Progress and prospects for the discovery of biomarkers for gastric cancer: a focus on proteomics

Introduction: Patient outcomes from gastric cancer vary due to the complexity of stomach carcinogenesis. Recent research using proteomic technologies has targeted components of all of these systems in order to develop biomarkers to aid the early diagnosis of gastric cancer and to assist in prognostic stratification.

Areas covered: This review is comprised of evidence obtained from literature searches from PubMed. It covers the evidence of diagnostic, prognostic, and predictive biomarkers for gastric cancer using proteomic technologies, and provides up-to-date references.

Expert commentary: The proteomic technologies have not only enabled the screening of a large number of samples, but also enabled the identification of diagnostic, prognostic and predictive biomarkers for gastric cancer. While major challenges still remain, to date, proteomic studies in gastric cancer have provided a wealth of information in revealing proteome alterations associated with the disease.     

Searching for consistently reported up- and down-regulated biomarkers in colorectal cancer: a systematic review of proteomic studies

The cumulative lifetime risk for the development of colorectal cancer in the general population is 6 %. In many cases, early detection by fecal occult blood test is limited regarding sensitivity. Therefore, there is an urgent need for improved diagnostic tests in colorectal cancer. The recent development of high-throughput molecular analytic techniques should allow the rapid evaluation of new diagnostic markers. However, researchers are faced with an overwhelming number of potential markers form numerous colorectal cancer protein expression profiling studies. To address the challenge, we have carried out a comprehensive systematic review of colorectal cancer biomarkers from 13 published studies that compared the protein expression profiles of colorectal cancer and normal tissues. A protein ranking system that considers the number of comparisons in agreement, total sample sizes, average fold-change and direction of differential expression was devised. We observed that some proteins were consistently reported by multiple studies as differentially expressed with a statistically significant frequency (P < 0.05) in cancer versus normal tissues comparison. Our systematic review method identified proteins that were consistently reported as differentially expressed. A review of the top four candidates revealed proteins described previously as having diagnostic value as well as novel candidate biomarkers. These candidates should help to develop a panel of biomarkers with sufficient sensitivity and specificity for the diagnosis of colorectal cancer in a clinical setting.     

Identification and validation of novel candidate protein biomarkers for the detection of human gastric cancer

The timely detection of gastric cancer will contribute significantly towards effective treatment and is aided by the availability and reliability of appropriate biomarkers. A combination of several biomarkers can improve the sensitivity and specificity of cancer detection and this work reports results from a panel of 4 proteins. By combining a validated preclinical mouse model with a proteomic approach we have recently discovered novel biomarkers for the detection of gastric cancer. Here, we investigate the specificity of four of those biomarkers (afamin, clusterin, VDBP and haptoglobin) for the detection of gastric cancer using two independent methods of validation: ELISA, and a non antibody based method: Multiple Reaction Monitoring with High Resolution Mass Spectrometry (MRM-HR). All four biomarkers reliably differentiated GC from benign patient serum, and also in a small cohort of 11 early stage cases. We also present a novel isoform specific biomarker alpha-1-antitrypsin (A1AT) that was identified using a mouse model for gastric cancer. This isoform is distinct in charge and mobility in a pH gradient and was validated using human samples by isoelectric focussing and Western-blot (IEF-WB). This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Characterization of the human gastric fluid proteome reveals distinct pH-dependent protein profiles: implications for biomarker studies

