Antibody-based Profiling of Expression Patterns using Cell and Tissue Microarrays

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1 Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 344 Antibody-based Profiling of Expression Patterns using Cell and Tissue Microarrays SARA STRÖMBERG ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2008 ISSN ISBN urn:nbn:se:uu:diva-8680

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3 List of papers This thesis is based on the following papers, which are referred to in the text by their roman numerals I Andersson A-C, Strömberg S, Bäckvall H, Kampf C, Uhlen M, Wester K, and Ponten F: Analysis of Protein Expression in Cell Microarrays: A Tool for Antibody-based Proteomics. J Histochem Cytochem 54: (2006). II Strömberg S, Gry M, Asplund C, Sköllermo A, Persson A, Wester K, Kampf C, Nilsson P, Andersson A-C, Uhlen M, Kononen J, Ponten F, and Asplund A: A high-throughput strategy for protein profiling in cell microarrays using automated image analysis. Proteomics. Jun;7(13): (2007). III Strömberg S, Gry M, Asplund A, Rimini R, Lundeberg J, Nilsson P, Ponten F, and Olsson MJ: Transcriptional profiling of melanocytes from patients with vitiligo vulgaris. Accepted for publication in Pigment Cell and Melanoma Research (published article online, 2007). IV Strömberg S, Agnarsdóttir M, Magnusson K, Bolander Å, Lundberg E, Asplund A, Ryan D, Rafferty M, Gallagher WM, Uhlen M, Bergqvist M, and Ponten F: Identification of the novel melanocytic markers Discs large homolog 5 and Syntaxin-7, in benign melanocytic lesions and malignant melanoma. Submitted V Strömberg S, Asplund A, Fagerberg L, El-Obeid A, Gry M, Nilsson K, Nilsson P, Rimini R, Uhlen M, and Ponten F: The proteomic landscape of in vitro growth: A comparative analysis using antibody-based proteomics. Manuscript All published material is reproduced with permission from the publishers.

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5 Contents Introduction...9 Proteomics...10 Antibody-based proteomics...12 Immunohistochemistry...14 Image capture and analysis in immunohistochemistry...16 Tissue microarray technology...18 Cell lines...20 Transcript profiling...23 DNA microarrays...23 Quantitative real-time PCR...24 Diseases of melanocytic origin...25 Vitiligo vulgaris...26 Malignant melanoma...27 Present Investigation...30 Aims...30 Results and discussion...31 Paper I...31 Paper II...32 Paper III...33 Paper IV...34 Paper V...35 Conclusions and future perspectives...36 Populärvetenskaplig sammanfattning...38 Acknowledgements...41 References...43

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7 Abbreviations 2-DE ALM CMA C t DAB ESI FFPE HPA HRP H-score ICAT Ig IHC LMM mab MALDI MS msab NMM OCT pab PCR PrEST PTM Rb recab SDS-PAGE SILAC SSM TMA two-dimensional gel electrophoresis acral lentiginous melanoma cell microarray cycle threshold 3,3 -diaminobenzidine electrospray ionization formalin-fixed paraffin-embedded human protein atlas horseradish peroxidase histochemical score isotope-coded affinity tag immunoglobulin immunohistochemistry lentigo malignant melanoma monoclonal antibody matrix-assisted laser desorption/ionization mass spectrometry monospecific antibody nodular malignant melanoma optimal cutting temperature polyclonal antibody polymerase chain reaction protein epitope signature tag post-translational modification retinoblastoma recombinant antibody sodium dodecyl sulfate polyacrylamide gel electrophoresis stable isotope labeling with amino acids in cell culture superficial spreading melanoma tissue microarray

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9 Introduction The flow of genetic information in a living cell can be described as a process, in which the DNA copies itself (replication), is transcribed into messenger RNA (mrna) (transcription), which transfers the genetic code to the ribosomes, where it is translated into proteins (translation) (Figure 1). In 2001, the International Human Genome Sequencing Consortium (IHGSC) 1 and Celera Genomics 2 each reported a first draft version of the human genome sequence. This first overview was updated in 2004, when the IHGSC reported the completion of the human genome sequence with 99% coverage 3. Since then, numerous studies have analyzed patterns of gene expression in normal and diseased tissues, primarily on a transcriptional level using microarray-based methods. Due to post-transcriptional and translational regulation of gene expression, the level of transcription of a gene only gives a rough estimate of its level of expression into a protein. Therefore, the mrna signature of a cell may not entirely mirror its protein profile. As for most genes, the gene function resides in the protein encoded by the gene, the logical next step is to explore the functional components of the human genome also on a protein level. Proteins are involved in a variety of processes, and constitute the functional elements of a cell, acting as building blocks, enzymes, transport and storage molecules, part of the immune system, growth regulators etc. Analysis of protein interactions and modifications, protein structure and site of expression as well as subcellular localization, will lead to a more comprehensive understanding of protein function. This thesis describes strategies for investigating gene and protein expression profiles in human cells and tissues, and for elucidating possible associations between protein signatures and disease. Replication Transcription AAAAA Translation DNA mrna PROTEIN Figure 1. The central dogma of molecular biology, involving the processes of replication, transcription and translation. 9

