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.

Deoxycytidine kinase modulates the impact of the ABC transporter ABCG2 on clofarabine cytotoxicity.

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.

Stromal aminopeptidase N expression: correlation with angiogenesis in non-small-cell lung cancer.

PURPOSE: 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. METHODS: We evaluated APN expression in non-small-cell lung cancer patients by immunohistochemistry. RESULTS: 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. CONCLUSION: 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.

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.

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.

Transporter-mediated protection against thiopurine-induced hematopoietic toxicity.

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.

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.

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.

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.

Expression of tissue inhibitor of metalloproteinase-3 (TIMP-3) and its prognostic significance in resected non-small cell lung cancer.

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.

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.

Influence of epidermal growth factor receptor (EGFR) gene mutations on the expression of EGFR, phosphoryl-Akt, and phosphoryl-MAPK, and on the prognosis of patients with non-small cell lung cancer.

BACKGROUND AND OBJECTIVES: In this paper we examined the influence of epidermal growth factor receptor (EGFR) gene mutations on EGFR expression, downstream mediators, and survival in patients with non-small cell lung cancer (NSCLC). METHODS: We retrospectively analyzed the tumors of 53 patients with completely resected pathological stage I-IIIA NSCLC for the presence of EGFR gene mutations, the expression of EGFR mRNA and protein, phosphoryl-Akt, and phosphoryl-mitogen-activated protein kinase (MAPK) using immunostaining, and patients' prognosis. RESULTS: EGFR mutations were associated with elevations in EGFR mRNA (P = 0.004) and protein (P = 0.029) expression, but not with the expression of phosphoryl-Akt or phosphoryl-MAPK. The 5-year survival rate for all patients who exhibited an EGFR mutation was similar to those who were free of such mutations (71% vs. 56%, P = 0.252). However, the 5-year survival rate of patients with either a stage I adenocarcinoma or large cell carcinoma who had an EGFR mutation was significantly greater than for those who did not have such a mutation (92% vs. 57%, P = 0.037). CONCLUSIONS: EGFR gene mutations were significantly associated with higher EGFR expression, but not with p-Akt or p-MAPK status. In early stage NSCLC, the presence of an EGFR gene mutation bode well for the patient's prognosis.

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

BACKGROUND: Maspin is a member of the serpin (serine protease inhibitor) superfamily, and its exact function in the development and progression of malignant tumors remains controversial, though some experimental studies have revealed potential tumor-suppressor activities. In addition, there have been only a few clinical studies on maspin expression in malignant tumors including non-small cell lung cancer (NSCLC). The purpose of this study was to assess maspin expression and its clinical significance in NSCLC. METHODS: A total of 210 consecutive patients with completely resected pathological (p-) stage I-IIIA NSCLC were retrospectively reviewed. Maspin expression along with intratumoral microvessel density, proliferative activity, and p53 status were evaluated immunohistochemically. The incidence of apoptotic cell death was also evaluated. RESULTS: The incidence of strong maspin expression was significantly higher in lung squamous cell carcinoma (56/76, 73.7%; P < .001) than in other histological types. The incidence of aberrant expression of p53 was significantly higher in maspin-strong than in maspin-weak tumors (56.2% and 35.8%, respectively; P = .005). There was no difference in prognosis according to maspin status for all patients. However, for squamous cell carcinoma patients, univariate analysis showed that enhanced maspin expression was a significant factor in predicting a favorable prognosis (5-year survival rates, 70.1% for maspin-strong tumors and 41.5% for maspin-weak tumors; P = .014), which was confirmed in a multivariate analysis (hazard ratio = .475, 95% confidence interval .241-.936; P = .032). CONCLUSIONS: Enhanced maspin expression was a significant and independent factor in predicting a favorable prognosis 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.

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.

A novel classification of MUC1 expression is correlated with tumor differentiation and postoperative prognosis in non-small cell lung cancer.

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.

Prognostic significance of reversion-inducing cysteine-rich protein with Kazal motifs expression in resected pathologic stage IIIA N2 non-small-cell lung cancer.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: RECK status was a novel prognostic factor in pathologic stage IIIA N2 NSCLC.