1

Mutagenesis, Malignancy and Genome Instability

Garth R. Anderson, Daniel L. Stoler, and Jeremy D. Bartos Roswell Park Cancer Institute, Buffalo, NY, USA

1 The Need for Genomic Instability 3 1.1 The Conceptual Framework 3 1.2 Multiple Forms of Genomic Damage 4

2 When Does Genomic Instability Begin? 7 2.1 Onset Early in Tumor Progression 7 2.2 The Leukemia Exception 8

3 The Maintenance of Genomic Integrity 9 3.1 DNA Damage Repair 9 3.2 High-fidelity DNA Replication 10 3.3 Chromosomal Segregation during Mitosis 10 3.4 Destabilized Chromosomes: Telomeres 11 3.5 When All Else Fails: Apoptosis. And without it, Trouble 11

4 In the Clinic 12 4.1 Heterogeneous Tumor Cell Populations and the Therapeutic Target Problem 12 4.2 Genomic Instability as a Clinical Prognostic Tool 13

5 A Difficult Future 14

Bibliography 14 Books and Reviews 14 Primary Literature 15

Encyclopedia of Molecular Cell Biology and Molecular Medicine, 2nd Edition. Volume 9 Edited by Robert A. Meyers. Copyright  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-30551-3 2 Mutagenesis, Malignancy and Genome Instability Keywords

Aneuploidy A cell containing other than the normal diploid number of chromosomes.

Arbitrarily primed PCR and Inter-(Simple Sequence Repeat) PCR Sampling methods to monitor genomic damage, in which polymerase chain reaction products are compared for tumor and corresponding normal tissue DNA. The former method utilizes arbitrary PCR primer pairs to copy the sampled set, while the latter method utilizes a single PCR primer to copy sequences (200–2000 bp) between relatively closely spaced repeat sites.

Intrachromosomal Instability A form of genomic instability in which alterations occur within individual chromosomes, but not particularly to repeat sequences.

Microsatellite Instability A form of genomic instability arising from defects in the DNA mismatch repair system, leading particularly to alterations of small numbers of bases within DNA-repeat sequences.

Spectral Karyotype (SKY) A technique by which individual chromosomes are labeled with distinctive fluorescent probes, enabling the direct viewing of both the number of chromosomes in a cell and the translocations between chromosomes.

Genomic instability is a widespread and essential feature of the common adult- onset cancers. For solid tumors to arise, several key genes must become mutated (to generate growth, facilitate invasion, enlist nutrient supply, evade the immune response, avoid apoptosis, and so on) and this degree of mutation requires ab- normally high mutation rates. To achieve such rates, genes whose normal role is to maintain genomic integrity become mutated, leading to ongoing genomic instability. Exogenous mutagens (carcinogens) additionally contribute to overall genomic damage, particularly in tobacco smokers. Genomic destabilization slowly leads to cancer as cells with mutations in genes advantageous for tumor develop- ment become selected, expand their populations, and evolve further. A considerable majority of human genes are involved in multicellular coordination and are not essential for maintaining the viability and reproductive potential of individual cells; thus, a window exists wherein an appropriate degree of genomic instability leads to tumor progression, as somatic evolution selects cells driven by self-interest. As with Darwinian evolution, such somatic evolution inherently leads to genomic diversity, Mutagenesis, Malignancy and Genome Instability 3

seen clinically as tumor heterogeneity. Although genomic instability and the ensuing genomic heterogeneity render therapeutic targeting difficult, measurement of the degree and form of genomic instability has considerable potential as a clinical prognostic tool.

