Carcinomas assemble a filamentous CXCL12-keratin-19 coating that suppresses T cell-mediated immune attack.

Cancer immunotherapy frequently fails because most carcinomas have few T cells, suggesting that cancers can suppress T cell infiltration. Here, we show that cancer cells of human pancreatic ductal adenocarcinoma (PDA), colorectal cancer, and breast cancer are coated with transglutaminase-2 (TGM2)-dependent covalent CXCL12-keratin-19 (KRT19) heterodimers that are organized as filamentous networks. Since a dimeric form of CXCL12 suppresses the motility of human T cells, we determined whether this polymeric CXCL12-KRT19 coating mediated T cell exclusion. Mouse tumors containing control PDA cells exhibited the CXCL12-KRT19 coating, excluded T cells, and did not respond to treatment with anti-PD-1 antibody. Tumors containing PDA cells not expressing either KRT19 or TGM2 lacked the CXCL12-KRT19 coating, were infiltrated with activated CD8+ T cells, and growth was suppressed with anti-PD-1 antibody treatment. Thus, carcinomas assemble a CXCL12-KRT19 coating to evade cancer immune attack.

Single Biomolecule Imaging by Electrochemiluminescence.

Herein, a single biomolecule is imaged by electrochemiluminescence (ECL) using Ru(bpy)32+-doped silica/Au nanoparticles (RuDSNs/AuNPs) as the ECL nanoemitters. The ECL emission is confined to the local surface of RuDSNs leading to a significant enhancement in the intensity. To prove the concept, a single protein molecule at the electrode is initially visualized using the as-prepared RuDSN/AuNPs nanoemitters. Furthermore, the nanoemitter-labeled antibody is linked at the cellular membrane to image a single membrane protein at one cell, without the interference of current and optical background. The success in single-biomolecule ECL imaging solves the long-lasting task in the ultrasensitive ECL analysis, which should be able to provide more elegant information about the protein in cellular biology.

Fluorometric immunoassay for the simultaneous determination of the tumor markers carcinoembryonic antigen and cytokeratin 19 fragment using two kinds of CdSe/ZnS quantum dot nanobeads and magnetic beads.

A method is described for the simultaneous determination of the carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA21-1). Two kinds of CdSe/ZnS quantum dot nanobeads (QBs), with emission maxima at 530 nm (green) and 585 nm (yellow), were used as labels, and magnetic beads (MBs) for separation. The MBs were used as substrates to couple CEA and CYFRA21-1 antibody for isolating the proteins. Then, the differently colored QBs were linked to the antibodies against CEA and CYFRA21-1, respectively. Following the formation of the immunocomplex, the intensities of the green and yellow emissions were measured at the same excitation wavelength of 340 nm. The detection limits are 0.1 ng⋅mL- 1 for CEA, and of 0.2 ng⋅mL- 1 for CYFRA21-1. The recoveries from spiked serum are 92.1 - 118.1% for CEA, and from 90.8% to 115.2% for CYFRA21-1, with the relative standard deviations of 6.3 - 12.3% and 7.1 - 11.8%. The method was successfully applied to the simultaneous determination of the two proteins in human serum sample (n = 45). The results correlated well with those of the chemiluminescent enzyme immunoassay kit. Graphical AbstractSchematic presentation of the fluorescence immunoassay for the simultaneous determination of carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA21-1) based on quantum dot nanobeads (QBs) and magnetic beads (MBs) is reported. The intensities of two kinds of CdSe/ZnS QBs, with the emission maxima at 530 nm (green) and 585 nm (yellow) were measured at the same excitation wavelength of 340 nm.

Imaging Features of Gadoxetic Acid-enhanced and Diffusion-weighted MR Imaging for Identifying Cytokeratin 19-positive Hepatocellular Carcinoma: A Retrospective Observational Study.

