Identification Of Potential Prognostic Biomarkers Research Articles

Significant changes in synovial fluid microRNAs after high tibial osteotomy in medial compartmental knee osteoarthritis: Identification of potential prognostic biomarkers.

High tibial osteotomy (HTO) is a well-established treatment for medial compartmental knee osteoarthritis. Several microRNAs (miRNAs) are involved in osteoarthritis progression and are useful as osteoarthritis-related biomarkers. In this prospective study, we investigated differentially expressed microRNAs in the synovial fluid (SF) before and after HTO in patients with medial compartmental knee osteoarthritis to identify microRNAs that can be used as prognostic biomarkers. We used miRNA-PCR arrays to screen for miRNAs in SF samples obtained preoperatively and 6 months postoperatively from 6 patients with medial compartmental knee osteoarthritis who were treated with medial open wedge HTO. Differentially expressed miRNAs identified in the profiling stage were validated by real-time quantitative PCR in 22 other patients who had also been treated with HTO. All patients radiographically corresponded to Kellgren-Lawrence grade II or III with medial compartmental osteoarthritis. These patients were clinically assessed using a visual analogue scale and Western Ontario McMaster Universities scores. Mechanical axis changes were measured on standing anteroposterior radiographs of the lower limbs assessed preoperatively and at 6 months postoperatively. Among 84 miRNAs known to be involved in the inflammatory process, 14 were expressed in all SF specimens and 3 (miR-30a-5p, miR-29a-3p, and miR-30c-5p) were differentially expressed in the profiling stage. These 3 miRNAs, as well as 4 other miRNAs (miR-378a-5p, miR-140-3p, miR-23a-3p, miR-27b-3p), are related to osteoarthritis progression. These results were validated in the SF from 22 patients. Clinical and radiological outcomes improved after HTO in all patients, and only 2 miRNAs (miR-30c-5p and miR-23a-3p) were significantly differentially expressed between preoperative and postoperative 6-month SF samples (p = 0.006 and 0.007, respectively). Of these two miRNAs, miR-30c-5p correlated with postoperative pain relief. This study provides potential prognostic miRNAs after HTO and further investigations should be considered to determine clinical implications of these miRNAs.

Screening and Identification of Potential Prognostic Biomarkers in Adrenocortical Carcinoma.

Objective: Adrenocortical carcinoma (ACC) is a rare but aggressive malignant cancer that has been attracting growing attention over recent decades. This study aims to integrate protein interaction networks with gene expression profiles to identify potential biomarkers with prognostic value in silico. Methods: Three microarray data sets were downloaded from the Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs) according to the normalization annotation information. Enrichment analyses were utilized to describe biological functions. A protein–protein interaction network (PPI) of the DEGs was developed, and the modules were analyzed using STRING and Cytoscape. LASSO Cox regression was used to identify independent prognostic factors. The Kaplan–Meier method for the integrated expression score was applied to analyze survival outcomes. A receiver operating characteristic (ROC) curve was constructed with area under curve (AUC) analysis to determine the diagnostic ability of the candidate biomarkers. Results: A total of 150 DEGs and 24 significant hub genes with functional enrichment were identified as candidate prognostic biomarkers. LASSO Cox regression suggested that ZWINT, PRC1, CDKN3, CDK1 and CCNA2 were independent prognostic factors in ACC. In multivariate Cox analysis, the integrated expression scores of the modules showed statistical significance in predicting disease-free survival (DFS, P = 0.019) and overall survival (OS, P < 0.001). Meanwhile, ROC curves were generated to validate the ability of the Cox model to predict prognosis. The AUC index for the integrated genes scores was 0.861 (P < 0.0001). Conclusion: In conclusion, the present study identifies DEGs and hub genes that may be involved in poor prognosis and early recurrence of ACC. The expression levels of ZWINT, PRC1, CDKN3, CDK1 and CCNA2 are of high prognostic value, and may help us understand better the underlying carcinogenesis or progression of ACC. Further studies are required to elucidate molecular pathogenesis and alteration in signaling pathways for these genes in ACC.

Genomic Landscapes of EBV-Associated Nasopharyngeal Carcinoma vs. HPV-Associated Head and Neck Cancer.