10 Proteomics The term proteome was originally used to describe the complete set of proteins expressed by a genome. Accordingly, the large-scale functional and structural analysis of all expressed proteins is called proteomics, which can be divided into to main approaches, separation-based and probe-based proteomics. Proteomic studies today do not only involve the characterization of all the proteins of a given cell, but also consider the dynamic entities of a proteome. Depending on cellular state and internal or external influences, proteins undergo regulatory modifications, change subcellular location, associate with other molecular components of a cell, become degraded etc. Thus, a proteome differs from cell to cell and also within the life cycle of a cell. In the beginning of 2008, the Ensembl database ( predicted the number of protein-coding genes in humans to be This estimation of proteome size relies on the assumption that a single representative protein is produced from every gene locus, which has been termed the non-redundant set of proteins 5. In a recent study, Clamp et al, estimated this number to , including evolutionary conserved protein-coding genes 6. If one includes the different protein isoforms produced by alternative splicing of mrna, the number of proteins increases. Several studies have reported a high rate of alternative splicing in the human genome, with 35-59% of human genes showing evidence of at least one splice form 1,7-10. With the use of microarrays, one study even suggests that at least 74% of human multi-exon genes are alternatively spliced 11. There are different routes to splicing, but exclusion of one or more exons probably constitute one of most common events of alternative splicing 12. Differential transcription start- and polyadenylation sites also contribute to different mrna isoforms 13. In a review by Uhlen and Ponten, the number of protein variants represented by splicing and proteolysis was estimated to be in a range of to In addition, different types of protein isoforms are formed through posttranslational modifications (PTMs). After translation, as a response to different extracellular or intracellular signals, PTMs change the properties of a protein by cleavage or addition of different functional subgroups to the amino acids of the protein. Some of the most common forms of PTMs are phosphorylations, acetylations, methylations and glycosylations 14. As a result of the range of regulatory events involved in the transcription and translation processes, an estimation of proteome size is obviously dependent on the definitions for different protein categories 5. 10

11 Separation-based proteomic analyses today involve a range of sophisticated technologies aiming at identification and characterization of proteins and their interactions. Already in the late 1970s, proteomics studies were initiated when protein mixtures were separated on polyacrylamide gels using two-dimensional gel electrophoresis (2-DE) DE couples isoelectric focusing (IEF) in the first dimension with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, enabling a separation of proteins according to isoelectric point and molecular weight. Following separation, proteins can be visualized using different staining protocols, such as Coomassie or silver staining, and the intensity of staining of the resolved proteins can be quantified with gel-imaging software. One limitation of the 2-DE separation method has been a limited dynamic detection range. With a dynamic range of abundance of proteins in biological samples as high as 10 6, only the most abundant proteins can be detected by 2-DE if a crude protein mixture is analyzed 16. Today, improvements of the 2-DE technology with more sensitive staining methods and higher-resolving gels have led to enhanced visualization of proteins with a range of molecular mass and different biochemical properties 17,18. The next step is to determine the identity of the gel-separated proteins, and for this purpose one of the most important tools for proteomics research, mass spectrometry (MS), can be employed. Selected spots on the gel are excised and subjected to proteolytic digestion, and the generated peptides are ionized and characterized using MS. A mass spectrometer consists of an ion source, a mass analyzer measuring the mass to charge ratio (m/z), and a detector registering the number of ions at each m/z value 19. There are two main approaches to volatize and ionize the proteins or peptides used for mass spectrometric protein identification, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). With MALDI, the protein sample is mixed with a matrix compound and the samples are ionized via laser pulses 20. MALDI is then usually coupled to a time of flight (TOF) analyzer that measures the mass of intact peptides, resulting in a peptide mass fingerprint of the investigated protein. With ESI the peptides are ionized directly from a solution 21. ESI is then commonly coupled to ion trap analyzers in which the ions are first captured for a certain time in a threedimensional trapping field, and then selectively ejected for detection of the generated mass spectrum. The obtained peptide mass spectra and sequence data can then be matched against available protein databases. Mass spectrometry can also be used for quantitative proteomic analysis, together with techniques based on stable isotope protein labeling. The ICAT (isotope-coded affinity tag) reagent strategy, involves labeling of cysteine residues of proteins with a biotin-labeled reagent containing either a heavy or a light isotope 22. Two protein mixtures are treated with heavy or light ICAT reagents, respectively, and then analyzed by MS. The relative protein composition of the two samples is determined by the ratio of heavy/light 11

