Anthea J. Stanley

The potential use of laser capture microdissection to selectively obtain distinct populations of cells for proteomic analysis — Preliminary findings

Proteomics‐based studies offer a powerful complementary approach to DNA/RNA‐based investigations and are now being applied to investigate aspects of many diseases including cancer. However, the heterogeneous nature of tissue samples often makes interpretation difficult. We have undertaken a study into the potential use of a novel laser capture microdissection (LCM) system to isolate cells of interest for subsequent proteomic analysis. Retrieval of selected cells is achieved by activation of a transfer film placed in contact with a tissue section, by a laser beam (30 or 60 μm diameter) which is focused on a selected area of tissue using an inverted microscope. The precise area of film targeted by the laser bonds to the tissue beneath it and these cells are then lifted free of surrounding tissue. Although the technique has been shown to be readily compatible with subsequent analysis of nucleic acids, little information is yet available regarding the application of protein‐based analyses to the captured tissue. We report here preliminary data regarding the potential use of the LCM system in combination with two‐dimensional electrophoresis to examine protein profiles of selected tissue areas. Electrophoretic profiles of proteins from normal and malignant renal tissue samples showed little change following LCM, nine selected proteins showed identical mass spectrometric sequencing profiles, and two selected proteins retained antigenicity. Dissection of epithelial tissue from a sample of normal human cervix resulted in enrichment of some proteins compared with analysis of the whole tissue. LCM will be a valuable adjunct to proteomic studies although further detailed validation is necessary.

Genetic and epigenetic analysis of von Hippel-Lindau (VHL) gene alterations and relationship with clinical variables in sporadic renal cancer.

Genetic and epigenetic changes in the von Hippel-Lindau (VHL) tumor suppressor gene are common in sporadic conventional renal cell carcinoma (cRCC). Further insight into the clinical significance of these changes may lead to increased biological understanding and identification of subgroups of patients differing prognostically or who may benefit from specific targeted treatments. We have comprehensively examined the VHL status in tissue samples from 115 patients undergoing nephrectomy, including 96 with sporadic cRCC. In patients with cRCC, loss of heterozygosity was found in 78.4%, mutation in 71%, and promoter methylation in 20.4% of samples. Multiplex ligation-dependent probe amplification identified intragenic copy number changes in several samples including two which were otherwise thought to be VHL-noninvolved. Overall, evidence of biallelic inactivation was found in 74.2% of patients with cRCC. Many of the mutations were novel and approximately two-thirds were potentially truncating. Examination of these and other published findings confirmed mutation hotspots affecting codons 117 and 164, and revealed a common region of mutation in codons 60 to 78. Gender-specific differences in methylation and mutation were seen, although not quite achieving statistical significance (P = 0.068 and 0.11), and a possible association between methylation and polymorphism was identified. No significant differences were seen between VHL subgroups with regard to clinicopathologic features including stage, grade, tumor size, cancer-free and overall survival, with the exception of a significant association between loss of heterozygosity and grade, although a possible trend for survival differences based on mutation location was apparent.

Urinary biomarker profiling in transitional cell carcinoma

Urinary biomarkers or profiles that allow noninvasive detection of recurrent transitional cell carcinoma (TCC) of the bladder are urgently needed. We obtained duplicate proteomic (SELDI) profiles from 227 subjects (118 TCC, 77 healthy controls and 32 controls with benign urological conditions) and used linear mixed effects models to identify peaks that are differentially expressed between TCC and controls and within TCC subgroups. A Random Forest classifier was trained on 130 profiles to develop an algorithm to predict the presence of TCC in a randomly selected initial test set (n = 54) and an independent validation set (n = 43) several months later. Twenty two peaks were differentially expressed between all TCC and controls (p < 10−7). However potential confounding effects of age, sex and analytical run were identified. In an age‐matched sub‐set, 23 peaks were differentially expressed between TCC and combined benign and healthy controls at the 0.005 significance level. Using the Random Forest classifier, TCC was predicted with 71.7% sensitivity and 62.5% specificity in the initial set and with 78.3% sensitivity and 65.0% specificity in the validation set after 6 months, compared with controls. Several peaks of importance were also identified in the linear mixed effects model. We conclude that SELDI profiling of urine samples can identify patients with TCC with comparable sensitivities and specificities to current tumor marker tests. This is the first time that reproducibility has been demonstrated on an independent test set analyzed several months later. Identification of the relevant peaks may facilitate multiplex marker assay development for detection of recurrent disease.

Sample size determination in clinical proteomic profiling experiments using mass spectrometry for class comparison.