Epstein-Barr virus-positive nasopharyngeal carcinoma (EBV(+) NPC), and human papillomavirus-positive head and neck squamous cell carcinoma (HPV(+) HNSCC) are two distinct types of aggressive head and neck cancers with early age onsets. Their recently identified genomic landscapes by whole-exome sequencing (WES) clearly reveal critical roles of: (1) inflammation via NF-kB activation, (2) survival via PI3K aberrations, and perhaps (3) immune evasion via MHC loss in these cancers as summarized in this review. Immediate outcomes of these WES studies include the identification of potential prognostic biomarkers, and druggable events for these cancers. The impact of these genomic findings on the development of precision medicine and immunotherapies will be discussed. For both of these cancers, the main lethality comes from metastases and disease recurrences which may represent therapy resistance. Thus, potential curing of these cancers still relies on future identification of key genomic drivers and likely druggable events in recurrent and metastatic forms of these intrinsically aggressive cancers of the head and neck.

Asparaginyl endopeptidase enhances pancreatic ductal adenocarcinoma cell invasion in an exosome-dependent manner and correlates with poor prognosis.

Pancreatic cancer is one of the most lethal types of cancer; owing to low early detection rates and high metastasis rates, it is associated with an extremely poor prognosis. Therefore, a better understanding of the molecular mechanisms that underlie its metastasis and the identification of potential prognostic biomarkers are urgently required. Although high expression levels of asparaginyl endopeptidase (AEP) have been detected in various types of solid tumor, the expression and functions of AEP in pancreatic carcinomas have yet to be determined. The present study aimed to examine the putative functions of AEP in pancreatic carcinoma. Immunohistochemical analysis revealed that AEP was highly expressed in pancreatic cancer tissues compared with adjacent normal tissues. Patients with high AEP expression exhibited a significantly shorter overall survival time. Results from multivariate Cox regression analysis revealed that AEP was an independent prognostic factor for overall survival. Gain- and loss-of-function experiments demonstrated that knockdown of AEP expression significantly reduced the invasive ability of pancreatic cancer cells, whereas overexpression of AEP increased the invasive ability. In addition, AEP was detected in exosomes that were derived from cultured pancreatic ductal adenocarcinoma cells (PDACs) and in the serum from patients with PDAC. The Matrigel-Transwell invasion assay revealed that exosomes enriched with AEP were able to enhance the invasive ability of PDAC cells, whereas exosomes lacking AEP decreased the invasive ability. Furthermore, results from the present study suggested that AEP may be crucial for activation of the phosphoinositide3-kinase/RAC‑α serine/threonine-protein kinase signaling pathway in PDAC cells. The present study data indicated that high AEP expression may be important for pancreatic carcinoma progression in an exosome-dependent manner, and that AEP may be an independent indicator of poor prognosis in patients with PDAC and may be a novel prognostic biomarker or therapeutic target in pancreatic carcinoma.

Abstract A33: γH2AX: A molecular marker of DNA damage response in smoking-induced pancreatic ductal adenocarcinoma

