Identification of Host-Immune Response Protein Candidates in the Sera of Human Oral Squamous Cell Carcinoma Patients

One of the most common cancers worldwide is oral squamous cell carcinoma (OSCC), which is associated with a significant death rate and has been linked to several risk factors. Notably, failure to detect these neoplasms at an early stage represents a fundamental barrier to improving the survival and quality of life of OSCC patients. In the present study, serum samples from OSCC patients (n = 25) and healthy controls (n = 25) were subjected to two-dimensional gel electrophoresis (2-DE) and silver staining in order to identify biomarkers that might allow early diagnosis. In this regard, 2-DE spots corresponding to various up- and down-regulated proteins were sequenced via high-resolution MALDI-TOF mass spectrometry and analyzed using the MASCOT database. We identified the following differentially expressed host-specific proteins within sera from OSCC patients: leucine-rich α2-glycoprotein (LRG), alpha-1-B-glycoprotein (ABG), clusterin (CLU), PRO2044, haptoglobin (HAP), complement C3c (C3), proapolipoprotein A1 (proapo-A1), and retinol-binding protein 4 precursor (RBP4). Moreover, five non-host factors were detected, including bacterial antigens from Acinetobacter lwoffii, Burkholderia multivorans, Myxococcus xanthus, Laribacter hongkongensis, and Streptococcus salivarius. Subsequently, we analyzed the immunogenicity of these proteins using pooled sera from OSCC patients. In this regard, five of these candidate biomarkers were found to be immunoreactive: CLU, HAP, C3, proapo-A1 and RBP4. Taken together, our immunoproteomics approach has identified various serum biomarkers that could facilitate the development of early diagnostic tools for OSCC.     

胰岛素抵抗在前列腺癌治疗前后动态变化的临床观察

目的:探讨胰岛素抵抗程度在前列腺癌治疗过程中动态变化及胰岛素抵抗程度与前列腺癌预后的关系。方法:选择前列腺癌惠者50名。平均年龄72岁。将前列腺癌患者根据治疗方法分为前列腺癌根治术组、手术去势组、药物去势组;根据有无复发转移分为复发转移组、无复发转移组。采用己糖激酶法测定空腹葡萄糖,放免法测定空腹胰岛素(Fasting serum insulin FINS)水平。运用HOMA模型中的胰岛素抵抗计算公式计算胰岛素抵抗指数(insulin resistance index IRI)。组间比较采用配对t检验。结果:前列腺癌患者在手术后的胰岛素抵抗指数明显下降,与手术前胰岛素抵抗指数存在统计学差异(P<0.05)。前列腺癌根治组的IRI值和手术去势组与药物去势组都存在统计学差异(P<0.05),但手术去势组和药物去势组两组之间不存在统计学差异(P>0.05)。前列腺癌治疗后无复发转移组的IRI值明显低于复发转移组,两组间IRI存在统计学差异(P<0.05)。结论:前列腺癌患者胰岛素抵抗程度与肿瘤治疗经过、效果有关。胰岛素抵抗指数有助于前列腺癌患者判断治疗效果,判断预后。     

Serum metallothionein in newly diagnosed patients with childhood solid tumours

Tumour markers are substances produced by malignant cells or by the organism as a response to cancer development. Determination of their levels can, therefore, be used to monitor the risk, presence and prognosis of a cancer disease or to monitor the therapeutic response or early detection of residual disease. Time-consuming imaging methods, examination of cerebrospinal fluid or tumour tissue and assays for hormones and tumour markers have been used for cancer diagnosis. However, no specific marker for diagnosis of childhood solid tumours has been discovered yet. In this study, metallothionein (MT) was evaluated as a prospective marker for such diseases. Serum metallothionein levels of patients with childhood solid tumours were determined using differential pulse voltammetry - Brdicka reaction. A more than 5-fold increase in the amount of metallothionein was found in sera of patients suffering from cancer disease, compared with those in sera of healthy donors. The average metallothionein level in the sera of healthy volunteers was 0.5 ± 0.2 μmol ? dm?3 and was significantly different (p<0.05, determined using the Schefe test) from the average MT level found in serum samples of patients suffering from childhood solid tumours (3.4 ± 0.8 μmol ? dm?3). Results found in this work indicate that the MT level in blood serum can be considered as a promising marker for diagnostics, prognosis and estimation of therapy efficiency of childhood tumours.     

