Annals of Oncology16 (Supplement 2): ii30 ii44, 2005 doi:10.

1093/annonc/mdi728

Genomics and proteomicsthe way forward

J. P. A. Baak1,2,3, E. A. M. Janssen1, K. Soreide1 & R. Heikkil4
Departments of 1Pathology and 4Medical Hemato-Oncology, Stavanger University Hospital, Stavanger, Gade Institute, 2University of Bergen, Norway; 3Free University, Amsterdam, The Netherlands

The Post-Genomic Era is characterized by huge numbers. This will only increase in the future when more will become known about proteins, which not only outnumber the genes, but also can be functionally altered in various ways. These "big numbers" have two consequences. Studies, which were despisingly called Fishing Expeditions only a few years ago are now respectfully dubbed Discovery Science, which answers questions and generate new hypotheses. Secondly, it is not always easy to select the way forward in Genomics and Proteomics. Therefore emerging technologies are updated and the hallmarks of cancer cells briey discussed. Promising applications for screening/early disease detection, diagnosis and tumour classication, prognostication, predictive response and tailoring therapy are described. Strategic choices as to certain types of study, which diseases or organ sites should be analyzed or techniques used, are mainly political and very much determined by loco-regional and national interests. However, in post-genomic research the following practical points must be considered. To avoid non-informative noise, homogeneous groups (preferably of small lesions) should be analyzed. Adequate sampling will be even more important than in the past to minimize the risk of non-informative benign cells inclusion. Accurate, biologically relevant, well reproducible quantitative morphological information is of the utmost importance to dene the lesions, as mixtures of functionally different cells will obscure important signals. RNA and proteins degrade quickly under hypoxic conditions, so that for reliable results the interval between tumour excision and freezing must be minimized and standardized. Promising results must always be independently validated as the use of relatively few patients combined with the enormous number of variables analyzed, carries a serious risk of selection bias and too optimistic results. An integrated collaboration structure of multiple disciplines becomes increasingly important.

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Introduction
A mere 50 years have passed since the 1953 landmark description of the DNA double helix [1], yet the parallel efforts of the Human Genome Project and Celera Genomics have successfully sequenced the human genome [2, 3] (ofcially completed on April 14, 2003), thus introducing the post-genomic era [4]. These are major achievements in biology and medicine in general, contributing not least to the understanding of carcinogenesis. New molecular insights and technologies in oncology have given clues to the initiation, early detection and possible prevention of neoplasia, prognostic and predictive markers, and targets for early detection and therapy. The search for cancer-causing alterations within the currently known genes of the whole genome (genomics, the study of the human genome) is complicated by the different ways the genes may be transcribed (transcriptomics) into a variety of functionally different proteins (proteomics, the analysis of the protein complement of the genome), which can themselves undergo essential functional changes.
q 2005 European Society for Medical Oncology

The denition of proteomics has changed greatly over time [5]. Originally, it was coined to describe the large-scale, highthroughput separation and subsequent identication of proteins resolved by 2-dimensional poly-acrimide gel electrophoresis (2DE). Currently, proteomics denotes nearly any type of technology focusing upon proteins analysis, ranging from a single protein to thousands in one experiment. Proteomics thus has replaced the phrase protein science. The post-genomic era is characterized by the generation of massive data numbers from studies with descriptive approaches. Although previously despisingly called shing expeditions, today the term discovery science is respectfully used for such large-scale studies, crediting the answers and new hypotheses being generated. Alongside the optimism fuelled by new discoveries, however, there is also the sensation of being overwhelmed by their complexity and size. Such a sensation is underlined by the notion that according to our existing knowledge, only 1.1% of the genome consists of exons coding for proteins, 24% is intronic sequences and the remaining 75% consists of intergenic DNA, currently without a known function in RNA transcription or protein translation.

ii31 Moreover, compared with evolutionary lower organisms, human beings have only twice to three times as many genes as the fruit y and the mustard plant. This indicates that the functional complexity, rather than the absolute number of genes, is essential for the human phenotype. To clarify where we are in the post-genomic era, we could say that we now have the letters (the sequence) and have thus far found a few sentences (genes that we know of), but we have only merely begun reading the chapter contents (how genes may be transcribed), while the books (the proteins and the metabolites) will keep human mankind reading for many centuries to come. In more scientic terms, the outline of the genome has enabled the study of gene products that are the focal point of proteomic studies, the effectors of the DNA [6]. Thus, considering the complexities of human nature and disease processes, to illustrate where cancer science is today: we have just become aware of the alphabet-code for a vast library of knowledge. We must therefore accept the idea that we are only at the beginning of a scientic discovery journey, a journey that may not be nished for many decades, or even centuries, to come. One may ask, how do we grasp this unfolding knowledge? This article will attempt to give a short overview of where we are and how the way forward might be chosen.