Abstract Introduction: Cigarette smoking is an important risk factor for the development pancreatic ductal adenocarcinoma (PDAC). Nicotine and nitrosamines cause pancreatic cell injury and contribute to a cascade of oncogenic events. Among multiple tobacco specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is considered to be the most carcinogenic and induces DNA double strand-breaks (DSBs). However, identification of potential prognostic biomarkers and experimental models of smoking-induced PDAC remain unknown. We have studied phosphorylated histone H2AX (γH2AX), a marker for double-stranded DNA breaks, as a new biomarker for detecting cigarette smoke-induced pathogenesis of PDAC. Experimental Procedure: We have characterized the expression of γH2AX protein in human pancreatic tissues, human pancreatic duct epithelial (HPDE)6, PDAC cell lines, and PanIn, primary PDAC (PDA) and liver metastasis (LMP) cell lines generated from a genetically engineered mice model of PDAC. Kaplan-Meier survival analysis was performed to determine the significance of γH2AX expression amongst smokers and non-smokers with PDAC. HPDE6 and PanIN cells were cultured for 4 days in the presence or absence of cigarette smoke (CS) (0-25μg/ml) or NNK (0-10μM). Cell cycle analysis and apoptosis were assessed through flow cytometry with the markers propidium iodide and annexin V respectively. HPDE6 and PanIN cells treated with CS or NNK for up to 50 days were analyzed for rapid activation of DNA damage response (DDR) pathway proteins γH2AX, RAD51, BRCA1, BRCA2, phospho-CHK2 and phospho-CHK1. Cells were treated with either CS or NNK along with a CHK2 inhibitor and analyzed for activation of γH2AX. To determine DNA integrity, accumulation of γH2AX was measured by ELISA in cells treated with CS or NNK. Immunofluorescence confocal microscopy was utilized to examine the role of γH2AX on nuclear foci formation with DNA repair complexes by quantifying the amount of co-localization. To replicate chronic smoke exposure, we treated HPDE6 and PanIn cells with 1 and 10 µg/ml CS or 10nM and 1µM NNK for 10 passages, and examined these cell lines for DSBs by comet assay, apoptosis markers by protein array and in vitro tumorigenicity by colony formation assay. Results: γH2AX expression was significantly higher (p&amp;lt;0.0001) in smokers when compared to non-smokers. Overall survival of patients with high γH2AX expression in their primary tumor was associated with significantly decreased median survival (11 months) when compared to low γH2AX expression (36 months, p=0.0007). HPDE6 cells exposed to CS or NNK, showed more than 80% survival. The surviving cells had altered morphology, acquired mesenchymal characteristics and displayed increased tumorigenicity suggesting that these cells acquire resistance to apoptosis after CS or NNK exposure. In addition, exposure of cells to CS or NNK resulted in generation of γH2AX and induction of phospho-CHK2 expression. RAD51 and phospho-CHK2 co-localize with γH2AX after DNA damage with CS or NNK exposure. Inhibition of CHK2 reduced expression of γH2AX. The product of the tumor suppressor gene BRCA2 co-localized with γH2AX indicating that γH2AX is necessary for the recruitment of DNA repair factors to the site of DNA damage. BRCA2 mutant cell line CAPAN1 exhibited a substantially reduced ability to form co-localization with RAD51 and γH2AX, consistent with a role of γH2AX in DNA damage repair. Conclusion: This work supports the role of γH2AX as a new molecular biomarker to assess the contribution of cigarette smoking-induced PDAC. γH2AX is necessary for DNA damage repair in PDAC and its activation is dependent on CHK2. Therapies that can block γH2AX expression by inhibiting upstream kinases may be a new potential therapeutic approach to treat and/or prevent PDAC. Citation Format: Kumaraswamy Honnenahally, Chanjuan Shi, Xi Chen, Jason Castellanos, Nipun Merchant, Timothy Blackwell, Nagaraj Nagathihalli. γH2AX: A molecular marker of DNA damage response in smoking-induced pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A33.

Proteomic identification of potential prognostic biomarkers in resectable pancreatic ductal adenocarcinoma

Pancreatic cancer is a devastating disease with a mortality rate almost identical with its incidence. In this context, the investigation of the pancreatic cancer proteome has gained considerable attention because profiles of proteins may be able to identify disease states and progression more accurately. Therefore, our objective was to investigate the changes in the proteome of patients suffering from pancreatic ductal adenocarcinoma (PDAC) by a comprehensive quantitative approach. Comparative proteomic profiling by label-free LC-MS/MS analysis of nine matched pairs of tumor and nontumor pancreas samples was used to identify differences in protein levels characteristic for PDAC. In this analysis, 488 proteins were quantified by at least two peptides of which 99 proteins displayed altered levels in PDAC (p < 0.01, fold change >1.3). Screening of data revealed a number of molecules that had already been related to PDAC such as galectin-1 (LEG1), major vault protein, adenylyl cyclase-associated protein 1 (CAP1), but also a potential new prognostic biomarker prolargin (PRELP). The Kaplan-Meier survival analysis revealed a significant correlation of protein abundance of PRELP with postoperative survival of patients with PDAC. For selected proteins the findings were verified by targeted proteomics (SRM), validated by immunohistochemistry and Western blotting and their value as candidate biomarkers is discussed.

