High expression of GADD-45alpha and VEGF induced tumor recurrence via upregulation of IL-2 after photodynamic therapy using NPe6.

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.

A possible relationship between the anti-cancer potency of photodynamic therapy using the novel photosensitizer ATX-s10-Na(II) and expression of the vascular endothelial growth factor in vivo.

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.

Breast cancer resistance protein (BCRP) affected acquired resistance to gefitinib in a "never-smoked" female patient with advanced non-small cell 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.

Basic and clinical research on photodynamic therapy at Tokyo Medical University Hospital.

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.

[Preoperative PDT for early stage lung cancer accompanied with infiltration to the central airway].

For the treatment of central type lung cancers, it is often necessary to perform bronchoplasty or bi-lobectomy even in early stage cases in which tumor invasion is found at the bronchial bifurcation. To solve this contradictory situation, we applied PDT for central type early stage lung cancer to reduce the extent of superficial infiltration, enabling a simple surgical technique or decreasing the amount of resected lung parenchyma. Among 7 patients, the simplification of surgical technique and reduction of the range of resection were possible in 2 cases each respectively. However, these objectives were not satisfied in the remaining 3 cases and the operations performed were those that had been originally scheduled before PDT. The current problems are the establishment of appropriate laser irradiation technique and accurate assessment for extension of invasion.

Increased cytotoxic effects of photodynamic therapy in IL-6 gene transfected cells via enhanced apoptosis.

PDT has been reported to induce cancer cell expression of cytokines, such as IL-6 and TNF-alpha, but it has been unclear whether cytokine expression by cancer cells is directly related to the antitumor effect of PDT. We treated Lewis lung carcinoma (LLC) cells with a new photosensitizer, mono-L-aspartyl chlorin e6 (NPe6) and light from a diode laser and found that expression of the mRNA of IL-2, IL-6, and TNF-alpha was increased by NPe6-mediated-PDT 6 hr later. To elucidate the mechanism of the direct anti-tumor effect of cytokine expression, we examined the photosensitivity of cytokine-gene-transfected cells, namely LLC-IL-2, LLC-IL-6, and LLC-TNF-alpha cells, by MTT assay. The IL-6 gene transfected, LLC-IL-6 cells were significantly more sensitive to cytotoxic effects than the parent LLC cells and other cytokine gene-transfected cells. This finding indicates that IL-6 expression modulates cellular sensitivity to PDT and that IL-2 and TNF-alpha expressions does not. In addition, the apoptosis of LLC-IL-6 cells induced by NPe6-PDT was greater than in the other cells as determined by DNA fragmentation and staining of apoptotic nuclei. Because IL-6 has been reported to induce apoptosis by downregulating expression of Bcl-2, we analyzed the expression of apoptosis-related Bcl-2, Bax, and cytochrome C by Western blot analysis. Decreased expression of Bcl-2 and cytochrome C was observed in both LLC cells and LLC-IL-6 cells. Bax protein increased in a time-dependent manner, and the ratio of Bax to Bcl-2 rose markedly after PDT in LLC-IL-6 cells. These results suggest that the increased sensitivity of LLC-IL-6 cells to PDT-induced cytotoxicity results from the high ratio of Bax to Bcl-2 in the IL-6-dependent apoptotic pathway. In conclusion, IL-6 expression plays a role in cellular sensitivity to PDT, and combination of IL-6 and PDT may provide a new strategy for cancer treatment.

[Bronchofiberscopy].

New diagnostic modalities have been used in conjunction with endoscopy for early detection of lung cancer. Videoendoscope is routinely used instead of fiberoptic bronchoscope. Fluorescence diagnosis has been proved to be useful in detecting subtle lesions which might be invisible by conventional endoscopy in central airway. Also, a number of small peripheral lesions has increased by the helical CT. CT guided transbronchial lung as well as needle cytology are indicated for definitive diagnosis of such lesions. Endobronchial Ultrasonography is employed to evaluate the depth of cancer invasion of the bronchus and lymph node swelling around the bronchus. It should be helpful in staging of lung cancer and selecting therapy.

Localization of experimental submucosal esophageal tumor in rabbits by using mono-L-aspartyl chlorin e6 and long-wavelength photodynamic excitation.

