Section 10 -

Appendix: Molecular Basis of Human Neoplasia Robert D. Odze, M.D. Jonathan Glickman, M.D., Ph.D. Mark Redston, M.D.

Classification of Human Cancer Genes There are two categories of human cancer genes: oncogenes and tumor supressor genes [1, 2, 3, 4, 5]. Some investigators also include a third category: genome-maintenance genes [6, 7]. Cancer genes may possess activity in more than one of these categories [8]. Cancer genes can be further sub-classified based on their normal cellular functions. Oncogenes. Oncogenes are activated during human tumorigenesis (the so-called "accelerators" of the cell) [9]. In general, oncogenes are normal cellular genes that are primarily involved in growth stimulation. Most oncogenes in human neoplasms are involved in signaling cascades that are important for cell division (proliferation) and survival (anti-apoptotic) [5]. Tumor Suppressor Genes Tumor supressors are genes that have a role in negatively regulating cell growth (the so-called "brakes" of the cell). These genes are inactivated in neoplasia, with subsequent loss of important regulatory check-points [10, 11]. Turning off a tumor supressor gene is a complex genetic event requiring inactivation of both copies, the classic "two-hit" hypothesis as proposed by Knudson (also known as bi-allelic inactivation) [12]. Tumor supressor genes are also at the root of many hereditary cancer syndromes, wherein one mutated/inactivated copy of a tumor supressor gene is inherited in the germ line in an autosomal dominant fashion [13]. Mutation Mechanisms General information on mutation mechanisms can be found in many standard textbooks [14], or subspecialty cancer genetics texts [5, 15]. Intragenic mutation. Small genetic alterations that affect a single gene are called intragenic mutations, and consist of "point" mutations (that alter only a single nucleotide), and small deletions or insertions of 1 or more nucleotides. There are three types of point mutations: missense mutations, which result in an amino acid substitution; nonsense mutations, which result in a stop codon; and silent mutations, which do not alter protein translation. Missense mutations are a common mechanism for oncogenic activation. Nonsense mutations generally result in loss of function, and are, therefore, mostly confined to tumor suppressor genes. Chromosomal deletions. Deletion of chromosomal regions, or entire chromosomes, are among the most common genetic alterations found in human tumors. These deletions are usually detected by examining polymorphic markers that differentiate the two parental alleles, typically by PCR amplification of polymorphic microsatellites in tumor and paired normal DNA. In tumorigenesis, loss of one of the parental alleles is called allelic loss or loss of heterozygosity (LOH). Allelic deletions have a number of "hot spots" in the genome that likely represent the locations of important tumor suppressor genes, and these regions differ by tumor type. Chromosomal amplifications. Small regions of a chromosome can undergo a dramatic increase in copy number, termed gene amplification. By cytogenetic methods, these amplifications appear as homogeneously staining regions (HSR) or multiple minute subchromosomal fragments (double minutes). The growth advantage of cells with amplifications is believed to be due to overexpression, and, thus, activation of important oncogenes that reside in this region. Chromosomal translocations or rearrangements. The translocation of a portion of one chromosome to another chromosome, or the rearrangement of a region within a chromosome, are both common events in human neoplasms. These alterations are thought to be responsible for activation of oncogenes by one of two mechanisms: 1) The new structure may place a growth-promoting gene under the control of a new promoter, which results in strong transcriptional activation. 2) The new structure may result in a new fusion gene, such as the joining of DNA binding and transcriptional activation domains that results in a novel activation factor. Occasionally, translocations and rearrangements can result in the inactivation of a tumor suppressor gene. DNA methylation. Methylation of human DNA can occur at the cytosine residue located within a CpG dinucleotide (reviewed in 16, 17). Methylation in normal tissue is involved in regulation of gene transcription, and is utilized in the inactivation one of the X chromosomes in female tissues. In human neoplasia, increased methylation in CpG islands located in gene promoter regions is a major mechanism of turning off gene expression and inactivating tumor suppressors. Methylation is not a structural change in DNA sequence, and is considered "epigenetic". Viral insertion. Some viruses can insert their genes into DNA, allowing expression of oncoproteins. In humans, the most common examples are the insertion and expression of the E6 and E7 oncoproetins in HPV-related cervical neoplasia. Aneuploidy. Aneuploidy refers to an abnormality in the relative chromosomal copy number in cells. Normal tissues are predominantly diploid, but have a subpopulation of tetraploid cells (representing G2 phase of the cell cycle), and a number of cells spanning diploid to tetraploid (representing S phase cells). By flow cytometry, neoplastic cells with gains and losses of entire chromosomes, identified as peaks of abnormal DNA content. Genomic Instability Evidence suggests that the basal mutation rate in normal cells is too low to account for the large number of mutations present in many human cancers [18]. It has therefore been hypothesized that neoplastic cells have a mutator phenotype – they acquire (and tolerate) mutations at a higher rate than normal cells [7]. Although the exact cause of this genome instability is not known in most cancers, there are at least two broad types of instability recognized, microsatellite instability and chromosomal instability [19, 20, 21, 22]. DNA Mismatch Repair Deficiency and Microsatellite Instability. The role of DNA mismatch repair in human tumorigenesis was uncovered by three independent groups [23, 24, 25]. Instead of loss of heterozygosity, these investigators found that a subset of cancers contained widespread alterations in the sizes of microsatellites. Termed microsatellite instability (MSI), it is now known