Purpose To determine the preoperative magnetic resonance (MR) imaging findings potentially most useful for predicting cytokeratin 19 (CK19)-positive hepatocellular carcinoma (HCC) and to evaluate the prognosis after curative resection in patients with a single HCC lesion positive for CK19 compared with patients with HCC who are negative for CK19. Materials and Methods The institutional review board approved this study and waived the requirement for informed consent. Two hundred four patients with CK19-negative HCC and 38 with CK19-positive HCC who underwent curative resection after gadoxetic acid-enhanced and diffusion-weighted MR imaging were retrospectively evaluated in a single institution. Two radiologists evaluated preoperative findings at MR imaging. Significant findings for differentiating the two groups were identified at univariate and multivariate analyses. By using receiver operating characteristic analysis, the optimal cut-off values for quantitative variables were determined. Recurrence-free survival rates after surgery were also compared between groups. Results At multivariate analysis, irregular tumor margin (P = .024), arterial rim enhancement (P < .001), lower tumor-to-liver signal intensity (SI) ratio on hepatobiliary phase (HBP) images (≤0.522; P = .01), and lower tumor-to-liver apparent diffusion coefficient (ADC) ratio (≤0.820; P < .001) were independent significant factors to predict CK19-positive HCC. When three of these four criteria were combined, 63.2% (24 of 38; 95% confidence interval: 46.0%, 78.2%) of CK19-positive HCCs were identified with a specificity of 90.7% (185 of 204; 95% confidence interval: 46.0%, 78.2%). When all four criteria were satisfied, specificity was 99.5% (203 of 204; 95% confidence interval: 97.3%, 100%). Recurrence-free survival rates were significantly lower in patients with CK19-positive HCCs compared with those with CK19-negative HCCs after curative resection (63.9% vs 90.0% at 1 year, 63.9% vs 79.9% at 2 years, and 54.8% vs 70.2% at 3 years, P = .001 by log-rank test). Conclusion At gadoxetic acid-enhanced and diffusion-weighted MR imaging, irregular margin, arterial phase rim enhancement, lower tumor-to-liver ADC ratio, and lower tumor-to-liver SI ratio at HBP imaging may be helpful to predict CK19-positive HCC with early recurrence (<2 years) after curative resection. © RSNA, 2017 Online supplemental material is available for this article.

Serum specific IgG response to toluene diisocyanate-tissue transglutaminase conjugate in toluene diisocyanate-induced occupational asthmatics.

BACKGROUND: Tissue transglutaminase (tTG) is a post-translational modifying enzyme located in airway epithelial cells. A potential contribution of serum specific IgG (sIgG) to tTG in airway inflammation of toluene diisocyanate (TDI)-induced occupational asthma (OA) has been suggested. OBJECTIVE: To prepare a TDI-tTG conjugate and detect serum specific antibodies in sera of patients with TDI-OA to understand this mechanism. METHODS: Ninety-nine patients with TDI-OA, 76 asymptomatic exposed controls, 208 patients with non-OA, and 74 unexposed controls were enrolled for this study. The TDI-tTG conjugate was prepared and confirmed by a native gel. Serum sIgG and/or sIgE antibodies to tTG, TDI-tTG, TDI conjugated to human serum albumin, cytokeratin 19, and serum cytokine levels, such as interleukin-8, transforming growth factor-ß1, and tissue inhibitor of metalloproteinase-1, were measured by enzyme-linked immunosorbent assay. The level of interleukin-8 produced from airway epithelial cells (A549) treated with tTG was evaluated to investigate the inflammatory effect of tTG and TDI-tTG. RESULTS: In the TDI-OA group, the prevalence of serum sIgG to TDI-tTG (17.2%) was higher than that of sIgG to tTG (11.1%), which were significantly higher than those of the 3 control groups (P < .05 for all groups). TDI-exposed subjects with high levels of serum sIgG to TDI-tTG had a high prevalence of sIgG to cytokeratin 19 and higher serum levels of transforming growth factor-ß1 and tissue inhibitor of metalloproteinase-1. The tTG and TDI-tTG dose-dependently increased interleukin-8 production from A549 cells. CONCLUSION: These findings suggest that TDI exposure in the workplace binds to tTG to form a conjugate that can induce serum sIgG antibody production, airway inflammation, and airway remodeling in patients with TDI-OA.

