Yasushi Horimasu

MUC5B promoter polymorphism in Japanese patients with idiopathic pulmonary fibrosis

A single nucleotide polymorphism (SNP) rs35705950 in the promoter of Mucin 5B (MUC5B) has been reported to be associated with idiopathic pulmonary fibrosis (IPF) mainly in Caucasian populations. This study was conducted to confirm the association between rs35705950 and IPF in a Japanese population.

Different MUC1 gene polymorphisms in German and Japanese ethnicities affect serum KL-6 levels

BACKGROUND KL-6 is a high-molecular-weight glycoprotein classified as human Mucin-1 (MUC1). KL-6 has been reported to be a sensitive biomarker for interstitial lung diseases (ILDs) in the Japanese population. It is also known that polymorphisms in the MUC1 gene affect serum levels of KL-6. This study was conducted to evaluate serum levels of KL-6 and MUC1 polymorphisms in both German and Japanese populations. METHODS Serum levels of KL-6 were measured in 267 patients with ILDs (152 German and 115 Japanese) and 186 healthy subjects (HS) (76 German and 110 Japanese). In addition, rs4072037 single nucleotide polymorphisms (SNPs) were genotyped by polymerase chain reaction. The optimal cutoff values for discriminating patients with ILDs from HS was determined by receiver operating characteristic analysis based on ethnicity and rs4072037 genotypes. RESULTS The serum KL-6 levels in patients with ILDs were significantly higher compared with HS in both the German and the Japanese cohorts (both p<0.001). The discriminating cutoff value of serum KL-6 in the German cohort was significantly higher than the value in the Japanese cohort. The difference in the serum levels of KL-6 was significantly associated with the rs4072037 genotype distribution. CONCLUSIONS Even in the German cohort, the serum KL6 levels were significantly higher in patients with ILDs than HS. Because of differences in the genotype distribution of rs4072037, the KL-6 cutoff value for the German cohort that discriminated patients with ILDs from HS was significantly higher than the value in the Japanese cohort.

Levels of surfactant proteins A and D and KL-6 are elevated in the induced sputum of chronic obstructive pulmonary disease patients: a sequential sputum analysis.

Background: Recent clinical studies have suggested that serum surfactant protein (SP) A, SP-D and Krebs von den Lungen-6 (KL-6) are potential biomarkers for interstitial lung diseases. Serum levels of SP-A and SP-D have also been found to be elevated in chronic obstructive pulmonary disease (COPD), but their significance has not been evaluated or compared in induced sputum samples obtained directly from the airways. Objective: A sequential sputum analysis was conducted to assess the value of SP-A, SP-D and KL-6 levels in COPD. Methods: The study material consisted of induced sputum samples from 61 subjects, 28 with COPD and 33 with prolonged cough (cough lasting >3 weeks and normal spirometry). Sputum was collected in 3 fractions (3 periods of 5 min each). Sputum levels of these proteins were measured, and receiver operating characteristic curve analysis was carried out to evaluate the sensitivity, specificity and area under the curve (AUC) for each fraction. Results: The levels of SP-A, SP-D and KL-6 were higher in patients with COPD than in those with prolonged cough in each of the fractions. Sputum levels of these proteins correlated inversely with obstruction and positively with ageing, smoking history, sputum macrophages and eosinophils. Sputum fractionation had a relatively minor effect on the levels and AUC of these proteins. Conclusion: Sequential sputum analysis from 3 consecutive fractions indicated a significant difference in the levels of SP-A, SP-D and KL-6 between COPD and prolonged cough. However, sputum fractionation itself had a relatively minor effect on the levels of these proteins.

Ageing and long-term smoking affects KL-6 levels in the lung, induced sputum and plasma

BackgroundKL-6 is a high-molecular-weight glycoprotein classified as a human MUC1 mucin. It was hypothesized that KL-6 could be detectable in the circulating blood and especially in airway secretions in lung diseases associated with mucus production such as chronic obstructive pulmonary disease (COPD). Additional aims of this study were to investigate whether the levels of KL-6 in plasma and sputum are related to ageing and smoking history.MethodsThe concentrations of KL-6 in plasma and induced sputum supernatants from young and/or middle aged/elderly non-smokers, smokers and patients with COPD were assayed by ELISA (n = 201). The subjects were classified into five groups according to age, smoking status and presence of COPD. In addition, KL-6 expression in control and diseased lung i.e. samples from patients with COPD (n = 28), were analyzed by immunohistochemistry and digital image analysis.ResultsThe plasma levels of KL-6 increased with age both in non-smokers and smokers. Among middle aged/elderly subjects, plasma KL-6 levels in all smokers regardless of COPD were significantly higher than in non-smokers, whereas sputum levels of KL-6 were significantly higher in COPD compared not only to non-smokers but also to smokers. KL-6 was more prominently expressed in the bronchiolar/alveolar epithelium in COPD than in the control lungs. Plasma and sputum KL-6 levels correlated inversely with obstruction and positively with smoking history and ageing. The linear multiple regression analysis confirmed that age and cigarette smoking had independent effects on plasma KL-6.ConclusionsKL-6 increases with ageing and chronic smoking history, but prospective studies will be needed to elucidate the significance of KL-6 in chronic airway diseases.

