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Background The important role of surgery in early-stage small cell lung cancer (SCLC) has been recognized, and curative surgical resection is recommended. However, the role of adjuvant chemotherapy for stage I SCLC has not yet been evaluated, and novel approaches focusing on the specific genomic characteristics of SCLC may be invaluable for customized therapy. In this study, we focused on the Klotho gene, which is an anti-aging gene known to be a potential tumor suppressor. We investigated whether the expression of Klotho, assessed by immunohistochemistry, can predict survival in patients with resected SCLC. Methods The medical records of patients diagnosed as having limited-disease (LD) SCLC and treated by surgical resection (n = 30) at Tokyo Medical University Hospital were retrospectively reviewed. The expression status of Klotho, and of the ATP-binding cassette (ABC) transporters MRP1, MDR and breast cancer resistant protein (BCRP), which can cause resistance to anticancer drugs, including irinotecan, was assessed by immunohistochemical analysis in resected surgical specimens of patients with early-stage SCLC. Results Of the 30 patients, Klotho expression was seen in the specimens from 18 patients (60.0%), but not in those of the remaining 12 patients (40.0%). The immunostaining for Klotho was mostly localized in the cytoplasm. The expression of Klotho was significantly associated with the overall survival (OS) (ratio 0.088; 95% confidence interval 0.019–0.409; P = 0.002). The administration of perioperative chemotherapy had no significant effect in improving the survival, as assessed by the Kaplan–Meier method. However, the patients showing Klotho expression in the resected specimens in p-stage I and II, may have benefited from perioperative chemotherapy. A multivariate analysis revealed no significant association between the expression status of MRP1, MDR or BCRP and the OS. Conclusion Expression of Klotho was predictive of a favorable outcome following resection in limited-disease SCLC patients, and the Klotho expression status may serve as a new biomarker for the need of additional therapies to be developed in the future.

Background and Objectives PDT induces apoptosis, inflammatory reactions, immune reactions, and damage to the microvasculature around the tumors. The mechanisms responsible for the anticancer effects of Photofrin-PDT and NPe6-PDT differ somewhat. To select a photosensitizer for lung cancer treatment and to improve the efficacy of PDT, the mechanisms of action for PDT using Photofrin or NPe6 must be elucidated and the phenomena validated by analyzing molecular determinants from clinical samples. Study Design/Materials and Methods We examined the role of immunological reactions in the anti-tumor effects of PDT using cytokine-overexpressing cells and investigated whether the anti-apoptotic protein Bcl-2 may be a molecular target. Moreover, we investigated the association between ATP-binding cassette transporter proteins such as breast cancer-resistant protein (BCRP), which can pump out some types of photosensitizer, and the efficacy of PDT using clinical samples from 81 early lung cancer lesions treated with PDT between 1998 and 2006 at the Tokyo Medical University Hospital. Results Photofrin-PDT damaged Bcl-2 and rapidly induced apoptosis, but NPe6-PDT did not damage Bc-2 nor did it induce morphologically typical apoptosis. However, NPe6-PDT exerted a strong anti-tumor effect, regardless of the overexpression of Bcl-2. By analyzing the BCRP-overexpressing cells, Photofrin, but not NPe6, was found to be a substrate of BCRP. All 81 lung cancer lesions were BCRP-positive; as Photofrin was found to be a substrate of BCRP, the expression of BCRP significantly affected the efficacy of Photofrin-PDT. However, NPe6-PDT exerted a strong antitumor effect regardless of BCRP expression, and the complete response rate after NPe6-PDT was much higher than that after Photofrin-PDT. Conclusions Our translational research suggests that NPe6-PDT may be superior to Photofrin-PDT for the treatment of lung caner, and individualized approaches to PDT based on the expression status of Bcl-2 and/or BCRP may improve the efficacy of PDT in patients with lung cancers. Lasers Surg. Med. 43:591–599, 2011. © 2011 Wiley-Liss, Inc.

Summary Background We had previously developed the possibility of use of a photodynamic diagnosis (PDD) system using a tumor-selective photosensitizer and laser irradiation for the early detection and photodynamic therapy (PDT) for centrally located early lung cancers. Recently, we established the autofluorescence diagnosis system integrated into a videoendoscope (SAFE-3000) as a very useful technique for the early diagnosis of lung cancer. Patients and methods Twenty-nine patients (38 lesions) with centrally located early lung cancer received PDD and PDT using the second-generation photosensitizer, talaporfin sodium (NPe6). Just before the PDT, we defined the tumor margin accurately using the novel PDD system SAFE-3000 with NPe6 and a diode laser (408 nm). Results Red fluorescence emitted from the tumor by excitation of the photosensitizer by the diode laser (408 nm) from SAFE-3000 allowed accurate determination of the tumor margin just before the PDT. The complete remission (CR) rate following NPe6-PDT in the cases with early lung cancer was 92.1% (35/38 lesions). We also confirmed the loss of red fluorescence from the tumors immediately after the PDT using SAFE-3000. We confirmed that all the NPe6 in the tumor had been excited and photobleached by the laser irradiation (664 nm) and that no additional laser irradiation was needed for curative treatment. Conclusions This novel PDD system using SAFE-3000 and NPe6 improved the quality and efficacy of PDT and avoided misjudgement of the dose of the photosensitizer or laser irradiation in PDT. PDT using NPe6 will become a standard option of treatments for centrally located early lung cancer.

