Complete solubilization of formalin-fixed,paraffin-embedded tissue may improve proteomic studies

Tissue-based proteomic approaches (tissue proteomics) are essential for discovering and evaluating biomarkers for personalized medicine. In any proteomics study, the most critical issue is sample extraction and preparation. This problem is especially difficult when recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, improving and standardizing protein extraction from FFPE tissue is a critical need because of the millions of archival FFPE tissues available in tissue banks worldwide. Recent progress in the application of heat-induced antigen retrieval principles for protein extraction from FFPE tissue has resulted in a number of published FFPE tissue proteomics studies. However, there is currently no consensus on the optimal protocol for protein extraction from FFPE tissue or accepted standards for quantitative evaluation of the extracts. Standardization is critical to ensure the accurate evaluation of FFPE protein extracts by proteomic methods such as reverse phase protein arrays, which is now in clinical use. In our view, complete solubilization of FFPE tissue samples is the best way to achieve the goal of standardizing the recovery of proteins from FFPE tissues. However, further studies are recommended to develop standardized protein extraction methods to ensure quantitative and qualitative reproducibility in the recovery of proteins from FFPE tissues.     

A well-based reverse-phase protein array applicable to extracts from formalin-fixed paraffin-embedded tissue

Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue offers significant diagnostic utility but is complicated due to the high level of covalently crosslinked proteins arising from formalin fixation. To address these challenges, we developed a reliable protein extraction method for FFPE tissue, based on heat-induced antigen retrieval within a pressure cooker. The protein extraction yield from archival FFPE tissue section is approximately 90% of that recovered from frozen tissue. This method demonstrates preservation of immunoreactivity and recovery of full-length proteins by Western blotting. Additionally, we developed a well-based RP protein array platform utilizing an electrochemiluminescence detection system. Protein samples derived from FFPE tissue by means of laser capture dissection, with as few as 500 shots demonstrate measurable signal differences for different proteins. The lysates coated to the array plate, remain stable over 1?month at room temperature. Theses data suggest that this new protein-profiling platform coupled with the protein extraction method can be used for molecular profiling analysis in FFPE tissue, and contribute to the validation and development of biomarkers in clinical studies.     

Impact of pre-analytical factors on the proteomic analysis of formalin-fixed paraffin-embedded tissue

Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a tremendous potential resource for biomarker discovery, with large numbers of samples in hospital pathology departments and links to clinical information. However, the cross-linking of proteins and nucleic acids by formalin fixation has hampered analysis and proteomic studies have been restricted to using frozen tissue, which is more limited in availability as it needs to be collected specifically for research. This means that rare disease subtypes cannot be studied easily. Recently, improved extraction techniques have enabled analysis of FFPE tissue by a number of proteomic techniques. As with all clinical samples, pre-analytical factors are likely to impact on the results obtained, although overlooked in many studies. The aim of this review is to discuss the various pre-analytical factors, which include warm and cold ischaemic time, size of sample, fixation duration and temperature, tissue processing conditions, length of storage of archival tissue and storage conditions, and to review the studies that have considered these factors in more detail. In those areas where investigations are few or non-existent, illustrative examples of the possible importance of specific factors have been drawn from studies using frozen tissue or from immunohistochemical studies of FFPE tissue.     

Semi-automated image processing system for micro- to macro-scale analysis of immunohistopathology: application to ischemic brain tissue

Immunochemical staining techniques are commonly used to assess neuronal, astrocytic and microglial alterations in experimental neuroscience research, and in particular, are applied to tissues from animals subjected to ischemic stroke. Immunoreactivity of brain sections can be measured from digitized immunohistology slides so that quantitative assessment can be carried out by computer-assisted analysis. Conventional methods of analyzing immunohistology are based on image classification techniques applied to a specific anatomic location at high magnification. Such micro-scale localized image analysis limits one for further correlative studies with other imaging modalities on whole brain sections, which are of particular interest in experimental stroke research. This report presents a semi-automated image analysis method that performs convolution-based image classification on micro-scale images, extracts numerical data representing positive immunoreactivity from the processed micro-scale images and creates a corresponding quantitative macro-scale image. The present method utilizes several image-processing techniques to cope with variances in intensity distribution, as well as artifacts caused by light scattering or heterogeneity of antigen expression, which are commonly encountered in immunohistology. Micro-scale images are composed by a tiling function in a mosaic manner. Image classification is accomplished by the K-means clustering method at the relatively low-magnification micro-scale level in order to increase computation efficiency. The quantitative macro-scale image is suitable for correlative analysis with other imaging modalities. This method was applied to different immunostaining antibodies, such as endothelial barrier antigen (EBA), lectin, and glial fibrillary acidic protein (GFAP), on histology slides from animals subjected to middle cerebral artery occlusion by the intraluminal suture method. Reliability tests show that the results obtained from immunostained images at high magnification and relatively low magnification are virtually the same.     