1 for complex multicellular organisms like The Need for Genomic Instability ourselves, with a wide variety of highly dif- ferentiated specialized tissues, spatial and 1.1 temporal control and coordination issues The Conceptual Framework come to predominate. Over the course of our evolutionary history, genes evolved, Cancer. What a strange disease! For in which enable cells to communicate with biology we are so used to seeing order, one another, responding to signals from stepwise differentiation and development, other cells in a highly specific manner, and precise control. Yet, with the common while also enabling those same cells to re- adult-onset solid tumors, we see impre- spond to other programs locked in during cision, unpredictable behavior, evasion of targeted therapies, disorganized growth, differentiation. Our cells must cooperate, and chaos. And death. But why is this so? proliferating only when and where ap- What is the nature of this disease, and why propriate, and willingly dying when and does it not behave like other life? Or does where appropriate; the necessary genes it, and we just need to recognize that can- to effect these behaviors evolved. Further cer has more in common with the random gene-based regulatory mechanisms arose, nature of evolutionary biology than it does which cause specific cells to adhere only with the fine coordination of organismic to appropriate partners, and move only in biology governed by precise processes of appropriate manners. molecular biology? The intricate, evolved coordination pro- To comprehend these cancers, we must cess generating this complex cellular be- first recognize the basic nature of multicel- havior is contained within the information lular organisms such as ourselves. Going stored in each duplicated copy of our from a unicellular organism to a mul- genome, present in each of our hundred ticellular organism was a huge leap in trillion or so cells, and this coordination evolutionary history. Single-celled organ- process continues, as most of these cells isms have to cope with the formidable are themselves turning over, at widely tasks of creating and replicating genetic varied rates. The complexities of this coor- information, controlling its expression, dination process are far more intricate than metabolically capturing energy to synthe- those fundamentally required for unicellu- size new components, and then coordi- lar life, and this is manifested within our nating the whole process to replicate their genomes. For the unicellular eukaryote entire selves. These cells have the essential yeast, only about 4000 genes are essen- genes and systems to allow self-replication, tial for its cellular growth and replication but their only concern about their neigh- (Saccharomyces Genome Database). In bors is to outcompete them. In contrast, contrast, humans contain approximately 4 Mutagenesis, Malignancy and Genome Instability