From genotype to phenotype: the hallmarks of human cancer cells

Research over the past decades has provided us with increased insight that cancer is a genetic disease. Knudson [12] formulated the so-called two-hits hypothesis to explain the development of retinoblastoma. A larger, stepwise number of acquired genetic mutations were later detected in the early colorectal cancer studies [1315]. Today, cancer is often perceived as a genetic disease driven by the time sequence of these DNA changes and is phenotypically determined by a limited number of underlying rules [16, 17]. These genetic alterations and rules are not the same for all tumors even within a certain organ site (e.g. lung cancer). This functional perception of cancer cells has clinical implications concerning the biological behavior, natural history and potential therapeutic tumor targets. Briey, the acquired set of functional capabilities of a cancer cell are: (i) self-sufciency in growth signals; (ii) insensitivity to anti-growth signals; (iii) evasion of apoptosis; (iv) limitless replicative potential; (v) sustained angiogenesis; and (vi) tissue invasion and metastasis [16]. Furthermore, extracellular matrix and its components (broblasts, stromal cells, signal substances, inammatory cells) inuence cancer cell proliferation, invasion and metastasis [1821]. Fibroblasts have a more profound inuence on the development and progression of carcinomas than was previously appreciated [19], e.g. in oral epithelium differentiation [22].

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The genome lays a foundation for the understanding of cancer as a genetic disease
The completion of the Human Genome Project gives us more insight into the genetic variations that are causally implicated in oncogenesis. A 2004 consensus report described that more than 1% of genes are causally involved in oncogenesis [7]. Some of these genetic mutations can be transmitted through the germ-line and result in hereditary cancers (510% of all). Many hereditary cancers have been extensively described, so that the focus now is more on the susceptibility for cancers in certain families or populations. This has resulted in a new approach, molecular epidemiology: searching for low penetrant cancer susceptibility genes that may give rise to much smaller increases in individual risk [8]. These genes may interact with environment and lifestyle factors and as a result, cancer risk is not equally elevated in all persons exposed to an environmental factor, and the risk can change. One example is the change in cancer incidence in Chinese and Japanese immigrants to the USA [9]. Another example is relatives of patients with early-onset lung cancer, who have a genetic predisposition and elevated risk for developing lung cancer, in contrast to relatives of patients that develop lung cancer later in life [10]. It was recently suggested that a T27C polymorphism in CYP17 (cytochrome P450 c17a) is associated with elevated sex hormone levels, and interacts with insulin levels and diet to affect breast density levels and potential breast cancer risk [11]. Such genetic susceptibility is currently being intensively investigated by DNA ngerprinting with the use of single nucleotide polymorphisms (SNPs) in large populations.

Molecular techniques
The molecular techniques for genomics and proteomics studies are developing rapidly (for an overview, see Baak et al. [23]). A short description follows here. Table 1 shows important widely established techniques with their respective strengths and weaknesses. Some newer promising methods will be described in detail.

Established DNA techniques

Conventional karyotyping. The development of chromosome banding techniques in 1969 was a breakthrough, as all chromosomes could be individually recognized (conventional karyotyping) and chromosomal rearrangements characterized (a classical example is the Philadelphia chromosome in chronic myeloid leukaemia). Fluorescence in situ hybridization. A small DNA fragment of known origin (a probe) is uorescently labeled and hybridized to a metaphase chromosome spread or interphase nuclei. The probe binds to homologous sequences within the chromosomes and this can be visualized by uorescence microscopy to identify chromosomes, centromeres, aberrations in interphase tumor nuclei and others (Figure 1). Comparative genomic hybridization (CGH). CGH provides information on the number of copies of chromosome parts throughout the whole tumor genome to identify genetic

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Table 1. Molecular techniques, their strong and weaker points Advantages Genomic techniques Karyotyping Fluorescence in situ hybridization Comparative genome hybridization Spectral karytyping Microarray Microsatellite instability Loss of heterozygosity Single nucleotide polymorphism (SNP) Whole chromosome analysis Gene specic Laborious Maximum four genes at a time Resolution limited to $ 10 Mbp Laborious Fresh tissue necessary for cDNA isolation Large numbers of microsatellites needed to be informative No correlation with mRNA expression Large number of SNPs needed to be informative SNPs map not nished yet Many false-negative and -positive results Hydrophobic and membrane proteins do not enter well Only 2000 spots per gel Not suitable for small clinical samples False positives Proteinligand interactions Gene function under well described circumstances Differential protein expression proling of complex protein mixtures Structural large chromosomal changes Gains and losses of specic genes in cells and tissue Gains and losses throughout the whole genome Structural chromosomal changes Differential expression of thousands of genes Chromosome stability, effectiveness of DNA repair mechanisms Losses at gene level Susceptibility for disease development and therapy response Limitations Provides data on

