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Background : Even if lung cancer is detected at an early stage, surgery may be difficult in patients with severe comorbidities, like interstitial pneumonia (IP). Radiation therapy cannot be performed due to the risk of acute IP exacerbation. Therefore, an effective alternative, such as photodynamic therapy (PDT), is required to prove that acute exacerbation is not induced after PDT in peripheral lung cancer. In this study, we investigated the effects of PDT on IP rat models. Methods : Bleomycin (BLM) was administered intratracheally. Seven days after administration, left thoracotomy was performed. Talaporfin sodium was injected, and diode laser irradiation (664 nm, 150mW, 100J/cm2) was performed. Seven days after PDT, the whole blood and left lungs were collected. A total of 23 rats, comprising BLM + PDT (n = 4), BLM + non-PDT (n = 10), non-BLM + PDT (n = 2), non-BLM + non-PDT (n = 5), and two rats that died immediately after PDT were observed. Serum levels of Krebs von den Lungen-6, surfactant protein-D, lactate dehydrogenase, and serum C-reactive protein were measured. Fibrosis and macrophage scorings, and the ​​collagen fibers percentage were examined by staining with hematoxylin and eosin, Elastica van Gieson, anti-α smooth muscle antibody, and anti-CD68 antibodies. Results : There was no remarkable difference in the values of each marker in fibrosis and macrophage scores with or without PDT. In case of death, fibrosis was mild, and PDT was not affected. Conclusions : In IP rat models, PDT did not induce lung fibrosis or acute exacerbation.

The aim of this study was to evaluate the feasibility of a combination therapy of photodynamic therapy (PDT) and airway stent placement using a transparent silicone stent (gold studded stent [GSS]). Laser irradiation (664 nm, continuous wave) was performed through the GSS using a straight and cylindrical fiber 1.0 cm away from a power meter. There are two types of GSS: the TD type for the trachea and the BD type for the bronchus. Laser outputs were set to 150 mW, 180 mW, 210 mW, 240 mW, 270 mW, and 300 mW. The laser powers passing through the both types of GSS were measured three times for each outputs and the averages were calculated. Based on the results, animal experiment was performed using two female pigs. Under general anesthesia, a GSS (BD type) was inserted into trachea of pigs, and PDT using NPe6 as a photosensitizer was performed by 100 J/cm2 laser irradiation on parts of the trachea with and without a GSS. Immediately after and 1 week after PDT, pig tracheas were harvested and histological analysis was performed. Histological analysis of areas with or without the stent showed edematous changes between the cartilage and submucosal layer immediately after PDT, and necrotic changes 1 week later. The effectiveness of NPe6-PDT for pigs’ trachea covered by the stent was same as trachea without the stent. The use of a GSS may enable PDT to be effective even in the area covered by the stent.

Introduction/Aim Photodynamic therapy (PDT) involves the use of a tumor-specific photosensitizer and laser irradiation, and is one of the treatment options recommended for early centrally located lung cancers, but not yet for peripheral-type lung cancers. We developed a new laser probe, the composite-type optical fiberscope (COF), which allows accurate laser irradiation of a cancer lesion with simultaneous visualization of the lesion. In this study, we attempted a new endobronchial PDT technique using the new laser probe, and evaluated the effectiveness and feasibility of this novel PDT technique for peripheral lung cancers. Methods This phase I study was conducted in 7 patients with peripheral lung cancers (primary tumor ≤20 mm in diameter). We performed endobronchial PDT for these patients using the new laser probe and talaporfin sodium as the photosensitizer. Results We performed PDT for 3 patients with peripheral lung cancer using a laser dose of 50 J/cm2 at 120 mW, and confirmed the feasibility of using this dose. Then, we escalated the laser dose to 100 J/cm2 in 4 additional patients. A total of 7 patients met our inclusion criteria. Evaluation at 2 weeks and 3 months after the PDT revealed no complication such as pneumonia or pneumothorax. At the evaluation conducted 6 months later, we found CR in 3 cases and SD in the remaining 4 cases. Conclusion PDT was found to be a feasible and non-invasive treatment modality for early peripheral-type lung cancer. In the future, PDT could become a standard treatment option for peripheral-type lung cancer.

