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We conducted a phase I study of the trans-bronchial injection of α-galactosylceramide (αGalCer)-pulsed antigen presenting cells (APCs) to evaluate their safety, immune responses, and anti-tumor activities. Patients with advanced or recurrent non-small cell lung cancer (NSCLC) refractory to standard treatments were eligible. αGalCer-pulsed APCs were administered intratumorally or intranodally by bronchoscopy. Twenty-one patients were enrolled in this study. No severe adverse events related to the cell therapy were observed during this study in any patient. After αGalCer-pulsed APCs were administrated, increased iNKT cell numbers were observed in PBMCs from eight cases, and IFN-γ producing cells were increased in the peripheral blood of 10 cases. Regarding clinical responses, one case exhibited a partial response and eight were classified as stable disease. In the tumor microenvironment, IFN-γ expression was upregulated after treatment in partial response or stable disease cases and TGF-β was upregulated in progressive disease cases., Competing Interests: Declaration of Competing Interest The authors declare no potential conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Invariant natural killer T (iNKT) cells produce copious amounts of cytokines in response to specific glycolipid antigens such as α-galactosylceramide (αGalCer) presented by CD1d-expressing antigen-presenting cells (APCs), thus orchestrating other immune cells to fight tumors. Because of their ability to induce strong antitumor responses activated by αGalCer, iNKT cells have been studied for their application in cancer immunotherapy. In our previous phase I/II trial in non-small cell lung cancer (NSCLC) patients who had completed the standard treatment, we showed a relatively long median survival time without severe treatment-related adverse events. Based on these results, we performed a phase II trial to evaluate clinical responses, safety profiles and immune responses as a second-line treatment for advanced NSCLC., Methods: Patients with advanced or recurrent NSCLC refractory to first-line chemotherapy were eligible. αGalCer-pulsed APCs were intravenously administered four times. Overall survival time was evaluated as the primary endpoint. The safety profile and immune responses after APC injection were also monitored. This study was an open label, single-arm, phase II clinical trial performed at Chiba University Hospital, Japan., Results: Thirty-five patients were enrolled in this study, of which 32 (91.4%) completed the trial. No severe adverse events related to the treatment were observed. The estimated median survival time of the 35 cases was 21.9 months (95% CI, 14.8 to 26.0). One case (2.9%) showed a partial response, 14 cases (40.0%) remained as stable disease, and 19 cases (54.3%) were evaluated as progressive disease. The geometric mean number of iNKT cells in all cases was significantly decreased and the mean numbers of natural killer (NK) cells, interferon-γ-producing cells in response to αGalCer, and effector CD8+ T cells were significantly increased after the administration of αGalCer-pulsed APCs., Conclusions: The intravenous administration of αGalCer-pulsed APCs was well-tolerated and was accompanied by prolonged overall survival. These results are encouraging and warrant further evaluation in a randomized phase III trial to demonstrate the survival benefit of this immunotherapy., Trial Registration Number: UMIN000007321., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Backgrounds: Overexpression of epidermal growth factor receptor (EGFR) has been established as a valid therapeutic target of non-small cell lung cancer (NSCLC). However, the clinical benefit of cetuximab as an EGFR-targeting drug is still controversial, partially due to the lack of effective means to identify suitable patients. This study aimed to investigate the potential of radiolabeled cetuximab as a non-invasive tool to predict cetuximab accumulation in NSCLC tumor xenografts with varying EGFR expression levels., Methods: The NSCLC tumors in model mice were subjected to in vivo biodistribution study and positron emission tomography (PET) imaging 48 h after injection of either 111 In- or 64 Cu-labeled cetuximab. The EGFR expression levels of NSCLC tumors were determined by ex vivo immunoblotting., Results: We found that tumors with high EGFR expression had significantly higher [ 111 In]In-DOTA-cetuximab accumulation than tumors with moderate to low EGFR expression (P < 0.05). Strong correlations were found between [ 111 In]In-DOTA-cetuximab tumor uptake and EGFR expression level (r = 0.893), and between [ 64 Cu]Cu-DOTA-cetuximab tumor uptake with EGFR expression level (r = 0.915). PET imaging with [ 64 Cu]Cu-DOTA-cetuximab allowed clear visualization of tumors., Conclusion: Our findings suggest that this immuno-PET imaging can be clinically translated as a tool to predict cetuximab accumulation in NSCLC cancer patients prior to cetuximab therapy.