Serum Amyloid A and Clusterin as Potential Predictive Biomarkers for Severe Hand,Foot and Mouth Disease by 2D-DIGE Proteomics Analysis

Hand, foot, and mouth disease (HFMD) affects more than one million children, is responsible for several hundred child deaths every year in China and is the cause of widespread concerns in society. Only a small fraction of HFMD cases will develop further into severe HFMD with neurologic complications. A timely and accurate diagnosis of severe HFMD is essential for assessing the risk of progression and planning the appropriate treatment. Human serum can reflect the physiological or pathological states, which is expected to be an excellent source of disease-specific biomarkers. In the present study, a comparative serological proteome analysis between severe HFMD patients and healthy controls was performed via a two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) strategy. Fifteen proteins were identified as differentially expressed in the sera of the severe HFMD patients compared with the controls. The identified proteins were classified into different groups according to their molecular functions, biological processes, protein classes and physiological pathways by bioinformatics analysis. The up-regulations of two identified proteins, serum amyloid A (SAA) and clusterin (CLU), were confirmed in the sera of the HFMD patients by ELISA assay. This study not only increases our background knowledge about and scientific insight into the mechanisms of HFMD, but also reveals novel potential biomarkers for the clinical diagnosis of severe HFMD.     

Identification of Autoantibodies against Transthyretin for the Screening and Diagnosis of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic, autoimmune, systemic and inflammatory rheumatic disease that leads to inflammation of the joints and surrounding tissues. Identification of novel protein(s) associated with severity of RA is a prerequisite for better understanding of pathogenesis of this disease that may also have potential to serve as novel biomarkers in the diagnosis of RA. Present study was undertaken to compare the amount of autoantigens and autoantibodies in the plasma of RA patients in comparison to healthy controls. Plasma samples were collected from the patients suffering from RA, Osteoarthritis (OA), Systemic lupus erythematosus (SLE) and healthy volunteers. The screening of plasma proteins were carried out using 2-dimensional gel electrophoresis followed by identification of differentially expressed protein by MALDI-TOF MS/MS. Among several differentially expressed proteins, transthyretin (TTR) has been identified as one of the protein that showed significantly up regulated expression in the plasma of RA patients. The results were further validated by Western blot analysis and ELISA. In comparison to OA synovium, an exclusive significantly high expression of TTR in RA has been validated through IHC, Western blotting and IEM studies. Most importantly, the increase in expression of TTR with the progression of severity of RA condition has been observed. The autoantibodies against TTR present in the RA plasma were identified using immunoprecipitation-Western methods. The significant production of autoantibodies was validated by ELISA and Western blot analysis using recombinant pure protein of TTR. Hence, these novel observations on increase in TTR expression with the increase in severity of RA conditions and significant production of autoantibodies against TTR clearly suggest that a systematic studies on the role TTR in the pathogenesis of RA is immediately required and TTR may be used as a serum diagnostic marker together with other biochemical parameters and clinical symptoms for RA screening and diagnosis.     

Search for potential markers for prostate cancer diagnosis, prognosis and treatment in clinical tissue specimens using amine-specific isobaric tagging (iTRAQ) with two-dimensional liquid chromatography and tandem mass spectrometry

This study aimed to identify candidate new diagnosis and prognosis markers and medicinal targets of prostate cancer (PCa), using state of the art proteomics. A total of 20 prostate tissue specimens from 10 patients with benign prostatic hyperplasia (BPH) and 10 with PCa (Tumour Node Metastasis [TNM] stage T1-T3) were analyzed by isobaric stable isotope labeling (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) approaches using a hybrid quadrupole time-of-flight system (QqTOF). The study resulted in the reproducible identification of 825 nonredundant gene products (p < or = 0.05) of which 30 exhibited up-regulation (> or =2-fold) and another 35 exhibited down-regulation (< or =0.5-fold) between the BPH and PCa specimens constituting a major contribution toward their global proteomic assessment. Selected findings were confirmed by immunohistochemical analysis of prostate tissue specimens. The proteins determined support existing knowledge and uncover novel and promising PCa biomarkers. The PCa proteome found can serve as a useful aid for the identification of improved diagnostic and prognostic markers and ultimately novel chemopreventive and therapeutic targets.     

