Post-analysis follow-up and validation of microarray experiments.

Measurement of gene-expression profiles using microarray technology is becoming increasingly popular among the biomedical research community. Although there has been great progress in this field, investigators are still confronted with a difficult question after completing their experiments: how to validate the large data sets that are generated? This review summarizes current approaches to verifying global expression results, discusses the caveats that must be considered, and describes some methods that are being developed to address outstanding problems.

Differential proteomic analysis of late-stage and recurrent breast cancer from formalin-fixed paraffin-embedded tissues.

The heterogeneity of breast cancer requires the discovery of more incisive molecular tools that better define disease progression and prognosis. Proteomic analysis of homogeneous tumor cell populations derived by laser microdissection from formalin-fixed, paraffin-embedded (FFPE) tissues has proven to be a robust strategy for conducting retrospective cancer biomarker investigations. We describe an MS-based analysis of laser microdissected cancerous epithelial cells derived from twenty-five breast cancer patients at defined clinical disease stages with the goal of identifying protein abundance characteristics indicative of disease progression and recurrence. Comparative analysis of stage 0 and stage III patients revealed 113 proteins that significantly differentiated these groups and included known factors associated with disease pathogenesis, such as CDH1 and CTNNB1, as well as those previously implicated in breast cancer, such as TSP-1. Similar analyses of patients presenting with stage II disease that did or did not exhibit recurrence two years postdiagnosis revealed 42 proteins that significantly differentiated these subgroups and included IRS-1 and PARK7. These data provide evidence supporting the utility of FFPE tissues for functional proteomic analyses and protein biomarker discovery and yielded protein candidates indicative of disease stage and recurrence in breast cancer that warrant further investigation for diagnostic utility and biological relevance.

mTRAQ-based quantification of potential endometrial carcinoma biomarkers from archived formalin-fixed paraffin-embedded tissues.

Formalin-fixed paraffin-embedded (FFPE) tissues are the primary and preferred medium for archiving patients' samples. Here we demonstrate relative quantifications of protein biomarkers in extracts of laser microdissected epithelial cells from FFPE endometrial carcinoma tissues versus those from normal proliferative endometria by means of targeted proteomic analyses using LC-multiple reaction monitoring (MRM) MS with MRM Tags for Relative and Absolute Quantitation (mTRAQ) labeling. Comparable results of differential expressions for pyruvate kinase isoform M2 (PK-M2) and polymeric Ig receptor were observed between analyses on laser microdissected epithelial cells from FFPE tissues and corresponding homogenates from frozen tissues of the same individuals that had previously been analyzed and reported. We also identified PK-M2 in the normal proliferative phase of the endometrium. Other biomarkers in addition to PK-M2 and polymeric Ig receptor were also observed but not consistently and/or were at levels below the threshold for quantification.

Proteomic analysis of laser-captured paraffin-embedded tissues: a molecular portrait of head and neck cancer progression.

PURPOSE: Squamous cell carcinoma of the head and neck (HNSCC), the sixth most prevalent cancer among men worldwide, is associated with poor prognosis, which has improved only marginally over the past three decades. A proteomic analysis of HNSCC lesions may help identify novel molecular targets for the early detection, prevention, and treatment of HNSCC. EXPERIMENTAL DESIGN: Laser capture microdissection was combined with recently developed techniques for protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues and a novel proteomics platform. Approximately 20,000 cells procured from FFPE tissue sections of normal oral epithelium and well, moderately, and poorly differentiated HNSCC were processed for mass spectrometry and bioinformatic analysis. RESULTS: A large number of proteins expressed in normal oral epithelium and HNSCC, including cytokeratins, intermediate filaments, differentiation markers, and proteins involved in stem cell maintenance, signal transduction, migration, cell cycle regulation, growth and angiogenesis, matrix degradation, and proteins with tumor suppressive and oncogenic potential, were readily detected. Of interest, the relative expression of many of these molecules followed a distinct pattern in normal squamous epithelia and well, moderately, and poorly differentiated HNSCC tumor tissues. Representative proteins were further validated using immunohistochemical studies in HNSCC tissue sections and tissue microarrays. CONCLUSIONS: The ability to combine laser capture microdissection and in-depth proteomic analysis of FFPE tissues provided a wealth of information regarding the nature of the proteins expressed in normal squamous epithelium and during HNSCC progression, which may allow the development of novel biomarkers of diagnostic and prognostic value and the identification of novel targets for therapeutic intervention in HNSCC.

