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Interleukin (IL)-18 plays an important role in enhancing cellular immunity against cancer and bacteria. We constructed retroviral and adenoviral vectors that show improved secretion of bioactive murine IL-18 that could further enhance antitumor immunity in a murine model. Secretion of bioactive IL-18 was facilitated by fusing the leader sequences of prepro-parathyroid hormone (PTH) or IL-1 receptor antagonist (IL-1ra) to the 5; end of the mature murine IL-18 cDNA. Transfectants established by the retroviral vector carrying IL-1ra/IL-18 hybrid showed about 100-fold more IL-18 production and interferon (IFN)-gamma induction from splenocytes when compared with those carrying PTH/IL-18 hybrid. Repeated intraperitoneal injection of an adenoviral vector with IL-1ra/IL-18 hybrid ligated to IL-18 (Ad.IL-1ra.IL-18) successfully prevented establishment of human colon cancer cells in the abdominal cavity of mice. Treatment with Ad.IL-1ra.IL-18 was associated with significantly elevated levels of serum IL-18 and IFN- gamma. IL-18 administration also enhanced the cytostatic activity of peritoneal exudate cells against cancer cells. These improved viral vectors, which efficiently produce bioactive IL-18, could be used as a useful tool for cancer gene therapy.

It has been reported that transforming growth factor (TGF)-beta, which plays an integral role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), suppresses proliferation of alveolar epithelial cells in vitro. Although hyperplastic lesions of alveolar lining epithelial cells (ALECs) are characteristic pathologic features of IPF, the mechanism of their involvement in the pathogenesis has not yet been extensively studied. On the assumption that the hyperplastic ALECs have escaped from the growth-inhibitory effects of TGF-beta, we searched for mutations in the microsatellite of the TGF-beta receptor type II (T beta RII) gene. To detect a deletion in the polyadenine tract in exon 3 of the T beta RII gene, cells were isolated by microdissection from lung sections of IPF patients, and DNA was extracted from these cells and amplified by high-fidelity polymerase chain reaction. A total of 121 sites of hyperplastic ALECs from 11 IPF patients were analyzed, and a one-base-pair deletion was detected in nine sites from five patients. The mutation was also detected in smooth muscle-like cells of the thickened pulmonary artery. In some tissue areas where the deletion was detected, low T beta RII expression was confirmed by immunohistochemical staining. These data suggest that microsatellite instability in the T beta RII gene occurred in some lesions of hyperplastic ALECs in IPF, although at a low incidence, and that this genetic disorder might play a partial role in the pathologic changes of IPF.

Interleukin (IL)-10 has been shown to reduce many inflammatory reactions. We investigated the in vivo effects of IL-10 on a bleomycin-induced lung injury model. Hemagglutinating virus of Japan (HVJ)-liposomes containing a human IL-10 expression vector (hIL10-HVJ) or a balanced salt solution as a control (Cont-HVJ) was intraperitoneally injected into mice on day −3. This was followed by intratracheal instillation of bleomycin (0.8 mg/kg) on day 0. Myeloperoxidase activity of bronchoalveolar lavage fluid and tumor necrosis factor-α mRNA expression in bronchoalveolar lavage fluid cells on day 7 and hydroxyproline content of the whole lung on day 21 were inhibited significantly by hIL10-HVJ treatment. However, Cont-HVJ treatment could not suppress any of these parameters. We also examined the in vitro effects of IL-10 on the human lung fibroblast cell line WI-38. IL-10 significantly reduced constitutive and transforming growth factor-β-stimulated type I collagen mRNA expression. However, IL-10 did not affect the proliferation of WI-38 cells induced by platelet-derived growth factor. These data suggested that exogenous IL-10 may be useful in the treatment of pulmonary fibrosis.