Gastric fluid is a source of gastric cancer biomarkers. However, very little is known about the normal gastric fluid proteome and its biological variations. In this study, we performed a comprehensive analysis of the human gastric fluid proteome using samples obtained from individuals with benign gastric conditions. Gastric fluid proteins were prefractionated using ultracentrifuge filters (3 kDa cutoff) and analyzed by two-dimensional gel electrophoresis (2-DE) and multidimensional LC-MS/MS. Our 2-DE analysis of 170 gastric fluid samples revealed distinct protein profiles for acidic and neutral samples, highlighting pH effects on protein composition. By 2D LC-MS/MS analysis of pooled samples, we identified 284 and 347 proteins in acidic and neutral samples respectively (FDR ≤1%), of which 265 proteins (72.4%) overlapped. However, unlike neutral samples, most proteins in acidic samples were identified from peptides in the filtrate (i.e., <3 kDa). Consistent with this finding, immunoblot analysis of six potential gastric cancer biomarkers rarely detected full-length proteins in acidic samples. These findings have important implications for biomarker studies because a majority of gastric cancer patients have neutral gastric fluid compared to noncancer controls. Consequently, sample stratification, choice of proteomic approaches, and validation strategy can profoundly affect the interpretation of biomarker findings. These observations should help to refine gastric fluid biomarker studies.     

Diverse proteomic alterations in gastric adenocarcinoma

Gastric adenocarcinoma is one of the most common cancers in Asian countries including China. Although its incidence rates in the West are lower than that in Asia, gastric cancer is still a major health problem worldwide, being second only to lung cancers in the number of deaths it causes. Helicobacter pylori infection has been identified as the major pathogen, but the detailed pathogenesis of gastric carcinoma remains elusive. Due to the lack of suitable and specific biomarkers for early detection, most cases of the disease are diagnosed at late stages and the survival rate is low. In this study, we used a proteomic approach to globally analyze the protein profiles of paired surgical specimens of primary gastric adenocarcinoma and nontumor mucosa aiming at identifying specific disease-associated proteins as potential clinical biomarkers and for carcinogenetic study. Compared to nontumor tissues, multiple protein alterations were found in tumor tissues. Some of these alterations involve variations in the expression of cytoskeleton proteins, including an increase in cytokeratin 8 and tropomyosin isoform and a decrease in cytokeratin 20. Co-up-regulations of heat-shock proteins and glycolytic enzymes were observed in tumor tissues, indicating self-protective efforts of cells and the growing energy requirement during malignant transformation. Diverse regulations also occurred with proteins involved in cell proliferation and differentiation, such as GMP reductase 2 and creatine kinase B, and proteins bearing potential tumor suppressor activities, including prohibitin and selenium binding protein 1. More interestingly, a human stomach-specific protein, 18 kDa antrum mucosa protein, was found to be dramatically under-expressed in cancer tissues, implicating a possible special pathological role for this protein in gastric carcinogenesis. Further comprehensive evaluation by globally considering the altered factors may result in the discovery of a biomarker index for effective assessment of the disease and may provide in-depth information for better understanding the pathogenesis of gastric cancer.     

Proteomic expression analysis of human colorectal cancer: Identification of soluble overexpressed proteins

Colorectal cancer is one of the most common malignancies in developed countries. Scarce clinical signs at the early stages of the disease and the lack of fast and sensitive diagnostic techniques based on the detection of tumor specific protein markers contribute greatly to the high mortality rate. The search for such markers is significantly complicated by the high levels of major structural and cytoskeletal proteins in normal and tumor tissues. Extraction with 0.2 M NaCl in the presence of the nonionic detergent NP-40 was performed to enrich the soluble protein fraction. This modification resulted in a considerably increased sensitivity of detection of minor proteins that may enter the circulation during carcinogenesis. The soluble protein profiles of the paired colon adenocarcinoma and normal tissue specimens were compared using 2D gel electrophoresis, which enabled the detection of 10 proteins whose levels in tumors were elevated at least 10-fold as compared to normal tissue. The proteins were identified by MALDI-TOF mass spectrometry, and two new protein markers of colon cancer, TAF9 and CISH, were discovered. Low levels of CISH synthesis in most normal human tissues and tumors other than colorectal cancer make it a prospective candidate diagnostic marker for this type of cancer.     