Application of a global proteomic approach to archival precursor lesions: deleted in malignant brain tumors 1 and tissue transglutaminase 2 are upregulated in pancreatic cancer precursors.

BACKGROUND: Pancreatic cancer is an almost uniformly fatal disease, and early detection is a critical determinant of improved survival. A variety of noninvasive precursor lesions of pancreatic adenocarcinoma have been identified, which provide a unique opportunity for intervention prior to onset of invasive cancer. Biomarker discovery in precursor lesions has been hampered by the ready availability of fresh specimens, and limited yields of proteins suitable for large scale screening. METHODS: We utilized Liquid Tissue, a novel technique for protein extraction from archival formalin-fixed material, and mass spectrometry to conduct a global proteomic analysis of an intraductal papillary mucinous neoplasm (IPMN). Tissue microarrays comprised of 38 IPMNs were used for validation of candidate proteins. RESULTS: The proteomic analysis of the IPMN Liquid Tissue lysate resulted in identification of 1,534 peptides corresponding to 523 unique proteins. A subset of 25 proteins was identified that had previously been reported as upregulated in pancreatic cancer. Immunohistochemical analysis for two of these, deleted in malignant brain tumors 1 (DMBT1) and tissue transglutaminase 2 (TGM2), confirmed their overexpression in IPMNs. CONCLUSION: Global proteomics analysis using the Liquid Tissue workflow is a feasible approach for unbiased biomarker discovery in limited archival material, particularly applicable to precursor lesions of cancer.

Liquid Tissue: proteomic profiling of formalin-fixed tissues.

Identification and quantitation of candidate biomarker proteins in large numbers of individual tissues is required to validate specific proteins, or panels of proteins, for clinical use as diagnostic, prognostic, toxicological, or therapeutic markers. Mass spectrometry (MS) provides an exciting analytical methodology for this purpose. Liquid Tissue MS protein preparation allows researchers to utilize the vast, already existing, collections offormalin-fixed paraffin-embedded (FFPE) tissues for the procurement of peptides and the analysis across a variety of MS platforms.

The granulin-epithelin precursor/PC-cell-derived growth factor is a growth factor for epithelial ovarian cancer.

PURPOSE: The role of growth factors in ovarian cancer development and progression is complex and multifactorial. We hypothesized that new growth factors may be identified through the molecular analysis of ovarian tumors as they exist in their native environment. EXPERIMENTAL DESIGN: RNA extracted from microdissected serous low malignant potential (LMP) and invasive ovarian tumors was used to construct cDNA libraries. A total of 7300 transcripts were randomly chosen for sequencing, and those transcripts were statistically evaluated. Reverse transcription-PCR and immunohistochemistry were used to validate the findings in tumor tissue samples. Ovarian cancer cell lines were used to test gene effects on monolayer growth, proliferative capacity, and density-independent growth. RESULTS: Analysis of the pooled library transcripts revealed 26 genes differentially expressed between LMP and invasive ovarian cancers. The granulin-epithelin precursor [GEP/PC-cell derived growth factor (PCDGF)] was expressed only in the invasive ovarian cancer libraries (P < 0.028) and was absent in the LMP libraries (0 of 2872 clones). All of the invasive tumor epithelia, 20% of the LMP tumor epithelia, and all of the stroma from both subsets expressed GEP by reverse transcription-PCR. Immunohistochemical staining for GEP was diffuse and cytosolic in invasive ovarian cancer tumor cells compared with occasional, punctate, and apical staining in LMP tumor epithelia. Antisense transfection of GEP into ovarian cancer cell lines resulted in down-regulation of GEP production, reduction in cell growth (P < 0.002), decrease in the S-phase fraction (P < 0.04), and loss of density-independent growth potential (P < 0.01). CONCLUSION: cDNA library preparation from microdissected tumor epithelium provided a selective advantage for the identification of growth factors for epithelial ovarian cancer. Differential granulin expression in tumor samples and the antiproliferative effects of its antisense down-regulation suggest that GEP may be a new autocrine growth factor and molecular target for epithelial ovarian cancer.

