Kengo Yanagita

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.

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.

Serum expression of S100A6 is a potential detection marker in patients with urothelial carcinoma in the urinary bladder

To investigate the level of serum S100A6 in patients with bladder cancer and in healthy controls and compare these levels with clinicopathologic findings, we evaluated the level of serum S100A6 in 30 healthy controls and 50 patients with bladder cancer diagnosed via transurethral resection of bladder tumor and/or radical cystectomy. S100A6 in sera was detected by employing automatic dot blot systems, and the micro Dot Blot array with a 256-solid pin configuration. The normalized signal of serum S100A6 expression in bladder cancer patients was significantly higher than that of healthy controls (P = 0.001). Serum S100A6 expression of non-muscle-invasive cancer (NMIC) was significantly higher than that of healthy controls (P = 0.04). Furthermore, the S100A6 serum level in patients with muscle-invasive bladder cancer was significantly higher than that in patient with NMIC (P = 0.004). The sensitivity and specificity were 48.0% (95% CI: 0.337-0.626) and 93.3% (95% CI: 0.779-0.992), respectively. The area under the curve was 0.727. Serum S100A6 expression is a potentially effective detection marker for bladder cancer. Applying this serum marker to clinical practice would require less-invasive examinations of patients and would help to detect life-threatening cancerous lesions earlier than current modalities.

Moesin and stress-induced phosphoprotein-1 are possible sero-diagnostic markers of psoriasis.

To identify diagnostic markers for psoriasis vulgaris and psoriatic arthritis, autoantibodies in sera from psoriasis vulgaris and psoriatic arthritis patients were screened by two-dimensional immunoblotting (2D-IB). Based on 2D-IB and MADLI TOF/TOF-MS analyses, eleven proteins each in psoriasis vulgaris and psoriatic arthritis were identified as autoantigens. Furthermore, serum levels of moesin, keratin 17 (K17), annexin A1 (ANXA1), and stress-induced phophoprotein-1 (STIP1), which were detected as autoantigens, were studied by dot blot analysis with psoriasis patients and healthy controls. The levels of moesin and STIP1 were significantly higher in sera from patients with psoriasis vulgaris than in the controls (moesin: P<0.05, STIP1: P<0.005). The area under the curve (AUC) for moesin and STIP1 between patients with psoraisis vulgaris and controls was 0.747 and 0.792, respectively. STIP1 and K17 levels were significantly higher in sera from patients with psoriatic arthritis than in those with psoriasis vulgaris (P<0.05 each). The AUC for STIP1 and K17 between patients with psoriatic arthritis and psoriasis vulgaris was 0.69 and 0.72, respectively. The STIP1 or moesin, CK17 serum level was not correlated with disease activity of psoriasis patients. These data suggest that STIP1 and moesin may be novel and differential sero-diagnostic markers for psoriasis vulgaris and psoriatic arthritis.

High expression level of preoperative serum Uroplakin III is associated with biologically aggressive bladder cancer.

BACKGROUND Uroplakins have been widely investigated as potential markers in patients with bladder cancer because these proteins are specific to the urothelium. However, the role of uroplakin proteins in bladder cancer remains unknown. In this study, preoperative serum levels of uroplakin III were measured in patients with urothelial carcinoma of the urinary bladder and examined for possible association with clinicopathological features and clinical outcomes. MATERIALS AND METHODS This study included 52 bladder cancer patients at various stages and 28 healthy controls. Uroplakin III levels were detected in preoperative sera using an automated dot blot system and a micro-dot blot array. RESULTS There was a significant increase in serum uroplakin III levels in patients with bladder cancer as compared to healthy controls (p<0.05). In addition, serum uroplakin III levels were associated with muscle-invasive status, high grade and lymphovascular invasion (p<0.02). Log-rank tests indicated high serum uroplakin III to be significantly associated with cancer-specific mortality. CONCLUSIONS Determination of serum uroplakin III level could be valuable for identifying patients with biologically aggressive bladder cancer.