Whole genome analysis Whole genome analysis Whole genome analysis Allele specic Allele specic Stable Abundant through genome

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RNA interference Proteomic techniques Two-dimensional SDSPAGE

Gene specic

Separation of complex protein mixtures Sensitivity (micromolar, 106) Posttranscriptional modications can be detected

Yeast two-hybrid

Detects new protein interactions in vivo High throughput

Laborious Protein microarray Data analysis Proteinligand interactions and protein expression in complex protein mixtures

Very diverse both function and detection Fluorescence microscopy (FRET, FLIP, FRAP, FLIM, BRET, PRIM) Mass spectometry and combinations with TOF Surface-enhanced laser desorption/ionization Very specic, under well described circumstances in vivo and in time High specicity and sensitivity (femtomolar, 1015) High speed, high resolution separations of complex protein mixtures Small samples Little sample preparation Tissue microarray High throughput

Limited amount of antibodies and recombinant proteins available Laborious Spatial and temporal dynamics of proteinligand interactions in the living cell Protein identication/characterization, mass ngerprinting Differential protein expression proling of complex protein mixtures

Often extensive sample preparation Detection >30 kDa

Low mass reproducibility Large protein prole CVs Formalin-xed material Protein expression and location in correlation with patient and therapy data

Morphologic information intact Possible on archival material

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Table 1. (Continued) Advantages Liquid chromatography nuclear magnetic resonance Quantitative Limitations Only metabolites [amino acids, organic acids and bases, nucleotides, carbohydrates, osmolytes, lipids (broad, non-specic resonances)] can be detected Expensive Provides data on Metabolite target analysis, metabolite proling, metabolic ngerprinting, metabolic proling

High throughput Complete sample recovery Little sample preparation Complex protein mixtures

changes (deletions or amplications). The resolution of chromosome CGH is limited to $ 10 million base pairs (Mbp). Array CGH. Array CGH does not require karyotyping and the resolution can be 0.5 Mbp or better. Figure 2 explains array

CGH and shows the results of chromosome and array CGH of the same chromosome combined in one gure. Spectral karyotyping (SKY). SKY gives all chromosomes a unique uorescent color by hybridizing with chromosomespecic probes each labeled with a different combination of

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Figure 1. Fluorescence in situ hybridization (FISH) examples. Top, left: interphase cytogenetics. Normal diploid (left, two spots per chromosome) and malignant (right, several spots per chromosome, indicating its aneuploidy) urothelial cells from a urinary bladder washing hybridized with centromere probes to chromosomes 3 (red), 7 (green) and 17 (white), and a locus-specic probe for p16 at 9p21 (yellow). Bottom: FISH using whole chromosome X and chromosome 3 probe on tumor cell line scc040, showing that a part of chromosome X is attached to chromosome 12, and showing that the addition on chromosome 3 is in fact other chromosome 3 material. Reprinted with permission from Baak et al. [23].

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Figure 2. Top left: schematic view of the array comparative genomic hybridization (CGH) technique and higher sensitivity and resolution of array CGH over chromosome CGH. Top right: example of a hybridization of green-labeled tumor DNA and red-labeled normal reference DNA onto an array of 2400 BAC clones representing the whole genome with an average spacing of 1 Mbp. Bottom: chromosome CGH detected low level gain of a large part of chromosome arm 20q; array CGH identied a narrow high level amplication in band 20q13.2. Reprinted with permission from Baak et al. [23].

Figure 3. Spectral karyotyping (SKY) karyogram of tumor cell line scc040. For every pixel in the image the spectrum from 450 to 750 nm is measured, and with this spectral information the unique color combination of each chromosome is identied. The addition on chromosome 3 is composed of chromosome 3 material, the addition on chromosome 12 comes from a part of chromosome X, and the addition on chromosome 18 consists of chromosome 8 material (the third chromosome 9 is lying in overlap with chromosome 4). Reprinted with permission from Baak et al. [23].

ii35 uorochromes, and is useful for the genome-wide detection of structural chromosomal changes. Translocations are readily visible (Figure 3). SKY does not require prior knowledge of chromosomal breakpoints. Gene expression (cDNA) arrays. High-density oligonucleotide cDNA microarrays measure many thousands of gene-specic mRNAs in a single tissue sample in parallel (Figure 4). Largescale gene expression analysis has proved to be a valid strategy for developing gene expression proles, or signatures, to classify prognostic subgroups. Unfortunately the method requires fresh tissue for optimal results. Loss of heterozygozity (LOH), microsatellite instability (MSI). One strategy for screening the genome used a panel of 150 polymorphic microsatellite markers from throughout the whole genome with LOH. By using microsatellites one can also check the ability of cells to repair DNA replication errors. In tumors with MSI, both genetic and epigenetic modications of mismatch repair genes were identied.