Laser printing of pluripotent embryonal carcinoma cells.

A technique by which to print patterns and multilayers of scaffolding and living cells could be used in tissue engineering to fabricate tissue constructs with cells, materials, and chemical diversity at the micron scale. We describe here studies using a laser forward transfer technology to print single-layer patterns of pluripotent murine embryonal carcinoma cells. This report focuses on verifying cell viability and functionality as well as the ability to differentiate cells after laser transfer. We find that when cells are printed onto model tissue scaffolding such as a layer of hydrogel, greater than 95% of the cells survive the transfer process and remain viable. In addition, alkaline comet assays were performed on transferred cells, showing minimal single-strand DNA damage from potential ultraviolet-cell interaction. We also find that laser-transferred cells express microtubular associated protein 2 after retinoic acid stimulus and myosin heavy chain protein after dimethyl sulfoxide stimulus, indicating successful neural and muscular pathway differentiation. These studies provide a foundation so that laser printing may next be used to build heterogeneous multilayer cellular structures, enabling cell growth and differentiation in heterogeneous three-dimensional environments to be uniquely studied.

Absolute quantitation of Met using mass spectrometry for clinical application: assay precision, stability, and correlation with MET gene amplification in FFPE tumor tissue.

BACKGROUND: Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for 'high Met' expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue. METHODS: After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH). RESULTS: Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/µg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (<150 amol/µg to 4669.5 amol/µg. High correlation was observed between SRM Met expression and both MET GCN and MET/CEP7 ratio as determined by FISH (n = 30; R2 = 0.898). IHC did not correlate well with SRM (n = 44; R2 = 0.537) nor FISH GCN (n = 31; R2 = 0.509). A Met SRM level of ≥1500 amol/µg was 100% sensitive (95% CI 0.69-1) and 100% specific (95% CI 0.92-1) for MET amplification. CONCLUSIONS: The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET tumors. These results demonstrate a novel clinical tool for efficient tumor expression profiling, potentially leading to better informed therapeutic decisions for patients with GEC.

Use of formalin-fixed, paraffin-embedded tissue for proteomic biomarker discovery.

Application of mass spectrometry to proteomic analysis of tissue is a highly desirable approach to discovery of disease biomarkers due to a direct correlation of findings to tissue/disease histology and in many respects obviating the need for model systems of disease. Both frozen and formalin-fixed, paraffin-embedded (FFPE) tissue can be interrogated; however, worldwide access to vastly larger numbers of highly characterized FFPE tissue collections derived from both human and model organisms makes this form of tissue more advantageous. Here, an approach to large-scale, global proteomic analysis of FFPE tissue is described that can be employed to discover differentially expressed proteins between different histological tissue types and thus discover novel protein biomarkers of disease.

Application of selected reaction monitoring for multiplex quantification of clinically validated biomarkers in formalin-fixed, paraffin-embedded tumor tissue.

One of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry-based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, human EGF receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue-SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry. The assays were preclinically validated by comparing Liquid Tissue-SRM quantitation of FFPE cell lines with enzyme-linked immunosorbent assay/electrochemiluminescence quantitation of fresh cells (R(2) > 0.95). Clinical performance was assessed on two cohorts of breast cancer tissue: one cohort of 10 samples with a wide range of HER2 expression and a second cohort of 19 HER2 IHC 3+ tissues. These clinical data demonstrate the feasibility of quantitative, multiplexed clinical analysis of proteomic markers in FFPE tissue. Our findings represent a significant advancement in cancer tissue analysis because multiplexed, quantitative analysis of protein targets in FFPE tumor tissue can be tailored to specific oncological indications to provide the following: i) complementary support for anatomical pathological diagnoses, ii) patient stratification to optimize treatment outcomes and identify drug resistance, and iii) support for the clinical development of novel therapies.

Selected Reaction Monitoring (SRM) Analysis of Epidermal Growth Factor Receptor (EGFR) in Formalin Fixed Tumor Tissue.

