Shinichiro Ryuge

Prognostic Significance of Nestin Expression in Resected Non-small Cell Lung Cancer

BACKGROUND Nestin is a class 6 intermediate filament protein expressed in stem/progenitor cells during CNS development. Nestin expression has been detected in many kinds of tumors and was reported in a recent small-scale study in non-small cell lung cancer (NSCLC). We investigated the relationships between nestin expression and clinicopathologic parameters and determined its prognostic significance concerning survival in patients with resected NSCLC. METHODS Nestin expression in tumor cells was studied immunohistochemically in 171 consecutive patients with NSCLC, and associations with clinicopathologic parameters were evaluated. Kaplan-Meier survival analysis and Cox proportional hazards models were used to estimate the effect of nestin expression on survival. RESULTS Nestin expression was observed in tumor cell samples in 27 of the 171 patients with NSCLC (15.8%). Nestin had only cytoplasmic expression. Clinicopathologically, nestin expression was significantly associated with squamous cell carcinoma (P = .001), poorer differentiation (P = .007), lymph node metastasis (P = .008), intratumoral vascular invasion (P = .003), intratumoral lymphatic invasion (P = .008), pleural invasion (P = .039), and poorer prognosis (P < .001). Multivariable analysis confirmed that nestin expression increased the hazard of death after adjusting for other clinicopathologic factors (hazard ratio, 2.75; 95% CI, 1.39-5.46). CONCLUSIONS The present study suggests that nestin expression is a prognostic indicator of poorer survival probability for patients with resected NSCLC and may be used as a potential marker for select patients who should receive adjuvant chemotherapy.

Circulating tumor cells as a prognostic factor in patients with small cell lung cancer

The detection of circulating tumor cells (CTCs) in peripheral blood is currently an important field of study. Detection of CTCs by the OBP-401 assay (TelomeScan®) has previously been reported to be useful in the diagnosis, prognosis and evaluation of therapeutic efficacy in breast and gastric cancer. The aim of the present study was to evaluate the OBP-401 assay as a novel method of detecting CTCs of small cell lung cancer (SCLC) patients and to evaluate whether CTC count is associated with prognosis. Prospectively, 30 consecutively diagnosed SCLC patients who had commenced chemotherapy or chemoradiotherapy were enrolled as subjects of the current study. Peripheral blood specimens were collected from the SCLC patients prior to and following the initiation of treatment and the viable CTCs were detected in the specimens following incubation with a telomerase-specific, replication-selective, oncolytic adenoviral agent, which was carrying the green fluorescent protein gene. CTCs were detected in 29 patients (96%). The group of 21 patients with a CTC count of <2 cells/7.5 ml prior to treatment (baseline) had a significantly longer median survival time than the group of eight patients with a CTC count of ≥2 cells/7.5 ml prior to treatment (14.8 and 3.9 months, respectively; P=0.007). The results of a multivariate analysis showed that the baseline CTC count was an independent prognostic factor for survival time (hazard ratio, 3.91; P=0.026). Among the patients that achieved a partial response to treatment, patients who had a CTC count of <2 cells/7.5 ml following two cycles of chemotherapy tended to have a longer median progression-free survival compared with patients who had a CTC count of ≥2 cell/7.5 ml (8.3 and 3.8 months, respectively; P=0.07). Therefore, CTCs may be detected via OBP-401 assay in SCLC patients and the CTC count prior to treatment appears to be a strong prognostic factor.

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.

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.

Pemetrexed for previously treated patients with non-small cell lung cancer and differences in efficacy according to thymidylate synthase expression.

The purpose of this study was to evaluate the efficacy of pemetrexed monotherapy in previously treated patients with advanced non-small cell lung cancer (NSCLC) including salvage treatment, and to evaluate whether thymidylate synthase (TS) expression is a predictor for pemetrexed efficacy. Hundred and four previously treated patients with advanced NSCLC who received pemetrexed monotherapy were retrospectively evaluated for clinical efficacy and toxicity. If available, tissue specimens of patients were also analyzed immunohistochemically for TS expression. The patients’ median age was 65 years (range: 43–82). An overall response rate of 9.6% and a median progression-free survival (PFS) time of 3.4 months were achieved. The response rates for the second-line, third-line, fourth-line or further treatments were 9.1, 9.3 and 10.2% (p = 0.33); the median PFS were 3.3, 3.2 and 3.8 months (p = 0.21). The median follow-up duration was 14.9 months; the median overall survival (OS) was 11.9 months. The median PFS and OS were significantly longer in the TS-negative group than in the TS-positive group (5.8 months vs. 1.6 months; p = 0.03, and 14.7 months vs. 8.6 months; p = 0.04, respectively). Pemetrexed monotherapy could be considered as an option in the fourth or later lines of treatment of previously treated patients with advanced NSCLC as well as a second- or third-line treatment, and TS expression may be a potentially predictive factor for pemetrexed efficacy in NSCLC patients.

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.

The balance between the expressions of hASH1 and HES1 differs between large cell neuroendocrine carcinoma and small cell carcinoma of the lung

To clarify the biological differences between small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC), we investigated the expression of two bHLH type transcription factors, human achaete-scute homolog 1 (hASH1) and hairy/enhancer of split 1 (HES1), which positively and negatively regulate the neuroendocrine differentiation of respiratory epithelial cells, respectively. Eighty-eight formalin-fixed and paraffin-embedded pulmonary carcinomas (32 SCLC, 32 LCNEC, 14 adenocarcinomas, and 10 squamous cell carcinomas) and 14 SCLC and 1 LCNEC derived cell lines were used. hASH1 and HES1 mRNA were detected using a highly sensitive in situ hybridization method with digoxigenin-labeled cRNA probes and biotinylated tyramide. The staining results were scored from 0 to 12 by multiplying the staining intensity by the percentage of positive tumor cells. The mean staining score of hASH1 mRNA was significantly higher in SCLC than in LCNEC (p<0.01); conversely, that of HES1 mRNA was lower in SCLC than in LCNEC (p<0.01). These findings reveal that SCLC more strongly expresses the neuroendocrine phenotype, while LCNEC shows characteristics more similar to the ciliated epithelium phenotype, suggesting that the biological characteristics of these two tumors are different.

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.