Rapid Detection of Free Cancer Cells in Intraoperative Peritoneal Lavage Using One-Step Nucleic Acid Amplification (OSNA) in Gastric Cancer Patients.

Cytokeratin-19 (CK19) has been proven to be commonly expressed by cancer cells in a variety of solid tumors and may serve as a suitable marker of metastases in gastric cancer (GC). Since objective assessment of peritoneal lavage or fluid for free cancer cells (FCC) is essential for clinical decision making in patients with GC, it is important to develop a quantitative and reproducible method for such evaluation. We assessed the possible application of One-Step Nucleic Acid amplification (OSNA) assay as a rapid method for FCC detection in intraoperative peritoneal lavage or fluid of GC patients. Seventy-eight intraoperative peritoneal lavage or fluid samples were eligible for the analysis by conventional cytology and OSNA examination. The concentration of CK19 mRNA in intraoperative peritoneal lavage and fluid was compared with the conventional cytological assessment. CK19 mRNA concentration was detected by OSNA assay. For peritoneal lavage samples, sensitivity and specificity were 83.3% and 87.8%, respectively. In peritoneal fluid, significantly higher CK19 values were observed in patients with serosal infiltration (medians: 100 copies/µL vs. 415.7 copies/µL; p = 0.0335) and lymph node metastases (medians: 2.48 copies/µL vs. 334.8 copies/µL). OSNA assay turns out to be an objective, fast, and reproducible quantitative method of FCC assessment.

Rapid and quantitative detection of urinary Cyfra21-1 using fluorescent nanosphere-based immunochromatographic test strip for diagnosis and prognostic monitoring of bladder cancer.

Bladder cancer is a common malignant tumour with high recurrence rate. Cytokeratin 19 fragments (Cyfra21-1) in urine has been regarded as a promising biomarker for the prognosis and diagnosis of bladder cancer due to the relevance of its high urinary level to the bladder cancer patients. However, currently detection methods of Cyfra21-1 have their limits, such as complicated steps, limited sensitivity or unsatisfying specificity. In this study, we developed a novel time-resolved fluoroimmuno test strip by using europium chelate microparticle (Eu-CM). Detection was performed in simple steps by carrying drops of sample into the well of the test strip, waiting for 15 min and inserting the strip into a fluorescence strip reader for quantitation. The standard curve equation of the test strip was y = 0.0177x + 0.01 (R2 = .9993). In the analysis of human urine samples (n = 115), it demonstrated a good performance (accuracy: CV < 10%, AUC: 0.989). With the cut-off value of 81 ng/mL, the sensitivity and specificity for bladder cancer were 92.86 and 100%, respectively. In comparison to ELISA and electrochemiluminescence methods, the Eu-CM based time-resolved fluoroimmuno test strip provided a rapid, sensitive and reliable method for monitoring bladder cancer. It may be applied as a non-invasive approach for in point-of-care for bladder cancer detection.

Magnetic resonance texture analysis for the identification of cytokeratin 19-positive hepatocellular carcinoma.