Mass spectrometric profiling approaches such as MALDI‐TOF and SELDI‐TOF are increasingly being used in disease marker discovery, particularly in the lower molecular weight proteome. However, little consideration has been given to the issue of sample size in experimental design. The aim of this study was to develop a protocol for the use of sample size calculations in proteomic profiling studies using MS. These sample size calculations can be based on a simple linear mixed model which allows the inclusion of estimates of biological and technical variation inherent in the experiment. The use of a pilot experiment to estimate these components of variance is investigated and is shown to work well when compared with larger studies. Examination of data from a number of studies using different sample types and different chromatographic surfaces shows the need for sample‐ and preparation‐specific sample size calculations.

An evaluation of a preparation of Mycobacterium vaccae (SRL172) as an immunotherapeutic agent in renal cancer

Two studies were carried out to evaluate heat-killed Mycobacterium vaccae SRL172 as an immunotherapeutic agent for patients with metastatic, post-nephrectomy, renal cell carcinoma. In the first study, 60 patients in France and the UK received injections of SRL172, and their survival was compared with that of historical controls who had been treated either with biological response modifiers (IL-2, IFN-alpha) or chemotherapy. In the second study, 36 patients were randomised to receive treatment with IL-2 alone or IL-2 plus SRL172. Survival and adverse events related to the treatments were assessed and compared between treatment groups. The first study showed that those treated with SRL172 alone survived equally as long as those receiving IL-2 or IFN-alpha and both treatment groups survived longer than those on chemotherapy (p<0.001), a result supported by Coxs proportional hazards regression analysis. The second study, stopped early due to drug supply issues, showed that the addition of SRL172 to IL-2 made no difference to survival compared to IL-2 alone, in the limited numbers treated. Adverse events occurring in those receiving SRL172 in the first study were mild and in the second study those receiving IL-2 alone had significantly more adverse events than those receiving SRL172 plus IL-2 (p<0.001). It is concluded that SRL172 may have activity in metastatic renal cancer and has very low toxicity, making it worthy of further study.

Serum biomarker discovery in renal cancer using 2-DE and prefractionation by immunodepletion and isoelectric focusing; increasing coverage or more of the same?

As an initial screen for novel markers of renal cancer and to minimise background heterogeneity, we have compared the within‐patient profiles of serum samples from seven patients pre‐ and post‐nephrectomy. Samples were depleted of six of the most abundant proteins using Agilents multiple affinity removal system (MARS) followed by solution‐phase IEF prior to separation by 2‐DE using narrow range IPG Strips, with a total of 84 gels. The reproducibility of the various steps was demonstrated and an approximate two‐fold increase (from 374 to 779) in the number of protein spots observed in the pH region 4.6–7.0 was obtained. However, the majority of additional proteins seen were further isoforms of existing proteins due to the higher resolution and the majority of protein spots identified were still moderate to highly abundant species. Only one protein spot (as yet unidentified) was found to change significantly in the same direction in at least four patients. Although this powerful prefractionation and analysis strategy allows the visualisation of multiple protein isoforms, it is insufficient to allow detection of lower abundance proteins in serum without the implementation of further strategies.

Pre-operative urinary cathepsin D is associated with survival in patients with renal cell carcinoma

Background:No circulating markers are routinely used for renal cancer. The objective of this pilot study was to investigate whether conditioned media (CM) from renal cancer cell lines contains potential biomarkers that, when measured in clinical fluids, have diagnostic or prognostic utility.Methods:Comparative 2D PAGE profiling of CM from renal cell carcinoma (RCC) and normal renal cultures identified cathepsin D that was subsequently validated in urine samples from 239 patients and healthy and benign disease subjects.Results:Urinary cathepsin D was found to be significantly associated with overall (OS) (hazard ratio, HR, 1.33, 95%CI [1.09–1.63], P=0.005) and cancer-specific survival (HR 1.36, 95%CI [1.07–1.74], P=0.013) in RCC patients on univariate analysis. An optimal cut point (211 ng ml−1 μmolCr−1) around which to stratify patients by OS was determined. Five-year OS equal to/above and below this value was 47.0% (95%CI 35.4%, 62.4%) and 60.9% (48.8%, 76.0%), respectively. On multivariable analysis using pre-operative variables, cathepsin D showed some evidence of independent prognostic value for OS (likelihood ratio test P-value=0.056) although requiring further validation in larger patient numbers with sufficient statistical power to determine independent significance.Conclusion:These data establish an important proof of principle and show the potential of proteomics-based studies. Cathepsin D may be of value as a pre-operative urinary biomarker for RCC, alone or in combination.

Intrinsic chemotherapy resistance to the tubulin-binding antimitotic agents in renal cell carcinoma.