Background The second-generation photosensitizer NPe6 has strong anti-tumor effects with a much shorter photosensitive period than the first-generation photosensitizer Photofrin. Although photosensitive period has been reduced, skin photosensitivity is still a major side effect of photodynamic therapy (PDT). Therefore, we conducted a prospective study to investigate whether the NPe6 fluorescence intensity in skin after PDT could be measured effectively in human patients to improve the management of a patient's photosensitive period. Methods The NPe6 fluorescence measurements using a constructed fluorescence sensing system at the inside of the arm were acquired prior to and 5 and 10 min after NPe6 administration as well as at the time of PDT (4–5 h after administration), at discharge (2 or 3 days after PDT), and at 1 or 2 weeks after PDT. Participants were interviewed as to whether they had any complications at 2 weeks after PDT. Results Nine male patients and one female patient entered this study. Nine patients were inpatients and one patient was an outpatient. All of the measurements of NPe6 fluorescence in the skin could be obtained without any complications. The spectral peak was detected at the time of discharge (2–3 days after administration) in most cases and it decreased at 1 or 2 weeks after PDT. Conclusions The fluorescence of NPe6 in the skin could be detected feasibly using the fluorescence sensing system in human patients. Measuring the relative concentration of NPe6 in the skin indirectly by measuring fluorescence intensity might be useful to predict the period of skin photosensitivity after PDT.

Background: Skin photosensitivity is a major side effect of photodynamic therapy (PDT). It is induced by the photosensitizer remaining in the skin. It is usually rapidly metabolized by the liver, but the pharmacokinetic profile varies widely among individuals. This makes it difficult to predict the incidence of skin photosensitivity. Therefore, we conducted a prospective study to investigate whether the NPe6 fluorescence intensity in skin after NPe6-PDT could be measured safely in human patients using a fluorescence sensing system for judging the risk of skin photosensitivity. Methods: The NPe6 fluorescence measurements using a constructed fluorescence sensing system at the inside of the arm were acquired prior to and 5 and 10 minutes after NPe6 administration as well as at the time of PDT (4-5 hours after administration), at discharge (2 or 3 days after PDT), and at 1 or 2 weeks after PDT. Participants were interviewed as to whether they had any complications at 2 weeks after PDT. Results: Nine male patients and one female patient entered this study. All of the measurements of NPe6 fluorescence in the skin could be obtained without any complications. The spectral peak was detected at the time of discharge (2-3 days after administration) in most cases and it decreased at 1 or 2 weeks after PDT. Conclusions: The fluorescence of NPe6 in the skin could be detected feasibly using the fluorescence sensing system in human patients. Measuring fluorescence intensity in the skin might be useful to predict the incidence of skin photosensitivity after PDT.

Bronchial thermoplasty (BT) is a novel, nonpharmacological procedure for treatment of severe asthma. Recently, the Asthma Intervention Research 2 clinical trial demonstrated asthmatics had fewer hospitalisations following BT, which persisted 5 years after therapy [1]. However, it is well recognised that asthma is a heterogeneous disease with distinct asthma phenotypes and, not surprisingly, not all asthmatics in that trial benefited from BT [2]. Optical coherence tomography small airway imaging may be used for better patient phenotyping and selection for BT We thank Myles McKinnon, Jennifer Campbell and Chulho Hyun (British Columbia Cancer Agency, Vancouver, BC, Canada) for their assistance with the bronchoscopy procedures.

We studied a 3-compartment dynamic model of talaporfin sodium pharmacokinetics in silico. Drug distribution might change after intravenous injection from plasma to interstitial space and then into myocardial cells. We have developed a new cardiac ablation using photosensitization reaction with laser irradiation shortly after talaporfin sodium injection. We think that the major cell-killing factor in our cardiac ablation would be an oxidation by singlet oxygen produced in the interstitial space in myocardium with laser irradiation shortly after the photosensitizer administration. So that the talaporfin sodium concentration change in time in the interstitial space should be investigated. We constructed the pharmacokinetics dynamic model composed by 3-compartments, that is, plasma, interstitial space, and cell. We measured talaporfin sodium fluorescence time change in human skin by our developed fluorescence measurement system in vivo. Using the measured concentration data in plasma and skin in human, we verified the calculation accuracy of our in silico model. We compared the simulated transition tendency of talaporfin sodium concentration from interstitial space to cells in our in silico model with the reported uptake tendency using cultured myocardial cell. We identified the transition coefficients between plasma, interstitial space, and cell compartment, and metabolization coefficient from plasma by the fitting with measured data.

