor cells and in endothelial cells in correlation with clinical outcomes in non–small cell lung cancer (NSCLC). Experimental Design: A total of 206 consecutive patients with resected pathological stage I-IIIA NSCLC were reviewed. Expression of Flt-4 was examined immunohistochemically, and Flt-4–positive microvessels were quantitatively evaluated (Flt-4–positive endothelial cell density). Results: There was no significant correlation between Flt-4–positive endothelial cell density and any characteristic of patients including nodal metastases. A significant correlation between Flt-4–positive endothelial cell density and Flt-4 status in tumor cells was documented (P < 0.001), but there was no significant difference in the mean Flt-4–positive endothelial cell density according to vascular endothelial growth factor-C or -D status in tumor cells. The 5-year survival rate for higher Flt-4–positive endothelial cell density tumor (56.4%) was significantly lower than that of lower Flt-4–positive endothelial cell density tumor (69.0%, P = 0.046); the prognostic significance was enhanced in pIIIA-N2 patients (5-year survival rates, 18.8% for higher Flt-4–positive endothelial cell density tumor and 50.0% for lower Flt-4–positive endothelial cell density tumor, respectively; P = 0.012). A multivariate analysis confirmed that higher Flt-4–positive endothelial cell density was a significant and independent prognostic factor (P = 0.019). CD34-positive vessel density or Flt-4 status in tumor cells was not a significant prognostic factor. Conclusions: Flt-4–positive endothelial cell density, not Flt-4 status in tumor cells, was a significant prognostic factor in NSCLC.

Purpose: The purpose is to assess clinical significance of matrix metalloproteinase (MMP)-2 and MMP-9 status, especially MMP-2 status, in stromal cells in non–small-cell lung cancer (NSCLC) because experimental studies have revealed that stromal MMP-2 plays important roles in progression of malignant tumors, but most clinical studies focused on tumoral MMP-2 expression, not stromal MMP-2 expression.Experimental Design: We conducted a retrospective study on MMP-2 and MMP-9 expression as evaluated immunohistochemically in a total of 218 consecutive patients with completely resected pathological stage I–IIIA, NSCLC.Results: Strong MMP-2 expression in tumor cells and stromal fibroblasts were documented in 54 (24.8%) and 132 (60.6%) patients, respectively. Strong MMP-2 expression in stromal fibroblasts was more frequently seen in squamous cell carcinoma (72.7%) than in adenocarcinoma (54.9%; P = 0.016). Tumors showing strong MMP-2 expression in stromal fibroblasts showed a significantly higher intratumoral microvessel density (IMVD) than weak stromal MMP-2 tumors (mean intratumoral microvessel density, 50.9 versus 32.4, P = 0.003). In addition, postoperative prognosis of strong stromal MMP-2 patients was significantly poorer than that of weak stromal MMP-2 patients (5-year survival rate, 77.5 versus 60.2%, P = 0.032), and the prognostic significance was enhanced in squamous cell carcinoma patients but disappeared in adenocarcinoma patients. Multivariate analyses confirmed that strong stromal MMP-2 expression was a significant factor to predict a poor prognosis in squamous cell carcinoma patients, not in adenocarcinoma patients. In contrast, MMP-2 or MMP-9 status in tumor cells was not a significant prognostic factor.Conclusions: MMP-2 status in stromal fibroblasts, not in tumor cells, was a significant prognostic factor associated with angiogenesis in NSCLC.

Purpose To evaluate the clinical usefulness of postoperative systemic inflammatory response syndrome (SIRS) as an index of surgical stress in patients undergoing thoracic surgery. Methods Forty-five consecutive patients who underwent thoracic surgery with thoracotomy were enrolled. The SIRS criteria were examined daily during the first 7 postoperative days. The serum interleukin-6 (IL-6) level, operation time, intraoperative blood loss, amount of thoracic drainage, and C-reactive protein levels were also measured. Results Sixteen cases were categorized into the SIRS group, whereas 29 cases were categorized into the non-SIRS group. Among the patients who underwent thoracic surgery, the physiological responses of the patients to the surgery, such as serum IL-6 levels and C-reactive protein levels, were significantly higher in the SIRS group than in the non-SIRS group ( P = .002 and .024, respectively). The serum IL-6 level on the first postoperative day was an independent factor associated with SIRS (95% CI 1.002-1.041; P = .030). Furthermore, there was a correlation between the number of SIRS days and the duration of the postoperative hospital stay ( r = 0.379, P = .012). Conclusions Our results demonstrated that SIRS reflected the degree of surgical stress, especially thoracotomic procedures, through the IL-6 levels, and affected the postoperative hospital stay. Systemic inflammatory response syndrome can be useful for the postoperative management of patients undergoing thoracic surgery.