12 signal intensities of the tagged peptide pairs. In the SILAC (stable isotope labeling with amino acids in cell culture) method, cells are grown in cell culture media containing isotopically labeled amino acids, leading to metabolic incorporation of the labeled amino acid into all proteins of the cells 23. By comparing the mass spectra between labeled cells and cells grown in normal growth media, mass differences of proteins can be detected, reflecting relative protein abundance. Antibody-based proteomics A probe-based strategy that can be used to study proteins and proteomes, is antibody-based proteomics, which is depending on the accessibility of protein-specific antibodies. Such affinity reagents can be used in a wide range of applications, each of which depends on the specific binding properties of the antibody. Antibodies, or immunoglobulins (Ig), are key components of the adaptive immune system, designed to recognize and bind to foreign substances (antigens) invading the body of a host species. Owing to their ability to bind with high specificity to a target molecule, antibodies have been extensively used in a variety of laboratory assays for studying biomolecules. For clinical use, antibodies are important diagnostic tools for various diseases and they have also emerged as an important class of human therapeutics 24. The natural antibody response evoked by the immune system, is utilized in the production of antibodies, which typically involves the injection of a specific immunogen into an animal, most commonly a mouse, rabbit, rat or goat. Today, four major types of antibodies exist, each of them with their own explicit characteristics, making them more or less suitable for different applications. Monoclonal antibodies (mabs), are generated through the fusion of antibody-producing B-cells from immunized mice with immortalized murine myeloma cells, rendering a hybridoma, a process first described by Köhler and Milstein in The combination of immortal growth of the tumor cell and the antibody production capacity of the B cell, allows for indefinite production of a specific antibody. Because each hybridoma is derived from a single antibody-producing B-cell, the cells within the hybridoma are identical and generate the same antibody molecule, recognizing the same single epitope of an antigen. This approach offers a reproducible and continuous supply of antibodies with exquisite specificity, making them attractive for therapeutic and diagnostic applications. However, to obtain antibody-producing hybridomas of correct specificity a time-consuming screening process is needed, rendering the use of mabs in large-scale efforts less cost efficient. The production of polyclonal antibodies (pabs) on the other hand is a more efficient process generating antiserum with multi-epitope binding 12

13 through immunization of animals, often rabbits. Because the antiserum contains a mixture of different antibodies recognizing different epitopes on the antigen, pabs recognize the antigen from different orientations. This is advantageous in multi-assay applications, when detection of proteins in both native and denatured forms is needed. On the other hand, pabs may exhibit cross reactivity to other proteins, since the strategy for generation of pabs generally involves no or limited purification procedures. Additionally, pabs suffer from lack of reproducibility, because re-immunization of the same antigen does not necessary yield equal antibody reactivity and titer. Monospecific affinity-purified antibodies (msabs) have both polyclonal and monoclonal properties. They are produced in rabbits through the immunization of suitable protein fragments, amino acids in length, which are selected to represent a part of the protein with lowest similarity to all other human proteins. These fragments, termed protein epitope signature tags (PrESTs), are designed with the aid of a computer program, which also selects primers to be used in the subsequent RT-PCR cloning of the PrEST 26. The DNA fragment is amplified using an mrna pool from human tissues and cloned into a plasmid vector where the human gene fragment is fused to a His 6 -tag and an albumin-binding domain. After expression in Escherichia coli (E.coli), the PrEST protein is purified using the His 6 -tag and evaluated using SDS-PAGE and MS, ensuring immunization of correct antigen 27. The resulting polyclonal antiserum is purified in a selection step, using the antigen as ligand, yielding antigen-specific multi-epitope binding msabs 28. This procedure has been proven to be an efficient approach for high-throughput generation of affinity reagents, which currently is used for a proteome-wide screening of protein expression 29,30. In this project, called the Swedish Human Protein Atlas (HPA) project, the antibodies are primarily used for immunohistochemical analyses of protein expression in samples from human normal and cancer tissues 31. Images of staining results are then displayed on a website ( which since the launch in 2005 has been updated every year and currently contains protein expression data from more than 3000 antibodies. For the production of recombinant antibody fragments the phage display technology can be employed, which involves the expression of proteins, including antibodies on the surface of a filamentous phage 32,33. Antibody genes of interest are fused to genes encoding a phage coat protein, thus being inserted into the phage genome. Since phages are viruses that infect a variety of Gram-negative bacteria, they can be used to infect E.coli bacteria. Upon transformation, E.coli starts to produce and secrete new phage particles, which express the encoded protein as a fusion product to the phage coat protein on its surface. In this way large phage libraries can be generated and subsequently screened to select specific phage particles with desired binding specificities 34,35. Recently, the Sanger Atlas of protein expression project described the generation of such a phage display library containing over

14 clones, which was used for high-throughput antibody generation 36. The generated antibodies were subsequently used for immunohistochemical analysis on frozen tissue microarrays consisting of normal adult human tissue and developmental and adult murine tissue. Besides using antibodies for in situ localization studies using immunohistochemistry, which will be described later, several other proteomic analyses depend on antigen-antibody recognition. Western Blot analysis can be used to detect a protein of interest in a protein lysate extracted from a cell or tissue sample. First, gel electrophoresis, typically SDS-PAGE is performed, enabling separation of proteins according to their molecular weight. Proteins are then transferred to a membrane, where a specific protein can be probed with an antibody and visualized using enzyme-based detection. Thus, using information about the molecular weight of a protein, western blot analysis can be used to measure relative amounts of proteins present in different samples. Based on the same principle, a method for transferring proteins from FFPE tissues slides to membranes has been described 37. Similarly, antibodies are applied for detection of proteins, but in contrast to western blot, this approach preserves the morphological perspective of protein expression. Protein array technologies are also available for detection of proteins in various sample types, such as serum, plasma and tissue lysates. Different assay formats are available, of which three standard types will be briefly described here. In the labeled-based assay, antibodies are attached to a solid support and captures chemically labeled proteins, allowing for direct detection of the interaction. This assay allows for a competitive two color approach, where two samples are labeled independently with different fluorophores, and mixed before applying them to the antibody array 38. Another format is the sandwich assay, in which immobilized antibodies capture unlabeled proteins in a biological sample, which subsequently can be detected using a second detection antibody 39,40. In contrast to previously described protein arrays, which immobilize the antibodies, reverse-phase protein arrays, immobilize the protein-containing cell lysates onto a surface 41. Presence of target proteins in the lysates is then determined by addition of specific antibodies. Immunohistochemistry Immunohistochemistry (IHC) provides information on the expression level and the localization of an antigen, and has therefore become the standard in situ assay to assess protein expression 42. IHC comprises the application of antibodies to tissue sections, and the subsequent binding of the antibody to its antigen. Enzyme-based or fluorescence-based color-producing reactions then yield information of the localization and expression of the antigen, which can be examined in a microscope. To arrest tissue decomposition and 14