10 times as many genes; some of these patterns, with disastrous consequences. additional genes generate more complex We therefore contain within each cell a cellular structures and functions, but most series of redundant safeguards necessary are utilized to coordinate multicellular to maintain the quality of our genetic in- growth and organization, giving rise to formation for many decades, enabling us differentiated cells and tissues signaling to survive as individuals to a point where and responding to one another. our progeny are fully capable of surviv- What happens when the quality of a cell’s ing and reproducing on their own. If a genetic information deteriorates? When single gene were sufficient to start the damage is severe and extensive, essential process of somatic evolution eventually gene functions will be lost and the cell leading to malignancy, then somewhere dies. But when genetic damage is less se- within our large population of cells that in- vere, and is ongoing over a long period of dividual gene loss would inevitably occur, time, cancer can slowly arise. For if ge- andwewouldneverbeabletosurvive netic damage occurs at a rate insufficient to adulthood. Redundant safeguards to to kill a cell, a population of mutant cells ensure replication fidelity and efficient has time to develop, expand, and acquire damage repair therefore exist to preserve additional mutations. With most human our genomic integrity. Additional apop- genes involved in multicellular coordina- totic safeguards destroy cells with badly tion, it becomes substantially more likely damaged genomes, reducing the proba- that a coordination gene will be lost than a bility of developing cancer to a tolerable gene essential for cellular survival. If both level. In spite of this, the disease man- alleles of a gene eventually become lost ages to eventually develop clinically in or mutated, less coordinated growth then about one-fourth of us, and is present in occurs. And these daughter cells in turn nearly all the elderly at a microscopic level. evolve further, and expand their popula- Low mutation rates acting over a long pe- tions. Selection will occur again and again riod of time provide a powerful means for cells with greater abilities to prolifer- for even the most stringent safeguards to ate, spread, and evade attack in the form be overcome by natural selection and cell of host defenses or therapeutic treatments. lineage expansion. Different daughter cells are free to evolve in different directions; unlike classical Dar- 1.2 winian evolution where natural selection Multiple Forms of Genomic Damage is stringent and competition fierce, the hu- man body is a hospitable environment for Unlike leukemias where a single translo- many of the successful diverging lineages. cation can complete the two basic steps This somatically evolved, genomically het- needed to produce this malignancy, solid erogeneous cell population becomes what tumors require several mutational events we see clinically presenting in the patient before a normal cell becomes an uncon- as a cancer. trollably proliferating invasive cancer cell; Human evolution occurred in spite of as described above, this slowly develops this same basic problem inherent in all through an ongoing process of cell pop- multicellular organisms, namely, where ulation expansion and natural selection loss of genomic stability will lead to of advantageous mutations. To a large cells evolving toward unicellular behavior degree, this arises from the mutation of Mutagenesis, Malignancy and Genome Instability 5 genes whose role is for the maintenance chromosome. Spectral karyotypic analysis of genomic integrity, yielding the genomic of multiple cells from a single tumor re- instability that underlies tumor progres- veals differences between cells, as chromo- sion; the continuing genomic instability somal instability continues during tumor of cultured tumor cells conclusively es- growth and genomic heterogeneity devel- tablishes a genetic component. Extrinsic ops (Fig. 1). carcinogens such as tobacco smoke can Intrachromosomal instability adds a initiate the process by mutating genes for third fundamental element to the genomic the preservation of genomic integrity, and complexity of solid tumors, in the form expedite the process by directly generating of numerous insertions, deletions, inver- numerous additional mutational events. sions, amplifications, and other smaller Genomic instability and ensuing tumor events. The estimated number of such progression thus become the combined events is increasing as our ability to detect result of endogenous (genetic) and exoge- and resolve them improves, with current nous factors. rough estimates of around ten thousand Genome instability in cancer is seen in events per tumor cell. The failure to de- multiple forms. Roughly 10% of adult- tect genomic damage in the forms of onset solid tumors exhibit abundant single microsatellite instability or chromosomal or oligobase events seen as microsatel- instability should not be taken to mean lite instability. As discussed below, this there is no genomic instability at all in arises from defects in DNA mismatch re- atumor. pair during DNA replication. Since repeat Inherent advantages exist for aneuploidy sequences are inherently more likely to to cooperate with intrachromosomal insta- generate small replication errors because bility during tumor progression. In order of slipped mispairing during replication, to remove both alleles of a key regula- the phenomenon is most readily seen tory gene through aneuploidy alone, both in repetitive microsatellite sequences. A small number of genes such as TGFβ- copies of the chromosome will have to RII contain coding repeat sequences, and be lost. But along with the gene of in- in tumors arising by microsatellite insta- terest, other genes essential for cellular bility, mutations in such genes become viability inevitably become lost, bringing selected for. These tumors have very few the somatic evolutionary process of tu- other mutations within nonrepeat cod- mor progression to an abrupt end. On ing sequences. the other hand, mechanisms, which alone Genome instability in cancer is more generate small events, also have problems; often seen on a larger scale, in the the probability of hitting both alleles of form of chromosomal instability or ane- a given gene become very low as the uploidy. Entire chromosomes are miss- event size shrinks. But when a process ing or present in extra copies, arising generating small events cooperates with a from inappropriate chromosomal segre- process generating chromosomal instabil- gation during mitosis generating aneu- ity, critical genes can readily be mutated ploidy. Translocations generating recom- and the normal allele lost, still without binant and/or truncated chromosomes killing the cell (Fig. 2). The evolutionary additionally occur, and multiple recom- process of tumor progression is then free bination events can arise within a single to slowly unfold. 6 Mutagenesis, Malignancy and Genome Instability

Colon 5 12 3 4 5

Cell carrying 92 6 7 8 9 10 11 12 chromosomes with various 13 14 15 16 17 18 translocations and derivatives 19 20 21 22 XY