Postoperative prognosis for patients with pathologic (p-) stage IIIA-N2 non-small-cell lung cancer (NSCLC) is poor, and significant factors that influence the prognosis remain unclear.A total of 99 patients who underwent complete resection for p-stage IIIA-N2 NSCLC without any preoperative therapy were retrospectively reviewed. Biological features such as tumor angiogenesis (intratumoral microvessel density [IMVD]), proliferative activity (proliferative index [PI]), and p53 status were also evaluated immunohistochemically.Univariate analysis revealed that the number of involved N2 stations was a significant prognostic factor; 5-year survival rates for a tumor with metastases in single N2 stations, tumor with metastases in two N2 stations, and tumor with metastases in 3 or more N2 stations were 41.6%, 35.3%, and 0.0%, respectively (P =.041) In addition, the 5-year survival rate for cN0-1 disease was significantly higher than that for cN2 disease (41.9% and 25.5%, respectively; P =.048) Tumor angiogenesis and proliferative activity were the most significant prognostic factors; 5-year survival rates for lower-IMDV tumor and higher-IMVD tumor were 53.6% and 15.9%, respectively (P =.002), and those for lower-PI tumor and higher-PI tumor were 47.0% and 20.4%, respectively (P =.019) There was no difference in the postoperative survival between tumor showing aberrant p53 expression and tumor showing no aberrant p53 expression. These results were confirmed by a multivariate analysis.P-stage IIIA-N2 NSCLC cases represented a mixture of heterogeneous prognostic subgroups, and the number of involved N2 stations, cN status, PI, and IMVD were significant predictors of the survival.

The reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) was initially isolated as a transformation-suppressor gene by expression cloning and found to encode a membrane-anchored regulator of the matrix metalloproteinases (MMPs). Experimental studies have shown that RECK can suppress tumour - invasion, metastasis and angiogenesis. However, the clinical impact of RECK remains unclear. To assess the clinical significance of RECK-expression in non-small cell lung cancer (NSCLC), a total of 171 patients with completely resected pathological stage (p-stage) I-IIIA NSCLC were retrospectively examined. Expression of RECK and vascular endothelial growth factor (VEGF) in tumour tissues was assessed by immunohistochemical staining (IHS). Intratumoural microvessel density (IMVD), a measurement of angiogenesis, was also determined by IHS using an anti-CD34 antibody. A significant inverse correlation between RECK-expression and tumour angiogenesis was documented; the mean IMVD in tumours with strong RECK-expression (157.1) was significantly lower than that observed in tumours with weak RECK-expression (194.5; P = 0.008). Interestingly, this inverse correlation was seen only when VEGF was strongly expressed, which suggests that RECK could suppress the angiogenesis induced by VEGF. The 5-year survival rate for patients with tumours with strong RECK-expression (75.8%) was significantly higher than that for patients with weakly expressing tumours (54.3%; P = 0.016). Subset analyses showed that the prognostic impact of RECK-status was evident in patients with either adenocarcinoma, poorly differentiated tumours, or p-stage IIIA disease. A multivariate analysis confirmed that reduced RECK-expression was an independent and significant factor in predicting a poor prognosis (P = 0.009; Hazard ratio (HR), 0.474 with a 95% Confidence interval (CI) of 0.271-0.830). In conclusion, RECK-status is a significant prognostic factor correlated with tumour angiogenesis in NSCLC patients.

