Ryo Nagashio

Expression of RACK1 is a novel biomarker in pulmonary adenocarcinomas

To develop useful early and/or differential diagnostic markers for pulmonary adenocarcinomas, we generated monoclonal antibodies using A549 cells derived from pulmonary adenocarcinomas as an immunogen. Hybridoma supernatants were immunohistochemically screened for antibody production by AMeX-fixed and paraffin-embedded A549 cell preparations. Positive clones were monocloned twice by limiting dilutions. From a group of obtained antibodies, an antibody designated as KU-Lu-3 showed cytoplasmic staining. The antigen recognized by KU-Lu-3 was detected by modified two-dimensional immunoblotting, and was determined to be the receptor of activated C kinase 1 (RACK1). To evaluate the utility of KU-Lu-3, we immunohistochemically studied 184 cases of pulmonary carcinoma and paired normal lung tissues, using formalin-fixed and paraffin-embedded tissue microarray sections. The expression was significantly high and frequent in adenocarcinomas but was barely detected in a few squamous cell carcinomas and large cell carcinomas (p<0.0001). Moreover, RACK1 expression was also significantly associated with the pathological stage, tumor size and lymph node status of adenocarcinoma patients, but not with tumor differentiation, or patient age and gender. These results suggest that RACK1 may be a novel differential diagnostic marker for pulmonary adenocarcinomas.

Detection of tumor-specific autoantibodies in sera of patients with lung cancer

The presence of autoantibodies (AAs) in sera from two pulmonary carcinoma patients, adenocarcinoma (AD) and small cell carcinoma (SCLC) was screened by immunoblotting using cell lysate of four cell lines (LCN1, large cell neuroendocrine carcinoma (LCNEC); N231, SCLC; A549, AD; RERF-LC-AI, squamous cell carcinoma (SCC)). To identify the antigens recognized by AAs, two-dimensional gel electrophoresis was immunoblotted and target spots were cut out from the membrane and gel. After trypsin digestion, the proteins were analyzed by mass-spectrometry using a liquid chromatography-tandem mass spectrometer. By this method, cytokeratin18 (CK18) and villin1 were identified with AAs in sera from patients with AD and SCLC, respectively. Thus, the expressions of CK18 and villin1 were further immunohistochemically studied on 124 formalin-fixed and paraffin-embedded pulmonary carcinomas of various histologic types (44 AD, 27 SCC, 29 SCLC, and 34 LCNEC) using commercially available CK18 and villin1 antibodies. Positive CK18 immunostaining was observed in almost all cases with staining intensities significantly higher in AD and LCNEC than in SCC and SCLC. Villin1 was detected in 17/44 (38.6%) of AD and 21/34 (61.8%) of LCNEC, respectively, while in only one each of SCLC and SCC. Thus, villin1 and CK18 may be useful markers to distinguish LCNEC/AD from SCLC/SCC, and the present method might be useful to identify specific tumor-associated molecules in sera from pulmonary carcinoma patients with different histologic types.

Proteomics of tumor‐specific proteins in cerebrospinal fluid of patients with astrocytoma: Usefulness of gelsolin protein

Changes in cerebrospinal fluid (CSF) composition have been shown to accurately reflect pathological processes in the CNS, and are potential indicators of abnormal CNS states, such as tumor growth. To detect biomarkers in high‐grade astrocytomas, the differential expression of proteins in the cerebrospinal fluid was analyzed from two cases each of diffuse astrocytoma (grade II), and glioblastoma (grade IV) using agarose 2‐D gel electrophoresis (2‐DE). It was found that the expression of gelsolin protein decreased with histological grade. To examine whether gelsolin is a useful indicator of tumor aggressiveness or patient outcome, its expression was further studied on immunohistochemistry in 41 formalin‐fixed and paraffin‐embedded astrocytomas. The positive cell rate of gelsolin in tumors was 59.4% in grade II, 30.0% in grade III and 29.4% in grade IV, respectively. Gelsolin expression was significantly lower in high‐grade astrocytomas (grade III or IV) than in low‐grade astrocytomas (grade II; P < 0.05). Moreover, in astrocytomas the overall survival of patients in the low‐expression group was significantly poorer than in the high expression group (P < 0.05). These data suggest that gelsolin is a prognostic factor in astrocytoma.

CAXII Is a sero-diagnostic marker for lung cancer.

