The Relationship between IGF Pathway and Acquired Resistance to Tyrosine Kinase Inhibitors in Cancer Therapy.

The tyrosine kinase signaling pathway is an important pathway for cell signal transduction, and is involved in regulating cell proliferation, cell cycle, apoptosis and other essential biological functions. Gene mutations involved in the tyrosine kinase signaling pathway often lead to the development of cancers. Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor-2 (HER2) are well known receptor tyrosine kinases (RTKs), which belong to the ERBB family and have high mutation frequency in cancers. Tyrosine kinase inhibitors (TKI) targeting EGFR and HER2 have been widely used in the clinical treatment of lung and breast cancers. However, after a period of treatment, patients will inevitably develop resistance to TKI. The insulin-like growth factor (IGF) receptor family, like the ERBB receptor family, belongs to the receptor tyrosine kinase superfamily, which also conducts an important cell signal transduction function. There is an overlap between IGF signaling and EGFR signaling in biological functions and downstream signals. In this review, we summarize the current state of knowledge of how IGF signaling interacts with EGFR signaling can influence cell resistance to EGFR/HER2-TKI. We also summarize the current drugs designed for targeting IGF signaling pathways and their research progress, including clinical trials and preclinical studies. Altogether, we aimed to discuss the future therapeutic strategies and application prospects of IGF signaling pathway targeted therapy.

Longitudinal detection of subcategorized CD44v6+ CTCs and circulating tumor endothelial cells (CTECs) enables novel clinical stratification and improves prognostic prediction of small cell lung cancer: A prospective, multi-center study.

Current management of small cell lung cancer (SCLC) remains challenging. Effective biomarkers are needed to subdivide patients presenting distinct treatment response and clinical outcomes. An understanding of heterogeneous phenotypes of aneuploid CD31- circulating tumor cells (CTCs) and CD31+ circulating tumor endothelial cells (CTECs) may provide novel insights in the clinical management of SCLC. In the present translational and prospective study, increased cancer metastasis-related cell proliferation and motility, accompanied with up-regulated mesenchymal marker vimentin but down-regulated epithelial marker E-cadherin, were observed in both lentivirus infected SCLC and NSCLC cells overexpressing the stemness marker CD44v6. Aneuploid CTCs and CTECs expressing CD44v6 were longitudinally detected by SE-iFISH in 120 SCLC patients. Positive detection of baseline CD44v6+ CTCs and CD44v6+ CTECs was significantly associated with enhanced hepatic metastasis. Karyotype analysis revealed that chromosome 8 (Chr8) in CD44v6+ CTCs shifted from trisomy 8 towards multiploidy in post-therapeutic patients compared to pre-treatment subjects. Furthermore, the burden of baseline CD44v6+ CTCs (t0) or amid the therapy (t1-2), the ratio of baseline CD31+ CTEC/CD31- CTC (t0), and CTC-WBC clusters (t0) were correlated with treatment response and distant metastases, particularly brain metastasis, in subjects with limited disease (LD-SCLC) but not in those with extensive disease (ED-SCLC). Multivariate survival analysis validated that longitudinally detected CD44v6+/CD31- CTCs was an independent prognostic factor for inferior survival in SCLC patients. Our study provides evidence for the first time that comprehensive analyses of CTCs, CTECs, and their respective CD44v6+ subtypes enable clinical stratification and improve prognostic prediction of SCLC, particularly for potentially curable LD-SCLC.

Identification of serum MiRNAs as candidate biomarkers for non-small cell lung cancer diagnosis.