LUN is a novel RING finger protein that is highly expressed in the lung and might be a transcriptional regulator of E-cadherin [J. Biol. Chem. 276 (2001) 14004]. It might be possible that LUN plays important roles in the development and progression of lung cancer through regulating expression of E-cadherin, but no clinical study on LUN expression has been reported. In the present study, we quantitatively examined gene expression of the LUN in surgical specimens resected from non-small cell lung cancer (NSCLC) patients. In normal lung tissues, the LUN gene expression was down-regulated in smokers (the mean LUN/GAPDH ratios, 0.222 for non-smokers and 0.144 for smokers; P = 0.030). In addition, the mean LUN/GAPDH ratio in lung cancer tissues was significantly lower than that in normal lung tissues (0.072 versus 0.162; P < 0.001). In addition, the LUN gene expression was slightly down-regulated along with progression of primary tumors, and strongly down-regulated along with nodal metastases (the mean LUN/GAPDH ratios, 0.091 for pN0, 0.073 for pN1, and 0.034 for pN2 diseases; P = 0.001). These results suggested that LUN might play important roles in inhibition of nodal metastases as well as in suppression of smoking-related oncogenesis in NSCLC.

Glomeruloid microvascular proliferation (GMP) is a focal proliferative budding of endothelial cells (ECs) resembling a renal glomerulus. Whereas some experimental and clinical studies have suggested recently that GMPs indicate an aggressive angiogenic phenotype, the incidence and clinical significance of GMPs remains unclear. Thus, we conducted a retrospective study on GMPs in a total of 236 patients with completely resected pathological (p-) stage I-IIIA NSCLC. ECs were highlighted with immunohistochemical staining using an anti-CD34 antibody, and GMPs were defined as focal glomerulus-like aggregates of closely associated and multilayer CD34-positive ECs. Expression of vascular endothelial growth factor, angiopoietin (Ang)-1, and Ang-2 was also examined immunohistochemically. GMPs were positive in 60 (25.4%) patients, and the incidence was not correlated with age, gender, histological type, or p-stage. The mean intratumoral microvessel densities for GMP-negative tumor and GMP-positive tumor were 178.2 and 184.1, respectively, showing that the incidence of GMPs was not correlated with intratumoral microvessel density (P = 0.676). There was no correlation between vascular endothelial growth factor expression and the incidence of GMPs, but GMPs were more frequently seen in Ang-1-positive tumor than in Ang-1-negative tumor. The 5-year survival rate of GMP-positive patients was 54.2%, which was significantly lower than that of GMP-negative patients (72.3%; P = 0.016). The 5-year survival rate of higher-MVD patients (71.5%) seemed to be lower than that of the lower-MVD patients (63.7%), but the difference did not reach a statistical significance (P = 0.137). A multivariate analysis confirmed that the presence of GMPs was a significant prognostic factor (P = 0.003), whereas MVD was not. In conclusion, GMPs indicate an aggressive angiogenic phenotype associated with a poor prognosis in NSCLC.

To reveal the incidence and clinical significance of mediastinal nodal metastases without N1-station nodal metastases ('skip-N2 metastases') in non-small cell lung cancer (NSCLC).A total of 450 NSCLC patients who underwent tumor resection with a systemic mediastinal nodal dissection were retrospectively reviewed. p53 status and proliferative activity represented as proliferative index (PI) were also examined immunohistochemically.Skip-N2 metastases were documented in 49 (13%) patients of all 450 patients; among 334 patients without N1-nodal involvement, 18% patients had skip-N2 metastases. The postoperative survival of skip-N2 patients was almost same as that for patients with metastases to both N1 and N2 nodes. Skip-N2 metastases were significantly more frequent in male patients and squamous cell carcinoma patients. In addition, the mean PI for tumor with skip-N2 metastases was significantly higher than that for any other pathologic nodal (pN)-status diseases. Combined with histologic type and PI, the incidences of skip-N2 metastases for adenocarcinoma showing lower PI were only 5% (7/137) of all patients and 7% (7/94) of patients without N1-nodal involvement.N1 nodal status is not a useful predictor of N2 nodal status in NSCLC, because skip-N2 metastases were documented in 18% patients showing no N1-nodal involvement. However, N1 node-guided dissection might be performed in patients with adenocarcinoma showing lower PI, because the incidence of skip-N2 metastases was extremely low.