M M M MMMMMMM

12 3 45

Cell carrying 84 6 7 8 9 10 11 12 chromosomes with various 13 14 15 16 17 18 translocations and derivatives

19 20 21 22 X Y

MMMMM

Spectral karyotypes of two cells from a single tumor Fig. 1 Genomic instability gives rise to genomic karyotyping. Chromosomes designated M heterogeneity within each tumor. Two cells from represent a variety of different translocations. the same tumor, colon 5, differ extensively at the Image is from Bartos et al. (2004). genomic level when analyzed by spectral

Multiple forms of instability occur sequentially Fig. 2 Multiple forms of genomic instability arise during tumor A. Early events: small progression. Inherent advantages exist for small-event instability to cooperate with large-event instability.

- Detected by inter-(simple sequence repeat) PCR, but not by spectral karyotyping (SKY) or Bac-arrays - Low probability of hitting both copies of a gene - Tumors are relatively benign

B. Later events: large

(Deletion)

- Detected by SKY, ordered Bac-arrays, inter-SSR PCR - Early silent mutations revealed - Tumors are more aggressive Mutagenesis, Malignancy and Genome Instability 7

This article focuses on genomic instabil- However, that viewpoint is still not uni- ity at the genetic level, where cells acquire versally accepted. Rubin, Duesberg, and permanent heritable changes. An addi- others postulated that normal mutation tional level of genomic instability occurs rates applied to a large, rapidly prolifer- at the epigenetic level, where changes in ating cell population, such as the stem DNA methylation patterns, particularly of cells in colonic crypts, would be likely CpG islands, can block the expression of to generate a rare cell, with exceptional otherwise normal genes. Clinically, this is bad luck, containing all the necessary mu- a significant source of microsatellite in- tations to produce malignancy. This cell stability, when the mismatch repair gene would then expand its population inex- MLH1 is silenced epigenetically. Compli- orably, generating the malignant tumor. cating the study of epigenetic events in By this reasoning, tumors should be essen- cancer is the finding by Smiraglia and tially homogeneous at the genomic level, colleagues that DNA methylation patterns but recent spectral karyotyping analyses of of cell lines grown in culture exhibit multiple tumor cells clearly show this not major differences from the patterns of tu- to be the case. mors themselves, presumably reflecting Verification of the Loeb model has come the silencing of genes superfluous to the from multiple directions, centering on artificial culture environment. demonstration of extensive genomic dam- age within tumor cells, the finding of extensive genomic heterogeneity within 2 tumors, and tissue culture assays of ge- When Does Genomic Instability Begin? nomic instability using tumor cell lines showing substantially elevated gene am- 2.1 plification rates. Since genomic instability Onset Early in Tumor Progression describes an ongoing process, it is impor- tant that these separate forms of evidence Lawrence Loeb was first to clearly point exist. Extensive genomic damage occur- out the need for genomic destabilization ring in a single burst, as can be seen to permit the several requisite steps to clinically when induced by radiation ther- convert a normal cell into a malignant tu- apy or chemotherapy, does not establish mor cell. This became more evident upon that instability exists; in contrast, true ge- the seminal demonstration by Land et al. nomic instability generates progressively that mutation of two cooperating onco- accumulating genomic alterations and ge- genes alone was insufficient to produce nomic diversity within a tumor. malignancy in mice, and the observations Measuring genomic damage is a com- by White, Vogelstein, and others that sev- plex task, reflecting the diversity of forms eral events in addition to suppressor gene of damage that may be present. Flow cy- loss occurred during human colorectal tu- tometry measurement of DNA content mor progression. On the basis of known per cell provides a rough measurement normal mutation rates and the need for of overall aneuploidy, but karyotyping is more than two mutational events, somatic essential for identification of specific chro- evolution to malignancy had to some- mosomal gains and losses. The advent of how be facilitated by decreasing genomic spectral karyotyping has greatly simplified stability. such analyses, and while also revealing 8 Mutagenesis, Malignancy and Genome Instability