It is important to detect mediastinal lymph node metastases in patients with lung cancer to improve outcomes, and it is possible that activatable fluorescence imaging with indocyanine green (ICG) can help visualize metastatic lymph nodes. Therefore, we investigated the feasibility of applying this method to mediastinal lymph node metastases in an epidermal growth factor receptor (EGFR)-positive squamous cell carcinoma of the lung. Tumors were formed by injecting H226 (EGFR-positive) and H520 (EGFR-negative) cell lines directly in the lung parenchyma of five mice each. When computed tomography revealed tumors exceeding 8 mm at their longest or atelectasis that occupied more than half of lateral lung fields, a panitumumab (Pan)-ICG conjugate was injected in the tail vein (50 μg/100 μL). The mice were then sacrificed 48 hours after injection and their chests were opened for fluorescent imaging acquisition. Lymph node metastases with the five highest fluorescent signal intensities per mouse were chosen for statistical analysis of the average signal ratios against the liver. Regarding the quenching capacity, the Pan-ICG conjugate had almost no fluorescence in phosphate-buffered saline, but there was an approximate 61.8-fold increase in vitro after treatment with 1% sodium dodecyl sulfate. Both the fluorescent microscopy and the flow cytometry showed specific binding between the conjugate and H226, but almost no specific binding with H520. The EGFR-positive mediastinal lymph node metastases showed significantly higher average fluorescence signal ratios than the EGFR-negative ones (n = 25 per group) 48 hours after conjugate administration (70.1% ± 4.5% vs. 13.3% ± 1.8%; p < 0.05). Thus, activatable fluorescence imaging using the Pan-ICG conjugate detected EGFR-positive mediastinal lymph node metastases with high specificity., Competing Interests: The authors have declared that no competing interests exist.

Near infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that combines the specificity of antibodies for targeting tumors with the toxicity induced by photosensitizers after exposure to near infrared (NIR) light. Herein we compare two NIR-light sources; light emitting diodes (LEDs) and Lasers, for their effectiveness in NIR-PIT. A photosensitizer, IRDye-700DX, conjugated to panitumumab (pan-IR700), was incubated with EGFR-expressing A431 and MDA-MB-468-luc cells. NIR-light was provided by LEDs or Lasers at the same light dose. Laser-light produced more cytotoxicity and greater reductions in IR700-fluorescence intensity than LED-light. Laser-light also produced more cytotoxicity in vivo in both cell lines. Assessment of super-enhanced permeability and retention (SUPR) effects were stronger with Laser than LED. These results suggest that Laser-light produced significantly more cytotoxic effects compared to LEDs. Although LED is less expensive, Laser-light produces superior results in NIR-PIT.