To develop sero-diagnostic markers for lung cancer, we generated monoclonal antibodies using pulmonary adenocarcinoma (AD)-derived A549 cells as antigens by employing the random immunization method. Hybridoma supernatants were immunohistochemically screened for antibodies with AMeX-fixed and paraffin-embedded A549 cell preparations. Positive clones were monocloned twice through limiting dilutions. From the obtained monoclonal antibodies, we selected an antibody designated as KU-Lu-5 which showed intense membrane staining of A549 cells. Based on immunoprecipitation and MADLI TOF/TOF-MS analysis, this antibody was recognized as carbonic anhydrase XII (CAXII). To evaluate the utility of this antibody as a sero-diagnostic marker for lung cancer, we performed dot blot analysis with a training set consisting of sera from 70 lung cancer patients and 30 healthy controls. The CAXII expression levels were significantly higher in lung cancer patients than in healthy controls in the training set (P<0.0001), and the area under the curve of ROC was 0.794, with 70.0% specificity and 82.9% sensitivity. In lung cancers, expression levels of CAXII were significantly higher in patients with squamous cell carcinoma (SCC) than with AD (P = 0.035). Furthermore, CAXII was significantly higher in well- and moderately differentiated SCCs than in poorly differentiated ones (P = 0.027). To further confirm the utility of serum CAXII levels as a sero-diagnostic marker, an additional set consisting of sera from 26 lung cancer patients and 30 healthy controls was also investigated by dot blot analysis as a validation study. Serum CAXII levels were also significantly higher in lung cancer patients than in healthy controls in the validation set (P = 0.030). Thus, the serum CAXII levels should be applicable markers discriminating lung cancer patients from healthy controls. To our knowledge, this is the first report providing evidence that CAXII may be a novel sero-diagnostic marker for lung cancer.

Expression of autocrine motility factor mRNA is a poor prognostic factor in high-grade astrocytoma

It has been reported that tumor infiltration is correlated with the expression of autocrine motility factor (AMF) and its receptor 78 kDa glycoprotein (gp78). The purpose of the present study was to detect AMF and gp78 mRNA expression levels and their localization in high‐grade astrocytomas (glioblastoma and anaplastic astrocytoma) and to determine whether AMF and gp78 are important prognostic factors. A total of 32 formalin‐fixed and paraffin‐embedded glioblastomas and 23 formalin‐fixed and paraffin‐embedded anaplastic astrocytomas was used. The expressions of AMF and gp78 mRNA were detected using the highly sensitive in situ hybridization method. The expression of AMF mRNA was detected in 27 of 32 glioblastomas (84.4%) and 11 of 23 anaplastic astrocytomas (47.8%). The positivity of AMF mRNA was significantly higher in glioblastomas than in anaplastic astrocytomas (P = 0.0094), but gp78 mRNA was detected in most cases and no statistical significance was observed. The overall survival of patients with AMF expression was significantly shorter than patients without AMF expression (P = 0.0175). In anaplastic astrocytomas, the overall survival of patients with AMF expression was also significantly shorter than in patients without AMF expression (P = 0.0058). This study demonstrated that AMF is a poor prognostic factor in high‐grade astrocytomas.

Significant high expression of cytokeratins 7, 8, 18, 19 in pulmonary large cell neuroendocrine carcinomas, compared to small cell lung carcinomas

The aim of the present study was to clarify protein profiling in small cell lung carcinoma (SCLC) and pulmonary large cell neuroendocrine carcinoma (LCNEC). The proteomic approach was used, and involved cell lysate from two cell lines (N231 derived from SCLC and LCN1 derived from LCNEC), with 2‐D gel electrophoresis (2‐DE). In the present study, 25 protein spots with greater than twofold quantitative differences between LCN1 and N231 cells on 2‐DE gels were confirmed. Within the 25 identified proteins, cytokeratins (CK) 7, 8, 18 and 19 were upregulated in LCN1 cells compared with N231 cells. The expression of CK7, 8, 18, and 19 was further studied on immunohistochemistry with 81 formalin‐fixed and paraffin‐embedded pulmonary carcinomas, which included 27 SCLC, 30 LCNEC, 14 adenocarcinomas, and 10 squamous cell carcinomas. Although the expression of CK7, 8, 18, and 19 was observed in all histological types, the mean immunostaining scores of CK7, 8, 18, and 19 were significantly higher in LCNEC than in SCLC (P < 0.001, P < 0.001, P < 0.01 and P < 0.001, respectively). These data suggest that the biological characteristics of LCNEC and SCLC may be different and the expression of CK may serve as differential diagnostic markers.

The clinicopathological significance of Lgr5 expression in lung adenocarcinoma

BACKGROUND The leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5), also known as Gpr49, has been identified as a marker of crypt basal columnar stem cells along the gastrointestinal tract and of bulge stem cells in the hair follicle. The aims of this study were to immunohistochemically examine Lgr5 expression in surgically resected non-small cell lung carcinomas (NSCLC), and evaluate the relationships between Lgr5 expression and the clinicopathological parameters and prognosis of patients. METHODS Lgr5 expression was immunohistochemically studied in 266 consecutive resected NSCLCs, and its associations with clinicopathological parameters including TTF-1 and CDX-2 expressions were evaluated. Kaplan-Meier survival analysis and Cox proportional hazards models were used to estimate the effect of Lgr5 expression on survival. RESULTS Lgr5 was detected only in tumors with adenocarcinoma histology, and 16 cases were judged as positive. Among lung adenocarcinomas, Lgr5 expression was significantly associated with a larger tumor size (> 5 cm) (P = 0.033), higher pathological TNM stage of the disease (stage II and III) (P = 0.025), TTF-1-negative adenocarocinoma (P = 0.042), and poorer prognosis (P = 0.026). However, Lgr5 expression was not an independent predictor of poorer survival after controlling for clinicopathological factors. CONCLUSIONS The present study reveals that Lgr5 is expressed in a subset of lung adenocarcinoma, and its expression is related to some clinicopathological parameters and a poorer prognosis, although further studies are required to clarify the biological function of Lgr5 in lung adenocarcinoma.