15 preserve tissue morphology, the majority of tissue samples in pathology archives are immersed in 10% formalin (4% formaldehyde), dehydrated through a series of graded alcohols, cleared with xylene and subsequently embedded in paraffin blocks, generating formalin-fixed paraffin embedded (FFPE) tissues. The exact mechanisms by which formaldehyde acts in fixing tissue are not fully clarified, but it involves cross-linking of reactive sites within and between proteins through the formation of methylene bridges 43,44. Thus, it is believed that formalin fixation alters the conformation of the antibody-contact sites, the epitopes, making them inaccessible to antibody binding, leading to poor antigenicity of FFPE tissues. In addition to formaldehyde, other fixatives are available, such as acetone and alcohol-based fixatives, which act by coagulating tissue proteins 45. Although some of these fixatives preserve antigenicity better than formaldehyde, they do not preserve morphology as well. In order to restore the formalin-modified antigenicity in FFPE tissue, antigen retrieval techniques are typically employed. The most widely used retrieval method is heat induced epitope retrieval (HIER), originally described by Shi and colleagues 46. Successful HIER is achieved by boiling tissue sections in aqueous solutions using water baths, ovens and microwaves 47. Using different retrieval solutions such as citrate buffers with different phs the extent of retrieval can be optimized 48,49, and specially designed pressure cookers allow a more reproducible retrieval, with exact temperature and time settings 50. Although the mechanism of HIER is not fully understood, it has been proposed by several investigators, that heat treatment disrupts the formalin-induced intra- and intermolecular crosslinks of proteins 49,51. Sompuran et al studied the protein epitopes for a number of antibodies, effective for formalin fixed tissues treated with antigen retrieval and found that these antibodies shared the characteristics of recognizing linear protein epitopes 52. Based on this data, they propose a model explaining the mechanism of HIER, in which the formalin-induced cross-links are reversed, thereby regenerating a linear peptide epitope. To obtain a strong and specific visualization of protein expression with IHC, different detection systems can be employed. In general, a primary antibody is allowed to incubate with the antigen of interest. Thereafter secondary reagents are applied, which bind to the primary antibody. The secondary reagents can constitute a secondary antibody, labeled with an enzyme such as horseradish peroxidase (HRP). The following visualization process depends on an enzyme-substrate reaction, which converts a colorless chromogen into a colored end product. Through addition of the substrate hydrogen peroxide (H 2 O 2 ) and a chromogen called 3,3 -diaminobenzidine (DAB) to the HRP-labeled secondary antibody, an enzyme-substrate complex will form leading to oxidation of DAB, which is converted into a brown color (Figure 2). This brown color can then be visualized in a microscope, representing antibody-antigen binding and thus site of protein expression. In addition to the reagents mentioned above, other enzyme-substrate complexes are 15

16 available as well as other chromogens. There are also detection systems that do not depend on enzyme-substrate reactions, but instead use fluorochromecoupled secondary antibodies. Today, several instruments are available that automate nearly all the inherent steps of IHC, from antigen retrieval to chromogen visualization. This not only increases the throughput of IHC, it also increases reproducibility, since all slides are treated identically and simultaneously, which is difficult to accomplish in manual IHC. DAB+H 2 O 2 brown color secondary antibody HRP primary antibody tissue antigen Figure 2. Simplified schematic illustration of the immunohistochemical staining procedure. Image capture and analysis in immunohistochemistry Analysis of immunohistochemical staining patterns is commonly performed by visual examination of tissue sections in a microscope. Parameters such as staining intensity are qualitatively interpreted using descriptive terms as weak, moderate and strong. Fraction positive cells is described in large interval estimates, for example as <25%, 25-50% or >75%. Manual microscopical analysis of IHC is the general standard for pathologist-based diagnostics of clinical lesions but its qualitative, subjective characteristics renders it less useful for quantitation of protein content. Different scoring systems aiming at a more quantitative measurement of immunoreactivity have been employed for IHC 53,54. The modified histochemical score (H-score) calculates the product of staining intensity and the percent immunostained cells, trying to translate nominal scores into a more continuous scale 55. Nevertheless, the H-score and similar systems still suffer from subjectivity and lack of scoring reproducibility. 16