Quantitative proteomic analysis of a paired human liver healthy versus carcinoma cell lines with the same genetic background to identify potential hepatocellular carcinoma markers

To comprehensively measure global changes in protein expression associated with human hepatocellular carcinoma (HCC), comparative proteomic analysis of two cell lines derived from the healthy and carcinoma tissue of a same donor respectively was conducted using quantitative amino acid-coded mass tagging /stable isotope labeling with amino acids in cell culture-based LC-MS/MS approach. Among a total of 501 proteins precisely quantified, the expressions of 128 proteins were significantly altered including 70 proteins up-regulated and 58 down-regulated in HCC cells. According to their previously characterized functions, the differentially expressed proteins were found associated with nine functional categories including glycolysis, stress response, cell communication, cell cycle, apoptosis/death, etc. For example, multiple enzymes involving glycolysis pathway were found differentially regulated in HCC cells, illustrating the critical participation of glycolysis in the HCC transformation. The accuracy of certain differentially expressed proteins identified through the amino acid-coded mass tagging-based quantification was validated in the paired cell lines, and later their pathological correlations were examined in multiple clinical pairs of normal versus tumor tissues from HCC specimen by using a variety of biological approaches including Western blotting and in situ immunoassays. These consistencies suggested that multiple proteins such as HSP27, annexin V, glyceraldehyde-3-phosphate dehydrogenase, nucleolin and elongation factor Tu could be the biomarkers candidates for diagnosis of HCC.     

Decreased annexin A3 expression correlates with tumor progression in papillary thyroid cancer

Purpose : The aim of this study is to identify the potential tumor markers that function in carcinogenesis and tumor progression, thus providing important diagnostic and prognostic information. Experimental design : We performed 2‐D gel electrophoresis and MALDI‐TOF MS to investigate the differentially expressed proteins in 25 papillary thyroid carcinoma tissues. For validation of candidate proteins and investigation of clinical significance, we performed Western, Northern blot analysis and immunohistochemical staining. Results : Our proteomic analyses revealed significantly decreased annexin A3 expression in papillary thyroid carcinoma at both the protein and mRNA levels, compared with normal thyroid tissue. ANXA3 immunoreactivity was not significantly correlated with lymph node metastasis, multifocality, capsular invasion or perithyroidal extension in thyroid cancer. However, the tumor subgroup with a lymph node metastasis score of >3 displayed significantly lower ANXA3 expression than did subgroups with negative and ≤3 scores (p=0.001). Moreover, ANXA3 expression was markedly lower in large tumors (>1 cm in diameter) than in microcarcinomas (p=0.001). Conclusion and clinical relevance : Decreased expression of ANXA3 in papillary thyroid cancer supports the idea that ANXA3 may be an effective marker of microcarcinoma, and a negative predictor of papillary thyroid cancer progression.     

Protein profiling of formalin fixed paraffin embedded tissue: Identification of potential biomarkers for pediatric brainstem glioma

Little is known about the molecular characteristics of pediatric brainstem gliomas (BSG), which continue to have a dismal prognosis. Targeted molecular strategies are limited due to rarity of biopsy BSG specimen coupled with obstacles associated with the analyses of formalin-fixed paraffin-embedded (FFPE) autopsies. The objective of this study was to develop methodologies to successfully identify the proteome profile from these archived FFPE specimens. Peptides were extracted from both tumor and adjacent normal FFPE brainstem specimen and quantified using (18) O proteolytic labeling strategy and LC-MS/MS analysis. The ingenuity pathway analysis software was used to elucidate interactions amongst differentially expressed proteins. We identified 188 proteins of which 54 (29%) were found up-regulated (≥1.5-fold) in BSG compared to normal sections. Of these, 15 (28%) proteins have previously been reported as potential biomarkers for supratentorial malignant gliomas, while the rest appear to be exclusive to pediatric BSG. Because the majority of differentially expressed proteins are unique to BSG, we conclude that pediatric BSG is distinct from supratentorial gliomas. To the best of our knowledge, this is the first proteome profile of pediatric BSG, which may facilitate discovery of novel therapeutic targets for early diagnostics and improving prognostics.     