Proteomics techniques
Two-dimensional electrophoresis. 2DE [16] is a powerful technique for protein separation. The digestion of spots in the gel makes it possible to further analyse proteins with mass spectrometry. Peptide spectra obtained in this way can be used to search protein sequence databases. Yeast two-hybrid system. The two-hybrid system can generate vast amounts of new data, but each and every complex has to be tested and conrmed separately afterwards. Protein microarrays. Most protein microarrays are afnitybased. Although powerful, these techniques have several drawbacks: lack of available antibodies, lack of puried recombinant proteins and cross-reactivity with afnity agents.
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Figure 4. Genes which distinguish normal from malignant endometrium (P, proliferative; S, secretory; T, tumors) endometrium (Permax <0.50, three-fold, 100 difference). Columns show individual tissues, rows represent genes. Color scale shows standard deviation from the mean expression value for each gene. Dendrograms on the margin show agglomerative hierarchical clustering (Wards linkage, euclidean distances) of genes (right) and tissues (bottom) (courtesy of Dr G. L. Mutter). Reprinted with permission from Baak et al. [23].

ii36 Fluorescence resonance energy transfer (FRET). FRET monitors macromolecular interactions and resolves the spatial and temporal dynamics of proteinprotein interactions in the living cell, but in principle it can also be applied to tissue sections of xed material. Surface-enhanced laser desorption/ionization (SELDI). SELDI is an afnity-based mass spectometry method in which proteins (<20 kDa) are selectively absorbed to a chemically modied surface, and impurities are removed by washing with buffer. Tissue microarrays (TMAs). TMAs (Figure 5) consist of hundreds of small core (0.6 2.0 mm) tissue sections arrayed on a glass slide for immunohistochemistry (IHC) (protein), in situ hybridization (DNA, RNA), and are useful for evaluation of new antibodies and large-scale outcome studies. Because of the very small sample size per case (with two cylinders of 1 mm diameter per tumor of 2 2 cm; typically <0.5% per section), TMAs only give a reliable impression of a tumor characteristic if large numbers of cases are analyzed, and even then, the signicance of rare events may easily be overlooked (see below under Critical remarks and need for validation). CA repeats (see below), and therefore SNPs are often less informative than other genetic markers such as simple sequence-length polymorphisms and microsatellites. On the other hand, SNPs occur abundantly in the whole genome and provide great potential for automated genotyping. SNPs can therefore be used as genetic markers to detect disease genes in genetic linkage studies. Thus high-density SNP mapping can be as informative as current strategies with simple sequencelength polymorphisms and microsatellites. As an example, genetic polymorphisms in T27C in CYP17 have been linked to the risk of developing breast cancer [24]. Loss of heterozygosity. Huang et al. [25] recently developed a high-density oligonucleotide array-based SNP genotyping method, whole genome sampling analysis (WGSA), to identify genome-wide chromosomal gains and losses at high resolution. WGSA simultaneously genotypes over 10 000 SNPs by allele-specic hybridization to perfect match and mismatch probes synthesized on a single array. The coupling of LOH analysis, via SNP genotyping, with copy number estimations using a single array provides additional insight into the structure of genomic alterations. With median inter-SNP euchro- matin distances of 199 kilobases, this method affords a resolution that is not easily achievable with non-oligonucleotide-based experimental approaches. RNA interference (RNAi). RNA silencing [26] is a posttranscriptional gene silencing process that is based on sequencespecic interactions of small interfering RNAs with targeted mRNA molecules. Silencing is initiated when double-stranded

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Evolving molecular methods

Single nucleotide polymorphisms. SNPs are the most abundant form of DNA polymorphisms in the human genome. Each SNP has a dened position in a chromosome at which base pairs differ among individuals with signicant frequency (>1%). Human SNPs are not very polymorphic, contrasting

Figure 5. An example of a tissue microarray (TMA) consisting of 1 mm diameter cylinders. Ki-67 staining. (A) Parafn block for TMA; (B) histological TMA section, with three control markers at the upper left; (C, D) examples of different immunostains.