Comparative Study of Circulating MMP-7, CCL18, KL-6, SP-A, and SP-D as Disease Markers of Idiopathic Pulmonary Fibrosis

Background. Recent reports indicate that matrix metalloproteinase-7 (MMP-7) and CC-chemokine ligand 18 (CCL18) are potential disease markers of idiopathic pulmonary fibrosis (IPF). The objective of this study was to perform direct comparisons of these two biomarkers with three well-investigated serum markers of IPF, Krebs von den Lungen-6 (KL-6), surfactant protein-A (SP-A), and SP-D. Methods. The serum levels of MMP-7, CCL18, KL-6, SP-A, and SP-D were evaluated in 65 patients with IPF, 31 patients with bacterial pneumonia, and 101 healthy controls. The prognostic performance of these five biomarkers was evaluated in patients with IPF. Results. The serum levels of MMP-7, KL-6, and SP-D in patients with IPF were significantly elevated compared to those in patients with bacterial pneumonia and in the healthy controls. Multivariate survival analysis showed that serum MMP-7 and KL-6 levels were independent predictors in IPF patients. Moreover, elevated levels of both KL-6 and MMP-7 were associated with poorer survival rates in IPF patients, and the combination of both markers provided the best risk discrimination using the C statistic. Conclusions. The present results indicated that MMP-7 and KL-6 were promising prognostic markers of IPF, and the combination of the two markers might improve survival prediction in patients with IPF.

Differences in serum SP-D levels between German and Japanese subjects are associated with SFTPD gene polymorphisms

BackgroundSurfactant protein A (SP-A) and SP-D are clinically established in Japan as serum biomarkers for diagnosing interstitial lung diseases (ILDs). Serum SP-D levels are affected by genetic variants. We conducted the present study to examine whether serum SP-A and/or SP-D levels in healthy subjects (HS) and patients with ILDs differ between populations with different genetic backgrounds.MethodsGerman subjects (n = 303; 138 patients with idiopathic interstitial pneumonias [IIPs] and 165 HS) and Japanese subjects (n = 369; 94 patients with IIPs and 275 HS) were enrolled. Serum SP-A and SP-D levels were measured using an enzyme-linked immunosorbent assay, and four single-nucleotide polymorphisms (SNPs) in the SFTPD gene were genotyped using genomic DNA extracted from blood samples.ResultsIn both the German and Japanese cohorts, serum SP-A and SP-D levels were significantly higher in patients with IIPs than in HS. There were no significant differences in SP-A levels between the German and Japanese cohorts; however, we found that serum SP-D levels were significantly higher in the German cohort, both in patients with IIPs and in HS (p < 0.001 and p = 0.005, respectively). Furthermore, the genotype distributions of the four SNPs in the SFTPD gene (rs721917, rs1998374, rs2243639, and rs3088308) were significantly different between German and Japanese cohorts (p < 0.001, p < 0.001, p = 0.022, and p < 0.001, respectively), and univariate linear regression analyses revealed that the genotypes of rs721917, rs1998374, and rs2243639 significantly correlated with serum SP-D levels (p < 0.001, p < 0.001, and p = 0.011, respectively). Furthermore, multivariate analyses revealed that the genotypes of these three SNPs correlated independently with serum SP-D levels (p < 0.001, p = 0.001, and p = 0.038, respectively), whereas ethnicity did not significantly correlate with serum SP-D levels.ConclusionsIn patients with IIPs and HS, serum SP-D, but not SP-A, levels were significantly higher in the German than in the Japanese cohort, in part, because of the different frequencies of SFTPD gene polymorphisms.

Interferon (alpha, beta and omega) receptor 2 is a prognostic biomarker for lung cancer.

Objectives: It has been reported that the type I interferon receptor subunit, interferon (alpha, beta and omega) receptor 2 (IFNAR2), is overexpressed in several malignancies, primarily adenocarcinomas (ADCs); however, the biological significance of IFNAR2 in human lung cancer has not yet been studied. Methods: Immunohistochemical analysis of 113 surgically resected lung specimens was performed, and the results were evaluated in association with clinical variables, including survival. Serum concentrations of IFNAR2 were also determined by an enzyme-linked immunosorbent assay in 157 lung cancer patients and 164 healthy volunteers. Results: IFNAR2 overexpression was observed in all histological types of lung cancer examined. Furthermore, strong IFNAR2 expression was associated with shorter progression-free survival (PFS) and overall survival (OS) (p < 0.0001 and p = 0.0110, respectively) in non-small cell lung cancer patients. Multivariate analyses confirmed its independent prognostic value for PFS and OS (p < 0.0001 and p = 0.0222, respectively). IFNAR2 serum levels were also significantly higher in lung cancer patients than in healthy volunteers (p < 0.0001). Conclusions: IFNAR2 overexpression was observed in various histological types of lung cancer, and appears to be associated with lung cancers that behave aggressively. The results of this study strongly support the potential of IFNAR2 to be a prognostic biomarker for lung cancer.