DNA methylation is an epigenetic modification that is highly disrupted in response to cigarette smoke and involved in a wide spectrum of malignant and nonmalignant diseases, but surprisingly not previously assessed in small airways of patients with chronic obstructive pulmonary disease (COPD). Small airways are the primary sites of airflow obstruction in COPD. We sought to determine whether DNA methylation patterns are disrupted in small airway epithelia of patients with COPD, and evaluate whether changes in gene expression are associated with these disruptions. Genome-wide methylation and gene expression analysis were performed on small airway epithelial DNA and RNA obtained from the same patient during bronchoscopy, using Illumina’s Infinium HM27 and Affymetrix’s Genechip Human Gene 1.0 ST arrays. To control for known effects of cigarette smoking on DNA methylation, methylation and gene expression profiles were compared between former smokers with and without COPD matched for age, pack-years, and years of smoking cessation. Our results indicate that aberrant DNA methylation is (1) a genome-wide phenomenon in small airways of patients with COPD, and (2) associated with altered expression of genes and pathways important to COPD, such as the NF-E2–related factor 2 oxidative response pathway. DNA methylation is likely an important mechanism contributing to modulation of genes important to COPD pathology. Because these methylation events may underlie disease-specific gene expression changes, their characterization is a critical first step toward the development of epigenetic markers and an opportunity for developing novel epigenetic therapeutic interventions for COPD.

In Japan, Photodynamic therapy (PDT) is recommended as a treatment option for centrally located early-stage lung cancers (CLELCs). It is a minimally invasive treatment with excellent anti-tumor effects. The 2nd generation photosensitizer, talaporfin sodium has strong anti-tumor effects with much less photosensitivity than porfimer sodium. Moreover, the laser equipment is compact and portable, and talaporfin sodium is now the current mainstay of PDT for lung cancer. For successful PDT, accurate evaluation of tumor extent and bronchial invasion is crucial. Detailed examination of the tumor using autofluorescence bronchoscopy and endobronchial ultrasonography or optical coherence tomography is extremely useful before PDT. At present, PDT has become the 1st choice of treatment for CLELC in institutions with the necessary equipment. It can also be effective for advanced lung cancer causing tracheobronchial obstruction. With such advances in PDT for CLELC, we are expanding the indications of PDT for not only CLELC, but also peripheral type lung cancer.

Next-generation sequencing (NGS) and digital PCR technologies allow analysis of the mutational profile of circulating cell-free DNA (cfDNA) in individuals with advanced lung cancer. We have now evaluated the feasibility of cfDNA sequencing for mutation detection in patients with non-small cell lung cancer at earlier stages. A total of 150 matched tumor and serum samples were collected from non-small cell lung cancer patients at stages IA–IIIA. Amplicon sequencing with DNA extracted from tumor tissue detected frequent mutations in EGFR (37% of patients), TP53 (39%), and KRAS (10%), consistent with previous findings. In contrast, NGS of cfDNA identified only EGFR, TP53, and PIK3CA mutations in three, five, and one patient, respectively, even though adequate amounts of cfDNA were extracted (median of 4936 copies/mL serum). Next-generation sequencing showed a high accuracy (98.8%) compared with droplet digital PCR for cfDNA mutation detection, suggesting that the low frequency of mutations in cfDNA was not due to a low assay sensitivity. Whereas the yield of cfDNA did not differ among tumor stages, the cfDNA mutations were detected in seven patients at stages IIA–IIIA and at T2b or T3. Tumor volume was significantly higher in the cfDNA mutation-positive patients than in the negative patients at stages T2b–T4 (159.1 ± 58.0 vs. 52.5 ± 9.9 cm3, P = 0.014). Our results thus suggest that tumor volume is a determinant of the feasibility of mutation detection with cfDNA as the analyte.

