Rachel A. Craven

Laser Capture Microdissection and Two-Dimensional Polyacrylamide Gel Electrophoresis: Evaluation of Tissue Preparation and Sample Limitations

Laser capture microdissection (LCM) is now well established as a tool for facilitating the enrichment of cells of interest from tissue sections, overcoming the problem of tissue heterogeneity. LCM has been used extensively in combination with analysis at the DNA and RNA levels, but only a small number of studies have employed LCM with subsequent protein analysis, albeit with promising results. This study focuses on the potential of LCM in combination with two-dimensional polyacrylamide gel electrophoresis. The effects of tissue section preparation and sample type were evaluated to fully determine the suitability of using LCM in global protein profiling. The effects of several histochemical stains (hematoxylin and eosin, methyl green and toluidine blue) and immunolabeling on subsequent two-dimensional polyacrylamide gel electrophoresis were investigated. Quantitative analysis was performed to establish the extent of changes in the relative intensity of protein species and their reproducibility. All staining protocols tested were found to be compatible with protein analysis although there was variation in protein recovery and the quality of the protein profiles obtained. LCM of renal and cervix samples indicated that protein yield after dissection was acceptable, although the extent of enrichment and dissection time was tissue-dependent, which may preclude the use of this approach with some tissue types. These results indicate that LCM has potential as a tool in proteomic research.

Analysis of VHL gene alterations and their relationship to clinical parameters in sporadic conventional renal cell carcinoma

Purpose: This study aimed to carry out a comprehensive analysis of genetic and epigenetic changes of the von Hippel Lindau (VHL) gene in patients with conventional (clear cell) renal cell carcinoma and to determine their significance relative to clinicopathologic characteristics and outcome. Experimental Design: The VHL status in 86 conventional renal cell carcinomas was determined by mutation detection, loss of heterozygosity (LOH), and promoter methylation analysis, extending our original cohort to a total of 177 patients. Data were analyzed to investigate potential relationships between VHL changes, clinical parameters, and outcome. Results: LOH was found in 89.2%, mutation in 74.6%, and methylation in 31.3% of evaluable tumors; evidence of biallelic inactivation (LOH and mutation or methylation alone) was found in 86.0% whereas no involvement of VHL was found in only 3.4% of samples. Several associations were suggested, including those between LOH and grade, nodal status and necrosis, mutation and sex, and methylation and grade. Biallelic inactivation may be associated with better overall survival compared with patients with no VHL involvement, although small sample numbers in the latter group severely limit this analysis, which requires independent confirmation. Conclusions: This study reports one of the highest proportions of conventional renal cell carcinoma with VHL changes, and suggests possible relationships between VHL status and clinical variables. The data suggest that VHL defects may define conventional renal cell carcinomas but the clinical significance of specific VHL alterations will only be clarified by the determination of their biological effect at the protein level rather than through genetic or epigenetic analysis alone. (Clin Cancer Res 2009;15(24):7582–92)

Laser capture microdissection and proteomics: possibilities and limitation.

Tissue heterogeneity has always limited the information available from analysis of biological samples in the study of disease. Several approaches have been developed to address this problem, with laser capture microdissection (LCM) emerging as one of the methods of choice. LCM has been extensively used in combination with mutation detection studies and analyses of gene expression at the mRNA level and its potential in proteomics‐based research is beginning to be realised. Here we review the progress made to date in the analysis of proteins in LCM‐captured material and evaluate the scope and limitations of this approach.

Proteomic Profiling Identifies Afamin as a Potential Biomarker for Ovarian Cancer

Purpose: To discover and validate serum glycoprotein biomarkers in ovarian cancer using proteomic-based approaches. Experimental Design: Serum samples from a “discovery set” of 20 patients with ovarian cancer or benign ovarian cysts or healthy volunteers were compared by fluorescence two-dimensional differential in-gel electrophoresis and parallel lectin-based two-dimensional profiling. Validation of a candidate biomarker was carried out with Western blotting and immunoassay (n = 424). Results: Twenty-six proteins that changed significantly were identified by mass spectrometric sequencing. One of these, confirmed by Western blotting, was afamin, a vitamin E binding protein, with two isoforms decreasing in patients with ovarian cancer. Validation using cross-sectional samples from 303 individuals (healthy controls and patients with benign, borderline, or malignant ovarian conditions and other cancers) assayed by ELISA showed significantly decreased total afamin concentrations in patients with ovarian cancer compared with healthy controls (P = 0.002) and patients with benign disease (P = 0.046). However, the receiver operating characteristic areas for total afamin for the comparison of ovarian cancer with healthy controls or benign controls were only 0.67 and 0.60, respectively, with comparable figures for CA-125 being 0.92 and 0.88 although corresponding figures for a subgroup of samples analyzed by isoelectric focusing for afamin isoform 2 were 0.85 and 0.79. Analysis of a further 121 samples collected prospectively from 9 patients pretreatment through to relapse indicated complementarity of afamin with CA-125, including two cases in whom CA-125 was noninformative. Conclusions: Afamin shows potential complementarity with CA-125 in longitudinal monitoring of patients with ovarian cancer, justifying prospective larger-scale investigation. Changes in specific isoforms may provide further information.

Circulating markers of biliary malignancy: opportunities in proteomics?