more complex chromosomal translocation carcinomas. Such results do not mean that events. Comparative genomic hybridiza- further genomic damage does not accu- tion to entire metaphase chromosomes re- mulate during progression, however. In veals amplifications and deletions of chro- the inter-SSR PCR analyses, DNA from mosomes and chromosomal fragments, million cell specimens were used, mak- ranging in size down to a few megabases. ing later events not present in a majority These may be integrated within other chro- of cells of the specimen undetectable by mosomes, or may be existing freely in this method. Karyotypic analyses of early the form of double minutes. Substantially adenomas, including spectral karyotypes, higher resolution is now achievable with confirm early extensive genomic damage the development of ordered Bac-arrays, in the forms of aneuploidy and translo- in which chromosomal regions as small cations. Recent work by Pretlow and col- as 150 kb may be analyzed. Finer anal- leagues, utilizing arbitrarily primed PCR yses down to single base size may be indicates that certain forms of genomic in- achieved through genomic sampling, in stability begin even prior to the adenoma which several smaller regions distributed stage, and can be detected within approx- throughout the genome are studied in de- imately one-fourth of aberrant crypt foci. tail. Arbitrarily primed PCR (polymerase These observations are highly important chain reaction) utilizes multiple primers in establishing that genomic instability be- with arbitrary sequences to amplify a small gins early, putting it in the proper position set of broadly distributed products. Inter- to facilitate the somatic evolutionary pro- (simple sequence repeat) PCR, inter-SSR cess of tumor progression. Early reports PCR, utilizes a single primer, anchored proposing that genomic instability might within repeat sequences at their 3
ends simply represent one of the numerous facing outward, to amplify those non- phenotypic differences between malignant repeat sequences between two repetitive and normal cells, being a consequence elements and generates roughly forty dif- and not a cause of malignancy, are no ferent genomic products per reaction. longer tenable. Both of these techniques provide means of readily sampling the genome to esti- 2.2 mate overall damage levels. Direct DNA The Leukemia Exception sequencing of the entire tumor genome is not currently practical, and inevitably Acuteleukemias,somechronicleukemias, becomes complicated by genomic hetero- and a few other uncommon child- geneity within the tumor cell population. hood/adolescent cancers present excep- But eventual comparisons of sequence tions to this model, affecting cell types data for individual tumor cell genomes when they are particularly vulnerable to tu- can be expected to yield fascinating data. mor development. If a normally proliferat- Examination of premalignant colorec- ing cell type in a nonlimiting environment tal adenomas demonstrated that extensive uncontrollably increases its proliferation genomic damage begins early in tumor rate, or loses the ability to differentiate, progression. Inter-(simple sequence re- mature, and ultimately die, the population peat) PCR analysis shows several thousand of this cell type will expand dramatically. genomic events have occurred in early ade- To generate these tumors, the number nomas, a level similar to that seen in frank of mutated genes can be as few as two, Mutagenesis, Malignancy and Genome Instability 9