BACKGROUND AND OBJECTIVE: To increase the applicability of photodynamic diagnosis with regard to deep-seated tumor, we illuminated tumors with a long-wavelength laser beam after photosensitization with mono-L-aspartyl chlorin e6 (NPe6). STUDY DESIGN/MATERIALS AND METHODS: Rabbits with VX2 esophageal tumors were divided into four groups. The control group was not treated, and the other three groups were injected with 1, 2.5, and 5 mg/kg mono-L-aspartyl chlorin e6 (NPe6), respectively. After excitation with a 664-nm laser beam (10 mW, 10 seconds), the fluorescence image and the relative fluorescence intensity (tumor/normal tissue) were recorded every 2 hours up to 8 hours by a newly developed diode laser endoscopic fluorescence imaging system. The tissue concentration of NPe6 was examined by high performance liquid chromatography at 2, 4, and 6 hours after injection with 1 and 5 mg/kg NPe6. RESULTS: The diode laser endoscopic fluorescence imaging system was able to selectively detect fluorescence from submucosal tumor by comparison with the surrounding normal mucosa after NPe6 injection. The fluorescence intensity correlated with NPe6 dose, selectively accumulated in the tumor tissue and relative intensity peaked at 6 hours after injection. No fluorescent images were detected in controls. CONCLUSION: Given intravenously, NPe6 at a dose of 5 mg/kg and excited with a 664-nm wavelength laser beam 6 hours later can define experimentally induced deep-seated esophageal carcinoma in rabbits, by using an endoscopic fluorescence imaging system.

Early detection of bronchial lesions using lung imaging fluorescence endoscope.

The performance of the Lung Imaging Fluorescence Endoscope (LIFE) system was compared with conventional bronchoscopy in 158 patients: 68 patients with invasive cancer, 42 patients with abnormal sputum cytology findings (12 early cancer and 26 dysplasia), 17 cases with resected lung cancer and 31 smokers with symptoms. The respective results of conventional bronchoscopy and LIFE for detection of dysplasia were; sensitivity 52% and 90% (biopsy basis), 62% and 92% (patient basis). Fluorescence bronchoscopy may be an important adjunct to conventional bronchoscopy to improve the localization of subtle lesions of bronchus.

Early Detection of Bronchial Lesions Using System of Autofluorescence Endoscopy (SAFE) 1000.

Recently several endoscopic fluorescence detection systems have been developed. In some of them, laser light was used for the excitation of autofluorescence, and sophisticated techniques were also necessary to amplify the fluorescence signal as well.The result of fluorescence diagnosis using a simple system with a conventional Xenon lamp excitation and an image intensifier is reported. The respective results of sensitivity and positive predictive values of cancer plus dysplasia were 66%, and 62% by standard bronchoscopy and 92% and 88% by the newly developed autofluorescence system. In this paper, developed endoscope for detection of tissue/mucosal autofluorescence without the application of any photosensitizing agents or use of any lasers is evaluated.

Lung cancers treated with photodynamic therapy and surgery.

Laser endoscopic surgery, especially the effectiveness of photodynamic therapy (PDT) using Photofrin as a photosensitizer, has now achieved a status as effective treatment modality for lung cancer. Twenty-six lung cancer patients received the preoperative PDT for the purpose of either reducing the extent of resection or increasing operability. Bronchoscopical PDT is performed with topical anesthesia approximately 48 h after the intravenous injection of 2.0 mg/kg body weight of Photofrin. Operation was performed 2-9 weeks after initial PDT. The initial purpose of PDT, i.e. either to reduce the extent of resection or convert inoperable disease to operable status, was achieved in 22 out of 26 patients treated. The survival rate of T3 (main bronchus invasion) cases treated by surgery alone increased significantly from 50.9% to 60.0% with the application of preoperative PDT. This remarkable result may imply that this new option of PDT as preoperative laser irradiation may contribute to the management of advanced lung malignancy.

Effectiveness of Photodynamic Therapy and Nd-YAG Laser Treatment for Obstructed Tracheobronchial Malignancies.

Since 1980, advanced lung carcinomas were treated with palliative laser therapy for the purpose of opening the endobronchial stenosis and obstruction by either photodynamic therapy (PDT) or Nd-YAG laser treatment at Tokyo Medical University. A total of 258 lesions were treated, 81 by PDT and 177 by Nd-YAG laser treatment. PDT achieved effective results in 61 (75%) of 81 lesions. In the Nd-YAG laser group, 143 (81%) of 177 lesions showed effective results. When the tumor was located in the trachea or main bronchi, effective results were obtained in 73% (19 of 26) of cases treated by PDT and in 93% of cases (64 of 69) treated by Nd-YAG laser. However, in cases in which the tumor was located in lobar or segmental bronchi, the tumor response was effective in 76% (42 of 55) of PDT-treated patients and 73% (79 of 108) of Nd-YAG laser-treated patients. With a mortality rate of 0%, the greatest advantage of PDT over Nd-YAG treatment was safety. Considering complications, PDT seems to be useful for obstruction of lobar and segmental bronchus. Nevertheless, when deciding among alternative therapies, physicians treating patients with advanced lung carcinoma should give careful consideration to the benefit and complications of both laser therapies and decide the most suitable modality.