Bronchoscopy is a minimally invasive method for diagnosis of diseases of the airways and the lung parenchyma. Standard bronchoscopy uses the reflectance/scattering properties of white light from tissue to examine the macroscopic appearance of airways. It does not exploit the full spectrum of the optical properties of bronchial tissues. Advances in optical imaging such as optical coherence tomography (OCT), confocal endomicroscopy, autofluorescence imaging and laser Raman spectroscopy are at the forefront to allow in vivo high-resolution probing of the microscopic structure, biochemical compositions and even molecular alterations in disease states. OCT can visualize cellular and extracellular structures at and below the tissue surface with near histological resolution, as well as to provide three-dimensional imaging of the airways. Cellular and subcellular imaging can be achieved using confocal endomicroscopy or endocytoscopy. Contrast associated with light absorption by haemoglobin can be used to highlight changes in microvascular structures in the subepithelium using narrow-band imaging. Blood vessels in the peribronchial space can be displayed using Doppler OCT. Biochemical compositions can be analysed with laser Raman spectroscopy, autofluorescence or multispectral imaging. Clinically, autofluorescence and narrow-band imaging have been found to be useful for localization of preneoplastic and neoplastic bronchial lesions. OCT can differentiate carcinoma in situ versus microinvasive cancer. Endoscopic optical imaging is a promising technology that can expand the horizon for studying the pathogenesis and progression of airway diseases such as COPD and asthma, as well as to evaluate the effect of novel therapy.

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.

Development of acquired resistance to gefitinib after an initial good response is common. Recently, it was reported that this acquired resistance is related to a secondary mutation associated with a substitution of threonine by methionine at codon 790 (T790M) of the epidermal growth factor receptor (EGFR) gene. In this report, we present a "never smoking" woman with advanced lung cancer who showed acquired resistance to gefitinib, and analysis of autopsy samples revealed no evidence of EGFR mutations in either exons 18-21 or codon 790, and positive immunostaining for breast cancer resistance protein (BCRP). We describe, for the first time, a case in which expression of BCRP was associated with acquired resistance to gefitinib, independent of EGFR mutations.

Background and Objectives We have been engaged in basic and clinical research on photodynamic therapy (PDT) and photodynamic diagnosis (PDD) for more than 25 years. Study Design/Materials and Methods PDT for 264 centrally located early-stage lung cancer lesions yielded an initial complete response (CR) rate of 84.8%. PDT is now becoming a standard option for centrally located stage 0 (TisN0M0) and stage I (T1N0M0) lung cancer. It is an attractive option for elderly patients in poor physical condition. Results Recent results of interstitial PDT for peripheral-type lung cancers suggest that it may be a promising local curative treatment modality for lesions less than 1.0 cm in diameter. Conclusions In this article, we introduce our recent clinical trials of PDT for lung cancers (both central and peripheral), and new techniques of PDD in sentinel node navigation biopsy for breast cancers. Moreover, we introduce basic research on cancers and infectious diseases in order to expand the clinical applications of PDT. Lasers Surg. Med. 38:371–375, 2006. © 2006 Wiley-Liss, Inc.

To identify a possible new treatment modality for malignant pleural mesothelioma (MPM), we examined whether combination treatment consisting of pemetrexed chemotherapy and photodynamic therapy (PDT) using the photosensitizer NPe6, enhanced the antitumor effect in both in vitro and in vivo models. We also investigated preclinical treatment schedules. Four human malignant mesothelioma cell lines (MSTO‑211H, H2052, H2452 and H28) were assayed using the WST assay after treatment with pemetrexed and NPe6‑PDT. The treatment schedule for the combination treatment was examined using nude mice. Pemetrexed pre‑treatment enhanced the lethal effect of NPe6‑PDT in the four malignant mesothelioma cell lines, but NPe6‑PDT followed by pemetrexed treatment did not enhance cell lethality in the in vitro assay. Pemetrexed pre‑treatment did not enhance the intracellular accumulation of NPe6, which is one of the determinants of the antitumor effect of PDT. In nude mice injected with MSTO‑211H cells and then treated using a combination of pemetrexed and NPe6‑PDT (10 mg/kg NPe6, 10 J/cm(2) laser irradiation), the tumor volume decreased by 50% but subsequently increased, reaching the pre‑treatment value after 14 days. Pemetrexed treatment followed by NPe6‑PDT resulted in an 80% reduction in the tumor size and inhibited re‑growth. NPe6‑PDT followed by pemetrexed treatment resulted in a 60% reduction in tumor size but did not inhibit re‑growth. NPe6‑PDT induced the expression of thymidylate synthase (TS), which confers resistance to pemetrexed, and NPe6‑PDT followed by pemetrexed treatment did not enhance the treatment outcome in vivo. In conclusion, combination treatment, consisting of pemetrexed followed by NPe6‑PDT, should be further investigated as a new treatment modality for MPM. In the future, this combination treatment may contribute to a reduction in local recurrence and a prolonged survival period in patients with MPM.