Protein arrays as tools for serum autoantibody marker discovery in cancer

Protein array technology has begun to play a significant role in the study of protein–protein interactions and in the identification of antigenic targets of serum autoantibodies in a variety of autoimmune disorders. More recently, this technology has been applied to the identification of autoantibody signatures in cancer.The identification of tumour-associated antigens (TAAs) recognised by the patient's immune response represents an exciting approach to identify novel diagnostic cancer biomarkers and may contribute towards a better understanding of the molecular mechanisms involved. Circulating autoantibodies have not only been used to identify TAAs as diagnostic/prognostic markers and potential therapeutic targets, they also represent excellent biomarkers for the early detection of tumours and potential markers for monitoring the efficacy of treatment. Protein array technology offers the ability to screen the humoral immune response in cancer against thousands of proteins in a high throughput technique, thus readily identifying new panels of TAAs. Such an approach should not only aid in improved diagnostics, but has already contributed to the identification of complex autoantibody signatures that may represent disease subgroups, early diagnostics and facilitated the analysis of vaccine trials.     

Serum-based protein biomarkers for detection of lung cancer

Lung cancer is one of the most common cancers in terms of both incidence and mortality.The major reasons for the increasing number of deaths from lung cancer are late detection and lack of effective therapies. To improve our understanding of lung cancer biology, there is urgent need for blood-based, non-invasive molecular tests to assist in its detection in a cost-effective manner at an early stage when curative interventions are still possible. Recent advances in proteomic technology have provided extensive, high throughput analytical tools for identification, characterization and functional studies of proteomes. Changes in protein expression patterns in response to stimuli can serve as indicators or biomarkers of biological and pathological processes as well as physiological and pharmacological responses to drug treatment, thus aiding in early diagnosis and prognosis of disease. However, only a few biomarkers have been approved by the FDA to date for screening and diagnostic purposes. This review provides a brief overview of currently available proteomic techniques, their applications and limitations and the current state of knowledge about important serum biomarkers in lung cancer and their potential value as prognostic and diagnostic tools.     

Bladder cancer associated glycoprotein signatures revealed by urinary proteomic profiling

Current methods in the noninvasive detection and surveillance of bladder cancer via urine analysis include voided urine cytology (VUC) and some diagnostic urinary protein biomarkers; however, due to the poor sensitivity of VUC and high false-positive rates of currently available protein assays, detection of bladder cancer via urinalysis remains a challenge. In the study presented here, a rapid, high-sensitivity technique was developed to profile the N-linked glycoprotein component in naturally micturated human urine specimens. Concanavalin A (Con A) affinity chromatography coupled to nanoflow liquid chromatography was utilized to separate the complex peptide mixture prior to a linear ion trap MS analysis. Of 186 proteins identified with high confidence by multiple analyses, 40% were secreted proteins, 18% membrane proteins, and 14% extracellular proteins. In this study, the presence of several proteins appeared to be associated with the presence of bladder cancer, including alpha-1B-glycoprotein that was detected in all tumor-bearing patient samples but in none of the samples obtained from non-tumor-bearing individuals. The combination of Con A affinity chromatography and nano-LC/MS/MS provides an initial investigation of N-glycoproteins in complex biological samples and facilitates the identification of potential biomarkers of bladder cancer in noninvasively obtained human urine.     

Upregulation of plasma C9 protein in gastric cancer patients

Gastric cancer is one of the leading causes of cancer‐related deaths worldwide. Current biomarkers used in the clinic do not have sufficient sensitivity for gastric cancer detection. To discover new and better biomarkers, protein profiling on plasma samples from 25 normal, 15 early‐stage and 21 late‐stage cancer was performed using an iTRAQ‐LC‐MS/MS approach. The level of C9 protein was found to be significantly higher in gastric cancer compared with normal subjects. Immunoblotting data revealed a congruent trend with iTRAQ results. The discriminatory power of C9 between normal and cancer states was not due to inter‐patient variations and was independent from gastritis and Helicobacter pylori status of the patients. C9 overexpression could also be detected in a panel of gastric cancer cell lines and their conditioned media compared with normal cells, implying that higher C9 levels in plasma of cancer patients could be attributed to the presence of gastric tumor. A subsequent blind test study on a total of 119 plasma samples showed that the sensitivity of C9 could be as high as 90% at a specificity of 74%. Hence, C9 is a potentially useful biomarker for gastric cancer detection.     