The role of transmembrane 4 superfamily (TM4SF) proteins during muscle cell fusion has not been investigated previously. Here we show that the appearance of TM4SF protein, CD9, and the formation of CD9–β1 integrin complexes were both regulated in coordination with murine C2C12 myoblast cell differentiation. Also, anti-CD9 and anti-CD81 monoclonal antibodies substantially inhibited and delayed conversion of C2C12 cells to elongated myotubes, without affecting muscle-specific protein expression. Studies of the human myoblast-derived RD sarcoma cell line further demonstrated that TM4SF proteins have a role during muscle cell fusion. Ectopic expression of CD9 caused a four- to eightfold increase in RD cell syncytia formation, whereas anti-CD9 and anti-CD81 antibodies markedly delayed RD syncytia formation. Finally, anti-CD9 and anti-CD81 monoclonal antibodies triggered apoptotic degeneration of C2C12 cell myotubes after they were formed. In summary, TM4SF proteins such as CD9 and CD81 appear to promote muscle cell fusion and support myotube maintenance.

The levels of soluble IL-2R were measured in pleural fluid from patients with tuberculosis pleurisy. There were significantly elevated soluble IL-2R values in tuberculous pleural fluid as compared with pleural fluid of nontuberculous etiology including malignant, bacterial and transudative pleural effusions. In patients with tuberculous pleurisy, the level of soluble IL-2R in pleural fluid was markedly greater than that in serum. Furthermore, a significant positive correlation was observed between soluble IL-2R levels and adenosine deaminase levels in tuberculous pleural fluid. These findings suggest that elevated levels of pleural fluid soluble IL-2R in tuberculous pleurisy could reflect the local proliferation of activated T-cells and may be clinically useful in the diagnostic procedures for patients with pleural tuberculosis.

SRPX2 (Sushi repeat-containing protein, X-linked 2) has recently emerged as a multifunctional protein that is involved in seizure disorders, angiogenesis and cellular adhesion. Here, we analyzed this protein biochemically. SRPX2 protein was secreted with a highly post-translational modification. Chondroitinase ABC treatment completely decreased the molecular mass of purified SRPX2 protein to its predicted size, whereas heparitinase, keratanase and hyaluroinidase did not. Secreted SRPX2 protein was also detected using an anti-chondroitin sulfate antibody. These results indicate that SRPX2 is a novel chondroitin sulfate proteoglycan (CSPG). Furthermore, a binding assay revealed that hepatocyte growth factor dose-dependently binds to SRPX2 protein, and a ligand–glycosaminoglycans interaction was speculated to be likely in proteoglycans. Regarding its molecular architecture, SRPX2 has sushi repeat modules similar to four other CSPGs/lecticans; however, the molecular architecture of SRPX2 seems to be quite different from that of the lecticans. Taken together, we found that SRPX2 is a novel CSPG that is overexpressed in gastrointestinal cancer cells. Our findings provide key glycobiological insight into SRPX2 in cancer cells and demonstrate that SRPX2 is a new member of the cancer-related proteoglycan family.

Objective The aim of this study was to evaluate the efficacy and toxicity of S-1, with adjusted doses based on body surface area (BSA) and creatinine clearance (Ccr), in chemonaive elderly patients with advanced non-small-cell lung cancer (NSCLC). Pharmacokinetic analysis was also carried out to evaluate the adequacy of the tailored S-1 dose. Methods Twenty-three patients with age 75 or older were enrolled from December 2007 to October 2009. S-1 was administered orally on days 1–28 twice daily (q42 days) until the first occurrence of disease progression, unacceptable toxicity or patient refusal. Doses of S-1 were adjusted based on individual Ccr and BSA. Pharmacokinetic studies were also carried out in 8 patients on day 7 of the first cycle. The primary objective is disease control rate (DCR; CR + PR + SD) and the secondary objectives are objective response rate (ORR), safety, overall survival (OS), progression-free survival (PFS) and clinical benefit rate (CBR; the rate of CR + PR + SD >24 weeks). Results The ORR was 8.7% (2/23), DCR was 69.6% (16/23) and the CBR was 30.4% (7/23). The median survival time (MST) was 228 days and the median PFS was 117 days. As for safety, grade 3/4 hematologic toxic effects have never been observed. As non-hematologic toxic effects, only one patient (4.3%) has experienced grade 3 anorexia and nausea/vomiting. In the pharmacokinetic study group (n = 8), the maximum plasma concentration (Cmax) and the area under the plasma concentration curve (AUC) of 5-FU were similar among eight patients irrespective of Ccr values which were comparable with the reported values in patients with normal renal function who received a standard dose of S-1 (80 mg/m2/day). Conclusions Our results suggest that tailored S-1 monotherapy is useful and tolerable as the first-line treatment in elderly patients with advanced NSCLC even when their renal functions are impaired.