ii37 RNA is processed into small RNAs, which triggers a number of enzymatic reactions resulting in the degradation of the targeted mRNA molecules [27]. RNAi-based genome-wide functional analysis for mammalian cells [28] is well underway. Combining RNAi with transfected cell array (TCA) holds enormous potential for drug-discovery oriented research and therapy. Transfected cell array. The principle of TCA is based on the transfection of DNA or RNA molecules. These are immobilized on a solid surface upon which cells are cultured. Cells growing on top of the DNA/RNA spots become transfected, resulting in the expression or silencing of specic genes/proteins in spatially distinctive groups of cells. Consequently, the physiological effects caused by the introduction of these foreign nucleic acids can be studied [29]. Densities of up to 8000 cell clusters per standard slide can be achieved [30]. Microuidics. Microuidics technology utilizes a network of channels and wells that are etched onto glass or polymer chips to build laboratories-on-chips. Pressure or electrokinetic forces move pico- or nanoliter volumes in a nely controlled manner through the channels. These microuidic circuits can be designed to accommodate virtually any analytic biochemical process. For example, a lab-on-a-chip for immunological assays could integrate sample input, dilution, reaction and separation; or one designed to map restriction enzyme fragments might have an enzymatic digestion chamber followed by a separation column. Labs-on-chips are well suited for highthroughput analyses. Their small dimensions reduce processing times and the amount of reagents necessary per assay, and have high reproducibility owing to standardization and automation [31]. the understanding of disease and development of new treatments may be very long indeed! The conclusion of the above is that we are at the very beginning of clinical genomic and proteomic applications. Nonetheless, the enormous expansion of molecular cancer research to date has created applications that are useful in the daily care of patients. The following description can by no means be complete, but aims to mention typical examples, including weak points, and how new discoveries have been made by the combined use of different technologies. Here we will concentrate on some established or promising applications in (i) early disease detection/screening, (ii) diagnosis and tumor classication, (iii) prognostication, (iv) prediction of response, and (v) tailoring of therapy.

Screening
In many countries, classical cytological screening for cervical cancer is being amplied with testing for human papilloma virus [34]. International genomic collaborations to detect minimal residual cancer have produced important results in leukemia [35]. Proteomics technologies have also been announced as future biomarkers for screening and early detection of cancer disease [3638]. However, the early promising results with near 100% sensitivity and specicity for detection of ovarian cancer with proteomics [39] have been seriously criticized, as Baggerly et al. [40] showed that the method performed no better than chance for classifying the second dataset. In agreement with this, the reproducibility of the proteomic proling approach remains to be established. Yet, others still hope that proteomic proling will enable protein-screening in adjunct with endoscopic surveillance for improved and more effective detection of early (pre)malignant disease [36, 41].
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Applications of genomics and proteomics

The long interval between a discovery and a clinical application
It takes a long time before the real impact of a new discovery is realized. An example is that the invention of the microscope by Leeuwenhoeck occurred in the 17th century, yet the era of cellular pathology was not fully introduced until the teaching of Virchow, nearly 200 years later! Similarly, even though p53 was discovered two decades ago, for the rst 10 years it was believed to be an oncogene. Only later was it vindicated as a tumor-suppressor gene and the guardian of the genome [32]. DNA contains four variables only: adenine, cytosine, guanine and thymidine, yet it took many decades to understand its biology, and that process is far from nished. Proteins are much more complex: they contain 20 different amino acids and the number of possible permutations thus is exponentially larger. In addition, there are more than 100 known different possible posttranslational modications [33] and each one will be able to change the function and location of a protein drastically over time and under different conditions. The interval between the development of genomic and proteomic technologies, and the actual paradigm shift in

Tumor classication
Histological typing and grading of tumors is widely used, but is notoriously subjective and lacks intra- and inter-observer reproducibility [42, 43]. This is unacceptable, as the therapeutic and social consequences of neighboring grades for the individual patient are often enormous: e.g. adjuvant chemotherapy or radiotherapy in FIGO 1 ovarian cancer grade III; and total colectomy in ulcerative colitis patients with some epithelial dysplasia [44]. Molecular analysis allows for subgrouping based on genomic or proteomic (including IHC) proles together with histopathology evaluation in colorectal cancer, breast cancer, lung cancer, lymphomas and others [4552]. A well known example is that BCL-2 (an anti-apoptotic protein) is overexpressed in follicular lymphomas, principally as a result of the t(14;18)(q32;q21), and useful in distinguishing follicular lymphoma (usually BCL-2-positive) from follicular hyperplasia (BCL-2-negative), although with certain exceptions [53]. Multiplex polymerase chain reaction (PCR) assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome

ii38 aberrations t(11;14) and t(14;18) [54], and clonality testing in lymphomas has been widely adopted as a routine diagnostic method. Moreover, undifferentiated (poor prognosis) laryngeal cancers and gastric appeared to be large-cell lymphomas (with good prognosis) when IHC panels were applied. The classication of the diagnosis unknown primary (in the past applied to 20% or more of metastases) can be reduced considerably with the todays application of accurate multi-panel immunopathology. Hopefully, molecular techniques will render even more exact subclassications in adjunct to histomorphological evaluation. Eventually, the diagnosis unknown primary may become obsolete [55]. An example is DNA methylation mapping, which showed that unique proles of hypermethylated CpG islands exist, dening each neoplasia [56], as in, for example, prostate cancer [57]. These results for subclassication of tumors are promising, but validation and an appropriate link to clinical outcomes is always mandatory. increasing the positive predictive value of PTEN-null glands (Figures 8 and 9). This again shows the importance of combined molecular and morphological technology to get the strongest prognostic information.