Integrative analysis identifies potential DNA methylation biomarkers for pan-cancer diagnosis and prognosis

DNA methylation status is closely associated with diverse diseases, and is generally more stable than gene expression, thus abnormal DNA methylation could be important biomarkers for tumor diagnosis, treatment and prognosis. However, the signatures regarding DNA methylation changes for pan-cancer diagnosis and prognosis are less explored. Here we systematically analyzed the genome-wide DNA methylation patterns in diverse TCGA cancers with machine learning. We identified seven CpG sites that could effectively discriminate tumor samples from adjacent normal tissue samples for 12 main cancers of TCGA (1216 samples, AUC > 0.99). Those seven potential diagnostic biomarkers were further validated in the other 9 different TCGA cancers and 4 independent datasets (AUC > 0.92). Three out of the seven CpG sites were correlated with cell division, DNA replication and cell cycle. We also identified 12 CpG sites that can effectively distinguish 26 different cancers (7605 samples), and the result was repeatable in independent datasets as well as two disparate tumors with metastases (micro-average AUC > 0.89). Furthermore, a series of potential signatures that could significantly predict the prognosis of tumor patients for 7 different cancer were identified via survival analysis (p-value < 1e-4). Collectively, DNA methylation patterns vary greatly between tumor and adjacent normal tissues, as well as among different types of cancers. Our identified signatures may aid the decision of clinical diagnosis and prognosis for pan-cancer and the potential cancer-specific biomarkers could be used to predict the primary site of metastatic breast and prostate cancers.     

A glance at the emerging diagnostic biomarkers in the most prevalent genitourinary cancers

Genitourinary cancers comprise of a heterogenous group of cancers of which renal cell carcinoma, urothelial bladder carcinoma, and prostate adenocarcinoma are the most commonly encountered subtypes. A lot of research is ongoing using various strategies for exploration of novel biomarkers for genitourinary cancers. These biomarkers would not reduce the need for invasive diagnostic techniques but also could be used for early and accurate diagnosis to improve the clinical management required for the disease. Moreover, selecting the appropriate treatment regimen for the responsive patients based on these biomarkers would reduce the treatment toxicity as well as cost. Biomarkers identified using various advanced techniques like next generation sequencing and proteomics, which have been classified as immunological biomarkers, tissue-specific biomarkers and liquid biomarkers. Immunological biomarkers include markers of immunological pathways such as CTLA4, PD-1/PDl-1, tissue biomarkers include tissue specific molecules such as PSA antigen and liquid biomarkers include biomarkers detectable in urine, circulating cells etc.The purpose of this review is to provide a brief introduction to the most prevalent genitourinary malignancies, including bladder, kidney, and prostate cancers along with a major focus on the novel diagnostic biomarkers and the importance of targeting them prior to genitourinary cancers treatment. Understanding these biomarkers and their potential in diagnosis of genitourinary cancer would not help in early and accurate diagnosis as mentioned above but may also lead towards a personalized approach for better diagnosis, prognosis and specified treatment approach for an individual.     

Heat-shock protein 27: a potential biomarker for hepatocellular carcinoma identified by serum proteome analysis

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and ranks second in China. The prognosis of HCC remains dismal mainly because of its late diagnosis, especially in patients with coexisting chronic liver diseases. To identify serum biomarkers for HCC, sera from 20 healthy volunteers, 20 hepatitis B virus (HBV) infected patients and 20 HCC patients were selected for screening study and same number of sera into the same three groups were used for validation study. A strategy including sonication, albumin and immunoglobulin G (IgG) depletion and desalting was optimized for screening differentially expressed proteins of low abundance in serum. By 2-DE image analysis and MALDI-TOF-MS/MS identification, eight proteins including heat-shock protein 27 (HSP27), alpha-fetoprotein (AFP), alpha-1 antitrypsin, clusterin, caeruloplasmin, haptoglobin alpha2 chain, tranferrin and transthyretin were found significantly changed among the healthy, HBV and HCC groups. Further validation study by Western blot showed the detection of HSP27 in 90% HCC sera and two HBV sera, but in none of normal sera. Thus, 2-DE based serum proteome analysis can be useful in the screening of serum biomarkers for HCC and HSP27 could aid in the diagnosis of HCC though further validation is needed.     

