Construction and validation of a novel IGFBP3-related signature to predict prognosis and therapeutic decision making for Hepatocellular Carcinoma.

Background: IGFBP3 plays a pivotal role in carcinogenesis by being anomalously expressed in some malignancies. However, the clinical value of IGFBP3 and the role of IGFBP3-related signature in HCC remain unclear. Methods: Multiple bioinformatics methods were used to determine the expression and diagnostic values of IGFBP3. The expression level of IGFBP3 was validated by RT-qPCR and IHC. A IGFBP3-related risk score (IGRS) was built via correlation analysis and LASSO Cox regression analysis. Further analyses, including functional enrichment, immune status of risk groups were analyzed, and the role of IGRS in guiding clinical treatment was also evaluated. Results: IGFBP3 expression was significantly downregulated in HCC. IGFBP3 expression correlated with multiple clinicopathological characteristics and demonstrated a powerful diagnostic capability for HCC. In addition, a novel IGRS signature was developed in TCGA, which exhibited good performance for prognosis prediction and its role was further validated in GSE14520. In TCGA and GSE14520, Cox analysis also confirmed that the IGRS could serve as an independent prognostic factor for HCC. Moreover, a nomogram with good accuracy for predicting the survival of HCC was further formulated. Additionally, enrichment analysis showed that the high-IGRS group was enriched in cancer-related pathways and immune-related pathways. Additionally, patients with high IGRS exhibited an immunosuppressive phenotype. Therefore, patients with low IGRS scores may benefit from immunotherapy. Conclusions: IGFBP3 can act as a new diagnostic factor for HCC. IGRS signature represents a valuable predictive tool in the prognosis prediction and therapeutic decision making for Hepatocellular Carcinoma.

MiR-942-5p inhibits tumor migration and invasion through targeting CST1 in esophageal squamous cell carcinoma.

INTRODUCTION: Cysteine Protease Inhibitor 1 (CST1), a cystatin superfamily protein with the effect on the inhibition of cysteine protease activity, is reported to be involved in the development of many malignancies. MiR-942-5p has been demonstrated its regulatory effects on some malignancies. However, the roles of CST1 and miR-942-5p on esophageal squamous cell carcinoma (ESCC) are still unknown up to now. METHODS: The expression of CST1 in ESCC tissues was analyzed by TCGA database, immunohistochemistry, and RT-qPCR, respectively. Matrigel-uncoated or-coated transwell assay was used to determine the effect of CST1 on migration and invasion of ESCC cells. Regulatory effect of miR-942-5p on CST1 was detected by dual luciferase assay. RESULTS: CST1 was ectopically highly expressed in ESCC tissues, and had the effect on promoting the migration and invasion of ESCC cells by upregulating phosphorylated levels of key effectors including MEK1/2, ERK1/2, and CREB in MEK/ERK/CREB pathway. Dual-luciferase assay results showed that miR-942-5p had a regulatory effect on targeting CST1. CONCLUSIONS: CST1 plays a carcinogenic role on ESCC, and miR-942-5p can regulate the migration and invasion of ESCC cells by targeting CST1 to downregulate MEK/ERK/CREB signaling pathway, suggesting that miR-942-5p/CST1 axis might be a promising target for diagnosis and treatment of ESCC.

Combination of serum CST4 and DR-70 contributes to early diagnosis of colorectal cancer.

BACKGROUND: Early diagnosis is of great significance for the prognosis of colorectal cancer (CRC) patients. Either serum cystatin S (CST4) or DR-70 has been demonstrated to play an important role on the diagnosis for CRC, however, the diagnostic performances of individual and combined detection of serum CST4 and DR-70 for the patients with CRC at early stage have still not been clarified up to now. METHODS: The performances of CST4 and DR-70 were evaluated by ELISA for the early diagnosis of CRC with 288 retrospective serum samples. A training set comprised of 64 patients with early CRC, 64 patients with colorectal benign lesions (CBL), and 64 healthy controls (HC) was used to develop the predictive model for early CRC. And then, data obtained from an independent validation set was applied to evaluate and validate the predictive model. RESULTS: In the training set, the levels of CST4 and DR-70 in early CRC group were significantly higher than that in CBL group/HC group (P < 0.001); serum CST4 presented the AUC of 0.927 for early CRC patients, with 57.8% sensitivity at 95.3% specificity; serum DR-70 presented the AUC of 0.725 for early CRC patients, with 29.7% sensitivity at 95.3% specificity; combination of serum CST4 and DR-70 presented the AUC of 0.941, with 68.8% sensitivity at 93.8% specificity. Additionally, the combination of serum CST4 and DR-70 showed the AUC of 0.940 for early CRC patients, with 71.9 % sensitivity at 89.1% specificity in the validation set. CONCLUSIONS: Both serum CST4 and DR-70 present the diagnostic value for the patients with early CRC, and the combination of CST4 and DR-70 contributes to the further improvement of the early diagnosis for CRC.