Targeted tumor therapies

Targeted therapies are now being developed that work at the level of the proteome. Examples of these agents include imatinib mesylate (Gleevecw) targeting the Bcr-ABL tyrosine kinase in chronic myelogenous leukemia and mutated c-KIT- or PDGFRa-tyrosine kinase in gastrointestinal stromal tumors [72], trastuzumab (Herceptinw) for (her2-neu amplied) breast cancer [73, 74] and getinib (Iressaw) for EGFR-mutated lung cancers [75]. These are all targeted therapies in that they are monoclonal antibodies (imatinib, trastuzumab) or small molecules (getinib) directed at distinct defects in the tumor cells. Even though early clinical trials have shown positive results, resistance patterns have been reported for getinib [76, 77], imatinib [78, 79] and trastuzumab [66, 67]. Consequently, several pathways should perhaps be simultaneously addressed if long-term response or remission is to be achieved. The problem is that such combinations would be expected to increase the toxicity, which undermines the whole idea of targeted therapy, but combination analysis may also prevent overtreatment. Her2-neu and proliferation testing is a good example. Volpi et al. [64] showed that in node-negative breast cancer patients, HER2 expression was a signicant discriminant of prognosis, but only in the subgroup of patients with rapidly proliferating cancers. PTEN-negative patients are more prone to trastuzumab resistance [66]. A problem with Her2-neu is the testing method and which patients to select for treatment. Generally accepted is to treat all IHC strongly positive (3+) cancers, although several have no gene overexpression while others with gene amplication do not show IHC [80]. The cause and the clinical value of these discordances are not yet known.

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Prognostic value, prediction of response and subgroup analysis

Many articles have had a prognostic goal, as staging (like grading and typing) is practically useful but may have a low prognostic accuracy. Prognostic gene expression signatures are promising, as, for example, in breast cancer [48]. CGH analysis in lymph node-negative breast cancers led to the discovery that a gain in chromosome 3q is a much stronger prognosticator than classical features [58]. The most common region of overlap in this study was 3q26. The PIK3CA gene [59] located here is mutated in up to 40% of primary breast cancers [60, 61] (Figure 6). Overexpression of PIK3CA can result in an increased activation of the Akt pathway after activation of ErbB2 upon binding with a growth factor (e.g. estradiol or heregulin) [28], and lead to excessively increased proliferation, invasion and cell motility (Figure 8). Proliferation is the strongest prognosticator in node-negative breast cancer [6264]. However, many other factors, like noey2 deletion, may also cause increased proliferation [65]. Noey2 is located on chromosome 1p, but 1p deletion was not prognostic [58]. We hypothesize that there are two types of high proliferation in breast cancer, a PIK3CA-dependent one (strongly prognostic) and a second caused by genetic changes in other genes (which are much less, or not at all, prognostic). It is important that the tumor suppressor gene PTEN opposes the action of PIK3CA. PTEN deciencies occur in up to 35% of breast tumors, and may be an important predictor of trastuzumab resistance in breast cancers with ErbB2 overexpression [66]. Loss of PTEN tumor suppressor function is observed in tumors of breast, prostate, thyroid and endometrial origin [6769]. Allelic losses in the proximity of the PTEN locus (10q23) also occur in sporadic colorectal cancers [70]. PTEN therefore may be a general key progression determinant in (pre)cancers. In endometrial hyperplasias (EHs), a frequent pre-neoplastic lesion, progression to cancer only occurs in PTEN-negative and not in the PTEN-positive EHs [68] (Figure 7). However, only those PTEN-null EHs with an unfavorable morphometric D-Score progressed [71], greatly

The way forward

What course shall we take with genomics and proteomics in cancer research and treatment? Which strategic and practical choices should be made?

Strategic choices
Just as All roads lead to Rome, so the answer to Genomics and ProteomicsThe Way Forward may be similarly easy, but at the same time complex, for there are certainly many possible ways to go in the post-genomic era. Eventually, many will hopefully lead to a better understanding of human disease, its causes, prevention and potential therapeutic targets. Clearly, one way will lead there sooner than another; we do not yet have a nal blueprint and need to rely on cooperation and experience in getting to our target. Strategic choices regarding types of study, which diseases or organ sites should be analyzed or which techniques used, are mainly political and very much determined by locoregional and national interests.

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Growth Factor

Receptor

Tyr Kinase P-Tyr

Ras GTP p85 SH2 p110 PIK3CA

PIP3 PTEN

PIP2

AKT

Phosporylation of BRCA1

Mitogenesis ACG kinases

Apoptosis Caspase 9, BAD

Cell Motility RAC, BTK kinase NFkappaB, uPa

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Figure 6. Schematic overview of some of the Akt pathway effectors. Overexpression of the catalytic subunit of PI-3 kinase may result in an increased activation of the Akt-pathway, in reaction to a growth factor binding to cell surface receptor ErbB2. PTEN promotes the transition from PIP3 to PIP2, thereby suppressing PIK3CA.