Renal cancer is one of the most chemoresistant tumor types. Using a panel of 10 established renal cancer cell lines that have not been subjected to prior drug selection, the range of functional resistance phenotypes to the tubulin‐binding agents paclitaxel, vinblastine, vincristine and patupilone (epothilone B, EPO906) was determined, together with expression of P‐glycoprotein (PgP), multidrug resistance associated protein‐2 (MRP2) and major vault protein (MVP) proteins. The IC50 values for vincristine correlated positively with PgP expression (r = 0.73; p = 0.031), with values for paclitaxel and vinblastine just failing to reach significance. A significant positive correlation was observed for sensitivity to paclitaxel and MRP2 expression only (r = 0.8; p = 0.013). MVP expression did not correlate with sensitivity to any of the drugs examined. All cell lines exhibited much greater sensitivity to patupilone, demonstrating for the first time the potential use of patupilone in this cancer. In tissue samples from chemotherapy‐naive renal cell carcinoma (RCC) patients, marked downregulation or absence of PgP in many tumor cells with expression levels more similar to sensitive cell lines rather than the resistant lines was seen. Similarly, MRP2 was absent or only weakly present in tumor cells, whereas MVP was very strongly upregulated in most tumor samples. This study illustrating discrepancies between results exclusively based on studies in cell lines and findings in vivo suggests that the role of PgP and MRP2 in intrinsic resistance in RCC in vivo may be less than expected from the in vitro findings and supports a potential role for MVP on the basis of in vivo expression studies.

Integrated multi-level quality control for proteomic profiling studies using mass spectrometry

BackgroundProteomic profiling using mass spectrometry (MS) is one of the most promising methods for the analysis of complex biological samples such as urine, serum and tissue for biomarker discovery. Such experiments are often conducted using MALDI-TOF (matrix-assisted laser desorption/ionisation time-of-flight) and SELDI-TOF (surface-enhanced laser desorption/ionisation time-of-flight) MS. Using such profiling methods it is possible to identify changes in protein expression that differentiate disease states and individual proteins or patterns that may be useful as potential biomarkers. However, the incorporation of quality control (QC) processes that allow the identification of low quality spectra reliably and hence allow the removal of such data before further analysis is often overlooked. In this paper we describe rigorous methods for the assessment of quality of spectral data. These procedures are presented in a user-friendly, web-based program. The data obtained post-QC is then examined using variance components analysis to quantify the amount of variance due to some of the factors in the experimental design.ResultsUsing data from a SELDI profiling study of serum from patients with different levels of renal function, we show how the algorithms described in this paper may be used to detect systematic variability within and between sample replicates, pooled samples and SELDI chips and spots. Manual inspection of those spectral data that were identified as being of poor quality confirmed the efficacy of the algorithms. Variance components analysis demonstrated the relatively small amount of technical variance attributable to day of profile generation and experimental array.ConclusionUsing the techniques described in this paper it is possible to reliably detect poor quality data within proteomic profiling experiments undertaken by MS. The removal of these spectra at the initial stages of the analysis substantially improves the confidence of putative biomarker identification and allows inter-experimental comparisons to be carried out with greater confidence.

Resistance to the Tubulin-Binding Agents in Renal Cell Carcinoma: No Mutations in the Class I β-Tubulin Gene but Changes in Tubulin Isotype Protein Expression

Purpose: The primary purpose of this study was to determine whether mutations of the class I β-tubulin gene may be implicated in the inherent resistance to tubulin-binding agents (TBA) in renal cancer, with a small number of samples and cell lines also being examined for class I and III β-tubulin isotype protein expression. Experimental Design: DNA was extracted from 90 renal tumors and the class I β-tubulin gene analyzed for mutations. For each sample, eight PCRs were used to cover the complete coding sequence with intronic primers ensuring highly homologous pseudogenes were not coamplified. Additionally, expression levels of class I and III β-tubulin isotypes in 17 matched normal and malignant renal samples and a panel of renal cell carcinoma cell lines with differing intrinsic resistance to the TBAs was examined by Western blotting. Results: Four polymorphic sequence changes of the class I β-tubulin gene were identified with no mutations. Class I protein expression levels were higher in tumor tissue versus normal tissue, whereas class III expression showed no consistent change. In renal cancer cell lines, a significant correlation between class III isotype expression and vinblastine sensitivity was observed. Conclusions: These results do not support a role for mutations in the class I β-tubulin gene in the intrinsic resistance of renal cancer to TBAs. Class III isotype expression may be implicated in resistance in vitro but in vivo, changes in class I isotype expression in renal cell carcinoma tissue may support a role in resistance to the TBAs and warrants further investigation.