Usefulness of AFP, PIVKA-II, and Their Combination in Diagnosing Hepatocellular Carcinoma Based on Upconversion Luminescence Immunochromatography.

OBJECTIVES: To evaluate the prognostic values of serum PIVKA-II (prothrombin induced by vitamin K absence-II) and α-fetoprotein (AFP) and the combination of these analytes for identifying hepatocellular carcinoma (HCC), and to analyze the correlation between biomarkers and clinicopathological features of HCC. METHODS: The levels of PIVKA-II and AFP in 331 case individuals were determined by upconverting phosphor technology-based immune lateral flow (UPT-LF) assay. We used the ROC curve to determine the diagnostic value; the relationships between the biomarkers and clinicopathological features of HCC also were analyzed. RESULTS: AFP and PIVKA-II have good diagnostic performance in the diagnosis of HCC; the best AUC was 0.76, 0.74. High levels of PIVKA-II were more advantageous than AFP in predicting tumor size, portal-vein embolism, and vascular invasion (all P <.05). CONCLUSION: Levels of PIVKA-II and AFP showed good diagnostic value for HCC, but the level of PIVKA-II was more closely related to the clinicopathological features of HCC.

Integration of IgG and IgA autoantibodies for early diagnosis of hepatocellular carcinoma.

BACKGROUND: Autoantibodes against tumor-associated antigens (TAAs) have been recommended for the early diagnosis of malignancies. In this study, we intend to comprehensively evaluate the performances of four autoantibodies including anti-p53, CTAG1A, TIF1γ-IgG and anti-TIF1γ-IgA for the early diagnosis of hepatocellular carcinoma (HCC), and then determine an optimal panel of autoantibodies for early HCC diagnosis. METHODS: The performances of four autoantibodies were evaluated by enzyme-linked immunosorbent assay (ELISA) for the early diagnosis of HCC with 380 retrospective serum samples. A training set comprised of 92 patients with early HCC, 72 patients with hepatic benign lesions (HBL), and 86 healthy controls (HC) was used to develop the predictive model for early HCC. And then, data obtained from an independent validation set was applied to evaluate and validate the predictive model to distinguish the early HCC from the controls (HBL + HC). RESULTS: The results of the training set showed the levels and positive rates of four autoantibodies in early HCC group were significantly higher than that in HBL group/HC group (P < 0.01), of which anti-p53-IgG exhibited the highest AUC of 0.679, with 33.7% sensitivity at 93.7% specificity; the panel comprised of four autoantibodies showed the highest AUC for the patients with early HCC, up to 0.814 (95%CI 0.760-0.860), with 72.8% sensitivity at 84.2% specificity among all possible combinations of four autoantibodies. Additionally, this four-autoantibody panel showed the AUC of 0.824, 70.8% sensitivity at 84.2% specificity in the validation set. CONCLUSIONS: Serum IgG autoantibodies against p53, CTAG1A and TIF1γ, and IgA autoantibody against TIF1γ present the diagnostic value for early HCC, of which anti-p53-IgG is a preferable biomarker. The panel comprised of four autoantibodies might contribute to early HCC diagnosis.

Development of a panel of autoantibody against NSG1 with CEA, CYFRA21-1, and SCC-Ag for the diagnosis of esophageal squamous cell carcinoma.

OBJECTIVE: Development of a panel of serum autoantibody against Neuron specific gene family member 1 (NSG1) with traditional tumor biomarkers of esophageal squamous cell carcinoma (ESCC) to further improve the diagnostic efficiency for ESCC patients. METHODS: Immunohistochemistry (IHC) staining was used to detect the expression of NSG1 protein in 40 pairs of ESCC tissues and matched paracancerous tissues. Serum anti-NSG1 levels of 203 patients with early ESCC, 103 patients with advanced ESCC, 135 patients with esophageal benign lesion (EBL), and 155 healthy controls (HCs) were detected by ELISA. The diagnostic performances of all possible combinations of serum anti-NSG1with CEA, CYFRA21-1 and SCC-Ag were assessed to develop an optimal panel for ESCC diagnosis. RESULTS: NSG1 protein expression in ESCC tissues was significantly higher than that in matched paracancerous tissues (p < 0.001). Serum anti-NSG1 expression in ESCC group was significantly higher than that in EBL group and HC group (p < 0.001). The AUC of serum anti-NSG1 for ESCC was 0.706, with 49.7% sensitivity at 93.5% specificity, superior to that of CEA, CYFRA21-1 and SCC-Ag. Of all possible combinations, serum anti-NSG1 combined with CEA, CYFRA21-1 and SCC-Ag showed the highest AUC of 0.758 and 67.3% sensitivity at 88.3% specificity for ESCC, with the highest NPV of 71.9% and the lowest NLR of 0.37. CONCLUSION: Aberrant NSG1 protein expression in ESCC tissues might be responsible for massive releases of autoantibody anginst NSG1 in sera of ESCC. A panel of anti-NSG1 with CEA, CYFRA21-1 and SCC-Ag contributes to further improving the diagnostic efficiency for ESCC.