Diagnostic and Prognostic Significances of MUC5B and TTF-1 Expressions in Resected Non-Small Cell Lung Cancer

To investigate the relationships between the expression of MUC5B and clinicopathological parameters, the expression of MUC5B was immunohistochemically studied. MUC5B expression was observed in 129 of 198 (65.2%) adenocarcinomas and in 4 of 49 (8.2%) squamous cell carcinomas (P < 0.00001). MUC5B expression was significantly associated with poorer differentiation (P = 0.0303), higher pathological TNM stage (p = 0.0153) and poorer prognosis of adenocarcinoma patients (P = 0.0017). Multivariable analysis with Cox proportional hazards models confirmed that MUC5B expression increased the hazard of death after adjusting for other clinicopathological factors (HR = 2.66; 95%CI, 1.26–5.61). We also immunohistochemically evaluated TTF-1 expression and found that the combination of MUC5B with TTF-1 is a useful marker for adenocarcinomas. The diagnostic accuracies of TTF-1 and MUC5B for adenocarcinoma were 83.8% and 70.4%, respectively. The accuracy increased to 94.3% when the two factors were combined. In survival analysis, the MUC5B(High)/TTF-1(−) group was significantly associated with a poorer outcome compared with the MUC5B(Low)/TTF-1(+) group (p < 0.0001). The present study suggested that the combination of MUC5B and TTF-1 expression is useful for discriminating adenocarcinomas from squamous cell carcinomas, yielding prognostic significance in patients with lung adenocarcinoma.

Impact of PD-L1 Expression in Patients with Surgically Resected Non-Small-Cell Lung Cancer

Background: Immunotherapy can become a crucial therapeutic option to improve the prognosis of patients with non-small-cell lung cancer (NSCLC). Here, we evaluated the impact of programmed cell death ligand-1 (PD-L1) expression in surgically resected NSCLCs. Methods: We estimated PD-L1 expression in 229 consecutive NSCLC specimens using rabbit polyclonal antibodies to human PD-L1 in a SP263 immunohistochemical assay and evaluated PD-L1 expression for potential associations with clinicopathological parameters and survival time. Results: PD-L1 expression was significantly higher in tumors from men or current smokers. Squamous cell carcinoma histology was independently associated with high PD-L1 expression according to multivariate analysis (p = 0.015). The 5-year survival rate of patients was 70%, and the difference in the 5-year survival rate according to PD-L1 expression was not statistically significant (high expression group [67%] vs. low expression group [68%]); however, the squamous cell carcinoma group exhibited significantly lower 5-year survival rates as compared to the non-squamous cell carcinoma group (53 and 71%, respectively; p = 0.026). Conclusion: Here, we revealed high PD-L1 expression and poor prognosis observed in patients with surgically resected squamous NSCLC as compared with non-squamous NSCLC. Our results support the identification of patient subsets that most likely respond to anti-PD-1 therapy as the first step in precision medicine.

Calnexin is a novel sero-diagnostic marker for lung cancer

To develop sero-diagnostic markers for lung cancer, we generated monoclonal antibodies using lung adenocarcinoma (AC)-derived A549 cells as antigens by employing the random immunization method. Hybridoma supernatants were immunohistochemically screened for antibodies with AMeX-fixed and paraffin-embedded A549 cell preparations. Positive clones were monocloned twice through limiting dilutions. From the obtained monoclonal antibodies, one designated as KU-Lad-001 was recognized as calnexin (CANX) based on immunoprecipitation and MADLI TOF/TOF-MS analysis. To evaluate the utility of this antibody as a sero-diagnostic marker for lung cancer, we performed reverse-phase protein array analysis with samples of 195 lung cancer patients and 100 healthy controls. The CANX expression levels were significantly higher in lung cancer patients than in healthy controls (P<0.0001), and the area under the curve of ROC was 0.980, with 96.9% specificity and 99.0% sensitivity. Furthermore, since CANX was also detected in stage I disease, the serum CANX levels should be applicable markers discriminating lung cancer patients from healthy controls and possibly used in the detection of early lung cancer. To our knowledge, the present results provide evidence that CANX may be a novel sero-diagnostic marker for lung cancer.