Change in serum KL-6 level from baseline is useful for predicting life-threatening EGFR-TKIs induced interstitial lung disease

BackgroundA high incidence of interstitial lung disease (ILD) has been reported in patients with advanced non-small cell lung cancer (NSCLC) treated with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), particularly in Japanese populations. A previous report from our laboratory demonstrated that KL-6 was a useful serum biomarker to assess the severity of drug-induced pneumonitis. Based on these observations, this study was conducted to evaluate the risk factors of EGFR-TKIs induced ILD and the usefulness of monitoring serum KL-6 levels in patients who developed EGFR-TKIs induced ILD in a large multi-institutional setting.MethodsWe retrospectively reviewed clinical records and radiographies of 341 patients with advanced NSCLCs who were treated with EGFR-TKIs, and analyzed risk factors for the development of EGFR-TKIs induced ILD. Changes of circulating levels of KL-6 were also evaluated in the patients who developed EGFR-TKIs induced ILD.ResultsAmong the 341 patients included in this study, 20 (5.9%) developed EGFR-TKIs induced ILD, and 9 (2.6%) died from ILD. Univariate analyses revealed that only preexisting pulmonary fibrosis was a significant risk factor for the development of EGFR-TKIs induced ILD (p = 0.003). Absolute levels of circulating KL-6 at neither baseline nor the onset of ILD could discriminate between life-threatening and non-life threatening EGFR-TKIs induced ILDs. However, we found that the ratios of serum KL-6 levels just after the onset of EGFR-TKIs induced ILD to those at baseline could quite precisely distinguish survivors from non-survivors (p = 0.006) as well as acute interstitial pneumonia (AIP) pattern from non-AIP pattern (p = 0.005).ConclusionsThe results of this study strongly support the potential of KL-6 as a diagnostic biomarker for life-threatening EGFR-TKIs induced ILD. Monitoring of KL-6 is also useful to evaluate the progression and severity of EGFR-TKIs induced ILD.

MT95-4, a fully humanized antibody raised against aminopeptidase N, reduces tumor progression in a mouse model

Aminopeptidase N (APN/CD13) is involved in tumor cell invasion and tumor angiogenesis and is considered a promising therapeutic target in the treatment of cancer. To develop a novel monoclonal antibody‐based cancer therapy targeting APN/CD13, we established a fully humanized anti‐APN/CD13 monoclonal antibody, MT95‐4. In vitro, MT95‐4 inhibited APN/CD13 enzymatic activity on the tumor cell surface and blocked tumor cell invasion. B16 mouse melanoma cells stably expressing human APN/CD13 were also established and were inoculated s.c. or injected i.v. into nude mice. We found that expression of human APN/CD13 in murine melanoma cells increased the size of subcutaneous tumors, extent of lung metastasis and degree of angiogenesis in the subcutaneous tumors; these tumor‐promoting and angiogenesis‐promoting characteristics were reduced by the i.p. administration of MT95‐4. To further verify the specificity of MT95‐4 for neutralization of APN/CD13 activity, MT95‐4 was administered into NOD/SCID mice inoculated s.c. with H1299 or PC14 cells, which exhibit high expression of APN/CD13, or with A549 cells, which exhibit weak expression of APN/CD13. MT95‐4 reduced tumor growth and angiogenesis in mice bearing H1299‐derived and PC14‐derived tumors, but not in mice bearing A549‐derived tumors. These results suggested that the antitumor and anti‐angiogenic effects of MT95‐4 were dependent on APN/CD13 expression in tumor cells. Given that MT95‐4 is the first fully humanized monoclonal antibody against APN/CD13, MT95‐4 should be recognized as a promising candidate for monoclonal antibody therapy against tumors expressing APN/CD13.

AGER gene polymorphisms and soluble receptor for advanced glycation end product in patients with idiopathic pulmonary fibrosis

The receptor for advanced glycation end product (RAGE) is a multiligand cell‐surface receptor abundantly expressed in the lung. RAGE/ligand interaction has been postulated to participate in the pathogenesis of inflammatory diseases, while soluble RAGE (sRAGE) might act as a decoy receptor. A functional polymorphism rs2070600 in the gene coding RAGE (AGER) might modulate its receptor function. The aim of this study was to investigate the association of AGER polymorphisms and circulatory sRAGE with the development and progression of idiopathic pulmonary fibrosis (IPF).