Clinical cancer proteomics: promises and pitfalls

Proteome analysis promises to be valuable for the identification of tissue and serum biomarkers associated with human malignancies. In addition, proteome technologies offer the opportunity to analyze protein expression profiles and to analyze the activity of signaling pathways. Many published proteomic studies of human tumor tissue are associated with weaknesses in tumor representativity, sample contamination by nontumor cells and serum proteins. Studies often include a moderate number of tumors which may not be representative of clinical materials. It is therefore very important that biomarkers identified by proteomics are validated in representative tumor materials by other techniques, such as immunohistochemistry. Proteome technologies can be used to identify disease markers in human serum. Tumor derived proteins are present at nanomolar to picomolar concentrations in cancer patient sera, 10(6)-10(9)-fold lower than albumin, and will give rise to correspondingly smaller spots/peaks in protein separations. This leads to the need to prefractionate serum samples before analysis. Despite various pitfalls, proteomic analysis is a promising approach to the identification of biomarkers, and for generation of protein expression profiles that can be analyzed by artificial learning methods for improved diagnosis of human malignancy. Recent advances in the field of proteomic analysis of human tumors are summarized in the present review.     

Identification and confirmation of increased fibrinopeptide a serum protein levels in gastric cancer sera by magnet bead assisted MALDI-TOF mass spectrometry

Gastric cancer is the second most common malignancy and prognosis remains dismal. The reasons for the poor prognosis are the lack of sensitive serum markers for early detection and screening of high-risk individuals as well as the limited treatment options in advanced cancer stages. Using MALDI-TOF mass spectrometry after prefractionation of sera with magnet hydrophobic C8 coated beads sera from 14 patients with gastric cancer and 14 healthy controls mass spectra were generated. A peptide fragment was found to be highly elevated in cancer sera and was identified as fibrinopeptide A. To confirm proteome analysis of gastric cancer sera, we then screened a larger series of patients with gastric cancer (n = 99), high-risk individuals (n = 13) and normal controls (n = 111) for fibrinopeptide A serum levels. Interestingly, the mean logarithmic concentrations of serum fibrinopeptide A levels were significantly higher in cancer patients (mean 3.636 +/- 0.3738; p < 0.0001) and high-risk individuals (mean 3.569 +/- 0.4722; p < 0.05) compared to normal controls (mean 3.303 +/- 0.4012). In contrast, we observed no association of fibrinopeptide A levels with tumor stage, tumor location, presence of regional or distant metastasis, and Lauren type of gastric cancer. In conclusion, MALDI-TOF mass spectrometry of prefractionated gastric cancer sera allows the identification of potential biomarkers that may lead to the development of serum based tests for screening of high-risk individuals.     

Serum differential protein identification of Xinjiang Kazakh esophageal cancer patients based on the two-dimensional liquid-phase chromatography and LTQ MS

The aim of this study was to investigate the impact of chemo-radiotherapy on serum protein expression of the esophageal cancer patients and discover potential biomarkers by detecting serum proteins mass spectrometry of the healthy Kazakh people in Xinjiang as well as the patients before and after their chemo-radiotherapy. In order to separate and compare the three serum samples (the healthy group’s, the patients’ before and after chemo-radiotherapy) with two-dimensional protein liquid chromatography system (Proteome LabTM PF-2D), then detect the differential protein spots with linear trap quadruple mass spectrometer (LTQ MS/MS). (1) The Kazakh esophageal cancer patients got 21 expressed protein spots peaks with significant difference after chemo-radiotherapy compared with before; before the treatment there were 10 different expressed protein spots compared with the healthy group, and after it there were four peaks in the expression of protein spots compared with the healthy group. (2) After LTQ mass spectrometric detection, 22 proteins were up-regulated in serum samples of the healthy group, 22 were up-regulated of the patients before medical treatment and 5 were up-regulated after chemo-radiotherapy. (3) 8 proteins including APOA1 can be served as serum markers in Kazakh esophageal cancer diagnosis, and proteins like CLU can be served as serum markers in judging the resistance and sensitivity towards chemo-radiotherapy. (4) The abnormal expressions of APOC2, APOC3, Antithrombin-III in esophageal cancer were discovered for the first time. Specific protein spots related to Xinjiang Kazakh esophageal cancer diagnosis and chemo-radiotherapy can be identified in the serum, which will probably become a maker in Kazakh esophageal cancer diagnosis and therapeutic evaluation.     

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).     