Background After gefitinib was approved in July 2002, we experience long-term surviving patients in the actual clinical setting. However, it is not clear how the factors or treatment strategy are contributing to the long-term surviving patients. We evaluated the effects of clinical backgrounds and treatment histories on overall survival (OS). Methods We extracted information on advanced NSCLC patients with the following inclusion criteria from the medical records: (i) patients who were diagnosed by October 2010 and treated with gefitinib after July 2002; (ii) performance status (PS) 0–2, (iii) PR, CR or long SD (6 months or more) by gefitinib treatment; (iv) patients who had not received curative surgical operation or curative radiation therapy. Primary objective is to evaluate the survival time of the patients who responded to gefitinib and clarify the relationship between clinical factors and survival time. We also conducted ‘Dynamic Treatment Regimen Analysis (DTRA)’ to explore the key treatment regimen and the sequence of regimens contributing to long-term survival. Results The medical records of total of 275 patients were extracted; 44% (122/275) were EGFR mutation examined and 93% (114/122) has shown the EGFR mutation positive. The mean age was 65, 72% (198/275) were women, 66% (182/275) were non-smokers and 90% (247/275) had adenocarcinoma histology. 20% (54/275) and 21% (58/275) underwent re-administration and beyond PD administration of gefitinib, respectively. The median survival time (MST) was 615 days (95% CI: 519–691). 10% patients survived for more than 5 years. The multivariate Cox analysis demonstrated that sex (P = 0.0108) and gefitinib re-administration (P = 0.0012) were significant independent factors for long-term OS. Grade 3/4 interstitial lung disease, skin, diarrhea and liver dysfunction were observed in 1.5, 4.4, 1.1 and 13.1% of the patients, respectively. Conclusions This study suggests that sex and gefitinib re-administration may have significant affects on OS in long survivors after responding gefitinib treatment.

Small Cell Lung Cancer (SCLC) easily acquires multidrug resistance after successful initial therapy. Overexpression of ATP-binding cassette (ABC) transporters is important for the multidrug resistance. Among them, ABCB1 and ABCG2 are known to be upregulated in chemoresistant SCLC. We found that human epidermal growth factor receptor 2 (HER2) expression is also upregulated in chemoresistant SBC-3/ETP, SBC-3/SN-38 and SBC-3/CDDP cells, compared to chemosensitive SBC-3 cells. Lapatinib, a tyrosine kinase inhibitor of HER2, could not suppress proliferation of these HER2-positive SCLC cells, but successfully restored chemosensitivity to etoposide and SN-38 in SBC-3/ETP and SBC-3/SN-38 cells, respectively. The reversal effect of lapatinib was thought to be caused by inhibition of drug efflux-pump functions of ABC transporters, and was producible with a clinically applicable concentration. Moreover, knocking down of HER2 by a small interfering RNA weakened the effect of lapatinib on ABCB1, indicating the involvement of HER2 in the inhibitory mechanisms. Notably, we demonstrated that caveolin-1 and Src play key roles in modulating ABCB1 function via HER2 inactivation. In SBC-3/ETP cells, dephosphorylation of HER2 by lapatinib activates Src and successively leads to increase caveolin-1 phosphorylation. Through this process, caveolin-1 dissociates from HER2 and strengthens association with ABCB1, and finally impairs the pump functions. Furthermore, we demonstrated that treatment by lapatinib in combination with etoposide or irinotecan significantly suppresses the growth of subcutaneous SBC-3/ETP and SBC-3/SN-38 tumors in mice, respectively. Collectively, these results indicate that combination therapy with lapatinib and cytotoxic agents could conquer ABC transporter-mediated chemoresistance in SCLC, especially in HER2-positive cases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 778. doi:1538-7445.AM2012-778