PDT induces apoptosis, inflammatory reactions, immune reactions, and damage to the microvasculature around the tumors. The mechanisms responsible for the anticancer effects of Photofrin-PDT and NPe6-PDT differ somewhat. To select a photosensitizer for lung cancer treatment and to improve the efficacy of PDT, the mechanisms of action for PDT using Photofrin or NPe6 must be elucidated and the phenomena validated by analyzing molecular determinants from clinical samples.We examined the role of immunological reactions in the anti-tumor effects of PDT using cytokine-overexpressing cells and investigated whether the anti-apoptotic protein Bcl-2 may be a molecular target. Moreover, we investigated the association between ATP-binding cassette transporter proteins such as breast cancer-resistant protein (BCRP), which can pump out some types of photosensitizer, and the efficacy of PDT using clinical samples from 81 early lung cancer lesions treated with PDT between 1998 and 2006 at the Tokyo Medical University Hospital.Photofrin-PDT damaged Bcl-2 and rapidly induced apoptosis, but NPe6-PDT did not damage Bc-2 nor did it induce morphologically typical apoptosis. However, NPe6-PDT exerted a strong anti-tumor effect, regardless of the overexpression of Bcl-2. By analyzing the BCRP-overexpressing cells, Photofrin, but not NPe6, was found to be a substrate of BCRP. All 81 lung cancer lesions were BCRP-positive; as Photofrin was found to be a substrate of BCRP, the expression of BCRP significantly affected the efficacy of Photofrin-PDT. However, NPe6-PDT exerted a strong antitumor effect regardless of BCRP expression, and the complete response rate after NPe6-PDT was much higher than that after Photofrin-PDT.Our translational research suggests that NPe6-PDT may be superior to Photofrin-PDT for the treatment of lung caner, and individualized approaches to PDT based on the expression status of Bcl-2 and/or BCRP may improve the efficacy of PDT in patients with lung cancers.

Background In terms of prognosis, large cell neuroendocrine carcinoma (LCNEC) differs distinctively from other non-small cell lung cancers, with the prognosis of LCNEC being poor, even for early-stage disease. Improvements in survival require a biomarker capable of defining a subset of patients destined to do poorly so that these patients can be targeted for additional therapies, including chemotherapy. In this study, we focused on the Klotho gene, which is an anti-aging gene known to be a potential tumor suppressor. We investigated whether the immunohistochemical expression of Klotho can predict survival patients with resected LCNEC. Methods The histological characteristics of patients receiving an initial diagnosis of LCNEC (n = 30) at Tokyo Medical University Hospital were retrospectively reviewed, and multiple variables including stage, lymphangioinvasion, lymph node status and the expression of Klotho as identified using an immunohistochemical analysis, were assessed. Results Immunostaining for Klotho was mostly cytoplasmic, and Klotho expression was seen in 10 patients (33.3%) but not in 20 patients (66.7%). The expression of Klotho was significantly associated with a good outcome of resected patients with LCNEC and Klotho(−) was associated with increased LCNEC risk by multivariate analysis (hazard ratio 4.92, 95% confidence interval 1.04–23.24, p = 0.044). Neither lymph node status nor lymphangioinvasion were significantly associated with a poor survival. However, among patients without lymph node metastasis or angioinvasion, the survival benefit of Klotho expression in the primary tumor was significantly higher, compared with that of patients without Klotho expression. Conclusion Klotho staining provides a new biomarker for a good outcome in patients with LCNEC, especially among patients without lymph node metastasis or lymphangioinvasion.

Background Patients with centrally located early lung cancer (CLELC) are often heavy smokers with a considerably high risk of multiple primary lung cancer (MPLC) lesions; treatment strategies for such patients must preserve the cardiopulmonary function. Methods Between July 2004 and July 2008, patients with CLELC underwent photodynamic therapy (PDT) using NPe6, second-generation photosensitizer at Tokyo Medical University Hospital. Among these patients, we retrospectively analyzed MPLC, which was treated by surgery plus PDT or PDT alone and examined the effectiveness of PDT, and we propose a treatment strategy for patients with MPLC. Results A total of 64 patients with CLECL received NPe6-PDT, and MPLCs were found in 22 patients (34.4%) using sputum cytology and a bronchoscopical examination using autofluorescence bronchoscopy. Among these 22 patients, 10 patients underwent surgery for primary lung cancer and underwent NPe6-PDT for the treatment of secondary primary CLELC, one patient underwent PDT for CLELC as a primary lesion followed by an operation for peripheral-type lung cancer as a secondary primary lesion, and 11 patients underwent PDT alone for MPLC lesions (28 lesions) that were roentgenographically occult lung cancers. Among these 22 patients with MPLC including peripheral-type lung cancers, which were resected by surgery, all 39 CLELC lesions exhibited a complete response after PDT, and all patients were alive. Conclusions For patients with lung cancer with a long-term history of smoking, careful follow-up examinations after surgical resection are needed considering the incidence of metachronous primary lung cancers. PDT can play an important role for the treatment strategy for MPLC.