17 Analysis of protein expression using automated IHC and tissue microarrays generates large amounts of information within a short time period, making tedious manual microscopical analysis a bottleneck. With the use of automated image capture and image analysis, the throughput and reproducibility of IHC analysis can effectively be increased. Today, various slidescanner systems are available which automatically load tissue slides into a color microscope, thereby generating high-resolution images of immunostained tissue sections. Lately, different technologies for automated examination of immunohistochemical staining patterns have been developed and utilized for analysis of subcellular localization and quantitation of protein expression. The automated cellular imaging system (ACIS) combines automated microscopy with image analysis, generating digitized images of tissue sections which are analyzed based on brown stain intensity levels 56. This method has successfully been used for detection and analysis of bone marrow metastases as well as for evaluation of a prognostic marker for prostate cancer 57,58. Another system which has been used for evaluation of protein expression in tissue microarrays, is the automated quantitative analysis system (AQUA), which depends on fluorescent tags to separate tumors from stroma and to define subcellular compartments 59. AQUA measures fluorescent signals from the target antigen on a continous 1000-point scale, enabling a quantitative analysis of relationship between levels of protein expression and prognostic factors in different cancer TMAs 60,61. The purpose of image analysis is to acquire information on staining parameters similar to that obtained by manual microscopical analysis. In order to attain such information, digital images have to be classified and labeled with meaningful information. Two available approaches for image classification into logical patterns are the classic pixel-based and the object-oriented image analysis approach. Pixels are the smallest units of digital images, constituting an area of color (color-images) or grayscale (black and white images). The pixel-based approach uses exclusively the value of each pixel to classify the image pixel-wise, and each pixel can only be classified into one class. In object-oriented image analysis information from surrounding pixels is combined and used to group pixels into meaningful areas. Instead of classifying single pixels, the digital image will be segmented into homogeneous image objects, which are being extracted for classification. The classification process is performed using a fuzzy classifier, which takes different attributes such as color, size, shape, etc into consideration. By combining multiple features using And, Or, Max, Min etc, different class descriptions are obtained. For histological images the object classification is a gradual and iterative process, starting from smaller structures in tissues such as nucleoli and proceeding into larger structures such as cells, glands etc. Finally a complex network consisting of all tissue structures in an image is generated. Since tissue samples are heterogeneous, with structures of varying shape and 17

18 size, an object-oriented approach is probably better for classifying its contents, as compared to classification based on single pixels. Tissue microarray technology In cellular pathology, microscopical study of tissue sections from excised formalin-fixed, paraffin-embedded tumors represents the golden standard for determining a diagnosis of cancer. Microscopical analysis of tissue sections together with immunohistochemistry provides an efficient tool to assess expression and localization patterns of proteins in virtually all human tissues and organs. However, traditional methods of cellular pathology are often tedious, time-consuming and relying on extensive manual interaction. With the introduction of the tissue microarray (TMA) technology and automated immunohistochemistry the analysis of protein distribution has efficiently been translated into a high-throughput procedure. As early as 1986 a forerunner to the modern tissue microarray technology was presented: the multitumor (sausage) tissue block. With this technique, 100 or more tissue samples were assembled together in a normal-sized paraffin block, allowing for simultaneous immunohistologic testing of numerous tissue samples on a single slide with one drop of antibody 62. In 1998, this technique was refined when Kononen et al introduced the TMA technology, described as the process where minute tissue cylinders (diameter 0.6 mm) were removed from hundreds of different donor tissue blocks to be placed in an orderly fashion in one recipient paraffin block 63. Together with automated immunohistochemistry this technique enables a time and costefficient procedure as well as a standardized method for studying gene expression at a cellular level (Figure 3). TMAs are constructed using manual or automated arraying instruments, which enable the accurate transfer of hundreds of donor tissue cores (diameter mm) from paraffin-embedded tissue blocks into a recipient block with a consistent core depth. The core depth is essential as it determines the number of representative sections that can be obtained from the complete array. The use of small core biopsies instead of complete tissue sections also allows for maximization of tissue resources. 18

19 Figure 3. A tissue microarray glass slide with magnifications of an immunohistochemically stained tissue core representing a malignant melanoma. The key to the construction of a successful TMA is the selection of appropriate areas from the donor blocks to ensure transfer of representative cores to the recipient block. This is achieved by reviewing hematoxylin and eosin (H&E) stained sections of each donor block. With the coordinated placement of each core into the recipient block, a link to the tissue of origin and its available clinical data is maintained, which can be used in subsequent prognostic clinical evaluation of potential biomarkers. One major concern with the use of TMAs is that mm small samples might not be representative for the entire tumor. By comparing staining results from TMAs with their corresponding tissue section, it has been shown that duplicate or triplicate tissue cores from each donor block is sufficient to obtain matching results 64,65. In a TMA consisting of breast tumor material, even a single sample from each tumor was sufficient to identify relationships between molecular markers and clinical outcome 66. However, because the intra-tumoral heterogeneity of protein expression might differ between tumor types, the number of cores needed to ensure representativity should optimally be determined for every tumor type. Although most TMA studies so far have used FFPE tissues, TMA constructions using other sources of donor material have been described. To avoid the compromised antigenicity and RNA degradation associated with FFPE tissues, frozen TMAs have been prepared, arraying frozen tissues into a recipient block consisting of the optimal cutting temperature (OCT) compound 67,68. Besides tissue samples, methods for arraying in vitro cultured cell lines and proteins 72 are also available. As described in the pioneer article for the TMA technology by Kononen et al, the major application of TMAs has been in cancer research, and typically to identify relevant associations between molecular markers and clini- 19