Endobronchial intervention has been well established in therapeutic and palliative methods for patients with malignant and benign airway obstruction. Central airway tumor obstruction could lead to significant dyspnea or hemoptysis and dramatically lower the quality of life. Such symptoms can be relieved immediately via bronchoscopic intervention. Endobronchial therapies such as neodymium yttrium aluminum garnet( Nd:YAG) laser therapy, argon plasma coagulation and microwave coagulation or stent placement are well-established treatment options. Each of these modalities has its specific advantages and disadvantages. Laser therapy is direct thermal ablation applied to endobronchial tumors. It has sufficient power to vaporize tissues and produces an excellent coagulation effect. However, the risk of perforation is high. On the other hand, photodynamic therapy has the curative potential for patients with early superficial lung cancer using low power laser. Moreover, it has recently been approved for the treatment of other cancers including advanced lung cancer. Stent placement is an effective treatment to re-establish airway patency and immediately relieve the patient from life-threatening dyspnea. Stent restenosis, migration, mucus plug and hemoptysis are common short-term and longterm problems. It is important to select the appropriate treatment method for each patient to avoid complications and achieve successful results.

Introduction: A recent ex-vivo study using micro-CT in patients with chronic obstructive pulmonary disease (COPD) showed that narrowing and disappearance of small conducting airways precedes the onset of emphysematous destruction in COPD. Until recently, the airway remodeling process could not be studied in detail in-vivo. In this study, we investigated the repeatability of navigating an Optical Coherence Tomography (OCT) catheter to image the same airways in smokers with and without COPD. Method: OCT imaging was performed by inserting the catheter through a sub-segmental airway to a small bronchiole. Three-dimensional OCT imaging of 5 cm of airway segments was obtained. The catheter was removed and reinsertion into the same airway was attempted. The number of airway generations and quantitative measurements of the airway wall area were investigated. Results: Sixty-three airways in 30 subjects were analyzed. Repeated insertion into the same airway was observed at 53.8 %, 92.3% and 70.8% of the time in the upper, middle and lower lobes respectively. The percentage differences of paired measurements of airway wall area between matched and unmatched airways in bronchioles were 5.8 ± 4.6 % and 7.3 ± 5.4 % respectively Conclusions: Repeated OCT imaging of airways is possible in the majority of cases except in the upper lobes. For airways that are not completely matched, some of the airway segments can still be used for comparison by careful alignment of the airway. OCT may be a useful method to study the remodeling process in small airways and the effect of therapeutic intervention.

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.

Photodynamic therapy (PDT), neodymium yttrium aluminum garnet (Nd-YAG) laser therapy, electrocautery and microwave coagulation therapy are therapeutic options available for management of endobronchial malignancies. All of these treatment modalities have been used for both palliation of late obstructing cancers, and more recently have been used as primary treatment of early stage lung cancers. Only PDT has the curative potential for patients with early superficial squamous cell carcinoma. Nd-YAG laser therapy is used for direct thermal ablation of tissue in endobronchial malignancy. This equipment is the most widely used type of laser for bronchoscopic interventions because it has sufficient power to vaporize tissues and produces an excellent coagulation effect. But the risks of perforation and bleeding are high. Endobronchial electrocautery is the use of high-frequency electrical current that generates heat due to tissue resistance, resulting in destruction of tissue. Argon plasma coagulation (APC) is a form of noncontact electrocoagulation. The risks of perforation and igniting are much lower than with the Nd-YAG laser therapy. Microwave coagulation therapy refers to the use of all electromagnetic methods for inducing tumor destruction by using devices with frequencies of 2450 MHz. It is important to select these treatment methods appropriately according to each case.