PURPOSE: To investigate potential findings associated with cytokeratin 19 (CK19)-positive HCC, with special emphasis on MR texture analysis. MATERIALS AND METHODS: Forty-eight patients with CK19-negative HCC and 38 patients with CK19-positive were retrospectively evaluated by texture analysis based on conventional MRI. Clinicalpathological characteristics, conventional MR imaging findings, and the MR texture analysis contained of 2415 texture features in the seven conventional sequences were compared. Significant features for differentiating were identified by univariate and multivariate analyses. Receiver operating characteristic analyses of the significant findings were performed and compared to evaluate their diagnostic performance. RESULTS: There was no significant difference between the top1 texture feature (three-dimensional standard deviation separation of intensity on T2-weighted original images, abbreviated as: StdSeparation 3D) and the combined top1-6 feature in identifying CK19-positive HCC(P = 0.660). Univariate and multivariate analyses indicated that serum alpha-fetoprotein (AFP) level ≥400 ng/mL(P = 0.013), arterial rim enhancement(P = 0.005), and StdSeparation 3D texture character(P = 0.002) were independent variables associated with CK19-positive HCCs. The combination of the three indices showed a better performance than AFP level(P = 0.0028), arterial rim enhancement(P < 0.0001), and their combination(P = 0.0098); while no significantly better than the StdSeparation 3D texture character alone(P = 0.0788). An acceptable discrimination(AUC = 0.765) with both sensitivity and specificity greater than 75% was achieved for StdSeparation 3D texture character. CONCLUSION: Serum AFP level ≥400 ng/mL, arterial rim enhancement, and the StdSeparation 3D texture character were independently associated with CK19-positive HCC. The StdSeparation 3D texture character may be a reliable imaging biomarker which can improve the diagnostic performance.

[Correlation between Serum Cytokeratin 19 Fragment and the Clinicopathological Features and Prognosis of Thymic Epithelial Tumors].

BACKGROUND: So far there's no tumor maker applied in diagnosis and treatment of thymic epithelial tumors. This study is to assess the correlation between serum cytokine 19 fragment (Cyfra 21-1) and clinicopathological features and prognosis of thymic epithelial tumors (TETs). METHODS: The clinical data of 159 patients with TETs in Shanghai Chest Hospital was retrospectively analysed. Patients were divided into groups according to different tumor stages and histotypes. Serum Cyfra 21-1 was thus compared. In addition, the possible relationship between perioperative serum Cyfra 21-1 level and the recurrent status was carrid out. RESULTS: Preoperative Cyfra 21-1 serum concentrations in patiants with advanced stage (T4) and thymic carcinomas were significantly higher than that in others (P<0.001, P<0.001, respectively). When the preoperative serum level exceeds the out-off of 1.66 ng/mL, it possibly indicates the recurrence during follow up. Furthermore, the sensitivity, specificity, and positive as well as negative predictive value (PPV and NPV) of postoperative Cyfra 21-1 to predict tumor recurrence were evaluated. At a cut-off of Cyfra 21-1 of 2.66 ng/mL, the sensitivity was 0.7, the specificity was 0.925, the PPV was 0.5 and the NPV was 0.966. CONCLUSIONS: The elevated level of preoperative serum Cyfra 21-1 indicates an advanced stage of tumor or a more malignant histotype (thymic carcinoma). It also probably suggests a higher risk of tumor recurrence. During the oncological follow up, in addition to regular imaging examinations, the blood test of serum Cyfra 21-1 is also suggested to improve the diagnosis of tumor recurrence in order to improve the prognosis.

Effect of Brownian motion on reduced agglomeration of nanostructured metal oxide towards development of efficient cancer biosensor.

We report results of the studies relating to fabrication of nanostructured metal oxide (NMO) based cancer biosensor. With the help of 2D electroactive reduced graphene oxide (RGO), we successfully inhibited the Brownian motion of NMO that led to reduced agglomeration of NMO. The nanostructured hafnium oxide (nHfO2) was used as a model NMO. The reduced agglomeration of nHfO2 was achieved through controlled hydrothermal synthesis and investigated via nanoparticles tracking analysis (NTA). X-ray diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscope (TEM) techniques were used for phase identification as well as morphological analysis of the synthesized nanohybrid (nHfO2@RGO) material. The 3-aminopropyl triethoxysilane (APTES) was used for the functionalization of nHfO2@RGO and electrophoretic deposition (EPD) technique was used for its deposition onto ITO coated glass electrode. Further, antibodies of cancer biomarker (anti-CYFRA-21-1) were immobilized via EDC-NHS chemistry and Bovine serum albumin (BSA) was used for blocking of the non-specific binding sites. The electrochemical response studies of fabricated immunoelectrode (BSA/anti-CYFRA-21-1/APTES/nHfO2@RGO/ITO) revealed higher sensitivity (18.24µAmLng-1), wide linear detection range (0 to 30ngmL-1), with remarkable lower detection limit (0.16ngmL-1). The obtained results showed good agreement with the concentration of CYFRA-21-1 obtained through enzyme linked immunosorbent assay (ELISA) in saliva samples of oral cancer patients.