20 cal endpoints such as disease-free and overall survival. TMAs have been used for identification of diagnostic markers specific for a certain tumor type, to associate molecular markers with cancer stage and in the prediction of treatment-responding patient groups. Furthermore, TMA technology has been widely used as a validation tool for data from gene expression microarrays 73,74. In the Swedish Human Protein Atlas project, TMAs consisting of 48 different normal tissues and 20 common cancer tissues are used for highthroughput tissue profiling using immunohistochemistry 75. The aim is to generate a comprehensive web-based protein atlas that displays digital images of IHC-stained tissues, providing information about protein expression in a range of human tissues. Using a recently developed search tool, queries can be specified to identify proteins expressed in a cell-, tissue- or cancerspecific manner 76. Proteins with a potential clinical interest can then be further investigated in TMAs containing a larger patient cohort material. However, there are aspects of TMA technology that present challenges in biomarker discovery, including issues related to tissue handling, the immunohistochemical staining procedure and evaluation of staining patterns. Although, IHC on TMAs reduce inter-experimental variability, several features of IHC, such as staining protocols and methods for antigen retrieval may vary between laboratories, resulting in difficulties in reproducing results. Dilution of the antibody, which typically is manually determined, has been shown to be crucial for evaluation of biomarker expression. McCabe et al, demonstrated that different antibody concentrations clearly affected the relationship between biomarker expression and clinical outcome 77. Also, the subjective microscopical scoring system remains an issue, generating nonquantitative results that suffer from inter-observer variability. Cell lines Cell cultures are widely used as basic research tools in biology and medicine, and particularly as in vitro models in cancer research. Usually, a distinction is made between cultured cells capable of unlimited amount of cell doublings and cultured cells with a limited lifespan. The former are defined as cell lines, which have undergone a change allowing them to grow indefinitely, while the latter are primary cultures that can be grown in vitro for extended periods of time in the presence of certain growth factors, but ultimately go into senescence. As first described by Hayflick, normal cells proliferate in culture until they reach a state where proliferation ceases 78. An advantage of primary cells is that they constitute direct representatives of their tissue of origin, but their finite lifespan may hamper long-term studies. Established cell lines on the other hand provide an almost unlimited supply of cells, allowing them to be used for continuous long-term studies. The term continuous cell line has been defined by the Terminology committee of the 20

21 tissue culture association as a culture which is apparently capable of an unlimited number of population doublings; often referred to as immortal cell culture 79. In contrast to cell lines derived from tumors, which show an indefinite life span and are immortal, normal diploid cells rarely spontaneously transform into immortal cell lines. Instead physical, chemical or viral agents have been used for immortalization of normal primary cells. Long-term treatment with carcinogenic agents has been widely used to immortalize human breast epithelial cells, resulting in chromosomal abnormalities, anchorage-independent growth and tumorigenicity in nude mice 80. Transfection of cells with the viral oncogene SV40 T antigen successfully generates cells with extended lifetime as a result of binding and inactivation of the growth suppressors p53 and retinoblastoma (Rb) 81. Other immortalization protocols involve human telomerase reverse transcriptase (htert) transfection with subsequent telomerase activation, which for some cells is sufficient for immortalization 82. Today a wide variety of different cell lines are available throughout the scientific community and repositories such as the American Type Culture Collection (ATCC) and Deutsche Sammlungen von Mikroorganismen und Zell kulturen (DSMZ), which acquire, preserve, authenticate and distribute reference cell lines for research use. Cancer cell lines have successfully been employed to investigate chemotherapeutic sensitivity 83,84. In the late 1980s, the US National Cancer Institute (NCI) 60 anticancer drug screen was initiated as an in vitro drug-discovery tool based on a panel of cell lines representing nine tumor types, leukemia, colon, breast, lung, CNS, renal, melanoma, ovarian and prostate 85. Early findings described mechanisms of action regarding growth inhibition and tumor cell killing for a variety of compounds and later the screen led to the identification of the anticancer drug bortezomib, used for treatment of myeloma 85. However, the validity of experimental observations made with in vitro cultured cell lines has been questioned, due to the discovery of crosscontaminated cell lines used under false descriptions 86,87. It has been shown, that many cell lines described in the literature over the years are actually derived from the first cell line ever established, the HeLa cell line 88. According to MacLeod and co-authors from the German DSMZ, 18% of cell lines donated to the cell bank were contaminated or misidentified 89. Later, they showed a similar frequency ( 15%) of cross-contamination in 550 human leukemia-lymphoma cell lines, independent of whether the cell lines were obtained from original investigators or secondary sources 90. Thus, to avoid use of false cell lines, there is a need to detect cross-contamination and regularly authenticate in vitro cultured cell lines. A combination of cytogenetic karyotyping and DNA fingerprinting have been used for cell line authentication 91, and more recently automated use of short tandem repeats (STR) was described as an efficient method for cell line identification 92. Another issue is the contamination of cell cultures with microorganisms, 21