BACKGROUND: Lung cancer is one of the most common solid tumors worldwide and the leading cause of cancer-associated death. Non-small cell lung cancer (NSCLC) is accounts for approximately 85% of all the lung cancers and lung squamous carcinoma (SCC) and adenocarcinoma (ADC) are the main subtypes of NSCLC. Early diagnose using serum biomarkers could improve the overall survival of patients. In this study, we aimed to identify miRNAs from serum with clinical utility in the diagnosis of NSCLC. METHODS: Ten patients with SCC, ten patients with ADC and five noncancerous individuals were enrolled in the screening cohort. miRNA expression levels in serum were measured by microarray analysis. Candidate miRNAs were validated by real-time quantitative polymerase chain reaction analysis in a validation cohort of 78 NSCLC patients and 44 noncancerous individuals. Receiver operating characteristic curves were used to assess the diagnostic performance of serum miRNAs for NSCLC. Logistic regression was used to evaluate the diagnostic value of the combination of markers. RESULTS: Six candidate miRNAs were differentially expressed between NSCLC patients and noncancerous individuals in the screening set (fold change > 2, p < 0.05). Among them, expression levels of miR-3149 and miR-4769.3p were confirmed to be significantly increased in tumor serum in the validation set. The area under the curve values of miR-3149 and miR-4769.3p in distinguishing NSCLC patients from noncancerous controls were 0.830 and 0.735, respectively. When combined with tumor markers CEA and Cyfra21-1, the joint diagnostic model increased the area under the curve to 0.898. CONCLUSION: Serum miRNAs miR-3149 and miR-4769.3p were up-regulated in NSCLC and may be potential biomarkers for early diagnosis of lung cancer.

VPS9D1-AS1 overexpression amplifies intratumoral TGF-ß signaling and promotes tumor cell escape from CD8+ T cell killing in colorectal cancer.

Efficacy of immunotherapy is limited in patients with colorectal cancer (CRC) because high expression of tumor-derived transforming growth factor (TGF)-ß pathway molecules and interferon (IFN)-stimulated genes (ISGs) promotes tumor immune evasion. Here, we identified a long noncoding RNA (lncRNA), VPS9D1-AS1, which was located in ribosomes and amplified TGF-ß signaling and ISG expression. We show that high expression of VPS9D1-AS1 was negatively associated with T lymphocyte infiltration in two independent cohorts of CRC. VPS9D1-AS1 served as a scaffolding lncRNA by binding with ribosome protein S3 (RPS3) to increase the translation of TGF-ß, TGFBR1, and SMAD1/5/9. VPS9D1-AS1 knockout downregulated OAS1, an ISG gene, which further reduced IFNAR1 levels in tumor cells. Conversely, tumor cells overexpressing VPS9D1-AS1 were resistant to CD8+ T cell killing and lowered IFNAR1 expression in CD8+ T cells. In a conditional overexpression mouse model, VPS9D1-AS1 enhanced tumorigenesis and suppressed the infiltration of CD8+ T cells. Treating tumor-bearing mice with antisense oligonucleotide drugs targeting VPS9D1-AS1 significantly suppressed tumor growth. Our findings indicate that the tumor-derived VPS9D1-AS1/TGF-ß/ISG signaling cascade promotes tumor growth and enhances immune evasion and may thus serve as a potential therapeutic target for CRC.

The isocitrate dehydrogenase 1 is a potential prognostic indicator for non-small cell lung cancer patients.

BACKGROUND: The serum isocitrate dehydrogenase 1(IDH1) level is significantly elevated in patients with non-small cell lung cancer (NSCLC) and has important clinical value as a marker for early diagnosis. This study examined the dynamic changes of serum IDH1 levels of patients with NSCLC undergoing surgery or medical treatment, to evaluate its potential prognostic value. METHODS: The study cohort included 83 NSCLC patients who underwent surgery, 37 NSCLC patients who underwent medical treatment, 50 healthy controls, and 52 disease controls. Serum levels of IDH1 were assayed by enzyme-linked immunoassay. Tumor biomarkers including carcinoembryonic antigen, squamous cell carcinoma, neuron-specific enolase, CYFRA21-1, and pro-gastrin-releasing peptide-which are currently used in clinical practice-were measured by automatic immunoanalyzers. RESULTS: Serum IDH1 was significantly higher in patients with NSCLC compared with healthy people or patients with benign lung diseases (p < 0.001). The area under the receiver operating characteristic curve for diagnosis and differential diagnosis were 0.897 and 0.879, respectively, which were superior to the five tumor markers. Serum IDH1 levels decreased in most patients after surgery, with the most dramatic changes in patients with stage I tumors compared with stage II and III. Analyses of changes in the serum IDH1 level of patients after receiving chemotherapy or targeted therapy revealed that for patients with progressive disease, serum IDH1 increased significantly after treatment; for patients with partial response or stable disease, it decreased steadily. CONCLUSION: IDH1 has potential prognostic value and may be used as a marker for the monitoring of treatment efficacy.

Role of aneuploid circulating tumor cells and CD31+ circulating tumor endothelial cells in predicting and monitoring anti-angiogenic therapy efficacy in advanced NSCLC.

