Takehiko Fujisawa

Somatic Mutations of the HER2 Kinase Domain in Lung Adenocarcinomas

Mutations in the epidermal growth factor receptor gene (EGFR) in lung cancers predict for sensitivity to EGFR kinase inhibitors. HER2 (also known as NEU, EGFR2, or ERBB2) is a member of the EGFR family of receptor tyrosine kinases and plays important roles in the pathogenesis of certain human cancers, and mutations have recently been reported in lung cancers. We sequenced the tyrosine kinase domain of HER2 in 671 primary non-small cell lung cancers (NSCLC), 80 NSCLC cell lines, and 55 SCLCs and other neuroendocrine lung tumors as well as 85 other epithelial cancers (breast, bladder, prostate, and colorectal cancers) and compared the mutational status with clinicopathologic features and the presence of EGFR or KRAS mutations. HER2 mutations were present in 1.6% (11 of 671) of NSCLC and were absent in other types of cancers. Only one adenocarcinoma cell line (NCI-H1781) had a mutation. All HER2 mutations were in-frame insertions in exon 20 and target the identical corresponding region as did EGFR insertions. HER2 mutations were significantly more frequent in never smokers (3.2%, 8 of 248; P=0.02) and adenocarcinoma histology (2.8%, 11 of 394; P=0.003). In 394 adenocarcinoma cases, HER2 mutations preferentially targeted Oriental ethnicity (3.9%) compared with other ethnicities (0.7%), female gender (3.6%) compared with male gender (1.9%) and never smokers (4.1%) compared with smokers (1.4%). Mutations in EGFR, HER2, and KRAS genes were never present together in individual tumors and cell lines. The remarkable similarities of mutations in EGFR and HER2 genes involving tumor type and subtype, mutation type, gene location, and specific patient subpopulations targeted are unprecedented and suggest similar etiologic factors. EGFR, HER2, and KRAS mutations are mutually exclusive, suggesting different pathways to lung cancer in smokers and never smokers.

A Phase I Study of α-Galactosylceramide (KRN7000)–Pulsed Dendritic Cells in Patients with Advanced and Recurrent Non–Small Cell Lung Cancer

Purpose: Human Vα24 natural killer T (NKT) cells bearing an invariant Vα24JαQ antigen receptor, the counterpart of murine Vα14 NKT cells, are activated by a specific ligand, α-galactosylceramide (αGalCer, KRN7000), in a CD1d-dependent manner. I.v. administration of αGalCer-pulsed dendritic cells (DC) induces significant activation and expansion of Vα14 NKT cells in the lung and resulting potent antitumor activities in mouse tumor metastatic models. We did a phase I dose escalation study with αGalCer-pulsed DCs in lung cancer patients. Experimental Design: Patients with advanced non–small cell lung cancer or recurrent lung cancer received i.v. injections of αGalCer-pulsed DCs (level 1: 5 × 107/m2; level 2: 2.5 × 108/m2; and level 3: 1 × 109/m2) to test the safety, feasibility, and clinical response. Immunomonitoring was also done in all completed cases. Results: Eleven patients were enrolled in this study. No severe adverse events were observed during this study in any patient. After the first and second injection of αGalCer-pulsed DCs, dramatic increase in peripheral blood Vα24 NKT cells was observed in one case and significant responses were seen in two cases receiving the level 3 dose. No patient was found to meet the criteria for partial or complete responses, whereas two cases in the level 3 group remained unchanged for more than a year with good quality of life. Conclusions: In this clinical trial, αGalCer-pulsed DC administration was well tolerated and could be safely done even in patients with advanced disease.

Endobronchial ultrasound: new insight for the diagnosis of sarcoidosis.

A diagnosis of sarcoidosis should be substantiated by pathological means in order to thoroughly exclude other diseases. The role of real-time endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in the diagnosis of sarcoidosis has not been reported. The purpose of the present study is to evaluate the diagnostic yield of EBUS-TBNA in demonstrating the pathological features of sarcoidosis. In total, 65 patients with suspected sarcoidosis, with enlarged hilar or mediastinal lymph nodes on computed tomography, were included in the study. Patients with a suspected or known malignancy or previously established diagnosis of sarcoidosis were excluded. Convex probe endobronchial ultrasonography integrated with a separate working channel was used for EBUS-TBNA. Surgical methods were performed in those in whom no granulomas were detected by EBUS-TBNA. Patients were followed up clinically. EBUS-TBNA was performed on a total of 77 lymph node stations in 65 patients. A final diagnosis of sarcoidosis was made for 61 (93.8%) of the patients. The remaining four patients were diagnosed as having Wegeners granulomatosis (n = 1) or indefinite (n = 3). In patients with a final diagnosis of sarcoidosis, EBUS-TBNA demonstrated noncaseating epithelioid cell granulomas in 56 (91.8%) of the patients. No complications were reported. Endobronchial ultrasound-guided transbronchial needle aspiration proved to be a safe procedure with a high yield for the diagnoses of sarcoidosis.