Background: The aim of this study is to investigate the underlying biologic mechanisms of 2-[18F]-fluoro-2-deoxy-d-glucose (18F-FDG) uptake on positron emission tomography (PET) in non-small cell lung cancer (NSCLC)., Methods: One-hundred forty patients with NSCLC who underwent 18F-FDG PET were included in the study. Tumor sections were stained by immunohistochemistry for glucose transporter 1 (GLUT1), GLUT3, hypoxia-inducible factor-1 alpha (HIF-1α), hexokinase I, vascular endothelial growth factor (VEGF), microvessels (CD34), epidermal growth factor receptor (EGFR), and molecules relevant to PI3K/Akt/mTOR signaling pathway (PTEN, p-Akt, p-mTOR and p-S6). We also conducted in vitro studies of 18F-FDG uptake and mTOR inhibition in NSCLC cells., Results: High 18F-FDG uptake was significantly associated with poor prognosis in NSCLC patients. 18F-FDG uptake was significantly correlated with GLUT1, hexokinase I, HIF-1α, VEGF, CD34, p-Akt, p-mTOR and EGFR. PTEN expression showed inverse correlation with 18F-FDG uptake. In in vitro study, 18F-FDG uptake was markedly decreased by the inhibition of GLUT1 and GLUT1 upregulation by the induction of HIF-1α increased the 18F-FDG uptake. Inhibition of both mTOR complex1 (mTORC1) and mTORC2 suppressed cell growth, but activity of mTORC1 regulated the 18F-FDG uptake. NCI-H1650 cells with PTEN loss showed the highest 18F-FDG uptake and the least sensitivity to mTOR inhibitors., Conclusion: The amount of 18F-FDG accumulation is associated with molecules relevant to glucose metabolism, hypoxia, angiogenesis and mTOR signaling pathway. Especially, PTEN status may affect not only 18F-FDG uptake but also effect of mTOR inhibitors on the growth of NSCLC., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Purpose: The intravenous administration of α-Galactosylceramide (α-GalCer)-pulsed antigen presenting cells (APCs) is well tolerated and the increased IFN-γ producing cells in the peripheral blood after the treatment appeared to be associated with prolonged survival. An exploratory study protocol was designed with the preoperative administration of α-GalCer-pulsed APCs to clarify the mechanisms of these findings, while especially focusing on the precise tumor site., Methods: Patients with operable advanced lung cancer received an intravenous injection of α-GalCer-pulsed APCs before surgery. The resected lung and tumor infiltrating lymphocytes (TILs) as well as peripheral blood mononuclear cells were collected and the invariant NKT (iNKT) cell-specific immune responses were analyzed., Results: Four patients completed the study protocol. We observed a significant increase in iNKT cell numbers in the TILs and augmented IFN-γ production by the α-GalCer-stimulated TILs., Conclusion: The administration of α-GalCer-pulsed APCs successfully induced the dramatic infiltration and activation of iNKT cells in the tumor microenvironment.

Purpose: We examined the possible efficacy of the yttrium-90 ((90)Y)-labeled anti-CDH3/P-cadherin mouse monoclonal antibody (MAb-6) in radioimmunotherapy (RIT) for lung and colorectal cancers that express CDH3/P-cadherin., Experimental Design: MAb-6 was established using genetic immunization. The biodistribution of MAb-6 in nude mice with lung and colorectal cancers was examined by administering indium-111((111)In)-labeled MAb-6 to mice. The mice were prepared by inoculation of CDH3/P-cadherin-positive (EBC1, H1373, and SW948) and CDH3/P-cadherin-negative (A549 and RKO) tumor cells. Therapeutic effects and toxicity were investigated by administration of (90)Y-labeled MAb-6 ((90)Y-MAb-6) to EBC, H1373, and SW948-inoculated mice., Results: Our in vivo results confirmed the specific binding of MAb-6 to tumor cells after intravenous injections of (111)In-labeled MAb-6 to mice with tumors expressing CDH3/P-cadherin. A single intravenous injection of (90)Y-MAb-6 (100 μCi) significantly suppressed tumor growth in mice with tumors expressing CDH3/P-cadherin. Furthermore, two injections of (90)Y-MAb-6 led to complete tumor regression in H1373-inoculated mice without any detectable toxicity., Conclusions: Our findings demonstrate that CDH3/P-cadherin-targeting RIT with (90)Y-MAb-6 is a promising strategy for the treatment for cancers expressing CDH3/P-cadherin.