Serum levels of soluble interleukin-2 receptor (IL-2R) were measured in patients with pulmonary tuberculosis, nontuberculous pulmonary diseases (bacterial pneumonia, lower respiratory tract infection, lung cancer), and in normal volunteers. Patients with tuberculosis had increased levels of soluble IL-2R compared to normal controls. Abnormally elevated levels were also shown in patients with nontuberculous pulmonary diseases, suggesting that elevations of soluble IL-2R are not specific for tuberculosis. In patients with tuberculosis, elevated levels of soluble IL-2R were steadily decreased to normal levels during successful treatment. Additionally, soluble IL-2R levels in tuberculosis were closely correlated with adenosine deaminase levels. Thus, it seems possible that measurements of soluble IL-2R may be beneficial in the diagnosis and the management of patients with tuberculosis. Furthermore, we demonstrated that elevated levels of soluble IL-2R in tuberculosis appear to be a consequence of cellular activation of mononuclear cells and not to be the result of cell death with subsequent IL-2R release.

We measured adenosine deaminase (ADA), lysozyme, fibronectin and carcinoembryonic antigen (CEA) in the pleural fluid of tuberculous and carcinomatous pleural effusion in order to discriminate these two groups. Tuberculous pleural effusion had significantly higher levels of ADA and lysozyme than did carcinomatous effusion. When ADA activity of more than 33 IU/ι is considered, diagnostic tests of tuberculous effusions showed a sensitivity of 100%, specificity of 95% and accuracy of 96%. A pleural fluid/serum ADA ratio (pl-ADA/s-ADA) above 1.1 was found in 100% of tuberculous and in 53% of carcinomatous effusions (sensitivity 100%, specificity 47%, diagnostic accuracy 70%). A lysozyme level above 12 μg/ml, selected as the discriminating limit, was found in 100% of tuberculous and in 17% of carcinomatous effusions (sensitivity 100%, specificity 83%, diagnostic accuracy 88%). Pleural fluid/serum lysozyme ratio (PL/SL) was also valuable in the discrimination of these two groups. When the cut-off level of 1.2 was considered, diagnostic tests of tuberculous effusions showed a sensitivity of 100%, specificity of 88% and accuracy of 93%, respectively. The mean fibronectin concentration in pleural fluid with tuberculous effusion was significantly higher than that in carcinomatous effusion, but there was a marked overlap between these two groups. On the other hand, CEA was significantly higher in carcinomatous effusions than in tuberculous effusions. At a cut-off level of 5 ng/ml, 53% of patients with carcinomatous effusion showed elevated pleural fluid CEA levels, while none of the tuberculous effusion did (sensitivity 53%, specificity 100%, diagnostic accuracy 65%). The combined assay of ADA, lysozyme, pl-ADA/s-ADA, PL/SL and CEA provide valuable information in the differential diagnosis between tuberculous and carcinomatous effusion, while fibronectin concentration can not be considered to have a definite diagnostic value.

A majority of small cell lung cancer (SCLC) cells lack a metastasis suppressor, tetraspanin CD9, and CD9 expression promotes their apoptosis. By a proteomics-based approach, we compared an SCLC cell line with its CD9 transfectant and found that a calcium-binding neuronal protein, calretinin, is upregulated in CD9-positive SCLC cells. Ectopic or anticancer drug-induced CD9 expression upregulated calretinin, whereas CD9 knockdown down-regulated calretinin in SCLC cells. When calretinin was knocked down, CD9-positive SCLC cells revealed increased Akt phosphorylation and decreased apoptosis. These results suggest that CD9 positively regulates the expression of calretinin that mediates proapoptotic effect in SCLC cells., Highlights • Most small cell lung cancer (SCLC) cells lack the metastatic suppressor CD9. • An SCLC cell line and its CD9 transfectant were compared by a proteomics approach. • Calretinin is upregulated in CD9 (+) SCLC cells. • Calretinin is a proapoptotic protein in CD9 (+) SCLC cells.