Identification of potential prognostic biomarkers for node-negative breast tumours by proteomic analysis: A multicentric 2004 national PHRC study

We used a 2D-electrophoresis (2-DE) proteomic approach to identify novel biomarkers in node-negative breast cancers. This retrospective study focused on a population of patients with ductal pN0M0 tumours. A subset of patients who developed metastases and in whose tumours were found high levels of uPA and PAI-1 (metastatic relapse, MR: n=20) were compared to another subset in whom no metastatic relapse occurred and whose tumours were found to have low levels of uPA and PAI-1 (no relapse, NR: n=21). We used a 2-DE coupled with MS approach to screen cytosol fractions using two pH-gradient scales, a broad scale (3.0-11.0) and a narrower scale focussing in on a protein rich region (5.0-8.0). This study was conducted on 41 cytosol specimens analyzed in duplicate on two platforms. The differential analysis of more than 2,000 spots in 2-DE gels, obtained on the two platforms, allowed the identification of 13 proteins which were confirmed by western blotting. Two proteins, GPDA and FABP4 were down-regulated in the MR subset whereas all the others were up-regulated. An in silico analysis revealed that GMPS (GUAA), GAPDH (G3P), CFL1 (COF1) and FTL (FRIL), the most informative genes, displayed a proliferation profile (high expression in basal-like, HER2+ and luminal B molecular subtypes). Inversely, similar to FABP4, GPD1 [GPDA] displayed a high expression in luminal A subtype, a profile characteristic of tumour suppressor genes. Despite the small size of our cohort, the 2-DE analysis gave interesting results which were confirmed by the in silico analysis showing that some of the corresponding genes had a strong prognostic impact in breast cancer, mostly because of their link with proliferation: GMPS, GAPDH, FTL and GPD1. A validation phase on a larger cohort is now needed before these biomarkers could be considered for use in clinical practice.

Identification of Potential Prognostic Biomarkers in Patients with Untreated, Advanced Pancreatic Cancer from a Phase III Trial (CALGB 80303)

Identification of Potential Prognostic Biomarkers in Patients with Untreated, Advanced Pancreatic Cancer from a Phase III Trial (CALGB 80303)

Identification of potential prognostic biomarkers in patients with untreated, advanced pancreatic cancer from a phase 3 trial (Cancer and Leukemia Group B 80303)

Patients with advanced stage adenocarcinoma of the pancreas have a poor prognosis. The identification of prognostic and/or predictive biomarkers may help stratify patients so that therapy can be individualized. Serum samples from patients enrolled in the Cancer and Leukemia Group B 80303 phase 3 trial, "Randomized Study of Gemcitabine With Versus Without Bevacizumab in Patients With Locally Advanced or Metastatic Adenocarcinoma of the Pancreas" were used to discover novel biomarkers. For the discovery phase, 40 sera were selected based on length of survival and type of therapy, and subjected to liquid chromatography coupled to tandem mass spectrometry analysis (LC-MS-MS). The top features (proteins) were then further selected for validation by enzyme-linked immunosorbent assay (ELISA). Quantification by nano-LC-MS-MS resulted in 1452 peptides mapping to 156 proteins across all 40 samples, 92 of which had 2 or more peptides. After curation of the data, the authors selected 1 putative prognostic protein, alpha 1-antichymotrypsin (AACT), and 2 putative predictive proteins, histidine-rich glycoprotein (HRG) and complement factor H (CFH), for validation by ELISA. AACT was found to be negatively correlated with overall survival (τ = -0.30 [-0.38, -0.22]; P < .00001). There was no evidence for interaction with bevacizumab and HRG, but there was some evidence for a weak positive correlation of HRG with overall survival (τ = 0.11 [0.03, 0.19]; P < .01). CFH was found to be neither a predictive nor a prognostic factor for overall survival. AACT may be a useful prognostic marker in patients with advanced stage pancreatic carcinoma, although additional validation studies are needed.

Prediction of the Clinical Outcome in Invasive Candidiasis Patients Based on Molecular Fingerprints of Five Anti-Candida Antibodies in Serum