Non-small cell lung carcinomas (NSCLC) overexpress the Her2/neu gene in approximately 59% of cases. Trastuzumab, a humanized monoclonal antibody, interferes with Her2 signaling and is approved for the treatment of Her2/neu overexpressing breast cancer. However, its therapeutic use in Her2/neu overexpressing NSCLC remains obscure. The present study aimed to determine the role of (64)Cu-labeled trastuzumab positron emission tomography (PET) for non-invasive imaging of Her2/neu expression in NSCLC. Trastuzumab was conjugated with the bifunctional chelator 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) and radiolabeled with (64)Cu. The molecular specificity of DOTA-trastuzumab was determined in NSCLC cell lines with Her2/neu overexpression (NCI-H2170) and negative expression (NCI-H520). Imaging of Her2/neu expression was performed in NCI-H2170 tumor-bearing mice with (64)Cu-DOTA-trastuzumab PET and (64)Cu-DOTA-IgG. In vitro studies revealed specific binding of DOTA-trastuzumab in the Her2/neu positive NCI-H2170 cells, while no binding was seen in the Her2/neu negative NCI-H520 cell line. Biodistribution and PET studies revealed a significantly high accumulation of (64)Cu-DOTA-trastuzumab in the Her2/neu overexpressing NCI-H2170 tumor at 24 and 48 h post-injection (21.4 +/- 1.4% and 23.2 +/- 5.1% injection dose/gram (% ID/g), respectively). PET imaging of Her2/neu negative NCI-H520 tumors showed much less uptake of (64)Cu-DOTA-trastuzumab (4.0% ID/g). The NCI-H2170 tumor uptake of (64)Cu-DOTA-trastuzumab was significantly higher than that of (64)Cu-DOTA-IgG (P < 0.0001). (64)Cu-DOTA-trastuzumab showed a very clear image of a Her2/neu positive tumor and appeared to be effective as a PET tracer for imaging of Her2/neu gene expression in NSCLC, suggesting its potential clinical use for identifying patients that might benefit from trastuzumab-based therapy.

To evaluate the safety, immune responses, and antitumor responses after the administration of alpha-galactosylceramide (alphaGalCer) KRN7000-pulsed PBMC cultured with IL-2 and GM-CSF (IL-2/GM-CSF-cultured PBMCs), a phase I-II study in patients with non-small cell lung cancer was conducted. Patients with advanced non-small cell lung cancer or recurrent lung cancer refractory to the standard therapy were eligible. alphaGalCer-pulsed IL-2/GM-CSF-cultured PBMCs (1 x 10(9)/m(2)) were i.v. administered four times. Immune responses were monitored weekly. Twenty-three patients were enrolled in this study and 17 cases (73.9%) completed. No severe adverse event related to the treatment was observed. After the injection of alphaGalCer-pulsed IL-2/GM-CSF-cultured PBMCs, an increased number of IFN-gamma-producing cells in the peripheral blood were detected in 10 patients (58.8%). Five cases remained as stable disease, and the remaining 12 cases were evaluated as progressive disease. The estimated median survival time (MST) of the 17 cases was 18.6 mo (range, 3.8 to 36.3 mo). Ten patients who displayed increased IFN-gamma-producing cells (> or =2-fold) showed prolonged MST (31.9 mo; range, 14.5 to 36.3 mo) as compared with poor-responder patients (n = 7) MST (9.7 mo; range, 3.8 to 25.0 mo) (log-rank test, p = 0.0015). The administration of alphaGalCer-pulsed IL-2/GM-CSF-cultured PBMCs was well tolerated and was accompanied by the successful induction of NKT cell-dependent immune responses. The increased IFN-gamma-producing cells that result from alphaGalCer stimulation in PBMCs were significantly associated with prolonged MST. These results are encouraging and warrant further evaluation for survival benefit of this immunotherapy.