Divide and conquer: subproteomic approaches toward gastric cancer biomarker and drug target discovery

The discovery of biomarkers for early detection and treatment for gastric cancer are two important gaps that proteomics have the potential to fill. Advancements in mass spectrometry, sample preparation and separation strategies are crucial to proteomics-based discoveries and subsequent translations from bench to bedside. A great number of studies exploiting various subproteomic approaches have emerged for higher-resolution analysis (compared with shotgun proteomics) that permit interrogation of different post-translational and subcellular compartmentalized forms of the same proteins as determinants of disease phenotypes. This is a unique and key strength of proteomics over genomics. In this review, the salient features, competitive edges and pitfalls of various subproteomic approaches are discussed. We also highlight valuable insights from several subproteomic studies that have increased our understanding of the molecular etiology of gastric cancer and the findings that led to the discovery of potential biomarkers/drug targets that were otherwise not revealed by conventional shotgun expression proteomics.     

Clinical proteomics of breast cancer

Despite the lifetimes that increased in breast cancers due to the the early screening programs and new therapeutic strategies, many cases still are being lost due to the metastatic relapses. For this reason, new approaches such as the proteomic techniques have currently become the prime objectives of breast cancer researches. Various omic-based techniques have been applied with increasing success to the molecular characterisation of breast tumours, which have resulted in a more detailed classification scheme and have produced clinical diagnostic tests that have been applied to both the prognosis and the prediction of outcome to the treatment. Implementation of the proteomics-based techniques is also seen as crucial if we are to develop a systems biology approach in the discovery of biomarkers of the early diagnosis, prognosis and prediction of the outcome of the breast cancer therapies. In this review, we discuss the studies that have been conducted thus far, for the discovery of diagnostic, prognostic and predictive biomarkers, and evaluate the potential of the discriminating proteins identified in this research for clinical use as breast cancer biomarkers.     

Identification of biomarkers for colorectal cancer through proteomics-based approaches

The early detection of colorectal cancer is one of the great challenges in the battle against this disease. However, owing to its heterogeneous character, single markers are not likely to provide sufficient diagnostic power to be used in colorectal cancer population screens. This review provides an overview of recent studies aimed at the discovery of new diagnostic protein markers through proteomics-based approaches. It indicates that studies that start with the proteomic analysis of tumor tissue or tumor cell lines (near the source) have a high potential to yield novel and colorectal cancer-specific biomarkers. In the next step, the diagnostic accuracy of these candidate markers can be assessed by a targeted ELISA assay using serum from colorectal cancer patients and healthy controls. Instead, direct proteomic analysis of serum yields predominantly secondary markers composed of fragments of abundant serum proteins that may be associated with tumor-associated protease activity, and alternatively, immunoproteomic analysis of the serum antibody repertoire provides a valuable tool to identify the molecular imprint of colorectal cancer-associated antigens directly from patient serum samples. The latter approach also allows a relatively easy translation into targeted assays. Eventually, multimarker assays should be developed to reach a diagnostic accuracy that meets the stringent criteria for colorectal cancer screening at the population level.     

Global and targeted quantitative proteomics for biomarker discovery

The extraordinary developments made in proteomic technologies in the past decade have enabled investigators to consider designing studies to search for diagnostic and therapeutic biomarkers by scanning complex proteome samples using unbiased methods. The major technology driving these studies is mass spectrometry (MS). The basic premises of most biomarker discovery studies is to use the high data-gathering capabilities of MS to compare biological samples obtained from healthy and disease-afflicted patients and identify proteins that are differentially abundant between the two specimen. To meet the need to compare the abundance of proteins in different samples, a number of quantitative approaches have been developed. In this article, many of these will be described with an emphasis on their advantageous and disadvantageous for the discovery of clinically useful biomarkers.     