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.

ATX-s10-Na(II) is a novel second-generation photo-sensitizer for photodynamic therapy (PDT). PDT using ATX-s10 and diode laser (670 nm) induces an apoptotic response, inflammatory reaction, immune reaction and damage to the microvasculature. In particular, the vascular shut-down effect plays an important role in the anti-tumor activity of ATX-s10-PDT. It has been reported that PDT induces hypoxia and expression of the vascular endothelial growth factor (VEGF) via the hypoxia-inducible factor 1 (HIF1)-alpha pathway. We hypothesized that the expression of VEGF may cause tumor recurrence after PDT and exert unfavorable effect against the anti-tumor activity of ATX-s10-PDT. In this study, we showed by DNA microarray analysis in vitro that VEGF mRNA expression was induced 3 h after laser irradiation in ATX-s10-PDT. We compared the anti-tumor activity of ATX-s10-PDT against lung cancer cell lines SBC-3 and SBC-3/VEGF, the latter overexpressing VEGF; there was no significant difference in the sensitivity to the PDT between the two cell lines as assessed by clonogenic assay. Furthermore, no statistically significant difference in the anti-tumor effect of PDT, as measured by tumor cures, was found between SBC-3 and SBC-3/VEGF tumors in female Balb/c-nu/nu nude mice in vivo. In conclusion, ATX-s10-PDT may prevent tumor recurrence despite induction of VEGF and promotion of tumor angiogenesis, which are known to enhance tumor proliferation and survival.

ATX-s10 is a novel and second-generation photosensitizer for photodynamic therapy (PDT). In order to conduct clinical trials of ATX-s10-PDT and/or extend its clinical applications, it is very important to elucidate the mechanisms of the action of ATX-s10-PDT. We examined the apoptic response against ATX-s10-PDT using a Bcl-2 or Bcl-2 mutant overexpressing cells. Using fluorescent microscopy, ATX-s10 localized not only to mitochondria but also to lysosomes and possibly other intracellular organelles, but not to the plasma membrane or the nucleus. These results suggest that ATX-s10-PDT can damage mitochondria and lysosomes. By Western blot analysis, ATX-s10-PDT damaged Bcl-2, which localized preferentially at mitochondrial membranes, and caused Bcl-2 to cross-link immediately after laser irradiation. However, ATX-s10-PDT was not able to rapidly induce morphologically typical apoptosis (i.e. chromatin condensation and fragmentation) as PDT using mitochondria targeted photosensitizers, such as phthalocyanine 4 (Pc 4). Pharmacological inhibitions of lysosomal cytokine protease cathepsins, such as cathepsin B and D, protected MCF-7c3 cells (human breast cancer cells expressing stably transfected procaspase-3) from apoptosis caused by ATX-s10-PDT. Overexpression of wild-type Bcl-2 or Bcl-2Delta33-54 resulted in relative resistance of cells to ATX-s10-PDT, as assessed by the degree of morphological apoptosis or loss of clonogenicity. We conclude that lysosomal damage by ATX-s10-PDT can initiate apoptotic response and this apoptotic pathway can be regulated by photodamage to Bcl-2 via mitochondrial damage.