Fig. 3 Extensive genomic heterogeneity Therapeutic pressure within a tumor underlies a considerable part of the eventual failure of pharmacologic therapies for the common, genomically unstable cancers. When therapeutic agents are first applied, susceptible cells are destroyed. But eventually, resistant cells are able to repopulate and further expand the tumor cell population. Selection of resistant cells which can be generated in a single step present in the undamaged DNA strand. by a translocation event that simultane- Larger events where breakage occurs can ously rearranges two genes. Such tumors exploit the genetic information present in arise rapidly, but due to their genomic a sister chromatid or homologous chro- homogeneity they also generally respond mosome, through homologous recombi- well to treatment; chronic myelogenous national repair, and still maintain full leukemia and its response to Glivec is integrity of the genetic information. While the classical example of a drug targeted this is feasible in a diploid cell or in a hap- to the translocation event, but childhood loid cell in cell cycle phase G2 using the acute leukemias also respond well to more sister chromatid, when allelic losses have conventional therapies. This picture can occurred or the sister chromatid has not yet abruptly change, as such a tumor pro- been synthesized, the process can fail be- gresses through blast crisis, giving rise cause of the absence of an intact template. to a genomically unstable heterogeneous When homologous sequences have been tumor cell population capable of evading lost or when breakage is extensive, repairs therapeutic interventions, and in doing so, of multiple double-stranded DNA break- leukemias can come to resemble the adult- age events can still utilize nonhomologous onset solid tumors (Fig. 3). end joining, but with the consequential loss of genomic integrity at the translo- cated repair sites. Further problems arise 3 if chromosomal fragments are not rein- The Maintenance of Genomic Integrity tegrated into whole chromosomes; either 3.1 the genetic information is lost or the small DNA Damage Repair fragment circularizes, with the risk of over- replication and amplification. DNA repair is a versatile process evolved In cancer, examples are known for de- to cope with diverse types of DNA dam- fects in each of these systems, as well age, which naturally occur. Single base as germ-line defects giving rise to hered- damage elicits base excision repair, while itary cancer syndromes. The first such dinucleotide damage (as occurs with UV- case identified was the causal relation- induced thymidine dimmers) utilizes nu- ship between mismatch repair defects and cleotide excision repair. Both of these hereditary nonpolyposis colorectal cancer. processes exploit the genetic information Although germ-line defects are found in 10 Mutagenesis, Malignancy and Genome Instability