BACKGROUND: Analysis of key therapeutic targets such as epidermal growth factor receptor (EGFR) in clinical tissue samples is typically done by immunohistochemistry (IHC) and is only subjectively quantitative through a narrow dynamic range. The development of a standardized, highly-sensitive, linear, and quantitative assay for EGFR for use in patient tumor tissue carries high potential for identifying those patients most likely to benefit from EGFR-targeted therapies. METHODS: A mass spectrometry-based Selected Reaction Monitoring (SRM) assay for the EGFR protein (EGFR-SRM) was developed utilizing the Liquid Tissue®-SRM technology platform. Tissue culture cells (n = 4) were analyzed by enzyme-linked immunosorbent assay (ELISA) to establish quantitative EGFR levels. Matching formalin fixed cultures were analyzed by the EGFR-SRM assay and benchmarked against immunoassay of the non-fixed cultured cells. Xenograft human tumor tissue (n = 10) of non-small cell lung cancer (NSCLC) origin and NSCLC patient tumor tissue samples (n = 23) were microdissected and the EGFR-SRM assay performed on Liquid Tissue lysates prepared from microdissected tissue. Quantitative curves and linear regression curves for correlation between immunoassay and SRM methodology were developed in Excel. RESULTS: The assay was developed for quantitation of a single EGFR tryptic peptide for use in FFPE patient tissue with absolute specificity to uniquely distinguish EGFR from all other proteins including the receptor tyrosine kinases, IGF-1R, cMet, Her2, Her3, and Her4. The assay was analytically validated against a collection of tissue culture cell lines where SRM analysis of the formalin fixed cells accurately reflects EGFR protein levels in matching non-formalin fixed cultures as established by ELISA sandwich immunoassay (R2 = 0.9991). The SRM assay was applied to a collection of FFPE NSCLC xenograft tumors where SRM data range from 305amol/µg to 12,860amol/µg and are consistent with EGFR protein levels in these tumors as previously-reported by western blot and SRM analysis of the matched frozen tissue. In addition, the SRM assay was applied to a collection of histologically-characterized FFPE NSCLC patient tumor tissue where EGFR levels were quantitated from not detected (ND) to 670amol/µg. CONCLUSIONS: This report describes and evaluates the performance of a robust and reproducible SRM assay designed for measuring EGFR directly in FFPE patient tumor tissue with accuracy at extremely low (attomolar) levels. This assay can be used as part of a complementary or companion diagnostic strategy to support novel therapies currently under development and demonstrates the potential to identify candidates for EGFR-inhibitor therapy, predict treatment outcome, and reveal mechanisms of therapeutic resistance.

Laser printing of single cells: statistical analysis, cell viability, and stress.

Methods to print patterns of mammalian cells to various substrates with high resolution offer unique possibilities to contribute to a wide range of fields including tissue engineering, cell separation, and functional genomics. This manuscript details experiments demonstrating that BioLP Biological Laser Printing, can be used to rapidly and accurately print patterns of single cells in a noncontact manner. Human osteosarcoma cells were deposited into a biopolymer matrix, and after 6 days of incubation, the printed cells are shown to be 100% viable. Printing low numbers of cells per spot by BioLP is shown to follow a Poisson distribution, indicating that the reproducibility for the number of cells per spot is therefore determined not by the variance in printed volume per drop but by random sampling statistics. Potential cell damage during the laser printing process is also investigated via immunocytochemical studies that demonstrate minimal expression of heat shock proteins by printed cells. Overall, we find that BioLP is able to print patterns of osteosarcoma cells with high viability, little to no heat or shear damage to the cells, and at the ultimate single cell resolution.

Proteomic analysis of formalin-fixed prostate cancer tissue.

Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue would enable retrospective biomarker investigations of this vast archive of pathologically characterized clinical samples that exist worldwide. These FFPE tissues are, however, refractory to proteomic investigations utilizing many state of the art methodologies largely due to the high level of covalently cross-linked proteins arising from formalin fixation. A novel tissue microdissection technique has been developed and combined with a method to extract soluble peptides directly from FFPE tissue for mass spectral analysis of prostate cancer (PCa) and benign prostate hyperplasia (BPH). Hundreds of proteins from PCa and BPH tissue were identified, including several known PCa markers such as prostate-specific antigen, prostatic acid phosphatase, and macrophage inhibitory cytokine-1. Quantitative proteomic profiling utilizing stable isotope labeling confirmed similar expression levels of prostate-specific antigen and prostatic acid phosphatase in BPH and PCa cells, whereas the expression of macrophage inhibitory cytokine-1 was found to be greater in PCa as compared with BPH cells.