Fluorescence-based immunosensor using three-dimensional CNT network structure for sensitive and reproducible detection of oral squamous cell carcinoma biomarker.

A hierarchical three-dimensional network of carbon nanotubes on Si pillar substrate (3DN-CNTs) was developed for the accurate detection of oral squamous cell carcinoma (OSCC) in clinical saliva samples. The 3DN-CNTs were uniformly coated with a layer of aluminum oxides to enhance structural stability during biomarker detection. Cytokeratin-19 antigen (Cyfra 21-1) was utilized as a model biomarker of OSCC for fluorescence-based immunosensor using 3DN-CNTs (3DN-CNTs sensor). The 3DN-CNTs sensor enhances the sensitivity of Cyfra 21-1 detection by increasing the density of immobilized antibody through high surface area of 3DN-CNTs and enhancing the accessibility of biomolecules through the ordered pathway of hierarchical structure. The reliable detection limit for sensing of Cyfra 21-1 was estimated as in the level of 0.5 ng/mL and the quantitative estimation of Cyfra 21-1 was analyzed by 4-parameter logistic (4-PL) model for curve-fitting analysis. Clinical applicability of 3DN-CNTs sensor was evaluated through correlation with the commercially available electrochemiluminescence (ECL) detection system in the hospital. The assay results of the two systems for clinical saliva samples showed a good linear correlation. The 3DN-CNTs sensor offers great potential for accurate diagnosis of OSCC using Cyfra 21-1 biomarker in clinical fluids.

Multifunctional substrate of label-free electrochemical immunosensor for ultrasensitive detection of cytokeratins antigen 21-1.

Poly(thionine)-Au, a novel multifunctional substrate with excellent redox signal, enzyme-like activity, and easy antibody immobilisation, was synthesised using HAuCl4 as the oxidising agent and thionine as the monomer. The prepared poly(thionine)-Au composite exhibited an admirable electrochemical redox signal at -0.15 V and excellent H2O2 catalytic ability. In addition, gold nanoparticles in this composite were found to directly immobilise antibodies and further improve conductivity. In addition, a label-free electrochemical immunosensor was developed using poly(thionine)-Au as the sensing substrate for ultrasensitive detection of cytokeratin antigen 21-1 (CYFRA 21-1), an immunoassay found in human serum. The prepared immunosensor showed a wide liner range from 100 ng mL-1 to 10 fg mL-1 and an ultralow detection limit of 4.6 fg mL-1 (the ratio of signal to noise (S/N) = 3). Additionally, this method was used to analyse human serum samples and yielded results consistency with those of ELISA, implying its potential application in clinical research. The poly(thionine)-Au composite can be easily extended to other polymer-based nanocomposites, which is significant for other electrochemical immunoassays.

L-cysteine capped lanthanum hydroxide nanostructures for non-invasive detection of oral cancer biomarker.