Carbon ion radiotherapy for stage I non-small cell lung cancer.

BACKGROUND AND PURPOSE Heavy ion radiotherapy is a promising modality because of its excellent dose localization and high biological effect on tumors. Using carbon beams, a dose escalation study was conducted for the treatment of stage I non-small cell lung cancer (NSCLC) to determine the optimal dose. MATERIALS AND METHODS The first stage phase I/II trial using 18 fractions over 6 weeks for 47 patients and the second one using nine fractions over 3 weeks for 34 patients were conducted by the dose escalation method from 59.4 to 95.4 Gray equivalents (GyE) in incremental steps of 10% and from 68.4 to 79.2 GyE in 5% increments, respectively. The local control and survival rates were obtained using the Kaplan-Meier method. RESULTS Radiation pneumonitis at grade III occurred in three of 81 patients, but they fully recovered. This was not a dose-limiting factor. The local control rates in the first and second trials were 64% and 84%, respectively. The total recurrence rate in both trials was 23.2%. The infield local recurrence in the first trial was significantly dependent on carbon dose. The doses greater than 86.4 GyE at 18 fractions and 72 GyE at nine fractions achieved a local control of 90% and 95%, respectively. The 5 year overall and cause-specific survivals in 81 patients were 42% and 60%, respectively. CONCLUSIONS With our dose escalation study, the optimum safety and efficacy dose of carbon beams was determined. Carbon beam therapy attained almost the same results as surgery for stage I NSCLC although this was a I/II study.

High magnification bronchovideoscopy combined with narrow band imaging could detect capillary loops of angiogenic squamous dysplasia in heavy smokers at high risk for lung cancer

Background: We investigated the use of high magnification bronchovideoscopy combined with narrow band imaging (NBI) for the detailed examination of angiogenic squamous dysplasia (ASD). This was carried out in relation to bronchial vascular patterns with abnormal mucosal fluorescence in heavy smokers at high risk for lung cancer. Methods: Forty eight patients with sputum cytology specimens suspicious or positive for malignancy were entered into the study. Conventional white light and fluorescence bronchoscopic examination was first performed. Observations by high magnification bronchovideoscopy with conventional white light were made primarily at sites of abnormal fluorescence, and then repeated with NBI light to examine microvascular networks in the bronchial mucosa. Spectral features on the RGB (Red/Green/Blue) sequential videoscope system were changed from the conventional RGB broadband filter to the new NBI filter. The wavelength ranges of the new NBI filter were B1: 400–430 nm, B2: 420–470 nm, and G: 560–590 nm. ASD tissues were also examined using a confocal laser scanning microscope equipped with argon-krypton (488 nm) and argon (514 nm) laser sources. Results: The microvessels, vascular networks of various grades, and dotted vessels in ASD tissues were clearly observed in NBI-B1 images. Diameters of the dotted vessels visible on NBI-B1 images agreed with the diameters of ASD capillary blood vessels diagnosed by pathological examination. Capillary blood vessels were also clearly visualised by green fluorescence by confocal laser scanning microscopy. There was a significant association between the frequency of dotted vessels by NBI-B1 imaging and tissues confirmed as ASD pathologically (p=0.002). Conclusions: High magnification bronchovideoscopy combined with NBI was useful in the detection of capillary blood vessels in ASD lesions at sites of abnormal fluorescence. This may enable the discrimination between ASD and another pre-invasive bronchial lesion.

Evidence for Immune Responses to a Self-Antigen in Lung Transplantation: Role of Type V Collagen-Specific T Cells in the Pathogenesis of Lung Allograft Rejection

We have reported that lung allograft rejection involves an immune response to a native protein in the lung, type V collagen (col(V)), and that col(V)-induced oral tolerance prevented acute and chronic rejection. In support of these findings col(V) fragments were detected in allografts during rejection, but not in normal lungs. The purpose of the current study was to isolate and characterize col(V)-specific allograft-infiltrating T cells and to determine their contribution to the rejection response in vivo. Two col(V)-specific T cell lines, LT1 and LT3, were isolated from F344 (RT1lv1) rat lung allografts during rejection that occurred after transplantation into WKY (RT1l) recipients. Both cell lines, but not normal lung lymphocytes, proliferated in response to col(V). Neither LT1 nor LT3 proliferated in response to alloantigens. LT1 and LT3 were CD4+CD25− and produced IFN-γ in response to col(V). Compared with normal CD4+ T cells, both cell lines expressed a limited V-β TCR repertoire. Each cell strongly expressed V-β 9 and 16, but differed in expression of other V-βs. Adoptive transfer of each cell line did not induce pathology in lungs of normal WKY rats. In contrast, adoptive transfer of LT1, but not LT3, caused marked peribronchiolar and perivascular inflammation in isograft (WKY) lungs and abrogated col(V)-induced oral tolerance to allograft (F344) lungs. Collectively, these data show that lung allograft rejection involves both allo- and autoimmune responses, and graft destruction that occurs during the rejection response may expose allograft-infiltrating T cells to potentially antigenic epitopes in col(V).