Serum Anti-Gal-3 Autoantibody is a Predictive Marker of the Efficacy of Platinum-Based Chemotherapy against Pulmonary Adenocarcinoma

BACKGROUND Identification of predictive markers for the efficacy of platinum-based chemotherapy is necessary to improve the quality of the life of cancer patients. MATERIALS AND METHODS We detected proteins recognized by autoantibodies in pretreated sera from patients with lung adenocarcinoma (AC) evaluated as showing progressive disease (PD) or a partial response (PR) after cisplatin-based chemotherapy by proteomic analysis. Then, the levels of the candidate autoantibodies in the pretreated serum were validated by dot-blot analysis for 22 AC patients who received platinum-based chemotherapy, and the expression of identified proteins was immunohistochemically analyzed in 40 AC biopsy specimens. RESULTS An autoantibody against galectin-3 (Gal-3) was detected in pretreated sera from an AC patient with PD. Serum IgG levels of anti-Gal-3 autoantibody were significantly higher in patients evaluated with PD than in those with PR and stable disease (SD) (p = 0.0084). Furthermore, pretreated biopsy specimens taken from patients evaluated as showing PD following platinum- based chemotherapy showed a tendency to have a higher positive rate of Gal-3 than those with PR and SD (p = 0.0601). CONCLUSIONS These results suggest that serum IgG levels of anti-Gal-3 autoantibody may be useful to predict the efficacy of platinum-based chemotherapy for patients with lung AC.

Prognostic significance of G6PD expression and localization in lung adenocarcinoma

Abnormal expressions of extracellular matrix (ECM) proteins are correlated with increased tumor progression, an advanced histologic grade, and metastasis. LCN1 cells derived from a pulmonary large cell neuroendocrine carcinoma were grown to form an Aegagropila-shaped conglomeration on a suspension culture dish (LCN1-sus). In contrast, LCN1 cells cultured in a type I collagen dish were adherent and tended to grow as spindle-shaped individual cells (LCN1-co). In this study, aiming at the discovery of predictive markers for tumor invasion, we performed protein profiling between LCN1-sus and LCN1-co cells using two-dimensional gel electrophoresis. Twenty-six protein spots with >1.2-fold quantitative differences between LCN1-sus and LCN1-co cells were detected. Among the identified proteins, we focused on and immunohistochemically investigated G6PD in lung cancer. G6PD expression was significantly associated with a higher pathological TNM stage (p = 0.0024), lymph node metastasis (p = 0.0187), poorer differentiation (p = 0.0046), pleural invasion (p = 0.0197), vascular invasion (p < 0.0001), lymphatic invasion (p = 0.0200) and poorer prognosis (p = 0.0005) in adenocarcinoma. Especially, G6PD-positive patients with overexpression at the invasive front had significantly poorer survival than those without overexpression (p = 0.0058). Moreover, multivariable analysis revealed that G6PD expression was an independent adverse-prognostic factor. These results suggest that G6PD may be a novel predictive prognostic marker for lung adenocarcinoma.

Fludrocortisone stimulates erythropoietin production in the intercalated cells of the collecting ducts

Erythropoietin has been thought to be secreted to plasma soon after the production because of the difficulty of Western blot analysis and immunohistochemistry. We established the new methods of Western blot analysis and immunohistochemistry. Using the new methods, we investigated the effects of aldosterone and fludrocortisone, an analogue of aldosterone on erythropoietin mRNA and protein production by the kidneys. Aldosterone stimulated Epo and HIF2α mRNA expressions in tubule suspensions and microdissected medullary thick ascending limbs and outer medullary collecting ducts. Western blot analysis showed a recombinant erythropoietin at 34-45 kDa and kidney erythropoietin at 36-40 and 42 kDa, both of which shifted to 22 kDa by deglycosylation. Erythropoietin protein expression was observed in the nephrons but not in the interstitial cells in control condition. Fludrocortisone stimulated erythropoietin mRNA and protein expressions in the distal nephrons, particularly in the intercalated cells of the collecting ducts. These data show that erythropoietin is produced by the nephrons by the regulation of renin-angiotensin-aldosterone system and not by the renal interstitial cells in control condition.