However, the following practical points must be considered in research on The way forward.

Analyze homogeneous groups

Large tumors are often geno- and phenotypically very heterogeneous, hypoxic and necrotic. The essential information one is searching for can be heavily blurred by the additional noise coming from these epiphenomenal properties. Thus, the utmost care is needed with the interpretation of results from large tumors. It is not always immediately evident that the material presented in an article is biased for large tumors, as, for example, in studies using frozen tissue for RNA and protein chips. With many years of daily practical experience in pathology laboratories, we can guarantee that frozen tumor

material often comes from large tumors! Other examples where heterogeneity has a major inuence are when tumors from different stages are included, or mixtures of carcinoma in situ, small and large cancers, or different histological subtypes or different age groups with varying genetic and prognostic information. Unfortunetely, including such mixtures may be the rule rather than the exception.

Sample accurately
Adequate sampling is always important, but even more so when the cancers are small, as is increasingly the case. In small samples there is the added risk of including proportionally signicant numbers of benign epithelial and other cells. Consequently, accurate sampling by means of, for

Figure 7. PTEN-expressing (brown) and -null glands (blue) in (A) normal proliferative and (B, C) hyperplastic endometrium. Note that adjacent glandular cells can be positive and negative (B). Stroma cells are invariably positive (reprinted from Baak et al. [104]).

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100 D-Score>1 or D-Score<1/PTEN+ Progression 0/89

80 % Without Cancer In Follow-Up

60 P<0.00001 40 D-Score<1 & PTEN Null Progression 7/14

20

0 0 24 48 72 96 120
Figure 10. The bench-to-bedside approach.

Follow Up (months)
Figure 8. Cancer-progression free survival curves of endometrial hyperplasia, according to combined application of the two different technologies: the morphometric D-Score and PTEN expression (reprinted from Baak et al. [104]).

breast cancer. A formal count is strongly prognostic but impressions are not (Baak J.P.A., van Diest P.J., Janssen E.A.M. and Voorhorst F., 2005, unpublished results).

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Poor reproducibility owing to different techniques or poor test methods

Up to 50% of breast cancers have been reported to present with loss of PTEN function [66], but others using IHC [84] found just 8% PTEN negativity and almost 70% showed weak positivity. This raises the important question: which determination technique best predicts the clinical outcome? A typical example of a poorly dened test method is the use of IHC without strict quality and assurance control or standardization; the international NEQAS network for IHC quality testing has shown that a high percentage of IHC determinations in different laboratories were suboptimal [85]. In the future, an international quality standard for IHC cancer studies should be introduced.

Figure 9. RNA and proteins are very sensitive to hypoxia, caused by excision of the tumor.

example laser dissection, becomes even more important than in the past. In pre-cancers, the precise localization in the mucosa of a prognostic factor is often highly important to extract its value. In a cervical squamous intraepithelial lesion, for example, the concentration of retinoblastoma protein is predictive of progression, but only in the deep layer of the epithelium. Mixtures of supercial, deep and basal cells (as occurs with biochemical tests) will blur the results [81].

Minimize and standardize the interval between excision of a tumor and freezing
This is essential for RNA and proteins, but less urgent for DNA (which is much more resistant to degradation) (Figure 9).

Critical remarks and need for validation

We have seen that post-genomic era studies are characterized by huge numbers [8691]. The traditional reductionist approach with hypothesis-driven research that focuses on one gene at a time is now being challenged by high-technology, hypothesis-generating Omic approaches [92]. The discoverybased research currently applied to the Omic-technologies calls for a change in reasoning [93]. The steps in development (Will it work?), evaluation (Does it work?), and explanation (How does it work?) are separate. Many of the current Omic-discoveries are based on large-scale detection of genes/gene products from where we do not yet know. One might argue that a test may be useful if patterns can reliably

Morphology, accurate quantitation and keeping the denitions

It is important to emphasize that in many areas, molecular insights are only possible with very careful morphological analyses, as for example in gastrointestinal lymphomas [52] and endometrial hyperplasias [82]. Accurate quantitation of microscopic features is also essential; qualitative impressions are often simply too indenite [83]. Not adhering to denitions of certain features may have enormous consequences for the prognostic value of a prognostic factor, where for example formal mitotic activity counts versus general mitotic impressions in