NPe6 is a novel second-generation photosensitizer used for photodynamic therapy (PDT). PDT using NPe6 and diode laser (664 nm) induces cell death, inflammatory reactions, immunological responses and damage to the microvasculature. In this study, we evaluated the influence of the immunological responses and of enhanced angiogenesis on the anti-tumor effect of NPe6-PDT using cytokine-overexpressing Lewis lung carcinoma (LLC), LLC-IL-2 cells both in vitro and in vivo. We showed by DNA microarray analysis in vitro that IL-2 and GADD-45alpha (growth arrest and DNA damage 45 alpha) mRNA expressions were induced by 3 h after NPe6-PDT applied at a dose killing 90% of the cells (LD90). IL-2-overexpressing cells (LLC/IL-2 cells) were resistant to the loss of clonogenicity as compared to the parental LLC cells in vitro. Furthermore, in female C57BL/6 mice, NPe6-PDT produced a cure rate of 66.7% in LLC tumors, whereas the cure rate was only 16.6% in LLC/IL-2 tumors, and overexpression of IL-2 caused failure of NPe6-PDT, with tumor recurrence, in vivo. These results suggest that IL-2 expression may play an unfavorable role in attenuation of the antitumor effect of NPe6-PDT. It has been reported that the expression of vascular endothelial growth factor (VEGF), in particular, may cause tumor recurrence after PDT and exert unfavorable effect in relation to attenuate the anti-tumor activity of PDT. Results of immunohistochemical analysis of LLC/IL-2 tumors have revealed that the expressions of GADD-45alpha and VEGF are induced in these tumors after PDT, and in particular, 12 h after PDT, the expression levels were much higher as compared with those in the LLC tumors. The results of our studies using in vitro and in vivo models suggest that the cell death caused by PDT was inhibited by induction of GADD-45alpha expression and that tumor recurrence was promoted by the enhancement of VEGF expression mediated by IL-2 upregulation. Therefore, it is speculated that the use of an IL-2 inhibitor may improve the efficacy of NPe6-PDT.

Photodynamic therapy (PDT) has now achieved the status of a standard treatment modality for centrally located early-stage lung cancer. In the last decade, CT screening for lung cancer has attracted much attention for its ability to detect early peripheral lung cancer. Extremely recently, treatment using PDT has been introduced for the first time in patients with peripheral lung cancer, who did not meet the previous criteria for surgery. The procedure was carried out with local anesthesia with xylocain infiltrated into the chest wall, 48 h after Photofrin administration. Needles (19 gauge) containing an internal catheter were inserted percutaneously under CT guidance. The needles were then extracted and a diffuser fiber with a 2 cm long tip for light delivery was positioned in the tumor through the catheter. Of the nine patients enrolled in this trial, seven achieved partial remission (PR). No serious complications, except for two cases of pneumothorax, were noted. As an increasing number of patients consider quality of life after therapy, the indications for PDT are expected to expand. We conclude that PDT is a promising new technique for curative treatment of localized, peripheral lung cancer less than 1 cm in size in patients who are unfit for surgery or radiotherapy.

It is very important to elucidate the mechanism of action and identify the molecular determinant of photodynamic medicine, in order to increase the number of clinical applications of photodynamic therapy (PDT) and perform personalized medicine. We have previously reported that PDT using some photosensitizers, such as phthalocyanine 4 (Pc 4) damages the anti-apoptotic protein Bcl-2, and that Bcl-2 is a molecular PDT target using a mitochondrion-targeting photosensitizer. In this study, we examined the molecular targets of Photofrin-PDT and NPe6-PDT, which are approved for early stage lung cancers by the Japanese Ministry of Health Labor and Welfare, by evaluating the photodamage to Bcl-2 using Western blot analysis. Our results showed that Photofrin-PDT damaged Bcl-2, induced morphologically typical apoptosis, and demonstrated equal sensitivity between MCF-7c3 cells (human breast cancer cells expressing stably transfected procaspase-3) and Bcl-2 overexpressing cells, MCF-7c3-GFP-Bcl-2 cells, with a clonogenic assay. However, NPe6-PDT did not damage Bcl-2 and took longer to induce typical apoptosis compared with Photofrin-PDT. MCF-7c3-GFP-Bcl-2 cells were considerably more resistant to the lethal effects of NPe6-PDT than parental MCF-7c3 cells. In conclusion, Photofrin-PDT damages different molecular targets, and our data indicate that the extent of Bcl-2 photodamage can determine the sensitivity of cancer cells to apoptosis and to overall cell killing caused by PDT using Photofrin, but not the lysosomal targeting NPe6. The application of these findings to clinical PDT may depend on the levels of the Bcl-2 proteins in the tumor being treated, and the tailor-made medicine based on the Bcl-2 photodamage may overcome any resistance afforded by elevated amounts of Bcl-2.