Cholangiocarcinoma, a primary liver tumour that arises from biliary epithelial cells, is increasing in incidence and has poor prognosis. Diagnosis is difficult, particularly in patients with primary sclerosing cholangitis, who are at risk of developing the disease. Timely diagnosis is essential because surgical resection in early disease remains the only cure. The lack of a sensitive and specific early diagnostic marker and of alternative treatments are the main reasons why patients have limited survival. The use of proteomic-based approaches, which analyse the physiological or pathological complement of proteins (ie, the proteome) in cells, tissues, or biological fluids, has received substantial interest in biomarker discovery. Proteomics complements genomic studies and examines functional end-units quantitatively and qualitatively, including post-translational modifications which might vary with disease and might have key roles in protein function or localisation. Major advances in technology and bioinformatics have enhanced proteomic studies, resulting in increased understanding of the pathogenesis of many diseases and in biomarker discovery with effective use of tissues, cell lines, and biological fluids. We review the current status and promise of proteomic-based approaches in biomarker discovery for cholangiocarcinoma.

Predicting Response to Bevacizumab in Ovarian Cancer: A Panel of Potential Biomarkers Informing Treatment Selection

Purpose: The aim of this study was to identify and validate novel predictive and/or prognostic serum proteomic biomarkers in patients with epithelial ovarian cancer (EOC) treated as part of the phase III international ICON7 clinical trial. Experimental Design: ICON7 was a phase III international trial in EOC which showed a modest but statistically significant benefit in progression-free survival (PFS) with the addition of bevacizumab to standard chemotherapy. Serum samples from 10 patients who received bevacizumab (five responders and five nonresponders) were analyzed by mass spectrometry to identify candidate biomarkers. Initial validation and exploration by immunoassay was undertaken in an independent cohort of 92 patients, followed by a second independent cohort of 115 patients (taken from across both arms of the trial). Results: Three candidate biomarkers were identified: mesothelin, fms-like tyrosine kinase-4 (FLT4), and α1-acid glycoprotein (AGP). Each showed evidence of independent prognostic potential when adjusting for high-risk status in initial (P < 0.02) and combined (P < 0.01) validation cohorts. In cohort I, individual biomarkers were not predictive of bevacizumab benefit; however, when combined with CA-125, a signature was developed that was predictive of bevacizumab response and discriminated benefit attributable to bevacizumab better than clinical characteristics. The signature showed weaker evidence of predictive ability in validation cohort II, but was still strongly predictive considering all samples (P = 0.001), with an improvement in median PFS of 5.5 months in signature-positive patients in the experimental arm compared with standard arm. Conclusions: This study shows a discriminatory signature comprising mesothelin, FLT4, AGP, and CA-125 as potentially identifying those patients with EOC more likely to benefit from bevacizumab. These results require validation in further patient cohorts. Clin Cancer Res; 19(18); 5227–39. ©2013 AACR.

Proteomic analysis of formalin-fixed paraffin-embedded renal tissue samples by label-free MS: assessment of overall technical variability and the impact of block age.

Protein profiling of formalin‐fixed paraffin‐embedded (FFPE) tissues has enormous potential for the discovery and validation of disease biomarkers. The aim of this study was to systematically characterize the effect of length of time of storage of such tissue blocks in pathology archives on the quality of data produced using label‐free MS.

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.

Proteomic identification of differentially expressed plasma membrane proteins in renal cell carcinoma by stable isotope labelling of a von Hippel‐Lindau transfectant cell line model

The von Hippel‐Lindau (VHL) tumour suppressor gene plays a central role in development of clear cell renal cell carcinoma (RCC). Using a cell line pair generated from the VHL‐defective RCC cell line UMRC2 by transfection with vector control or VHL (−/+VHL) and stable isotope labelling with amino acids in cell culture (SILAC) followed by enrichment of plasma membrane proteins by cell surface biotinylation/avidin‐affinity chromatography and analysis by GeLC‐MS/MS, VHL‐associated changes in plasma membrane proteins were analysed. Comparative analysis of ‐/+VHL cells identified 19 differentially expressed proteins which were confirmed by reciprocal SILAC labelling. These included several proteins previously reported to be VHL targets, such as transferrin receptor 1 and the α3 and β1 integrin subunits and novel findings including upregulation of CD166 and CD147 in VHL‐defective cells. Western blotting confirmed these changes and also revealed VHL‐dependent alterations in protein form for CD147 and CD166, which in the case of CD166 was shown to be due to differential glycosylation. Analysis of patient‐matched normal and malignant renal tissues confirmed these differences were also present in vivo in a subset of clear cell RCCs. These results illustrate the potential of this approach for identifying VHL‐dependent proteins that may be important in tumorigenesis.

Key clinical issues in renal cancer: a challenge for proteomics

Renal cancer has many clinical challenges which proteomics is ideally placed to address. The issues cover all aspects of the disease including diagnosis, prognosis, treatment selection and monitoring to detect metastatic disease. In all cases novel biomarkers would considerably help in clinical management and with the relative resistance to conventional chemotherapy and radiotherapy, a better understanding of the underlying pathogenesis may contribute to the much needed development of novel therapeutic targets and the better use of promising new anti-angiogenic treatments. This review briefly highlights some of the clinical issues and describes proteomics-based approaches generally, before focussing on reviewing the proteomic studies to date in this area.