The ABCC4 membrane transporter modulates platelet aggregation.

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.

Human immunodeficiency virus protease inhibitors interact with ATP binding cassette transporter 4/multidrug resistance protein 4: a basis for unanticipated enhanced cytotoxicity.

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.

Impact of KRAS and EGFR gene mutations on recurrence and survival in patients with surgically resected lung adenocarcinomas.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

D2-40-positive lymphatic vessel density is a poor prognostic factor in squamous cell carcinoma of the lung.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Substrate overlap between Mrp4 and Abcg2/Bcrp affects purine analogue drug cytotoxicity and tissue distribution.

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 [(125)I]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.

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

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.

Perimembrane Aurora-A expression is a significant prognostic factor in correlation with proliferative activity in non-small-cell lung cancer (NSCLC).

PURPOSE: Aurora-A, also known as STK15/BTAK, is a member of the protein serine/threonine kinase family, and experimental studies have revealed that Aurora-A plays critical roles in cell mitosis and in carcinogenesis. However, no clinical studies on Aurora-A expression in non-small-cell lung cancer (NSCLC) have been reported. Thus, the present study was conducted to assess the clinical significance of Aurora-A status. EXPERIMENTAL DESIGN: A total of 189 consecutive patients with resected pathologic (p-)stage I-IIIA, NSCLC were retrospectively reviewed, and immunohistochemical staining was used to detect Aurora-A expression. RESULTS: Aurora-A expression was negative in 31 patients (16.4%); among Aurora-A positive patients, 124 patients showed pure diffuse cytoplasmic Aurora-A expression and the other 34 patients showed perimembrane Aurora-A expression. Perimembrane Aurora-A tumors showed the highest proliferative index (PI) (mean PIs for negative, diffuse cytoplasmic, and perimembrane tumors: 49.2, 41.7, and 63.5, respectively; P < .001). Five-year survival rates of Aurora-A negative, diffuse cytoplasmic, and perimembrane patients were 67.8%, 66.7%, and 47.6%, respectively, showing the poorest postoperative survival in perimembrane patients (P = .033). Subset analyses revealed that perimembrane Aurora-A expression was a significant factor to predict a poor prognosis in squamous cell carcinoma patients, not in adenocarcinoma patients. A multivariate analysis confirmed that perimembrane Aurora-A expression was an independent and significant factor to predict a poor prognosis. CONCLUSIONS: Perimembrane Aurora-A status was a significant factor to predict a poor prognosis in correlation with enhanced proliferative activity in NSCLC.

Schedule-dependent synergistic effect of pemetrexed combined with gemcitabine against malignant pleural mesothelioma and non-small cell lung cancer cell lines.

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 (IC(50), 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 ratio of membrane-bound form Flt-1 mRNA to VEGF mRNA correlates with tumor angiogenesis and prognosis in non-small cell lung cancer.

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.

Maspin gene expression is a significant prognostic factor in resected non-small cell lung cancer (NSCLC). Maspin in NSCLC.

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.

Expression of MAGE-D4, a novel MAGE family antigen, is correlated with tumor-cell proliferation of non-small cell lung cancer.

PURPOSE: 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). EXPERIMENTAL DESIGN: Expression of MAGE-D4 protein was estimated by immunohistochemistry and MAGE-D4 mRNA expression was investigated using quantitative reverse transcription-PCR (RT-PCR). RESULTS: 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; p<0.001). 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). CONCLUSIONS: MAGE-D4 plays some roles in tumor cells proliferation in NSCLC, but MAGE-D4 expression status did not provided a prognostic significance.

Expression of MDA-7/IL-24 and its clinical significance in resected non-small cell lung cancer.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: MDA-7/IL-24 status was a significant prognostic factor in lung adenocarcinoma, not in lung squamous cell carcinoma.

Systemic inflammatory response syndrome and surgical stress in thoracic surgery.

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.

Glomeruloid microvascular proliferation is superior to intratumoral microvessel density as a prognostic marker in non-small cell lung cancer.

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.

Carbonyl reductase expression and its clinical significance in non-small-cell lung cancer.

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, 3.288x10(-2) for pT1, 1.628x10(-2) for pT2, and 1.175x10(-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, 1.446x10(-2) and 2.531x10(-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.

Flt-4-positive endothelial cell density and its clinical significance in non-small cell lung cancer.

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.

Matrix metalloproteinase-2 status in stromal fibroblasts, not in tumor cells, is a significant prognostic factor in non-small-cell lung cancer.

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.

Expression of a novel matrix metalloproteinase regulator, RECK, and its clinical significance in resected non-small cell lung cancer.

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.

Expression of LUN gene that encodes a novel RING finger protein is correlated with development and progression of non-small cell lung cancer.

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.

Endobronchial closure of postoperative bronchopleural fistula using vascular occluding coils and n-butyl-2-cyanoacrylate.

We report herein 2 patients with intractable postoperative bronchopleural fistula with empyema after lobectomy or subsegmentectomy. The patients underwent several treatments including thoracotomy, but the fistula closure was not successful. Finally, the bronchopleural fistula was successfully treated by endobronchial closure using vascular occluding coils and n-butyl-2-cyanoacrylate (Histoacryl).