the family cancer syndrome, with mu- functional gene products themselves, two tations predominantly in hMLH1 and keys are the ability to monitor the qual- hMSH2, microsatellite unstable sporadic ity of the replication product, repairing colon cancers (about 10% of sporadic damage immediately, and the ability to colon cancers) also exhibit defects in temporarily halt replication through check- this same system, either arising from so- point activation, waiting to resume until matic mutations or epigenetic silencing. themoremajordamageisrepaired.And Identification of BRCA1 and BRCA2 as as a final safeguard, the cellular suicide the major genes behind familial breast process of apoptosis is ready to eliminate cancer, and the subsequent elucidation any cell with irreparable damage to its of the roles of these genes in homolo- genomic integrity. gous recombinational repair, reinforced While DNA repair draws most attention the concept of defective repair genes un- as a foundation for genomic instability derlying both genomic instability and the and tumor progression, uncorrected er- subsequent cancer arising after decades of rors introduced during DNA replication somatic evolution. are equally serious and are known to un- derlie some familial cancers. Mismatched 3.2 repair errors, in particular, give rise to High-fidelity DNA Replication hereditary nonpolyposis colorectal cancers and endometrial cancers. Errors arising DNA replication is the point where during normal DNA replication are in genomic integrity inherently becomes general within the capability of repair sys- most vulnerable, and most malignancies tems to process, but high-level error rates correspondingly arise out of the relatively have the potential to exceed normal re- rapidly proliferating cell populations such pair capacities. as those lining the gut or in the basal layer of the skin. During replication, the three 3.3 billion base pairs of the human genome Chromosomal Segregation during Mitosis must be copied accurately, completely, and only once, during the few hours that The first known form of genomic insta- constitute S-phase. This may be occurring bility in cancer was aneuploidy, ranging in an environment contaminated with from extra or missing copies of single chro- carcinogens, reactive oxygen species, or mosomes up through tetraploid or higher other agents that may damage DNA. states, where entire additional copies of the And once the DNA is replicated, it complete genome are present. These phe- must be accurately segregated to daughter nomena arise out of inaccurate segregation cells during mitosis. In spite of these of daughter chromosomes during mitosis. difficulties, and the fact that billions of One common diagnostic hallmark of solid cells endure these constraints over seven tumors is the presence of multipolar mi- decades, most of us complete our lives toses leading to inappropriate segregation. never having developed a malignancy. Centrosomal anomalies typically under- How is such precision achieved? DNA lie such inaccurate segregation; these in replication involves over two hundred turn can be a consequence of oncogene separate gene products, in a most intri- activation as shown by Duensing et al. cate orchestration. Aside from the highly Thus, a stimulus for overproliferation can Mutagenesis, Malignancy and Genome Instability 11 promote further tumor progression by small circular chromosomal structures. giving rise to this particular form of chro- This entire process is known as bridge- mosomal instability in turn leading to breakage-fusion, and represents one means the loss of suppressor or other growth of producing translocations and deletions. inhibitory genes, extra copies of growth- Amplifications can also arise as circu- promoting genes, and the like. Qualitative lar structures overreplicate and randomly changes in these genes, however, do not reintegrate. arise out of aneuploidy; for this reason Telomere deficiency is able to promote there is a selective advantage during tumor tumor development in mice, and human progression to bring intrachromosomal cells with severely shortened telomeres instability into play. Apparent centroso- are associated with tumor formation. mal abnormalities can also arise from Since telomerase is reactivated in many improper separation of sister chromatids tumor cells as part of the acquisition during mitosis, as is seen with sepa- of cellular immortality, telomere-mediated rate defects. instability is likely to play a larger role relatively early in tumor progression. 3.4 Attempts at treating cancer by interfering Destabilized Chromosomes: Telomeres with this reactivated telomerase may be counterproductive as genomic instability Chromosomal ends are capped with is driven higher, although this would unique structures, telomeres, which are relate to the relative rate of increase recognized as end structures, and thus in genomic instability compared to the are not subjected to ligation-mediated re- relative rate at which the cells then pair. Human telomeres contain eight-base progress to senescence. DNA-repeat sequences, totaling around 10 kb in length in young individuals. 3.5 Telomeres shorten in somatic cells as they When All Else Fails: Apoptosis. And without undergo repeated rounds of replication it, Trouble as part of the normal maturation pro- cess, since the telomerase that generates Maintenance of genomic integrity includes these cap structures is only active dur- the fail-safe mechanism of apoptosis to ing embryonic development and in germ cause the self-destruction of cells, which cell generation. in spite of their several DNA damage re- When telomeres are short or absent, pair systems and replication checkpoints, chromosomes become very sticky at their still manage to contain significant unre- ends as repair is attempted through lig- paired or incompletely repaired damage. ation. This generates a dicentric chro- Apoptosis utilizes sensors to detect DNA mosome, with two centromeres. Alterna- damage; these particularly rely on the tively, exonuclease processing may play ataxia telangiectasia mutated (ATM) pro- a significant role in generating dicentric tein family to sense broken DNA and chromosomes. During mitosis, dicentric trigger a kinase cascade leading through chromosomes are broken randomly as the capase activation to cell destruction. An- daughter cells separate. In turn, these tiapoptotic mechanisms exist to suppress breakpoints are highly unstable, fusing uncalled-for suicide, utilizing in particular into other chromosomes or generating the Bcl2 system. Overexpression of Bcl2 12 Mutagenesis, Malignancy and Genome Instability