Prognosticating the efficacy of anti-angiogenic therapy through longitudinal monitoring and early detection of treatment resistance in cancer patients remain highly challenging. In this study, co-detection and comprehensive phenotypic and karyotypic molecular characterization of aneuploid circulating tumor cells (CTCs) and circulating tumor endothelial cells (CTECs) were conducted on non-small cell lung cancer (NSCLC) patients receiving bevacizumab plus chemotherapy. Prognostic values of the cell-based significant univariate risk factors identified by Cox regression analyses were progressively investigated. Subjects showing an increase in total post-therapeutic platelet endothelial cell adhesion molecule-1 (CD31)- CTCs and CD31+ CTECs exhibited a significantly reduced median progression-free survival (mPFS) and overall survival. Further stratification analyses indicated that pretherapeutic patients bearing vimentin (Vim)+ CTECs (mesenchymal M-type) at baseline revealed a significantly shortened mPFS compared with patients with Vim- CTECs. Post-therapeutic patients harboring epithelial cell adhesion molecule (EpCAM)+ CTCs and CTECs (epithelial E-type), regardless of Vim expression or not, showed a significantly reduced mPFS. Post-therapeutic patients possessing de novo EpCAM+ /Vim+ (hybrid E/M-type) CTECs displayed the shortest mPFS. Patients harboring either pre- or post-therapeutic EpCAM- /Vim- null CTECs (N-type) exhibited a better response to therapy compared to patients harboring EpCAM+ and/or Vim+ CTECs. The presented results support the notion that baseline Vim+ CTECs and post-therapeutic EpCAM+ CTCs and CTECs are predictive biomarkers for longitudinal monitoring of response to anti-angiogenesis combination regimens in NSCLC patients.

IGFBP7 overexpression promotes acquired resistance to AZD9291 in non-small cell lung cancer.

AZD9291 (osimertinib) is the third-generation EGFR-TKI treat for EGFR mutated NSCLC patients. Despite its encouraging efficacy in clinical, acquired resistance is still inevitable. The mechanism of drug resistance needs to be further explored. In a previous study, we established an AZD9291-resistant cell strain named HCC827/AZDR. We found that insulin-like growth factor binding protein 7 (IGFBP7) expression was markedly increased in HCC827/AZDR cells and AZD9291-resistant patients by RNA sequencing and immunohistochemical analysis, respectively. Reduced IGFBP7 in HCC827/AZDR cells by si-RNA interference recovered the sensitivity to AZD9291 partially and increased AZD9291-induced cell apoptosis. Enhancing IGFBP7 expression in EGFR-mutated non-small cell lung cancer (NSCLC) cells using lentiviruses infection reduced their sensitivity to AZD9291. This study is the first to discover that high IGFBP7 expression could occur following treatment with AZD9291. This might be one of the mechanisms underlying AZD9291 resistance and a potential therapeutic target following AZD9291 resistance.

[CD45RO⁺ Memory T Lymphocytes As A Candidate Marker for Non-small Cell Lung Cancer].