Analysis of the Cost-effectiveness of Photodynamic Therapy in Early Stage Lung Cancer.

Methods A cost-effectiveness analysis was carried out for photodynamic therapy (PDT) performed in early stage lung cancer cases, which by definition have no lymph node metastasis. The alternative treatment method was lobectomy, which conventionally would have been the first choice of treatment. Costs (C) and effectiveness (E) both of the PDT group and operation group were compared. Effectiveness was determined using quality adjusted life years saved (QALYs) which is the 5-year survival rate adjusted in terms of the quality of life of the patient, and the cost-effectiveness rate was obtained based on the costs of treatment methods during the patient's stay in the hospital. Health care costs, including drugs, were calculated according to the 1992 National Health Insurance list in yen. Costs which were non-reimbursable by the public insurance system, such as for special rooms and sun block cream, were also expressed in yen.Results The total cost of the operated group was yen1,793,832 and that for the PDT group was yen1,017,104. The cost-effectiveness rate of the operated group, that is the average cost of treatment per postoperative living month, was yen37,537, while that of the entire PDT group was yen30,003. This indicates that the cost-effectiveness rate for the operated group is apparently 1.3 times higher than that of the PDT group. The monthly cost-effectiveness rate for the PDT group of lesions smaller than 2 cm was yen25,533. Therefore the cost in the operated group is 1.5 times higher.Conclusions This study demonstrated the merits of PDT for early stage lung cancer from the point of view of cost-effectiveness.

Fibrin plugging as a cause of microcirculatory occlusion during photodynamic therapy.

An experimental system that allows the white light observation of rapid changes in vessels without disturbance by red laser light was used. Mice were injected with mono-L-aspartyl chlorin-e-6 (Npe-6) i.v. via the tail vein and were immediately exposed to laser light. White emboli were observed forming on the inside of the vessel walls within seconds after commencement of light exposure. Emboli adhered to vessel walls and caused vascular obstruction. Light microscopy of the exposed material using fibrin staining was performed. Electron microscopy on the same material was also carried out. The embolisation time was influenced by both drug dose and laser power. With low laser power, it took a long time to stop the blood flow. Fibrin staining revealed the white emboli to be composed of fibrin. Electron microscopy findings revealed damage to endothelial cells and platelet aggregation. This study suggests that two main mechanisms (direct cellular damage and vascular shut-down ) might actually be complementary and synergistic in the production of vascular lesions using photodynamic therapy.

The effects of serum on cellular uptake and phototoxicity of mono-L-aspartyl chlorin e6 (NPe6) in vitro.

Previous reports showed that the photosensitizer mono-L-aspartyl chlorin e6 (NPe6) binds to serum proteins. However, the influence of this binding on the cellular uptake and photodynamic therapy (PDT) phototoxicity of NPe6 is still undefined. In this paper, we studied how serum in medium affected the P388 cellular uptake and PDT phototoxicity of NPe6 in vitro. This was assessed by (1) detection of the red shift (654 nm Q band peak of absorption) induced by protein binding NPe6; (2) detection of intracellular concentration of NPe6 by HPLC and (3) measurements of the cell survival ratio after PDT by MTT assay. The 654 nm Q band peak of NPe6 shifted to 665 nm after binding of NPe6 and serum proteins. The protein-bound NPe6 cannot be uptaken by cells, thus there was no PDT phototoxicity. Nevertheless, phototoxicity recovered when the concentration of NPe6 excessed the serum protein binding ability or there was free serum protein in the medium. These data suggested that the cellular uptake of NPe6 is inhibited by serum components in the medium, and that only free NPe6 is accumulated by P388 cells even during relatively long incubations. The cytotoxicity of PDT mainly depends on the free NPe6 level in the medium.

[Photodynamic therapy for bronchogenic carcinoma].