In this paper, we present the result of studies related to the in situ synthesis of amino acid (L-Cysteine) capped lanthanum hydroxide nanoparticles [Cys-La(OH)3 NPs] towards the fabrication of efficient immunosensor for non-invasive detection of oral cancer. The characterization of Cys-La(OH)3 NPs was carried out by different techniques including X-ray diffraction, scanning electron microscopy, transmission electron microscopy, fourier transform infrared spectroscopy and electrochemical techniques. These Cys-La(OH)3 NPs were electrophoretically deposited onto an indium-tin-oxide glass substrate and used for immobilization of anti-cytokeratin fragment-21-1 (anti-Cyfra-21-1) for the electrochemical detection of Cyfra-21-1. This immunosensor shows a broad detection range of 0.001-10.2ngmL-1, the low detection limit of 0.001ngmL-1, and high sensitivity of 12.044µA (ng per mL cm-2)-1 with a response time of 5min. This immunosensor was found to be more advanced in terms of high sensitivity and low detection limit as compared to previously reported biosensors and commercially available ELISA kit (Kinesis DX).

Nanostructured zirconia decorated reduced graphene oxide based efficient biosensing platform for non-invasive oral cancer detection.

We report results of the studies relating to fabrication of a non-invasive, label-free and an efficient biosensing platform for detection of the oral cancer biomarker (CYFRA-21-1). One step hydrothermal process was used for uniform decoration of nanostructured zirconia (average particle size 13 nm) on reduced graphene oxide (ZrO2-RGO) to avoid coagulation of the zirconia nanoparticles and to obtain enhanced electrochemical performance of ZrO2-RGO nanocomposite based biosensor. Further, ZrO2-RGO has been functionalized using 3-aminopropyl triethoxy saline (APTES) and electrophoretically deposited on the indium tin oxide coated glass substrate at a low DC potential.The APTES/ZrO2-RGO/ITO electrode exhibits improved heterogeneous electron transfer (more than two times) with respect to that of the APTES/ZrO2/ITO electrode indicating faster electron transfer kinetics. The -NH2 containing APTES/ZrO2-RGO/ITO platform is further biofunctionalized with anti-CYFRA-21-1. The structural and morphological investigations of the ZrO2-RGO based biosensing platform have been accomplished using X-ray diffraction (XRD), electrochemical, transmission electron microscopy (TEM), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FT-IR) studies. This immunosensor exhibits a wider linear detection range (2-22 ng mL(-1)), excellent sensitivity (0.756 µA mL ng(-1)) and a remarkable lower detection limit of 0.122 ng mL(-1). The observed results have been validated via enzyme linked immunosorbent assay (ELISA).

Serum cytokeratin 19 fragment, CK19-2G2, as a newly identified biomarker for lung cancer.

BACKGROUND: CK19-2G2, a new fragment of cytokeratin 19, is a potential tumor marker for diagnosing lung cancer. The preoperative level of serum CK19-2G2 has been demonstrated to be associated with tumor metastasis and survival of breast cancer patients. This study investigated the postoperative dynamic changes in serum CK19-2G2 levels and its clinical significance in lung cancer patients. MATERIALS AND METHODS: Preoperative serum CK19-2G2 levels were measured in 630 lung cancer patients and were compared with individuals with benign pulmonary diseases (n = 134) and healthy volunteers (n = 263). In 352 cases, the patients underwent surgery. In these patients, in addition to preoperative assays, serum CK19-2G2 was also monitored at 1 week and 1 month after the operation. RESULTS: The preoperative baseline levels of serum CK19-2G2 was significantly higher in lung cancer patients than patients with benign diseases and healthy controls (P<0.001). The postoperative levels of CK19-2G2 declined significantly within 1 week after tumor resection. Hereafter, a further decrease was observed in the patients who underwent palliative operations, while for the patients in the radical resection group, their CK19-2G2 levels stabilized. CONCLUSION: CK19-2G2 may be a candidate marker for diagnosing and monitoring a patient's response to lung cancer treatment. In addition, CK19-2G2 may be an indicator for micrometastases in lung cancer patients.

Lung cancer circulating tumor cells isolated by the EpCAM-independent enrichment strategy correlate with Cytokeratin 19-derived CYFRA21-1 and pathological staging.