Tissue proteomics for cancer biomarker development: laser microdissection and 2D-DIGE

Novel cancer biomarkers are required to achieve early diagnosis and optimized therapy for individual patients. Cancer is a disease of the genome, and tumor tissues are a rich source of cancer biomarkers as they contain the functional translation of the genome, namely the proteome. Investigation of the tumor tissue proteome allows the identification of proteomic signatures corresponding to clinico-pathological parameters, and individual proteins in such signatures will be good biomarker candidates. Tumor tissues are also a rich source for plasma biomarkers, because proteins released from tumor tissues may be more cancer specific than those from non-tumor cells. Two-dimensional difference gel electrophoresis (2D-DIGE) with novel ultra high sensitive fluorescent dyes (CyDye DIGE Fluor satulation dye) enables the efficient protein expression profiling of laser-microdissected tissue samples. The combined use of laser microdissection allows accurate proteomic profiling of specific cells in tumor tissues. To develop clinical applications using the identified biomarkers, collaboration between research scientists, clinicians and diagnostic companies is essential, particularly in the early phases of the biomarker development projects. The proteomics modalities currently available have the potential to lead to the development of clinical applications, and channeling the wealth of produced information towards concrete and specific clinical purposes is urgent.     

早期胃癌患者外周血清差异蛋白表达谱的建立及其生物学分析

目的:分析早期胃癌患者外周血中低丰度蛋白的表达差异,以筛选诊断早期胃癌血清多肽或蛋白标志物。方法:应用高通量的AAH-BLG-1000蛋白芯片分别检测3例早期胃癌患者和3例正常对照成人的外周血清,建立早期胃癌患者外周血清差异蛋白表达谱,分析其相关生物学信息,以筛选早期胃癌的血清肿瘤标志物。结果:与正常对照组比较,早期胃癌患者外周血清中有10种蛋白表达显著上调(P0.05),52种蛋白表达显著下调(P0.05)。生物信息学分析发现差异蛋白质集中于血管生成、信号调控,免疫调节、酶联受体蛋白信号,细胞凋亡等生物进程,而差异蛋白中的VEGI、CD40L、SMAD7、PLUNC、NTN、LTβR和HEVM7个差异蛋白的特征性改变有望成为早期胃癌血清学的肿瘤标志物。结论:应用蛋白芯片技术所得的早期胃癌患者血清中的差异表达蛋白有望成为诊断早期胃癌的血清肿瘤标志物。     

Sieving through the cancer secretome

Cancer is among the most prevalent and serious health problems worldwide. Therefore, there is an urgent need for novel cancer biomarkers with high sensitivity and specificity for early detection and management of the disease. The cancer secretome, encompassing all the proteins that are secreted by cancer cells, is a promising source of biomarkers as the secreted proteins are most likely to enter the blood circulation. Moreover, since secreted proteins are responsible for signaling and communication with the tumor microenvironment, studying the cancer secretome would further the understanding of cancer biology. Latest developments in proteomics technologies have significantly advanced the study of the cancer secretome. In this review, we will present an overview of the secretome sample preparation process and summarize the data from recent secretome studies of six common cancers with high mortality (breast, colorectal, gastric, liver, lung and prostate cancers). In particular, we will focus on the various platforms that were employed and discuss the clinical applicability of the key findings in these studies. This article is part of a Special Issue entitled: An Updated Secretome.     

Proteomics in Parkinson’s disease: current trends,translational snags and future possibilities

Proteomic technologies are widely used to understand the molecular mechanism of Parkinson’s disease (PD) and to develop biomarkers for its early diagnosis. The differential expression patterns of brain, cerebrospinal fluid and blood proteins of patients or chemically induced animal models are used to identify protein fingerprints for developing diagnostic and therapeutic strategies for PD. A number of differentially expressed proteins associated with energy metabolism, oxidative stress, signal transduction, electron transport and detoxification pathways are identified using proteomic strategies. Proteomics immensely contributed to the detection of qualitative and quantitative changes of expressed proteins and their post-translational modifications. An update on proteomics-driven research for developing early biomarkers and understanding the molecular aspects of PD, along with their translational snags, challenges and future possibilities, are discussed in this review.