Purpose: Human Valpha24 natural killer T (Valpha24 NKT) cells bearing an invariant Valpha24JalphaQ antigen receptor are activated by a glicolipid ligand alpha-galactosylceramide (alphaGalCer; KRN7000) in a CD1d-dependent manner. The human Valpha24 NKT cells activated with alphaGalCer and interleukin-2 have been shown to produce large amounts of cytokines, such as IFN-gamma, and also exerting a potent killing activity against various tumor cell lines. We did a phase I study with autologous activated Valpha24 NKT cell therapy., Experimental Design: Patients with advanced or recurrent non-small cell lung cancer received i.v. injections of activated Valpha24 NKT cells (level 1: 1 x 10(7)/m2 and level 2: 5 x 10(7)/m2) to test the safety, feasibility, and clinical response of this therapeutic strategy. Immunomonitoring was also done in all cases., Results: Six patients were enrolled in this study. No severe adverse events were observed during this study in any patients. After the first and second injection of activated Valpha24 NKT cells, an increased number of peripheral blood Valpha24 NKT cells was observed in two of three cases receiving a level 2 dose of activated Valpha24 NKT cells. The number of IFN-gamma-producing cells in peripheral blood mononuclear cells increased after the administration of activated Valpha24 NKT cells in all three cases receiving the level 2 dose. No patient was found to meet the criteria for either a partial or a complete response., Conclusions: The clinical trial with activated Valpha24 NKT cell administration was well tolerated and carried out safely with minor adverse events even in patients with advanced diseases.

Purpose: The purpose of this work was to evaluate the ability of breath-hold gadolinium-enhanced three-dimensional (3D) MR angiography to assess the invasion of the pulmonary vein and the left atrium by lung cancer., Method: Gadolinium-enhanced 3D MR angiography was performed in 20 consecutive patients with lung cancer., Results: At two sites with left atrial invasion shown by MR angiography, associated partial resection of the left atrium was performed. At five sites with invasion of the proximal pulmonary vein within 1.5 cm from the left atrium on MR, partial resection of the left atrium was performed at one site, and the pulmonary vein was resected at the intrapericardial portion at three sites. At two sites with invasion of the proximal pulmonary vein 1.5 cm more distal to the left atrium, the pulmonary vein was resected at the extrapericardial portion., Conclusion: Breath-hold gadolinium-enhanced 3D MR angiography is suitable for assessing invasion of the pulmonary vein and the left atrium by lung cancer.

Using proton magnetic resonance spectroscopy (1H MRS) spectra were obtained in vitro from extracts of four types of lung cancer (squamous cell, adenocarcinoma, large cell, small cell) and normal lung. The hydrophilic phase of the chloroform/methanol-water extracts yielded several distinct peaks. Among them the peak areas for cholines, creatines, glycine, and alanine, and their ratios were calculated and used as parameters to characterize different lung tissues. The ratios, cholines/alanine and glycine/alanine, were significantly (P < 0.001 to P < 0.05) higher for the normal lung than lung cancers. Creatines/glycine and creatines/cholines generally provided good discrimination (P < 0.001 to P < 0.05) between any two types of lung cancer. When data were further analyzed by discriminant factor analysis, there was 81.5 to 90.7% accuracy in predicting between normal lung and each cancer type, or among the four types of lung cancer. These results suggested that 1H MRS might be useful as an adjunct modality in the differential diagnosis of lung cancers.

As an initial step to elucidate the nature of the unique characteristics of the crystalloids of alveolar soft part sarcoma, a three-dimensional model of the crystalloids was prepared by digital image analysis of electron micrographs by computer. It was revealed that occult periodicities are present at two intervals, 60 A and 380 A, in the filamentous structure of the crystalloid; and it was also revealed by the observation of each cross-section that two globular substances with a diameter of 60 A are arranged in a dumbbell pattern in each filamentous structure. The model prepared based on these data showed the double strands crossing each other at intervals of 380 A, each of which consists of successive arrangement of the globular substances with a diameter of 60 A. This structure is clearly similar to that of actin. The similarities and differences between these results and the well-known studies of the organization of naturally occurring actin bundles are discussed.