An experimental strategy for quantitative analysis of the humoral immune response to prostate cancer antigens using natural protein microarrays

The identification of human tumor antigens has potential utility in the diagnosis and treatment of cancers. We demonstrate here a complete strategy to profile immunoreactivity and identify tumor antigens from proteins derived from tumor cell lines. Microarrays of proteins produced from 2-D LC fractionation of prostate tumor cell-line lysates were used to profile immunoreactivity in the sera of prostate cancer patients and control subjects. Cancer-associated immunoreactivity to distinct groups of chromatography fractions was present in about 50% of the patients, with greater immunoreactivity present in patients with non-organ-confined cancer than in patients with organ-confined cancer. We grouped the immunoreactive fractions by similarities in elution order and patterns of immunoreactivity to guide and interpret the MS analysis of selected fractions, which was used to identify the proteins that may be responsible for the immunoreactivity. As a complementary method to further characterize and validate the immunoreactivity of the proteins identified by mass spectrometry, we demonstrate the use of focused microarrays of recombinant proteins. Disease-associated immunoreactivity was confirmed for one of the identified proteins, human Kallikrein 11. These results demonstrate a practical approach to screening, identifying, and validating immunoreactive proteins that could be applied to diverse studies on humoral immune responses.     

Discovery of stage-related proteins in esophageal squamous cell carcinoma using proteomic analysis

Esophageal squamous cell carcinoma (ESCC) is the major subtype of esophageal cancers in China, and characterized with high morbidity and mortality. So far, the diagnosis of ESCC is mainly dependent on the alterations in esophageal histology, but most cases of ESCC with low stage do not display visible histological abnormalities. Therefore, a deep understanding of the mechanism of ESCC progression and seeking stage-specific molecules might improve the diagnosis and therapy for ESCC. In this study, we used proteomics to analyze ESCC tissues with classification by TNM stage, and determined the proteomic features correlated with ESCC progression (from stages I to III). Proteins that exhibited significantly different expression patterns between ESCC and corresponding normal esophageal tissues were identified using MS. The identified proteins with differentiated expression mainly fell into three protein categories (i.e. cytoskeleton system-associated proteins, metabolism enzymes, and heat shock proteins). In addition, real-time PCR highlighted some molecules that were associated with tumor stages at the mRNA level, such as enolase 1, chromosome 1 ORF 10, elastase inhibitor, α B crystalline, stress-induced phosphoprotein 1, and squamous cell carcinoma antigen 1. Altogether, these data provided further information on ESCC progression and potential drug targets for ESCC clinical therapy.     

Modulated T-complex protein 1 ζ and peptidyl-prolyl cis-trans isomerase B are two novel indicators for evaluating lymph node metastasis in colorectal cancer: Evidence from proteomics and bioinformatics

Lymph node metastasis (LNM) is an important indicator for systematic therapy, which could increase the survival of colorectal cancer (CRC) patients. However, effective clinical evaluation for LNM is still absent to date. In this study, protein expression profiles of CRC tissues were compared between patients with and without LNM. Based on average expression level, 12 proteins were found to be differentially expressed in the CRC tissues with LNM, whose discrimination reliability was confirmed by PCA. With stepwise linear discriminant analysis, T-complex protein 1 ζ subunit and peptidyl-prolyl cis-trans isomerase B (PPIB) were identified as two main contributors for separating CRC tissues with positive LNM from those negative ones in both original-grouped and cross-validated-grouped cases, which was also supported in subsequent linear support vector machine analysis. In addition, the expression alterations of the two proteins were verified by Western blot and immunohistochemistry. Functional studies also confirmed the role of PPIB in migration and invasion of cancer cells. Taken together, the down-regulated T-complex protein 1 ζ subunit and up-regulated PPIB were identified as two promising indicators for the clinical evaluation of LNM in CRC patients.     

IVIM及纹理分析在术前预测宫颈癌类型和淋巴结转移研究进展

本文综述了体素内不相干运动扩散加权成像（intravoxel incoherent motion diffusion-weighted imaging,IVIM-DWI）及纹理分析技术在术前鉴别宫颈癌组织学亚型及淋巴结转移的临床应用进展。MRI（magnetic resonance imaging）是宫颈癌临床术前最常用的影像学诊断和分期方法。宫颈癌的组织学类型及有无淋巴结转移与患者的生存及预后紧密相关。IVIM-DWI为MRI新型功能成像技术,其D值反映组织内单纯水分子的布朗运动信息、间接反映恶性肿瘤组织细胞密集度;其D^*值和f值能提供肿瘤组织内血流灌注的信息。影像组学的纹理分析技术（texture analysis,TA）通过提取肿瘤组织的纹理特征进行客观、定量分析,能检测人眼不能识别的肿瘤组织的微观改变,揭示更多肿瘤组织的灰度分布与定量数据特征,为临床术前预测宫颈癌不同组织学类型及转移淋巴结提供了可能。     