22 such as mycoplasma. In a study of 462 leukemia cell lines, mycoplasma infection was detected in 28% of the investigated cell lines 93. Data is available that mycoplasma infections not only affect the metabolism of the cells, but also induce cytogenetic changes 94,95. For detection of mycoplasma contamination of cell cultures, a number of techniques can be employed, one of which is the polymerase chain reaction, providing a fast and accurate analysis of mycoplasma infection 96. Scientific interpretations of results based on assays using cell lines, also depend on how well any given cell line serves as a representative for its tissue of origin and whether the cells have retained the features of the cells from which they were derived. Many cell lines have been in culture for a long period of time, possibly affecting the genetic and phenotypic stability of the cells. Different immortalization methods may also confer altered genoand phenotype to the immortalized cells. In a comparative study of MCF-7 breast cancer cell lines obtained from different laboratories, differences in growth rate, karyotype and hormone receptor activity was demonstrated between the cell lines, although a similar morphology was observed 97. Conversely, identical gene configurations of the tumor suppressors p15 and p16 were found in paired leukemia/lymphoma cell lines and corresponding primary cell material, suggesting that these alterations were not acquired in vitro 98. Since many cell lines have defects in genes involved in detection and repair of DNA damage, their genotype can change over time in culture. Therefore, to minimize genetic drift and phenotypical change during in vitro culture, the United Kingdom Coordinating Committee on Cancer Research (UKCCCR) guidelines for the use of cell lines in cancer research, suggest that cells should not be grown continuously, but instead be replenished from frozen stocks after a maximum of ten passages or three months continuous culture 99. Recently, several investigators have used gene expression profiling of cell lines and corresponding in vivo tumor tissues to evaluate the use of cell lines as models for cancer tissue. Using gene expression profiles of the NCI-60 cell lines, Sandberg and Ernberg created a tissue similarity index (TSI), measuring the similarity of gene expression between cell lines and different tumor types 100. A high TSI score toward their tumor of origin was detected for 34 of the cell lines, but the analysis also identified seven of the cell lines as being of another origin than presumed. In a similar analysis of cervical cancer cell lines, it was shown that many of the investigated cell lines retained a high expression correlation to cervical cancer, but cell lines with poor correlation was also identified, suggesting that these should be avoided as models for cervical cancer 101. To summarize, the validity of results derived from research on in vitro cultured cell lines is highly dependent on the use of appropriate cell lines, cultured under standard conditions and free from microbial infection and cross-contamination. 22

23 Transcript profiling Measurements of the mrna molecules in a cell reflect the real-time activity of its genome, what genes that are expressed and to what extent. A widely used technique to measure gene expression is DNA microarray technology, which allows for global analysis of thousands of genes simultaneously. To assess the reliability of microarray results, a common approach is to validate mrna expression levels with an alternative method. Thanks to its advantages in detection sensitivity and high precision quantification this can be performed with quantitative real-time PCR 102,103. DNA microarrays DNA microarrays are used to quantify tens of thousands of DNA or RNA sequences in a single assay. Since the introduction of the technology for more than a decade ago, it has become the most commonly used method for measuring gene expression. The principle of the method depends on the hybridization between DNA or RNA molecules to its complementary sequence amongst a mixture of molecules, the latter attached at specific locations to a solid support. Since the samples of interest are fluorescently labeled, the hybridization signal can be measured, and is dependent on amount of sample bound to its complementary probe. Today, the two basic described microarray technologies are spotted cdna microarrays 104,105 and high-density oligonucleotide arrays 106. Spotted cdna arrays use bp long cdna probes, placed on a regular pattern on a glass support. cdna microarray experiments typically involve hybridizing two mrna target samples, each of which is labeled with its own fluorescent dye (usually a red fluorescent dye cyanine-5 (Cy5) and a green fluorescent dye cyanine-3 (Cy3)). These are mixed in equal proportions and competitively hybridized to the spots on a slide. By scanning the array at different wavelengths and calculating the ratio of the red and green intensities for each spot, the relative abundance of a given transcript in the two target samples can be determined 107. Highdensity oligonucleotide arrays represent a quite different technology, compared to spotted cdna arrays. The oligo-chip technique, developed at Affymetrix, uses short (20-25 nucleotides in length) oligonucleotides, attached on glass chips as multiple probes (15-20) per gene 108. For each probe on the array that perfectly matches its target sequence, i.e. a perfect match (PM) 23