BACKGROUND: Lung cancer is the most common malignancy world-wide. There are a variety of immune infiltrating cells in tumor microenvironment, which is an important component of tumor immunity and has clinical significance for the prognosis of patients. CD45RO is a surface marker of memory T cells. The expression of CD45RO⁺ tumor infiltrating lymphocytes (TILs) is associated with the prognosis of many tumors. The purpose of this study was to evaluate the relationship between the density of CD45RO⁺ TILs in tumor and stromal area and the clinical characteristics of patients with non-small cell lung cancer (NSCLC) and its impact on the prognosis of patients. We aimed to explore the clinical value of CD45RO⁺ TILs and programmed cell death ligand 1 (PD-L1) as prognostic markers. METHODS: Multiple fluorescent immunohistochemical staining was used to stain the tissue microarray chips of 167 patients with NSCLC, marking CD45RO, cytokeratin (CK) and PD-L1. Using artificial intelligence image recognition technology and tumor cell-specific CK staining, divide the tumor and stromal area in the tissue, evaluate the density of CD45RO⁺ TILs in the tumor and stromal area, and the expression level of PD-L1 in tumor cells. The non-parametric test was used to analyze the relationship between CD45RO⁺ TILs and the clinical characteristics of patients, and the Kaplan-Meier method and Cox risk ratio model were used to analyze the relationship between CD45RO⁺ TILs independently or in combination with PD-L1 and tumor prognosis. RESULTS: The density of CD45RO⁺ TILs was significantly associated with patient age, smoking, tumor stage, and pathological type. Single-factor survival analysis showed that NSCLC (P=0.007) stromal region and lung adenocarcinoma (LUAD) (P<0.001) with CD45RO⁺ TILs high density had better OS. Multivariate survival analysis showed that the high density of CD45RO⁺ TILs in the stromal region of NSCLC (HR=0.559, 95%CI: 0.377-0.829, P=0.004) and lung adenocarcinoma (HR=0.352, 95%CI: 0.193-0.641, P=0.001) were independent prognostic factors for overall survival time (OS). Combined with PD-L1 score of tumor cells in tumor tissues and infiltration score of CD45RO⁺ TILs in all tumor tissues, the patients were divided into 4 groups: patients with PD-L1⁺/CD45RO⁺ had the longest disease-free survival (DFS) time, and patients with PD-L1⁺/CD45RO- had the shortest DFS time. Multivariate Cox regression analysis showed that PD-L1⁺/CD45RO- was an independent prognostic factor for DFS and had a higher risk of poor prognosis compared to the other three groups (HR=2.221, 95%CI: 1.258-3.919, P=0.006). CONCLUSIONS: In tumor tissues, the density of CD45RO⁺ TILs, as well as the combination of CD45RO⁺ TILs and PD-L1 in tumor areas, significantly correlated with clinicopathological features and prognosis of NSCLC, which can be used as a new prognosis marker.

[Research Progress of Single Cell Sequencing in Lung Cancer].

Lung cancer is the malignant tumor with the highest mortality rate in the world. Heterogeneity of lung cancer, usually studied by sequencing technology, is considered to have important clinical significance in current studies. However, general sequencing technology can only explain the differences between samples integrally and its resolution is not enough to describe the differences between the individual cells. Therefore, people urgently hope to understand the cell type, state, subgroup distribution in the tumor microenvironment and the communication behavior between cells in the single cell level. Single-cell sequencing technology solves this problem. Using this technique will contribute to further understanding the mechanism of the occurrence and development of lung cancer, discovering new diagnostic markers and therapeutic targets, and providing theoretical references for the precise treatment of lung cancer patients in the future. This article reviews the progress of single-cell sequencing technology and focuses on its research on lung cancer tumor heterogeneity, tumor microenvironment, invasion and metastasis, treatment response, and drug resistance.
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Comprehensive Comparative Molecular Characterization of Young and Old Lung Cancer Patients.

BACKGROUND: Young lung cancer as a small subgroup of lung cancer has not been fully studied. Most of the previous studies focused on the clinicopathological features, but studies of molecular characteristics are still few and limited. Here, we explore the characteristics of prognosis and variation in young lung cancer patients with NSCLC. METHODS: A total of 5639 young lung cancer samples (NSCLC, age ≤40) were screened from the SEER and the same number of the old (NSCLC, age ≥60) were screened by propensity score matching to evaluate the prognosis of two groups. 165 treatment-naïve patients diagnosed with NSCLC were enrolled to explore the molecular feature difference between two age-varying groups. CCLE cell line expression data was used to verify the finding from the cohort of 165 patients. RESULTS: The overall survival of the young lung cancer group was significantly better than the old. Germline analysis showed a trend that the young group contained a higher incidence of germline alterations. The TMB of the young group was lower. Meanwhile, the heterogeneity and evolutionary degrees of the young lung cancer group were also lower than the old. The mutation spectrums of two groups exhibited variance with LRP1B, SMARCA4, STK11, FAT2, RBM10, FANCM mutations, EGFR L858R more recurrent in the old group and EML4-ALK fusions, BCL2L11 deletion polymorphism, EGFR 19DEL, 20IN more recurrent in the young group. For the base substitution, the young showed a lower fraction of transversion. Further, we performed a pathway analysis and found the EGFR tyrosine kinase inhibitor resistance pathway enriched in the young lung cancer group, which was validated in gene expression data later. CONCLUSIONS: There were significantly different molecular features of the young lung cancer group. The young lung cancer group had a more simple alteration structure. Alteration spectrums and base substitution types varied between two groups, implying the different pathogenesis. The young lung cancer group had more potential treatment choices. Although young lung patients had better outcomes, there were still adverse factors of them, suggesting that the young group still needs more caution for treatment choice and monitoring after the treatment to further improve the prognosis.