Laser endoscopic surgery has now achieved a status as effective therapeutic modality for lung cancer. Especially increasing attention has been focused on photodynamic therapy (PDT) using Photofrin and excimer dye laser. PDT obtained government approval in October 1994 and finally obtained national insurance reimbursement status in April 1996. Over the past decade, 248 patients (296 lesions) with central type lung cancers have been treated in our hospital. Overall complete remission was obtained in 42.5% of the 125 lesions, partial remission in 56.8% and no remission was obtained in 1.0%. Indications of PDT are follows, 1. Early stage lung cancer as a curative purpose: among 104 early stage lesions CR was obtained in 87 (83.7%) and 61 cases were disease free at 2 to 178 months. 2. Advanced lesions for opening of bronchi: overall, "effective" opening of bronchi was achieved in 61 out of 81 lesions (75%) for the PDT group, as opposed to 143 of 177 (81%) for the Nd-YAG laser therapy group. 3. Preoperative laser irradiation for the propose of increasing operability and reducing the extent of resection area: the initial purpose of PDT, i.e., either reduction of extent of resection of conversion of inoperable disease to operable status, was achieved in 21 out of 21 patients treated. 4. Multiple primary lung cancer. The success from clinical trials using PDT for treatment of cancers offers encouragement for its future use. More stable, definitive and more successful results will be obtained if new dyes which distribute more equally in the tumor tissue and deeper tissue penetration by longer wavelength beams are used.

Endoscopic diagnosis and treatment of a case of respiratory papillomatosis.

Respiratory papilloma is a rare lesion that arises in the larynx, trachea and bronchus. We describe a patient with laryngeal papilloma that spread to the trachea and which was effectively treated by Nd-YAG laser. Two years after the initial treatments of the laryngeal and tracheal papillomas, a recurrent lesion (solitary papilloma) was observed on the membranous portion of the trachea. We examined the recurrent lesion by bronchoscopy including bronchoscopic ultrasound (US), helical computed tomography (CT) and tracheal biopsies. Respiratory papillomatosis sometimes shows either malignant transformation or invasion to tracheal wall without displaying cytohistological atypia. Therefore, we concluded that bronchoscopic US and helical CT were useful for deciding on therapeutic strategy in cases of recurrence of tracheal papilloma.

Early localization of bronchogenic cancerous/precancerous lesions with lung imaging fluorescence endoscope.

The result of the clinical trial using the lung imaging fluorescence endoscope (LIFE) was reported. A total of 77 biopsy confirmed sites from 30 patients were evaluated. The sensitivity for metaplasia detection by the LIFE system was 96% compared to 28% by conventional bronchoscopy. The LIFE system was found to be useful in detecting subtle cancerous/precancerous lesions.

Photodynamic Therapy With YAG-OPO Laser for Early Stage Lung Cancer.

Photodynamic therapy (PDT) utilizing Photofrin is proving to be effective for the treatment of early stage lung cancers. The effect of PDT utilizing YAG-OPO laser as new light source was evaluated in 26 patients (29 lesions) with early stage lung cancers. YAG-OPO laser is solid state tunable laser which is easy to change wavelength between 620 and 670 nm exciting various kinds of photosensitizers. Moreover, YAG-OPO laser is more reliable, smaller and has less consumables than argon-dye laser or excimer-dye laser. As the result of PDT with YAG-OPO laser, complete remission (CR) was obtained in 82.6% of the 29 lesions, partial remission (PR) in 13.8% and no change (NC) was obtained in 3.4%. We conclude that PDT utilizing YAG-OPO laser is efficacious in the treatment of early stage lung cancers and can achieve complete remission.

[Laser photodynamic therapy for central-type lung cancer].

Laser endoscopic surgery is recognized as an effective treatment for lung cancer. Attention has increasingly been focused on photodynamic therapy (PDT) with dihermatoporphyrin ethers. The Japanese government approved the use of this therapy in October 1995, and reimbursement through the national health insurance system began in April 1996. Over the past decade, 140 patients (283 lesions) with central type lung cancers have been treated at our hospital. Overall complete remission was obtained in 39.6% of 112 lesions, partial remission in 59.4% and no remission in 1.0% The indications for PDT are as follows: 1. Early stage lung cancer curative; among 95 early stage lesions complete remission was obtained in 79 (83.2%), and 71 patients were disease free at 3 to 176 months. 2. Advanced lesions opening of bronchi; overall, "effective" opening of bronchi was achieved in 61 of 81 lesions (75%), in the PDT group, and in 143 of 177 (81%) in the Nd-YAG laser therapy groups. 3. Preoperative laser irradiation to increase operability and reduce the extent of operation; the extent of resectin was reduced, or inoperable lesions were made operable in 21 of 24 patients treated. 4. Multiple primary lung cancer. The success of clinical trials of PDT for treatment of lung cancers bodes well for its future use. More stable, definitive, and successful treatment will become possible with the use of new dyes that distribute more evenly in tumor tissue, and with the deeper tissue penetration made possible by longer-wavelength beams.