Development of a panel of serum IgG and IgA autoantibodies for early diagnosis of colon cancer.

Purpose: Our pilot study in a small cohort by ELISA showed that the levels and positive rates of serum IgG autoantibodies against p53, HRAS and NSG1, and IgA autoantibody against TIF1γ in early colon cancer (CC) group were significantly higher than that of colon benign lesion (CBL) group / healthy control (HC) group (P <0.01), which suggested that four autoantibodies might be valuable for the diagnosis of patients with CC at early stage. On the basis of pilot study, we intend to comprehensively elucidate the performance of four autoantibodies for the early diagnosis of CC in a large sample cohort, and explore the optimal panel of autoantibodies in the diagnosis of patients with CC at early stage. Methods: Western blot was used to define the ELISA results of serum anti-p53, HRAS, NSG1-IgG and anti-TIF1γ-IgA. The performances of anti-p53, HRAS, NSG1-IgG and anti-TIF1γ-IgA were evaluated by ELISA for the early diagnosis of CC with 601 serum samples of 157 patients with CC at early stage, 144 patients with CC at advanced stage, 130 patients with CBL, and 170 HC, and then the performances of different combinations of four autoantibodies were analyzed for the development of an optimal panel for the early diagnosis of CC. Results: The results of anti-p53, HRAS, NSG1-IgG and anti-TIF1γ-IgA in western blotting were consistent with that in ELISA. The levels and positive rates of anti-p53, HRAS, NSG1-IgG and anti-TIF1γ-IgA in early CC group were significantly higher than that in CBL group/HC group (P <0.01), while had no significant difference from that in advanced CC group (P >0.05), of which anti-TIF1γ-IgA showed the highest area under the receiver operating characteristic curve (AUC) of 0.716 for the patients with CC at early stage, with 25.5% sensitivity and specificity at 96.7%. Additionally, a panel of anti-p53, HRAS-IgG and anti-TIF1γ-IgA showed the highest AUC among all possible combinations of four autoantibodies, up to 0.737, with 47.1% sensitivity at 92.0% specificity. Conclusions: Serum IgG autoantibodies against p53, HRAS and NSG1, and IgA autoantibody against TIF1γ show the diagnostic value for the patients with CC at early stage, of which anti-TIF1γ-IgA is demonstrated to be a preferable biomarker, and an optimal panel comprised of anti-p53, HRAS-IgG and anti-TIF1γ-IgA might contribute to the further improvement of early diagnosis for CC.

The Combination of IgA and IgG autoantibodies against Transcriptional Intermediary Factor-1γ contributes to the early diagnosis of Lung Cancer.

Objective: The aberrant expression of tumor-associated antigens (TAAs) is responsible for the release of large amounts of autoantibodies in sera, and serum autoantibody detection has been demonstrated to contribute to the early diagnosis of malignancies. Recent studies showed the closely correlation of transcriptional intermediary factor-1γ (TIF1γ) with some malignancies. In our pilot study, we found aberrantly high expression of TIF1γ protein existed in cancer tissues other than matched paracancerous tissues of patients with lung cancer (LC) at early stage by immunohistochemistry (IHC) staining. As a result, this study aims to detect the expression of autoantibodies against TIF1γ in sera of patients with LC at early stage by using enzyme-linked immunosorbent assay (ELISA) and investigate its potential value for the early diagnosis of LC. Methods: The expressions of TIF1γ protein in 60 pairs of LC tissues and matched paracancerous tissues were detected by IHC staining. The levels of anti-TIF1γ-IgA, IgG, IgM, and IgE in the sera of 248 patients with LC at early stage, 200 patients with lung benign lesions (LBL), and 218 healthy controls (HC) were detected by ELISA, respectively. Western blot was used to validate the ELISA results of serum autoantibodies against TIF1γ. Results: The positive rate of TIF1γ protein expression in LC tissues was 83.33%, which was significantly higher than 25.00% in paracancerous tissues (P<0.01). The levels and positive rates of serum anti-TIF1γ-IgM and anti-TIF1γ-IgE in early LC group had no significant difference from that in LBL group and HC group (P>0.05), while the levels and positive rates of anti-TIF1γ-IgA and anti-TIF1γ-IgG were significantly higher than that in LBL group and HC group (P<0.01), of which anti-TIF1γ-IgA showed the area under the receiver operating characteristic curve (AUC) of 0.704 for the patients with LC at early stage, with 28.20% sensitivity at 95.93% specificity, and anti-TIF1γ-IgG showed the AUC of 0.622 for the patients with LC at early stage, with 18.54% sensitivity at 94.25% specificity. The results of anti-TIF1γ-IgA and anti-TIF1γ-IgG in western blot were consistent with that in ELISA. Additionally, the combination of anti-TIF1γ-IgA and anti-TIF1γ-IgG improved the AUC to 0.734, with 38.31% sensitivity at 92.34% specificity. Conclusions: There is a strong humoral immune response to autologous TIF1γ existing in patients with early LC. Both serum anti-TIF1γ-IgA and anti-TIF1γ-IgG show the diagnostic value for the patients with LC at early stage, of which anti-TIF1γ-IgA is donstrated to be a preferable biomarker, and the combined detection of anti-TIF1γ-IgA and anti-TIF1γ-IgG might contribute to the further improvement of early diagnosis for LC.