Television-based densitometric analysis of proteins separated by two-dimensional gel electrophoresis

A program is described for a computer-aided, television-based system which can quantify the densities of stained proteins on two-dimensional electrophoretic gels. The system is designed to detect and graphically reproduce the edges of spots which are often located in nonuniform levels of background stain and to integrate the density of each spot detected. Tests of the system are described which show that it responds linearly to optical densities encountered on the electrophoretic gels and is sufficiently stable and sensitive to permit quantitative analysis of most, if not all, proteins separated on a gel.     

iTRAQ‐Based Quantitative Proteomic Analyses of High Grade Esophageal Squamous Intraepithelial Neoplasia

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide and is the fourth most lethal cancer in China. Little is known about the proteome of high grade esophageal squamous intraepithelial neoplasia (HGN), which is a premalignant lesion of ESCC. A quantitative proteomic analysis using an isobaric tag for relative and absolute quantification (iTRAQ) approach is used to characterize the protein expression profiles in HGN. Among the 3156 identified proteins, a total of 236 proteins are discovered to be differentially expressed. Compared with paired normal esophageal epithelial tissues, 138 proteins are upregulated and 98 proteins are downregulated in HGN. Bioinformatics analyses are performed according to gene ontology, clusters of orthologous groups, and kyoto encyclopedia of genes and genomes enrichment analyses. Six differentially expressed proteins are chosen and validated by Western blotting. The results of the study increase our understanding of early tumorigenesis during ESCC, and provide insights into the proteome at the initial stages of the disease that can be used to identify potential biomarkers for early diagnosis and for therapeutic targets.     

NMR spin relaxation methods for characterization of disorder and folding in proteins

The flexibility and dynamics of proteins directly influence the processes of protein folding, recognition, and function. NMR spin relaxation methods are used to assess the dynamics and mobility of proteins, for fast ps and ns motions as well as slower microsecond and ms events. The degree of protein flexibility and disorder as well as the changes in protein flexibility can be assessed by NMR spin relaxation methods at individual residues within the protein. In addition to probing protein dynamics, the changes in the NMR-derived order parameters can be used to estimate the entropic contributions of order-disorder transitions. Furthermore, kinetic processes in the ms time regime may be directly investigated to extract the rates of conformational interconversion, ligand binding, and protein folding processes. We show how a variety of dynamical information can be obtained from NMR relaxation measurements. We present examples that illustrate the use of NMR spin relaxation analysis for investigation of folding and disorder in proteins.     

Ultrasound image analysis technology under deep belief networks in evaluation on the effects of diagnosis and chemotherapy of cervical cancer

The purpose of this study was to explore the value of extraction of tumor features in contrast-enhanced ultrasonography (CEUS) images based on the deep belief networks (DBN) for the diagnosis of cervical cancer patients and realize the intelligent evaluation on effects of diagnosis and chemotherapy of the cervical cancer. An automatic extraction algorithm with the time-intensity curve (TIC) was proposed based on Sparse nonnegative matrix factorization (SNMF) in this study, and was applied to the framework of automatic analysis of cervical cancer tumors based on the deep belief networks, to assist doctors in the analysis of cervical cancer tumors. The framework was applied to the real clinical diagnostic data, and the feasibility of the method was verified by comparing the accuracy, sensitivity, and specificity. Later, the parameters of patients’ time to peak (TP), peak intensity (PI), mean transit time (MTT), and area under the curve (AUC) were obtained by drawing TICs, and the changes of p53 protein and ki-67 protein obtained by pathological section staining were analyzed to evaluate the therapeutic effect in the patients. It was found that the proposed model of tumor feature extraction based on the DBN had the higher accuracy (86.36%), sensitivity (83.33%), and specificity (87.50%). The related parameters of TIC curve obtained based on SNMF showed that there was a significant difference in p53 content between tissues with different degrees of disease (p?<?0.05), the PI of poorly differentiated tissues was significantly higher than that of those with high to medium differentiation (p?<?0.05). In addition, PI and AUC of patients after chemotherapy were significantly lower than that before chemotherapy (p?<?0.05), while MTT was significantly higher than that before chemotherapy (p?<?0.05). Therefore, the proposed TIC feature extraction of CEUS images based on SNMF and the automatic tumor classification based on deep learning can be used in the diagnosis and efficacy evaluation of cervical cancer patients.