Associations between gastro-oesophageal reflux disease and a range of diseases: an umbrella review of systematic reviews and meta-analyses.

OBJECTIVE: Numerous meta-analyses have revealed the association between gastro-oesophageal reflux disease (GORD) and a range of diseases; however, the certainty of the evidence remains unclear. This study aimed to summarise and assess the certainty of evidence derived from meta-analyses. METHODS: Embase, PubMed, Web of Science, Cochrane Databases of Systematic Reviews, CNKI and Wangfang databases from their inception to 22 February 2020 were queried for systematic reviews and meta-analyses on the association between GORD and various diseases. The methodological quality of the included studies was assessed using A Measurement Tool to Assess Systematic Reviews 2 (AMSTAR 2), and evidence certainty was evaluated using the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE) system. Statistical analysis was conducted using Stata V.15. RESULTS: Ten publications with associations between GORD and different types of diseases were included. There was high heterogeneity (I2 >75%) among seven independent meta-analyses. Evidence for publication bias in two independent meta-analyses was also observed. According to the AMSTAR 2 approach, the methodological quality was high for 20% of meta-analyses, moderate for 10%, low for 40% and critically low for 30%. Based on GRADE approach, the certainty of evidence was high for the association between GORD and higher risk of chronic obstructive pulmonary disease (COPD) exacerbation (OR 5.37; 95% CI 2.71 to 10.64) and higher prevalence of oesophageal adenocarcinoma (OR 4.57; 95% CI 3.89 to 5.36), and it was moderate for the association between GORD and higher chronic rhinosinusitis prevalence (OR 2.16; 95% CI 1.37 to 3.48). CONCLUSION: The association between GORD and a range of diseases was extensively studied, and our findings revealed a high certainty of evidence of the association between GORD and an increased risk of COPD exacerbation as well as increased prevalence of oesophageal adenocarcinoma. Further investigations using systematic reviews and meta-analyses of high methodological quality that include prospective large cohort studies and adjusted confounders are warranted. PROSPERO REGISTRATION NUMBER: CRD42019122264.

Association between H. pylori infection and health Outcomes: an umbrella review of systematic reviews and meta-analyses.

OBJECTIVE: Systematic reviews and meta-analyses have revealed the associations between H. pylori infection and various health outcomes. We aimed to evaluate the strength and breadth of evidence on the associations. DESIGN: Umbrella review of systematic reviews and meta-analyses. SETTING: No settings. PARTICIPANTS: No patients involved. DATA SOURCES: Embase, PubMed, Web of Science, Cochrane Library Databases, CNKI, VIP database and Wangfang database from inception to February 1, 2019. OUTCOMES MEASURES: Diverse diseases (such as cancer and ischaemic heart disease). RESULTS: Sixty articles reporting 88 unique outcomes met the eligible criteria. 74 unique outcomes had nominal significance (p<0.05). Of the outcomes with significance, 61 had harmful associations and 13 had beneficial associations. Furthermore, 73% (64) of the outcomes exhibited significant heterogeneity . Of the these meta-analyses, 32 had moderate to high heterogeneity (I2=50%-75%) and 24 had high heterogeneity (I2>75%). Moreover, 20% exhibited publication bias (p<0.1). In addition, 97% of the methodological qualities were rated 'critically low'. 36% of the evidence qualities of outcomes were rated 'low', 56% of the evidence qualities were rated 'very low' and 8% of the evidence qualities were rated 'moderate'. H. pylori infection may be associated with an increased risk of five diseases and a decreased risk of irritable bowel syndrome. CONCLUSION: Although 60 meta-analyses explored 88 unique outcomes, moderate quality evidence only existed for six outcomes with statistical significance. H. pylori infection may be associated with a decreased risk of irritable bowel syndrome and an increased risk of hypertriglyceridemia, chronic cholecystitis and cholelithiasis, gestational diabetes mellitus, gastric cancer and systemic sclerosis. TRIAL REGISTRATION: CRD42019124680.

Cyclophilin B overexpression predicts a poor prognosis and activates metastatic pathways in colon cancer.