24 probe, a mismatch (MM) probe is added, which is an identical probe except for a single base mismatch in a central position of the sequence. These MM probes serve as internal specificity controls and allow for subtraction of both background and cross-hybridization signals. In contrast to cdna arrays, one fluorescently labeled target sample is hybridized to the array, making it possible for quantitative estimates of the number of transcript per cell by averaging the signal from multiple probes for each transcript 108. Quantitative real-time PCR Quantitative real-time PCR relies on the detection and measurement of products generated during each cycle of the polymerase chain reaction (PCR). Since the PCR process involves an exponential generation of DNA copies, a quantitative relationship exists between the amount of starting material and amount of PCR product generated at a specific cycle. To be able to monitor the amplification process, specific fluorescent probes are included in the PCR reaction, which directly or indirectly associate with the amplified target. DNA binding dyes such as SYBR green emit fluorescence when bound to double-stranded DNA. For every amplification cycle more dye can bind to DNA and the fluorescence intensity increases proportionally to DNA concentration 109. The Taqman system utilizes a complementary sequencespecific probe, labeled with a reporter dye and a quencher dye at the 5 and 3 end, respectively 110. As long as the probe is intact the quencher fluorochrome reduces the fluorescence signal from the reporter. As the Taq DNA polymerase begins the replication of a new strand of DNA, its 5 nuclease activity starts to degrade the attached probe at the 5 end. This allows for separation of the reporter dye from the quencher dye, resulting in increased emission of fluorescence from the reporter. Depending on the number of copies one starts with, a certain number of cycles are needed to make a specific number of products. By determining the number of cycles required for the amplification-associated fluorescence to reach a certain cycle threshold (c t ) level of detection, the initial amount of template can be determined. Consequently, the more copies one starts with, the fewer cycles of amplification are needed to reach this threshold. Two methods, absolute or relative quantification, are available to quantify real-time PCR results 111,112. Absolute quantification of mrna determines the input copy number of a transcript, through generation of a standard curve using serially diluted samples of known concentration. A linear relationship is created between the c t values and initial amounts of total RNA or cdna, and the c t values of unknown samples can be used to determine their concentrations. Relative quantification assays analyze changes in gene expression in a given sample relative to another reference sample, and results are expressed as a ratio between target and reference sample. 24

25 Diseases of melanocytic origin Melanocytes are neural crest-derived cells residing in the basal layer of the epidermis (Figure 4), hair follicle and eye. The principle function of melanocytes is to synthesize melanin pigments that provide the skin, hair and eye with color and photoprotection against ultraviolet radiation (UVR). Inside the melanocytes, melanin biosynthesis occurs in specialized membranebound organelles termed melanosomes. Pigmentation is achieved through the active transport of melanosomes along the melanocyte dendrites, with subsequent transfer to growing hair or surrounding keratinocytes, where melanin protects from UV-induced DNA damage. Loss of melanocytes or their ability to produce pigments, or defects in the formation, maturation and trafficking of melanosomes is associated with depigmentation disorders Under normal conditions, melanocytes only divide intermittently, but as a response to UV light exposure, the mitotic rate increases. Sun exposure is also considered as an etiologic factor for malignant melanoma, which is a malignancy arising from melanocytes. Melanoma is, like most cancers, very complex, and may be the result of many different genetic alterations and environmental factors, leading to enhanced proliferation of melanocytes and development of malignancy. Figure 4. Schematic drawing of the skin, with a magnified view of the placement of melanocytes in the basal layer of the epidermis. Adapted with permission from Kari C. Toverud MS CMI. 25

26 Vitiligo vulgaris Generalized vitiligo (vitiligo vulgaris) is an acquired cutaneous disorder, characterized by a localized and progressive loss of skin pigmentation. The clinical presentation consists of symmetrically distributed areas of depigmented spots (Figure 5). Population studies have shown a worldwide incidence ranging from 0.5 to 2%, in different geographical regions and ethnic groups 117. Today, causative treatment of vitiligo is not available. Instead current regimes are directed toward inhibition of disease progression and restoration of pigmentation. Figure 5. Generalized vitiligo on the hands of a young woman, demonstrating the symmetrical distribution of depigmented lesions. Courtesy of Dr. Mats J. Olsson, Uppsala, Sweden. The pathogenesis of vitiligo has not yet been elucidated, and various hypotheses for the etiology of the disease exist 118. Several investigators consider vitiligo as an autoimmune disease, with destruction of epidermal melanocytes as a result of an autoimmune attack 119,120. A number of findings have been described in support for an autoimmune origin of generalized vitiligo, one of which is the association with other autoimmune diseases in vitiligo patients. Vitiligo is a component of the multiple autoimmune polyendocrinopathy syndromes I and II, (APS1 and APSII) 121, and associations to autoimmune thyroid disease, pernicious anemia, Addison s disease and systemic lupus erythemathosus have also been described 122. Histological examination of skin biopsies from vitiligo patients has revealed presence of cytotoxic and helper T-cells in the infiltrates associated with the vitiligous lesions, indicating that alterations in cellular immunity result in the destruction of melanocytes 123,124. Evidence is also presented of humoral immune responses in vitiligo, with detection of autoantibodies towards melanosomal proteins, such as the tyrosinase family of proteins 125,126. Other studies propose oxidative stress as the initial pathogenic event in melanocyte degeneration, demonstrated by H 2 O 2 accumulation in the epidermis of patients with active disease 127. Moreover, an imbalance of intracellular antioxidants, particularly catalase, has been found associated with elevated levels of H 2 O 2 26