that these tumors have defects in one of the genes that mediate DNA mismatch repair (reviewed in 26). Hereditary germ line DNA mismatch repair defects are also the underlying cause of about two thirds of hereditary non-polyposis colorectal cancer (HNPCC) [27]. Loss of mismatch repair leads to a dramatic increase in mutation rate, which is characterized by frameshift mutations in repetitive microsatellite DNA sequences. When identified in a PCR test, these alterations are called high frequency microsatellite instability (MSI-H) [23, 24, 25, 28, 29]. Mutations are also occur in short repetitive DNA sequences within coding regions of genes, such as the TGRBRII gene [30], and therefore the molecular pathogenesis of mismatch repair deficient cancers follows a genetically distinct pathway. Chromosomal Instability. In cancers without microsatellite instability, the most common types of genetic alterations are gains and losses of entire chromosomes, resulting in changes in DNA copy number (aneuploidy), subchromosomal allelic gains and deletions, and often chromosomal translocations and rearrangements. This form of genome instability is broadly referred to as chromosomal instability, and includes most aneuploid tumors [21, 22, 31]. Although the exact biologic basis of this process is not clearly defined, abnormalities in cell division checkpoints, including regulation of double strand break repair, chromosomal segregation and mitotic spindle assembly, are leading candidates [32, 32]. CpGIsland Methylator Phenotype. Methylation of CpG islands in gene promoter regions is a major mechanism of shutting down tumor suppressors in human neoplasia (reviewed in 16, 17, 33). Some investigators have observed a subset of tumors in which this phenomenon is particularly prevalent, suggesting an underlying defect in methylation (akin to a form of genome instability) [34, 35]. The underlying cause of methylation abnormalities are not understood, and it remains unknown whether these tumors are truly distinct. Telomerase and Telomere Erosion. Telomeres are terminal DNA sequences at the end of chromosomes that play a critical role in maintenance of chromosomal integrity (reviewed in 36). Telomeres are difficult to replicate, and become shorter over time with ongoing cell division (telomere erosion), eventually leading to a state where cells can no longer divide (senescence). In cells that have lost cellular checkpoints provoked by DNA damage (such as p53 and Rb deficient cells), cell division may persist despite telomere erosion, resulting in a burst of genome instability that may be important in neoplastic progression [36]. In mouse models and human tissues, such a burst of genome instability has been seen to accompany the transition through high grade dysplasia to cancer in the colorectum [38]. Genetic versus Epigenetic. In addition to genetic alterations, there are widespread gene expression abnormalities that accompany neoplastic progression. Referred to as epigenetic changes, these alterations are often more difficult to implicate biologically because it is hard to demonstrate that they have undergone clonal expansion (reviewed in 39, 40) . Molecular Diagnostic Approaches and Limitations Immunohistochemistry. The simplest way to understand the complex events in genetic progression may be to reduce them to their smallest elements (a reductionist approach). So, rather than addressing whether a gene is oncogenic, tumor suppressing, or related to genome instability, one can simply determine whether a gene is up or down regulated, and how this relates to biologic and clinical outcomes. mRNA expression. The advent of microarray expression profiling, in particular, has led to the identification of putative biomarkers with no available antibodies. Furthermore, in some studies, mRNA expression levels have correlated with clinical outcome, while protein expression has been less predictive. Among the methods currently utilized in mRNA expression analysis, quantitative RT-PCR (using real time PCR technology) and in-situ hybridization have the most relevance to pathology practice. Mutation detection. There are a variety of methods to detect mutations in clinical samples that are beyond the scope of the presentation (reviewed in 41, 42) . A number of steps are involved, including nucleic acid extraction, PCR, and some sort of mutation screening (including sequencing, SSCP, and others). These methods are usually utilized in molecular diagnostic labs, and application to GI biopsy samples has the added difficulties of heterogeneous cell populations, small sample size, and poor quality of nucleic acid obtained from paraffin samples. Allelic deletion (loss of heterozygosity) and amplification. Identification of these alterations has particular relevance to biomarkers of progression in GI dysplasia. Unfortunately, these analyses require fairly sophisticated methodologies, including PCR, fluorescence in situ hybridization (FISH; reviewed in 43), and comparative genomic hybridization (CGH; reviewed in 44). Use of microarray based methods may facilitate future clinical use (reviewed in 45, 46) . Rare mutation detection from shed cells. Tumor cells may be shed into a number of body fluids and cavities, including blood, urine, sputum, feces, bile and others. One approach to neoplasia screening has been to utilize the identification of rare DNA mutations within these samples as a marker of occult malignancy [47]. Gene Expression Profiling. The development of high through-put expression (transcriptional) profiling has opened a new window in the descriptive analysis of human neoplasms (reviewed in 45, 48, 49) . DNA microarrays have up to 50,000 DNA sequences or genes spotted onto glass slides or fabricated into "chips" allowing for simultaneous comparative analysis of the expression levels of a vast number of mRNAs. When combined with complex biostatistical computation methods, these approaches have identified subsets (or clusters) of human tumors that share major gene expression profiles. DNA mismatch repair. Microsatellite instability is detected by extracting DNA from normal and tumor tissue, amplifying selected microsatellites by PCR, and analyzing fragment size by gel electrophoresis or automated sequencer [28]. 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