BACKGROUND: Cyclophilin B (CypB) has been found overexpressed in various malignant tumors. To date, there are few studies on CypB in colon cancer. In this study, we aimed to analyze the CypB expression pattern and to further evaluate its clinical significance, especially its prognostic value for colon cancer. METHODS: CypB expression was investigated in colon cancer tissue microarrays (TMA) by RNAscope in situ hybridization and immunohistochemical (IHC) staining. The correlation between CypB and clinicopathological characteristics was analyzed. The Cancer Genome Atlas (TCGA) RNA-seq dataset of colon adenocarcinoma (COAD) was further analyzed to validate our main findings. Gene Set Enrichment Analysis (GSEA) and Search Tool for the Retrieval of Interacting Genes (STRING) analysis were performed to enrich CypB related biological pathways. In vitro experiments by knockdown of CypB in colon cancer cell HCT116 were performed to verify the bioinformatics results and analyze its role in the metastatic pathways in colon cancer. RESULTS: We found that CypB expression was highly upregulated in colon cancer tissues (P<0.05). Importantly, the overall survival (OS) time of patients with high CypB expression was significantly shorter than those with low CypB expression, and overexpressed CypB was identified as an independent prognostic indicator for poor survival (P=0.015). Subgroup analysis indicated that a high level of CypB was associated with a shorter OS time, especially for advanced cancer patients, such as later T stage, lymph node metastasis, larger tumor size (P<0.05). Analysis of TCGA RNA-seq dataset of COAD provided us with a larger clinical sample verification. Bioinformatics analysis and the following in vitro study revealed that CypB was involved in tumor metastatic associated signaling pathways. CONCLUSIONS: CypB overexpression predicts a poor prognosis and may activate metastatic pathways in colon cancer.

Abnormally localized DLK1 interacts with NCOR1 in non-small cell lung cancer cell nuclear.

Delta-like homolog 1 (DLK1) regulates noncanonical Notch signaling pathway as ligand. DLK1 was abnormally expressed in a variety of tumors, affecting tumorigenesis and developments. The biological function of DLK1 toward cell proliferation and signaling activation was controversial across different cell types. Two currently known isoforms of DLK1, which are membrane-tethered isoform and soluble isoform, are believed to be the key of DLK1 dual behaviors. While these isoforms are not enough to explain the phenomena, our observations offer the possibility of a third isoform of DLK1. In the present study, we verified the nuclear localization of DLK1 in lung cancer cells. The nuclear localized DLK1 was observed in 107 of 351 non-small cell lung cancer (NSCLC) samples and was associated with tissue differentiation and tumor size. Through co-immunoprecipitation (co-IP) combined mass spectrometry (MS), we identified nuclear receptor corepressor 1 (NCOR1) as DLK1's novel interaction protein and confirmed their interaction in nuclear. We analyzed the expression of NCOR1 in two independent cohorts and demonstrated that NCOR1 is a tumor suppressor and has prognosis potential in lung squamous carcinomas. At last, we analyzed the colocalization of DLK1 and NCOR1 in 147 NSCLC samples by immunohistochemistry (IHC). The result indicated NCOR1 might participate with nuclear localized DLK1 in regulating cell differentiation.

FAM83H-AS1 is upregulated and predicts poor prognosis in colon cancer.

Long noncoding RNAs (LncRNAs) have been proven to participate in many principal pathways during colonic mucosa tumourigenesis. FAM83H-AS1 has been identified as one of the LncRNAs that is dysregulated in multi-type cancers. Here, our purpose was to determine the expression pattern and clinical significance of FAM83H-AS1 and further analyse its prognostic value in colon cancer. FAM83H-AS1 expression was examined in 90 patients with colon cancer by RNAscope in situhybridization, and the expression levels were analysed with the overall survival (OS) rates for patients with colon cancer. TCGA datasets derived from colon cancer (COAD) were used to further validate the main findings. We demonstrated that the expression of FAM83H-AS1 was significantly higher in cancer tissues than in paired normal mucosa from colon cancer patients (P < 0.001). The prognostic analysis indicated that the OS rates for colon cancer patients with high levels of FAM83H-AS1 were significantly lower than those for patients with low levels of FAM83H-AS1 (P = 0.013). The higher levels of FAM83H-AS1 were found to be associated with a lower expression of SMAD1/5/9, which is involved in TGF-ß signalling in colon cancer tissues (P = 0.0174). In HCT116 and SW480 cell lines, the down regulation of FAM83H-AS1 promoted the expression of SMAD1. Our investigations suggest that the upregulation of FAM83H-AS1 serves as a promising predictor and might be conducive for clinicians to estimate OS time. FAM83H-AS1 might serve as an inhibitor of TGF-ß signalling.