Combination of Serum miRNAs with Serum Exosomal miRNAs in Early Diagnosis for Non-Small-Cell Lung Cancer.

PURPOSE: Circulating microRNAs (miRNAs) have shown the potential for non-invasive diagnosis of various types of malignancies at an early stage. The aim of the study was to explore the feasibility of a combination of 8 serum miRNAs related to non-small-cell lung cancer (NSCLC) with the corresponding serum exosomal miRNAs in early diagnosis for the patients with NSCLC. METHODS: We measured 8 serum miRNAs and the corresponding serum exosomal miRNAs including miR-21-5p, miR-126-3p, miR-141-3p, miR-146a-5p, miR-155-5p, miR-222-3p, miR-223-3p, and miR-486-5p in 48 patients with early NSCLC at stages I/II, 32 patients with lung benign lesion (LBL), and 48 healthy control (HC) by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The expression levels of 4 serum miRNAs including miR-21-5p, miR-141-3p, miR-222-3p, and miR-486-5p, and 2 serum exosomal miRNAs including miR-146a-5p and miR-486-5p in the early NSCLC group were significantly different from that in the LBL group and the HC group (P < 0.01). The areas under the receiver operating characteristic curves (AUC) of the 4 serum miRNAs and 2 serum exosomal miRNAs in the early NSCLC group were ≥0.697, of which serum exosomal miR-146a-5p and miR-486-5p were 0.813 and 0.886, respectively, and higher than that of the 4 serum miRNAs. Additionally, a combination of 4 serum miRNAs with 2 serum exosomal miRNAs improved the AUC to 0.960 for the patients with NSCLC at early stages, with a sensitivity of 85.42% and a specificity of 92.50%. CONCLUSION: This study suggests that serum exosomal miRNAs other than serum miRNAs might be preferable biomarkers for the patients with NSCLC at early stages, and a combination of serum miRNAs with serum exosomal miRNAs contributes to the further improvement of early diagnosis for NSCLC.

Development of up-converting phosphor technology-based lateral flow assay for quantitative detection of serum PIVKA-II: Inception of a near-patient PIVKA-II detection tool.

BACKGROUND: Development of a test card based on up-conversion phosphor technology-based immune lateral flow (UPT-LF) assay as a near-patient detection tool for serum Prothrombin induced by vitamin K absence or antagonist II (PIVKA-II). METHODS: Up-converted phosphor nanoparticles (UCPs) were used to bind to PIVKA-II monoclonal antibodies as labeled probes to develop the test card for detecting serum PIVKA-II. The UPT-LF test card was evaluated by the limit of detection, linearity, stability, recovery rate, precision and interference. Preliminary clinical validation was conducted by detection of 498 serum samples from 228 patients with hepatocellular carcinoma (HCC), 170 patients with liver benign lesion (LBL) and 100 healthy controls (HC). Additionally, the correlation of serum PIVKA-II detection between UPT-LF assay and Chemiluminescence enzyme immunoassay (CLEIA) assay were performed. RESULTS: Modified and activated UCPs bound to monoclonal antibodies powerfully to form the luminescent labeled probes. Limit of detection and linear range of UPT-LF test card for serum PIVKA-II were 2.66 and 4.8-20,000 ng/ml, respectively. Test card had good 25 °C thermal and 4 °C validity period stability, 93.1%-99.2% of recovery rate, 2.6-5.8% and 5.4-8.9% of intra-assay and inter-assay CVs, and strong anti-interference ability for 8 common serum analytes. The sensitivity and specificity (vs LBL + HC group) of test card for HCC were 71.5% and 88.9%, respectively. The R2 between UPT-LF assay and CLEIA assay was 0.901. CONCLUSIONS: UPT-LF assay provides a reliable, rapid and convenient test for quantitative detection of serum PIVKA-II as well as diagnosis of HCC by a point of care testing way.