(Introduction) We have reported that the klotho gene, which is an anti-aging gene known to be a tumor suppressor, is a prognostic factor for lung cancer patients. In this study, we determined whether klotho expression may have a predictive value of the efficacy of anti-cancer agents for cancer treatment. (Methods) We performed co-transfection of klotho and GFP-plasmid into MCF-7c3 cells, which stably express procaspase-3, and treated the cells with various kinds of anti-cancer agents. We observed typical apoptosis morphologically using fluorescent microscopy and evaluated the apoptotic response. (Results) Western blot analysis revealed high klotho expression in the cells transfected with the klotho gene plasmid. There was no evidence of klotho expression in the normal MCF-7c3 cells. The anti-cancer drug sensitivity test showed that docetaxel significantly induced more typical apoptosis in the cells with klotho than in the cells without klotho (41.2% and 18.3%, respectively; p < 0.05). Paclitaxel also significantly induced more typical apoptosis in the cells with klotho than in the cells without klotho (34.9% and 14.9%, respectively; p < 0.05). However, there was no significant difference in the apoptosis rates induced by other anti-cancer drugs, such as cisplatin, carboplatin, pemetrexed, gemicitabine, vinorelbine, etoposide, and gefitinib. (Conclusion) A high expression of the klotho protein can markedly increase the apoptotic response of MCF-7c3 cells to docetaxel and paclitaxel. These results suggest that klotho expression may indicate the predictive value of chemotherapy and play an important role in cancer treatment. Apoptosis induced by taxane-induced stratified expression of klotho proteinRate of apoptosisDocetaxelPaclitaxelA: Klotho(-)/GFP(-)14.8%19.3%A vs B: p>0.05A vs B: p>0.05B: Klotho(-)/GFP(+)18.3%14.9%B vs C: p=0.01B vs C: p=0.03C: Klotho(+)/GFP(+)41.2%35.9% Citation Format: Yujin Kudo, Jitsuo Usuda, Hideyuki Furumoto, Sachio Maehara, Keishi Ohtani, Tatsuya Inoue, Taichiro Ishizumi, Yasufumi Kato, Masatoshi Kakihana, Naohiro Kajiwara, Tatsuo Ohira, Norihiko Ikeda. Expression of klotho enhances the apoptotic response of cancer cells to anticancer agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1351. doi:10.1158/1538-7445.AM2014-1351

Examining and quantifying changes in airway morphology is critical for studying longitudinal pathogenesis and interventions in diseases such as chronic obstructive pulmonary disease and asthma. Here we present fiber-optic optical coherence tomography (OCT) as a nondestructive technique to precisely and accurately measure the 2-dimensional cross-sectional areas of airway wall substructure divided into the mucosa (WAmuc), submucosa (WAsub), cartilage (WAcart), and the airway total wall area (WAt). Porcine lung airway specimens were dissected from freshly resected lung lobes (N = 10). Three-dimensional OCT imaging using a fiber-optic rotary-pullback probe was performed immediately on airways greater than 0.9 mm in diameter on the fresh airway specimens and subsequently on the same specimens post-formalin-fixation. The fixed specimens were serially sectioned and stained with H&E. OCT images carefully matched to selected sections stained with Movat's pentachrome demonstrated that OCT effectively identifies airway epithelium, lamina propria, and cartilage. Selected H&E sections were digitally scanned and airway total wall areas were measured. Traced measurements of WAmuc, WAsub, WAcart, and WAt from OCT images of fresh specimens by two independent observers found there were no significant differences (p>0.05) between the observer's measurements. The same wall area measurements from OCT images of formalin-fixed specimens found no significant differences for WAsub, WAcart and WAt, and a small but significant difference for WAmuc. Bland-Altman analysis indicated there were negligible biases between the observers for OCT wall area measurements in both fresh and formalin-fixed specimens. Bland-Altman analysis also indicated there was negligible bias between histology and OCT wall area measurements for both fresh and formalin-fixed specimens. We believe this study sets the groundwork for quantitatively monitoring pathogenesis and interventions in the airways using OCT.

For the first time, the use of fiber-optic color Doppler optical coherence tomography (CDOCT) to map in vivo the three-dimensional (3-D) vascular network of airway segments in human lungs is demonstrated. Visualizing the 3-D vascular network in the lungs may provide new opportunities for detecting and monitoring lung diseases such as asthma, chronic obstructive pulmonary disease, and lung cancer. Our CDOCT instrument employs a rotary fiber-optic probe that provides simultaneous two-dimensional (2-D) real-time structural optical coherence tomography (OCT) and CDOCT imaging at frame rates up to 12.5 frames per second. Controlled pullback of the probe allows 3-D vascular mapping in airway segments up to 50 mm in length in a single acquisition. We demonstrate the ability of CDOCT to map both small and large vessels. In one example, CDOCT imaging allows assignment of a feature in the structural OCT image as a large (∼1 mm diameter) blood vessel. In a second example, a smaller vessel (∼80 μm diameter) that is indistinguishable in the structural OCT image is fully visualized in 3-D using CDOCT.

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.