Proteomics in prostate cancer biomarker discovery

Despite advances in molecular medicine, genomics, proteomics and translational research, prostate cancer remains the second most common cause of cancer-related mortality for men in the Western world. Clearly, early detection, targeted treatment and post-treatment monitoring are vital tools to combat this disease. Tumor markers can be useful for diagnosis and early detection of cancer, assessment of prognosis, prediction of therapeutic effect and treatment monitoring. Such tumor markers include prostate-specific antigen (prostate), cancer antigen (CA)15.3 (breast), CA125 (ovarian), CA19.9 (gastrointestinal) and serum α-fetoprotein (testicular cancer). However, all of these biomarkers lack sensitivity and specificity and, therefore, there is a large drive towards proteomic biomarker discovery. Current research efforts are directed towards discovering biosignatures from biological samples using novel proteomic technologies that provide high-throughput, in-depth analysis and quantification of the proteome. Several of these studies have revealed promising biomarkers for use in diagnosis, assessment of prognosis, and targeting treatment of prostate cancer. This review focuses on prostate cancer proteomic biomarker discovery and its future potential.     

Banking of biological fluids for studies of disease-associated protein biomarkers

With the increasing demand of providing personalized medicine the need for biobanking of biological material from individual patients has increased. Such samples are essential for molecular research aimed at characterizing diseases at several levels ranging from epidemiology and diagnostic and prognostic classification to prediction of response to therapy. Clinically validated biomarkers may provide information to be used for diagnosis, screening, evaluation of risk/predisposition, assessment of prognosis, monitoring (recurrence of disease), and prediction of response to treatment and as a surrogate response marker. Many types of biological fluids or tissues can be collected and stored in biorepositories. Samples of blood can be further processed into plasma and serum, and tissue pieces can be either frozen or fixed in formalin and then embedded into paraffin. The present review focuses on biological fluids, especially serum and plasma, intended for study of protein biomarkers. In biomarker studies the process from the decision to take a sample from an individual to the moment the sample is safely placed in the biobank consists of several phases including collection of samples, transport of the samples, and handling and storage of samples. Critical points in each step important for high quality biomarker studies are described in this review. Failure to develop and adhere to robust standardized protocols may have significant consequences as the quality of the material stored in the biobank as well as conclusions and clinical recommendations based on analysis of such material may be severely affected.     

Soluble programmed death-1 (sPD-1) and programmed death ligand 1 (sPD-L1) as potential biomarkers for the diagnosis and prognosis of glioma patients

BackgroundThis study aimed at investigating the feasibility of testing for soluble programmed death-1 (sPD-1) and soluble programmed death ligand 1 (sPD-L1) in serum samples of glioma patients and to evaluate the diagnostic and prognostic value of these two soluble molecules.MethodsSerum samples collected from 70 glioma patients before surgery were designated as the pre-operative (Pre) group, samples obtained from 90 post-surgery glioblastoma patients were designated as the Post group, and samples from 20 healthy volunteers were used as controls. Peripheral blood sPD-1 and sPD-L1 levels were detected by using ELISA kits and compared among the groups. The associations of these soluble molecule levels with clinicopathological variables and tumour progression were investigated.ResultsAmong the three groups, the Pre group had the highest sPD-1 levels, whereas the median sPD-L1 level was significantly lower in the Post group than in the other two groups. The area under the curve (AUC) of sPD-1 (0.762) for diagnosis was similar to that of sPD-L1 (0.718). Higher serum levels of sPD-1 and sPD-L1 were present in samples of patients with more advanced brain tumours. Kaplan-Meier analysis showed that higher serum levels of sPD-1 (>11.14 pg/mL) and sPD-L1 (>63.03 pg/mL) might predict shorter progression-free survival times of glioma patients.ConclusionsThis study showed that sPD-1 and sPD-L1 might be promising predictive biomarkers for the diagnosis and prognosis of glioma patients.     

Proteomics of human prostate cancer biospecimens: the global,systems-wide perspective for Protein markers with potential clinical utility

Despite intense global efforts, no new clinical and/or viable biomarkers have been established to overcome the limitation of the prostate specific antigen in the early diagnosis and prognosis of prostate cancer (PCa). The current proteomic approaches to PCa biomarker discovery, each have distinct advantages and disadvantages, yet when combined hold real promise in the coming years. One key approach to this effort is the development of non-targeted, depletion-free and quantitative liquid chromatography–ultra high resolution tandem mass spectrometry (LC–MS) pipelines for the systems-wide interrogation of the diverse proteomes encompassed in whole tissue and blood serum or plasma. Derived quantitative proteomes can be decoded for their biomedical relevance with advanced bioinformatics and bibliographic mining to yield promising ‘molecular portraits’ that can gauge prostatic disease at the serological level. Their functional annotation, although potentially useful, is beyond our current level of biological understanding and should not be requisite for their effective use in the clinical monitoring of prostatic disease.