Better prognostic predictors for invasive candidiasis (IC) are needed to tailor and individualize therapeutic decision-making and minimize its high morbidity and mortality. We investigated whether molecular profiling of IgG-antibody response to the whole soluble Candida proteome could reveal a prognostic signature that may serve to devise a clinical-outcome prediction model for IC and contribute to known IC prognostic factors. By serological proteome analysis and data-mining procedures, serum 31-IgG antibody-reactivity patterns were examined in 45 IC patients randomly split into training and test sets. Within the training cohort, unsupervised two-way hierarchical clustering and principal-component analyses segregated IC patients into two antibody-reactivity subgroups with distinct prognoses that were unbiased by traditional IC prognostic factors and other patients-related variables. Supervised discriminant analysis with leave-one-out cross-validation identified a five-IgG antibody-reactivity signature as the most simplified and accurate IC clinical-outcome predictor, from which an IC prognosis score (ICPS) was derived. Its robustness was confirmed in the test set. Multivariate logistic-regression and receiver-operating-characteristic curve analyses demonstrated that the ICPS was able to accurately discriminate IC patients at high risk for death from those at low risk and outperformed conventional IC prognostic factors. Further validation of the five-IgG antibody-reactivity signature on a multiplexed immunoassay supported the serological proteome analysis results. The five IgG antibodies incorporated in the ICPS made biologic sense and were associated either with good-prognosis and protective patterns (those to Met6p, Hsp90p, and Pgk1p, putative Candida virulence factors and antiapoptotic mediators) or with poor-prognosis and risk patterns (those to Ssb1p and Gap1p/Tdh3p, potential Candida proapoptotic mediators). We conclude that the ICPS, with additional refinement in future larger prospective cohorts, could be applicable to reliably predict patient clinical-outcome for individualized therapy of IC. Our data further provide insights into molecular mechanisms that may influence clinical outcome in IC and uncover potential targets for vaccine design and immunotherapy against IC.

Identification of Potential Prognostic Biomarkers in Mantle Cell Lymphoma by Array-Based Quantitative Nuclease Protection Assay

AbstractAbstract 2007 Background:The clinical course of patients with mantle cell lymphoma (MCL) is highly variable and there is an urgent need for individual patient risk stratification to facilitate accurate interpretation of clinical trials and rational development of alternative treatments for those with high risk disease. Despite the identification of potential prognostic biomarkers through various screening studies including gene expression profiling, these findings have yet to be translated into a clinical application. Array-based quantitative nuclease protection assay (qNPA) can assess gene expression in formalin-fixed paraffin-embedded (FFPE) tissue in a simple, robust manner and may have potential to become a clinical laboratory test. The aim of this study was to determine the ability of qNPA to identify prognostic mRNA biomarkers in routinely processed FFPE diagnostic biopsies of MCL. Design:The expression of 42 genes with potential prognostic significance was analyzed using qNPA in a plate based, low density array format on FFPE tissue from 57 patients with MCL diagnosed between 1999 and 2010. Gene expression was normalized to two housekeeping genes, TBP and B2M. Results:56 (98%) of the cases were successfully analyzed with an average co-efficient of variation (CV) of gene expression between triplicate specimens for all genes in each case ranging from 7% to 16% and median CV of 11%. By univariate analysis with the Cox proportional hazard model, expression of the MYC, TNFRSF10B, CDKN2A, and CCND1 genes were significantly associated (p < 0.05) with progression free survival.Of relevance, MYC and TNFRSF10B were amongst 5 genes out of 33 screened by Hartmann et al. (J Clin Oncol 26:4966-72, 2008) that produced the best survival predictor in a previous qRT-PCR-based assay performed on both fresh-frozen and FFPE MCL tumor samples. In addition, homozygous deletions of the CDKN2A locus on 9p21 has been implicated as a key component in aggressive disease manifestation and is postulated to be a result of the loss of two key regulatory elements encoded at this locus, the cell cycle inhibitor INK4a and the p53 regulator ARF. Finally, expression of a more stable, truncated form of CCND1 lacking the 3'UTR, was first described by Wiestner et al. (Blood 109:4599-4606, 2007) and has been associated with shorter survival. We designed the CCND1 probe in the qNPA assay to target the distal 3'UTR in order to specifically measure non-truncated CCND1. By qNPA, 7 of the 57 MCL cases lacked CCND1 expression and the 3-year overall survival for these cases was 42% compared to 70% for positive cases. Conclusion:qNPA can reliably and reproducibly assess the expression of specific genes from FFPE tissue with relatively small CVs and identified four genes of possible prognostic significance in archived MCL specimens, suggesting this test may have potential to be developed into a clinical assay to guide individualized therapy in patients with MCL.Table 1Association between gene expression and progression free survival by Cox proportional hazard modelGeneHazard Ratio95% lower confidence limit95% upper confidence limitp-valueMYC2.171.353.51<0.01TNFRSF10B0.460.220.960.04CDKN2A0.650.431.000.05CCND10.720.520.980.04 Disclosures:No relevant conflicts of interest to declare.