A Phase I Study of In vitro Expanded Natural Killer T Cells in Patients with Advanced and Recurrent Non–Small Cell Lung Cancer

Purpose: Human Vα24 natural killer T (Vα24 NKT) cells bearing an invariant Vα24JαQ antigen receptor are activated by a glicolipid ligand α-galactosylceramide (αGalCer; KRN7000) in a CD1d-dependent manner. The human Vα24 NKT cells activated with αGalCer and interleukin-2 have been shown to produce large amounts of cytokines, such as IFN-γ, and also exerting a potent killing activity against various tumor cell lines. We did a phase I study with autologous activated Vα24 NKT cell therapy. Experimental Design: Patients with advanced or recurrent non–small cell lung cancer received i.v. injections of activated Vα24 NKT cells (level 1: 1 × 107/m2 and level 2: 5 × 107/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 Vα24 NKT cells, an increased number of peripheral blood Vα24 NKT cells was observed in two of three cases receiving a level 2 dose of activated Vα24 NKT cells. The number of IFN-γ-producing cells in peripheral blood mononuclear cells increased after the administration of activated Vα24 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 Vα24 NKT cell administration was well tolerated and carried out safely with minor adverse events even in patients with advanced diseases.

Anti-Type V Collagen Lymphocytes that Express IL-17 and IL-23 Induce Rejection Pathology in Fresh and Well-Healed Lung Transplants

Immunity to collagen V [col(V)] contributes to lung ‘rejection.’ We hypothesized that ischemia reperfusion injury (IRI) associated with lung transplantation unmasks antigenic col(V) such that fresh and well‐healed lung grafts have differential susceptibility to anti‐col(V)‐mediated injury; and expression of the autoimmune cytokines, IL‐17 and IL‐23, are associated with this process. Adoptive transfer of col(V)‐reactive lymphocytes to WKY rats induced grade 2 rejection in fresh isografts, but induced worse pathology (grade 3) when transferred to isograft recipients 30 days post‐transplantation. Immunhistochemistry detected col(V) in fresh and well‐healed isografts but not native lungs. Hen egg lysozyme‐reactive lymphocytes (HEL, control) did not induce lung disease in any group. Col(V), but not HEL, immunization induced transcripts for IL‐17 and IL‐23 (p19) in the cells utilized for adoptive transfer. Transcripts for IL‐17 were upregulated in fresh, but not well‐healed isografts after transfer of col(V)‐reactive cells. These data show that IRI predisposes to anti‐col(V)‐mediated pathology; col(V)‐reactive lymphocytes express IL‐17 and IL‐23; and anti‐col(V)‐mediated lung disease is associated with local expression of IL‐17. Finally, because of similar histologic patterns, the pathology of clinical rejection may reflect the activity of autoimmunity to col(V) and/or alloimmunity.

A Phase I-II Study of α-Galactosylceramide-Pulsed IL-2/GM-CSF-Cultured Peripheral Blood Mononuclear Cells in Patients with Advanced and Recurrent Non-Small Cell Lung Cancer

To evaluate the safety, immune responses, and antitumor responses after the administration of α-galactosylceramide (αGalCer) 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. αGalCer-pulsed IL-2/GM-CSF-cultured PBMCs (1 × 109/m2) 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 αGalCer-pulsed IL-2/GM-CSF-cultured PBMCs, an increased number of IFN-γ-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-γ-producing cells (≥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 αGalCer-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-γ-producing cells that result from αGalCer stimulation in PBMCs were significantly associated with prolonged MST. These results are encouraging and warrant further evaluation for survival benefit of this immunotherapy.

Smoking before surgery predicts poor long-term survival in patients with stage I non-small-cell lung carcinomas.

PURPOSE The majority of lung carcinoma patients requiring resection have smoking habits prior to surgical treatment, and the correlation of smoking with postoperative complications is well known. However, few studies have investigated the correlation between long-term survival and cigarette smoking in patients with primary, resected lung carcinoma. We analyzed the relationship between clinical factors, including cigarette smoking before surgery, and 10-year survival in stage I non-small-cell lung carcinoma (NSCLC). PATIENTS AND METHODS Cigarette smoking habit and other factors influencing either the overall survival or the disease-specific survival rates of patients with stage I primary, resected NSCLC were evaluated according to the Cox proportional hazards model using a total of 369 patients with stage I-NSCLC. RESULTS Comparison of the cause of death in patients with 30 or more pack-years and patients with less than 30 pack-years showed significant differences in the prevalence of recurrent disease and onset of nonmalignant disease. Multivariate analysis demonstrated significant correlations between overall survival and age and pack-years. Disease-specific survival showed significant correlations with age, tumor classification, and visceral pleural invasion. CONCLUSION Smoking pack-years is an important clinical prognostic factor in evaluating overall long-term survival in patients with stage I primary, resected NSCLC.