     

Identification of nasopharyngeal carcinoma antigens that induce humoral immune response by proteomic analysis

A proteomics-based approach has been used to identify proteins that commonly elicit a humoral immune response in nasopharyngeal carcinoma (NPC). Sera from 19 newly diagnosed NPC patients and 19 healthy individuals were analyzed for IgG autoantibodies against NPC proteins resolved by 2-DE. Protein spots that exhibited selective reactivity with sera from NPC patients were identified by MS. Among nine identified proteins, cytokeratin 19 (CK19), Erb3 binding protein (EBP1), and Rho GDP dissociation inhibitor-beta (Rho-GDI-2) induced autoantibodies in more than 36.8% of NPC patients but not in healthy individuals. Furthermore, Western blot analysis and immunohistochemical staining were performed to determine the expression and localization of CK19, EBP1, and Rho-GDI-2 in NPC and normal nasopharyngeal mucosal tissues. Up-regulated CK19 and EBP1, but not Rho-GDI-2, were observed in NPC vs. normal tissue. Subcellular localization of the three proteins in NPC tissue was same as that in the normal tissue. Thus, overexpression of CK19 and EBP1 may be one of the mechanisms for their autoantibody development in NPC. To validate the findings of a proteomic analysis, occurrence of autoantibodies against these three proteins was detected by immunoprecipitation and Western blot analysis in additional 30 NPC patients, 23 other types of cancer patients and 20 healthy individuals. Results showed that frequency of autoantibodies against CK19, EBP1 and Rho-GDI-2 in NPC patients was significantly higher than that in other types of cancer patients and healthy individuals. We conclude that CK19, EBP1 and Rho-GDI-2 may have utility in NPC screening and diagnosis.     

MALDI mass spectrometry imaging: A cutting‐edge tool for fundamental and clinical histopathology

Histopathological diagnoses have been done in the last century based on hematoxylin and eosin staining. These methods were complemented by histochemistry, electron microscopy, immunohistochemistry (IHC), and molecular techniques. Mass spectrometry (MS) methods allow the thorough examination of various biocompounds in extracts and tissue sections. Today, mass spectrometry imaging (MSI), and especially matrix‐assisted laser desorption ionization (MALDI) imaging links classical histology and molecular analyses. Direct mapping is a major advantage of the combination of molecular profiling and imaging. MSI can be considered as a cutting edge approach for molecular detection of proteins, peptides, carbohydrates, lipids, and small molecules in tissues. This review covers the detection of various biomolecules in histopathological sections by MSI. Proteomic methods will be introduced into clinical histopathology within the next few years.     

Formalin-fixed paraffin-embedded tissue: The holy grail of clinical proteomics?

Tissue is the most relevant biological material to gather insight in disease mechanisms by means of omics technologies. However, fresh frozen tissue, which is generally regarded as the best imaginable source for such studies, is often not available. In case it is available, the different ways of storage (e.g. −20°C, −80°C, liquid nitrogen, etc.) hamper the conduction of reproducible multicenter studies because of different protein degradation rates. Formalin-fixed paraffin-embedded (FFPE) tissue on the contrary is considered as a valuable alternative for fresh frozen tissue, because only a few standard operation procedures are applied worldwide for the preparation of these tissues and because they are all stored in the same way. However, a study on the impact of the different preparation protocols for FFPE tissue was still lacking. Therefore, Bronsert et al. in this issue [Bronsert, P., Weißer, J., Biniossek, M. L., Kuehs, M. et al., Proteomics Clin. Appl. 2014, 8 786–804] conducted such a study that provides proof that there is no significant effect between these sample preparations procedures, and thereby they further open the gate for FFPE tissues to enter the field of clinical proteomics.     

Determination of myosin light chain phosphorylation using astronomical image analysis programs on autoradiographs of electrophoretic gels

Computer image analysis programs developed by astronomers for celestial photometry were used for the analysis of autoradiographs of electrophoretic slab gels. Oxytocin-stimulated myosin light chain phosphorylation in mammary myoepithelial cells was studied by incubating cells with [32P]orthophosphate followed by oxytocin addition, polyacrylamide gel electrophoresis of cellular protein, and autoradiography of electrophoretic slab gels. After scanning and digitization of autoradiographs, [32P]phosphate incorporation into the myosin light chain was measured by the determination of radiation flux recorded on the X-ray film. Within seconds after oxytocin addition, the myosin light chain was fully phosphorylated. The large library of astronomical computer programs has applications for the quantitative analysis of both one- and two-dimensional electrophoretic gels.