Long noncoding RNA VPS9D1-AS1 overexpression predicts a poor prognosis in non-small cell lung cancer.

Long noncoding RNAs (LncRNAs) have been reported to play vital roles in non-small cell lung cancer (NSCLC). Recently, LncRNA/VPS9D1-AS1 has been reported to be overexpressed in various cancers. In this study, we aimed to investigate its expression pattern and clinical significance and further evaluate its prognostic value for NSCLC. VPS9D1-AS1 expression was examined in 184 NSCLC patients using a highly sensitive in situ hybridization protocol (RNAscope), and the expression values were correlated with the clinicopathological features. Another cohort including 12 NSCLC patients was used to validate the differential expression of VPS9D1-AS1 by qRT-PCR. TCGA datasets were further used to validate the main findings. We found that the levels of VPS9D1-AS1 were significantly higher in cancer tissues than in paired normal tissues from both lung squamous cell carcinoma (SCC) and adenocarcinoma (ADC) (P < 0.001). Importantly, the levels of VPS9D1-AS1 in patients with lung SCC were significantly higher than those in patients with lung ADC. The high levels of VPS9D1-AS1 were found to be associated with cancer lymph node metastasis (P = 0.020). Prognostic analysis revealed that the survival time for SCC patients with high levels of VPS9D1-AS1 was significantly shorter than that of patients with low levels of VPS9D1-AS1 (P = 0.007). Therefore, our findings suggest that the overexpression of VPS9D1-AS1 serves as a promising biomarker to predict the prognosis of NSCLC.

Utility of dominant epitopes derived from cell-wall protein LppZ for immunodiagnostic of pulmonary tuberculosis.

BACKGROUND: Serological antibodies tests for tuberculosis (TB) are widely used in developing countries. They appear to have some advantages- faster, simple and could be used for extrapulmonary TB. However, most of current commercial TB serological tests are failed to provide sufficient sensitivity and specificity. Improved serological biomarkers were essential. In this study, we present an approach using peptide array to discover new immunodiagnostic biomarkers based on immunodominant epitopes of TB antigens. RESULTS: The Probable conserved lipoprotein LppZ, which is difficult to express and purify in vivo was selected as the model antigen. We use two-step screening for dominant epitope selection. Based on peptide array data from 170 TB patients and 41 control samples, two dominant epitopes were identified to have diagnostic value for TB patients. Truncation assay was used to identify the core reactive sequence. Peptide- based ELISA was used to evaluate the diagnostic ability of pep-LppZ-1 and pep-LppZ-13. Pep-LppZ-1 has a sensitivity of 49.2% and a specificity of 83.3% in TB diagnose. Pep-LppZ-13 has a sensitivity of 43.3% and a specificity of 88.5% in TB diagnose. CONCLUSIONS: Our result demonstrated that peptide array screening would be an advantage strategy of screening TB diagnostic peptides.

Multispectral imaging reveals hyper active TGF-ß signaling in colorectal cancer.

Advances in multiplex immunohistochemistry (IHC) techniques and digital pathology platforms allow quantification of multiple proteins at same tissue section and produce continuous data. TGF-ß signaling plays crucial and complex roles in colorectal cancer (CRC). We here aimed to investigate clinical pathological relevant of proteins involved in TGF-ß signaling at CRC tissues. Multiplex fluorescent IHC was used to quantitative analysis. The levels of eight proteins (TGF-ß1, TGFBRI, TGFBRII, SMAD4, SMAD2/3, p-SMAD2/3, SMAD1/5/9, and p-SMAD1/5/9) were determined in TMA sections. Quantitative analysis was carried out by a scoring system by InForm software. It revealed that TGF-ß signaling was hyper active. The levels of TGF-ß1, TGFBRI, TGFBRII, SMAD4, SMAD1/5/9 and p-SMAD2/3 were significantly increased in cancer tissues when compared their levels in normal tissues. Furthermore, the levels of eight proteins in stroma were significantly lower than the levels that in cancer tissues. Clinical pathological relevant analysis exhibited that TGF-ß signaling inclined to suppress the progression of tumor. SMAD1/5/9, TGFBRII, SMAD2/3 were confirmed as significant predictors for overall survival. In conclusion, we established a method based on multispectral imaging to extensively explore the clinical relevant of TGF-ß signaling proteins. These results provided an opportunity to consider the novel application for proteins involving TGF-ß signaling that used as diagnostic or prognostic biomarkers to conduct tumor therapy.