BACKGROUND: Cytokeratin 19-derived CYFRA21-1 is an acceptable lung cancer biomarker. However, whether CYFRA21-1 correlates with lung cancer circulating tumor cells (CTCs) remains unclear. METHODS: CTCs in 42 lung cancer patients and 10 nonmalignant pulmonary disease patients were isolated by means of an EpCAM-independent enrichment strategy. Correlation of lung cancer CTCs with serum concentration of CYFRA21-1 and pathological staging was investigated. RESULTS: Among lung cancer patients in this study, 39% (7/18) of those with normal CYFRA21-1 (≤3.3 ng/ml) and 62% (13/21) of high CYFRA21-1 (>3.3 ng/ml) patients were found to have ≥3 CTCs/7.5 ml blood. The CTCs-positive rate of stage I to IV lung cancer patients was 20% (2/10), 45% (5/11), 54% (6/11) and 70% (7/10), respectively. Comparing M0 vs M1 patients, the CTCs-positive rate was 43% (13/30) and 70% (7/10), respectively. All M1 patients (10/10) had one or more CTCs detected, whereas none of the nonmalignant pulmonary disease patients had detectable CTCs. CONCLUSION: Lung cancer CTCs isolated by the EpCAM-independent enrichment approach correlate with CYFRA21-1 and TNM staging. Correlation of CTCs and CYFRA21-1 in lung cancer patients is of potential clinical utility in terms of early diagnosis and predicting prognosis.

Cytokeratin19-2g2, a novel fragment of cytokeratin19 in serum, indicating a more invasive behavior and worse prognosis in breast cancer patients.

BACKGROUND: Various studies have been searching for new tumor biomarkers for breast cancer for years. However, so far, few markers have been proved clinically useful except CA153. Based on knowledge that most adenocarcinomas including breast carcinoma expressed Cytokeratin19, the authors studied CK19-2G2,a novel fragment of cytokeratin19 shedding into serum in breast cancer patients. PATIENTS AND METHODS: The serum samples of four hundred and seventeen patients including three hundred and three (fifty-four DCIS and two hundred and forty-nine stage I-III) PBC patients and one hundred and fourteen MBC patients, eighty-one healthy controls and twenty-one breast benign disease patients were provided for measurement of CK19-2G2, CEA and CA153.The correlation between clinicopathological characters, prognosis and CK19-2G2 levels was further studied. RESULTS: The serum CK19-2G2 levels in breast cancer patients were significantly higher than that in healthy and benign controls. For breast cancer patients, CK19-2G2 levels in MBC were significantly higher than that in PBC patients. The sensitivities of CK19-2G2 for breast carcinoma are as high as CEA and CA153, and up to 71% in MBC patients. Serum CK19-2G2 levels (≥2 mU/mL) were associated with pathological stages, tumor size (≥2 cm), lymph node involvement, and HER2 status. Multivariate analysis revealed that high serum CK19-2G2 level was an independent factor for relapse (P = 0.029) and death (P = 0.040) in breast cancer patients. CONCLUSION: Serum CK19-2G2 may be an independent indicator for prognosis and a candidate marker for monitoring metastasis in breast cancer.

Diagnostic value of mesothelin in pleural fluids: comparison with CYFRA 21-1 and CEA.