Introduction Erlotinib and pemetrexed have been approved for the second-line and maintenance treatment of non-small cell lung cancer (NSCLC). With the recommended doses determined by our previous phase I study, we conducted a phase II study to evaluate the efficacy and safety of combination of the two agents in pretreated non-squamous NSCLC patients. Methods This study was performed in patients with stage IIIB/IV or post-surgically recurrent non-squamous NSCLC whose disease had progressed on or after receiving first-line chemotherapy. Patients received 500 mg/m2 of intravenous pemetrexed every 21 days and 150 mg of oral erlotinib on days 2–16 until disease progression, unacceptable toxicity, or withdrawal of consent. The expected response rate and threshold were defined as 33.5% and 10%, respectively. Assuming a one-sided alpha of 5%, a power of 80%, the possible deviation from assessment, 26 patients were necessary. Results A total of 27 patients, 16 males and 11 females were recruited. Patients had the median age of 70 years (range, 48–80 years) and included 21 stage IV diseases, 22 adenocarcinomas. Epidermal growth factor receptor (EGFR) mutations were examined in all patients. One patient had positive EGFR mutation, but the other 26 patients had wild-type EGFR. The median number of treatment courses was 3 (range, 1 to over 19). The best overall response rate and disease control rate were 11.1% and 63.0%, respectively. The median progression-free survival and overall survival were 2.8 months (95% confidence interval (CI); 1.9–7.5 months) and 15.8 months (95% CI; 9.3 months to not available), respectively. Dermal, hepatic, gastrointestinal and hematological disorders were the frequently observed adverse events. One patient experienced grade 3 drug-induced interstitial lung disease. Conclusions We could not demonstrate the add-on effect of intermittent erlotinib on pemetrexed in a second-line setting for patients with non-squamous NSCLC without EGFR mutations.

Background Erlotinib and pemetrexed have been approved for the second-line treatment of non-small cell lung cancer (NSCLC). These two agents have different mechanisms of action. Combined treatment with erlotinib and pemetrexed could potentially augment the antitumor activity of either agent alone. In the present study, we investigated the safety profile of combined administration of the two agents in pretreated NSCLC patients. Methods A phase I dose-finding study (Trial registration: UMIN000002900) was performed in patients with stage III/IV nonsquamous NSCLC whose disease had progressed on or after receiving first-line chemotherapy. Patients received 500 mg/m2 of pemetrexed intravenously every 21 days and erlotinib (100 mg at Level 1 and 150 mg at Level 2) orally on days 2–16. Results Twelve patients, nine males and three females, were recruited. Patient characteristics included a median age of 66 years (range, 48–78 years), stage IV disease (nine cases), adenocarcinoma (seven cases) and activating mutation-positives in the epidermal growth factor receptor gene (two cases). Treatment was well-tolerated, and the recommended dose of erlotinib was fixed at 150 mg. Dose-limiting toxicities were experienced in three patients and included: grade 3 elevation of serum alanine aminotransferase, repetitive grade 4 neutropenia that required reduction of the second dose of pemetrexed and grade 3 diarrhea. No patient experienced drug-induced interstitial lung disease. Three patients achieved a partial response and stable disease was maintained in five patients. Conclusions Combination chemotherapy of intermittent erlotinib with pemetrexed was well-tolerated, with promising efficacy against pretreated advanced nonsquamous NSCLC.

e18056 Background: Erlotinib and pemetrexed have been approved in second-line treatment for non-small cell lung cancer (NSCLC) and have different mechanisms of action. Their combination could potentially augment the antitumor activity brought by either agent alone. We investigated the safety in combination of erlotinib and pemetrexed in pretreated NSCLC patients. Methods: A phase I dose-finding study was performed in patients with stage III/IV nonsquamous NSCLC whose disease had progressed on or after receiving first-line chemotherapy. This study was registered as UMIN000002900. Patients received 500 mg/m2 of pemetrexed intravenously every 21 days and erlotinib (100 mg in Level 1, 150 mg in Level 2) orally on days 2-16. Results: Twelve patients were recruited. Patient characteristics included median age of 66 (range, 48-78), 9 males, 9 stage IV, 7 adenocarcinoma, and 2 activating mutation-positives in the gene of epidermal growth factor receptor. Treatment was well-tolerated and the recommended dose of erlotinib was fixed on 150 mg. Dose-limiting toxicities were experienced in 3 patients: grade 3 elevation of serum alanine aminotransferase, repetitive grade 4 neutropenia that required the second dose reduction of pemetrexed, and grade 3 diarrhea. None experienced drug-induced interstitial lung disease. Three patients achieved partial response and 5 kept stable disease. Conclusions: Combination chemotherapy of intermittent erlotinib with pemetrexed was well-tolerated with promising efficacy against pretreated advanced nonsquamous NSCLC.