Combined detection of dickkopf-1 subtype classification autoantibodies as biomarkers for the diagnosis and prognosis of non-small cell lung cancer.

PURPOSE: This study aims to identify the clinical significance of serum autoantibodies against dickkopf-1 (DKK1) and evaluate their feasibility in the immunodiagnosis and prognosis of non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Epitope mapping by peptide microarray-based serum screening of NSCLC patients (n=72) and healthy controls (n=16) was performed. Indirect ELISA with peptides was used to measure the serum levels of autoantibodies in 206 NSCLC patients and 99 healthy controls. A 3-year follow-up was monitored to evaluate the correlation between serological levels of autoantibodies and overall survival (OS) and progression-free survival (PFS). RESULTS: Four highly reactive epitopes were identified, which included peptides 67-84 (Pep A), 37-54 (Pep B), 145-156 (Pep C) and 247-261 (Pep D). The autoantibodies levels were considerably higher in sera of NSCLC patients compared with controls (P<0.001), and a highly significant correlation with distant metastases was observed (Pep A: P=0.09, Pep B: P<0.01, Pep C: P<0.01 and Pep D: P<0.01). High levels of antibody subtype to Pep B were remarkably associated with better OS (P=0.004) and PFS (P=0.006). Subsequent Cox regression analysis disclosed that antibody to Pep B was an independent prognostic factor for NSCLC (OS: P=0.008, HR =0.435, 95% CI 0.236-0.802; PFS: P=0.032, HR =0.533, 95% CI 0.322-0.950). CONCLUSION: Identified linear epitopes of antigens by peptide microarray are easily available, and subtype classification of DKK1 autoantibodies as novel biomarkers for the diagnosis and prognosis of NSCLC. Our results also highlight the antibody subtype to Pep B as the most valuable biomarker for favorable prognosis of NSCLC.

Pregnancy-specific glycoprotein 9 (PSG9), a driver for colorectal cancer, enhances angiogenesis via activation of SMAD4.

PSG9 is a member of the pregnancy-specific glycoprotein (PSG) family and has been shown to contribute to the progression of colorectal cancer (CRC) and cancer-related angiogenesis. Here, we aim to investigate abnormal PSG9 levels in patients with CRC and to emphasize the role of PSG9 in driving tumorigenesis. Serum from 140 patients with CRC and 125 healthy controls as well as 74 paired tumors and adjacent normal tissue were used to determine PSG9 levels. We discovered that PSG9 was significantly increased in serum (P<0.001) and in tumor tissues (P<0.001) from patients with CRC. Interestingly, the increased PSG9 levels correlated with poor survival (P=0.009) and microvessel density (MVD) (P=0.034). The overexpression of PSG9 strongly promoted the proliferation and migration of HCT-116 and HT-29 cells. However, PSG9 depletion inhibited the proliferation of SW-480 cells. Using a human umbilical vein endothelial cell tube-forming assay, we found that PSG9 promoted angiogenesis. The overexpression of PSG9 also increased the growth of tumor xenografts in nude mice. Co-immunoprecipitation experiments revealed that PSG9 was bound to SMAD4. The PSG9/SMAD4 complex recruited cytoplasmic SMAD2/3 to form a complex, which enhanced SMAD4 nuclear retention. The PSG9 and SMAD4 complex activated the expression of multiple angiogenesis-related genes (included IGFBP-3, PDGF-AA, GM-CSF, and VEGFA). Together, our findings illustrate the innovative mechanism by which PSG9 drives the progression of CRC and tumor angiogenesis. This occurs via nuclear translocation of PSG9/SMAD4, which activates angiogenic cytokines. Therefore, our study may provide evidence for novel treatment strategies by targeting PSG9 in antiangiogenic cancer therapy.