CYFRA 21-1 and CEA have been applied for the differential diagnosis of malignant pleural mesothelioma (MPM). The soluble mesothelin-related peptide (SMRP) has been proposed as a specific marker for distinguishing MPM from benign diseases and other malignancies in pleural effusions (PEs). In this study, we evaluated the usefulness of SMRP in PEs in the detection of mesotheliomas by comparing it with that of CYFRA 21-1, CEA, and with cytological examination. One hundred and seventy-seven consecutive patients (57 MPM, 64 metastatic tumors, and 56 benign diseases) were evaluated using commercial tests. The performance of the markers was analyzed by standard ROC analysis methods, using the area under a ROC curve (AUC) as a measure of accuracy. CYFRA 21-1 better differentiated malignant from benign effusions. The corresponding area under the receiver operating characteristic curve was 0.87, while it was 0.74 for SMRP and 0.64 for CEA (p < 0.001). Conversely, SMRP differentiated MPM from all other PEs better than both CYFRA 21-1 and CEA (AUC = 0.84, 0.76, and 0.32, respectively, p = 0.003). Low levels of CEA were associated with a MPM diagnosis. The AUC for differentiating MPM from metastases was 0.81 for SMRP, 0.61 for CYFRA 21-1, and 0.20 for CEA (p < 0.001). In cases with negative or suspicious cytology, SMRP and CYFRA 21-1 identified 36/71 and 46/66 malignant PEs (29 and 31 MPM, respectively). Only 1 MPM showed a high CEA concentration. No single marker showed the best performance in any comparison. Results suggest that SMRP could improve CYFRA 21-1 and CEA accuracy in the differential diagnosis of MPM.

Tumor-related gene changes in immunosuppressive Syrian hamster cholangiocarcinoma.

The results of a previous study demonstrated that prednisolone enhanced cholangiocarcinogenesis. Therefore, to clarify molecular changes during immunosuppressive cholangiocarcinogenesis, Syrian hamsters were divided into 8 groups: uninfected controls; immunosuppressed Syrian hamsters using prednisolone (P); normal Syrian hamsters administered N-nitrosodimethylamine (ND); immunosuppressed Syrian hamsters administered N-nitrosodimethylamine (NDis); normal Syrian hamsters infected with Opisthorchis viverrini (OV); immunosuppressed Syrian hamsters infected with O. viverrini (OVis); normal Syrian hamsters infected with O. viverrini and administered N-nitrosodimethylamine (CCA); and immunosuppressed Syrian hamsters infected with O. viverrini and administered N-nitrosodimethylamine (CCAis). Syrian hamster livers were used for analysis of tumor-related gene expression and immunohistochemistry through cytokeratin 19 (CK19) and proliferating cell nuclear antigen (PCNA) staining. The tumor-related gene expression results show that CCAis groups at all time points exhibited upregulation of COX-2, IL-6, SOD1, CAT and iNOS and downregulation of p53, which correlated with the predominant expression of CK19 and PCNA in liver tissue. These results suggest that prednisolone enhances cholangiocarcinoma development, which was confirmed by molecular changes.

Characterization of in vivo keratin 19 phosphorylation on tyrosine-391.

BACKGROUND: Keratin polypeptide 19 (K19) is a type I intermediate filament protein that is expressed in stratified and simple-type epithelia. Although K19 is known to be phosphorylated on tyrosine residue(s), conclusive site-specific characterization of these residue(s) and identification potential kinases that may be involved has not been reported. METHODOLOGY/PRINCIPAL FINDINGS: In this study, biochemical, molecular and immunological approaches were undertaken in order to identify and characterize K19 tyrosine phosphorylation. Upon treatment with pervanadate, a tyrosine phosphatase inhibitor, human K19 (hK19) was phosphorylated on tyrosine 391, located in the 'tail' domain of the protein. K19 Y391 phosphorylation was confirmed using site-directed mutagenesis and cell transfection coupled with the generation of a K19 phospho (p)-Y391-specific rabbit antibody. The antibody also recognized mouse phospho-K19 (K19 pY394). This tyrosine residue is not phosphorylated under basal conditions, but becomes phosphorylated in the presence of Src kinase in vitro and in cells expressing constitutively-active Src. Pervanadate treatment in vivo resulted in phosphorylation of K19 Y394 and Y391 in colonic epithelial cells of non-transgenic mice and hK19-overexpressing mice, respectively. CONCLUSIONS/SIGNIFICANCE: Human K19 tyrosine 391 is phosphorylated, potentially by Src kinase, and is the first well-defined tyrosine phosphorylation site of any keratin protein. The lack of detection of K19 pY391 in the absence of tyrosine phosphatase inhibition suggests that its phosphorylation is highly dynamic.