ii41 discriminate disease from no disease, regardless of whether they can be understood or explained. However, before such proles are to be clinically implicated the results need to be validated, according to good laboratory practice guidelines [44, 94]. In other words, microarray techniques have made it easier to nd the needle in the haystack. However, as these results often come up with tens or hundreds of genes that are differentially expressed in tumors compared with normal cells, we now nd ourselves looking at a haystack of needles. The challenge for the future is to sift out the genetic information from non-informative noise and nd clinically useful data by means of sound experimental design [95]. Testing very many variables at the same time has a serious risk, i.e. that the learning (training) sets are very small compared with the large number of features analyzed. This can result in far too optimistic results that cannot be reproduced in subsequent new patient groups (a phenomenon called overtraining of the initial learning set). For example, the differences in the same tumor types can be more striking than similar, as shown in microarray studies for lung cancer [46, 96]. We have mentioned above that serum proteomics tests [39] reported to be nearly 100% sensitive and specic for ovarian cancer, 3 years later are yet to be duplicated. Verication by an independent method [92] or in independent patient groups (see Baak [44]) is mandatory. Recently, Michiels et al. [97] reanalyzed data from the seven largest published studies that have attempted to predict prognosis of cancer patients on the basis of DNA microarray analysis. By using multiple random sets the stability of the molecular signature and the proportion of misclassications were studied. They found that the list of genes identied as predictors of prognosis was highly unstable; molecular signatures depended strongly on the selection of patients in the training sets. For all but one study, the proportion misclassied decreased as the number of patients in the training set increased. Because of inadequate validation, the chosen seven studies published overoptimistic results. Five of the seven studies did not classify patients better than chance [97]. In agreement with Baggerly et al. [40] and Michiels et al. [97] we believe that any genomic and proteomic study should be considered prone to error before it is further validated by methods or patient groups that are independent of the original study. Large scale analyses on many patients may be required. One such analysis used a statistical method, comparative metaproling, which identies and assesses the intersection of multiple gene expression signatures from a diverse collection of microarray datasets [98]. The results were encouraging. From 40 published cancer microarray datasets, comprising 38 million gene expression measurements from >3700 cancer samples, a common transcriptional prole emerged that is universally activated in most cancer types relative to the normal tissues from which they arose, likely reecting essential transcriptional features of neoplastic transformation [98].

Consequences of the post-genomic era

The variety of consequences of the post-genomic era is not yet clear, but certain points have emerged. Will genomics and proteomics replace morphological science? When gene expression arrays became available some 6 years ago, it was predicted that the end of morphological science was near, but it now seems unlikely that this will happen soon. The studies by Mutter et al. on endometrial pre-cancers are classical examples of the development as follows. In 1985, the basis was laid for the WHO94 classication of endometrial hyperplasias, but a molecular basis was lacking. By 1995, studies could not detect a strong correlation between WHO94 and genetic clonality. However, subsequent computerized morphometrical analysis revealed a strong correlation between clonality and the prognostic morphometric D-Score [82]. Further analyses comparing the prognostic value of the WHO94 and D-Score showed that the latter was superior [71, 99], but the morphometric technology required for the D-Score is not widely available. This led to a subjective variant, the Endometrial Intraepithelial Neoplasia (EIN) classication [100], which in our study is well reproducible [101]. Thus, pathologists come back to the standard microscopic image, but with renewed thinking, and are now armed with new knowledge about reactive endometrial changes (called benign hyperplasias) versus genetically monoclonal expansive growing lesions with a high cancer risk (called EIN). This new approach greatly strengthens the prognostic evaluations and therapeutic choices. Of course, compared with genetic testing microscopic evaluations are currently very cost-effective and time-efcient, but this may change over time when more efcient automated molecular tests become available. It is beyond the purpose of this article to discuss in detail the expected changes in the curricula of medical students, basic scientists, clinicians and technicians, and the consequences for the staff members of cancer-related departments. Bioinformatics (the application of computer science and informatics to molecular biology) becomes essential for the study of DNA, RNA and proteins, and will help to map sequences to databases, create models for molecular interaction, evaluate structural compatibility, nd differences between host and pathogen DNA and identify conservation motifs in protein structure [102]. For research, the development of a cooperative framework among basic researchers, medical specialists, bio-mathematicians, technology experts and producers is essential for realizing the revolutionary promise that genomics and proteomics hold for drug development, regulatory science, medical practice and public health. The hospital structure may also change from speciality driven (surgery, radiology, medical oncology, pathology) to specialized multidisciplinary treatment teams encompassing certain technologies, e.g. anti-angiogenesis or gene transfer. The legal aspects of biobanks are becoming rapidly more important. Publications on obscure material may soon no

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ii42 longer be acceptable. The ownership of the material and permission from the patient to use the material is already legalized in a number of countries.
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Concluding remarks
It follows from the above that we agree with Hall and Lowe [103] that modern cancer research is about the interplay between disciplines. New ndings must be carefully validated. A lesson from several successful studies is that genomic and proteomic insights only came through very careful morphological studies, analysis of homogeneous groups and the use of accurate, reproducible quantitative methods. Finally, a piece of sound advice to everyone on the way forward: dont sell your microscope!

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