can promote hematologic malignancies to the initiating event. And targeting a to- simply by preventing proper numerical tally lost gene, such as with adenovirus control of specific cell populations. cytocidal to p53-negative cells, is proving For solid tumors accumulating extensive to be even more difficult than targeting a DNA damage during their years of progres- specific alteration. sion, defects in the apoptotic pathway are This problem can be further illustrated widely seen and play their own crucial role. with two antibody-based therapies. Her- As genomic damage accumulates, these ceptin is used in the treatment of breast cells survive and progress further, instead cancer, directed against the frequently am- of altruistically eliminating themselves. plified Her2-neu homolog of the epidermal growth factor receptor, which confers a proliferative advantage. Therapeutic fail- 4 ure occurs after several months, largely In the Clinic as a subpopulation of existing resistant 4.1 tumor cells become selected for and repop- Heterogeneous Tumor Cell Populations and ulates the tumor (Fig. 3). Antiangiogenic the Therapeutic Target Problem therapies have had considerable theoreti- cal appeal, in that they are directed against Medical oncology has achieved notable the genomically stable endothelial cells of curative success in the treatment of the tumor vasculature. Experimental stud- acute childhood leukemias responding to ies of mouse models with two peptides agents targeting proliferating cells, and (angiostatin and endostatin) were highly excellent if not curative responses have successful, but have so far yielded disap- been achieved in a few adult tumors such pointing results in human clinical trials. as chronic myelogenous leukemia, where This has been followed by new approaches the etiologic Philadelphia Chromosome using monoclonal antibodies targeted at translocation provides a specific target in the major angiogenic factor, vascular en- the recombined Abl kinase. dothelial growth factor and its receptor. However, medical oncologists have But even here therapeutic failure oc- achieved far less success in the treatment curs within a few months. Heterogeneous of adult-onset solid tumors, where death tumor cells produce several different an- may be delayed but cure is rarely achieved. giogenic factors, and the elimination of Thesamegenomeinstabilitynecessaryfor one system soon selects for cells utilizing tumor progression to occur has an addi- other factors. tional negative consequence as genomic It now appears reasonable to conclude diversity permeates the tumor cell popula- that therapeutic rehabilitation of adult- tion. Genetic events frequently seen in a onset solid tumor cells is likely to be tumor type are not present in all cells of an exercise in futility. And based on the tumor, unless the event occurred very, results seen up to the present with very early in progression and all tumor even the newest classes of tumor cell cells have descended from a cell with that targeted agents, the same could relate event. Apc evidently represents such a tar- to therapies, even combination therapies, get for colorectal tumor progression, but targeted at specifically killing the tumor even here alternative mutations occurring cells. For within each such tumor there later in progression may create alternatives is a genomically diverse cell population, Mutagenesis, Malignancy and Genome Instability 13 exploiting a variety of pathways, and with with more extensive damage typically will a broad spectrum of genomic defects reflect more rapidly evolving populations, in individual cells. Common events will when comparisons are made on a stage- be present in various lineages, akin to for-stage basis and patients with prior DNA the branches of a tree, and various damaging therapies are excluded. Multiple recurring means of effecting proliferation biopsies can reveal genomic heterogeneity and evading host defenses will be selected, within the tumor cell population, which but diversity and heterogeneity will almost in turn further reflect the likelihood that always be present to some extent. cells resistant to any given therapy are So how can cancer ever be cured? We already present. must never forget that currently almost The form of genomic instability present half of all cancers are indeed cured, and also has major clinical implications, in those successes are principally the result terms of genomic diversity. Microsatel- of surgical resection. If the patient is phys- lite instability generates approximately ically separated from the heterogeneous 600 000 events, but since few genes con- tumor cell population, the heterogeneity tain coding repetitive sequences, relatively arising out of tumor cell genome instabil- little damage to genetic information oc- ity no longer matters. Surgery, although curs. And such tumors generally have a relatively costly and associated with its good prognosis. With hematologic malig- own morbidities and mortalities, is the nancies, acute childhood diseases driven most effective approach to curing cancer, by a single translocation provide a homoge- and efforts directed at improving surgical neous target, and also respond to therapy. capabilities have genuine potential. Im- But once aneuploidy and intrachromoso- provements in diagnosis and detection mal damage becomes extensive, as in blast through imaging, molecular diagnostics, crisis, therapeutic outcome becomes poor. and patient-friendly screening approaches, The selection of a clinically practicable can significantly improve the ability of the assay methodology is not a trivial matter. surgeon to cure cancer patients. This, com- A few approaches have demonstrated their bined with proven prevention strategies practicability for providing genome-wide directed at tobacco and other environmen- information in a clinical setting. Spec- tal factors, should not be neglected in tral karyotyping, in which chromosomes favor of glamorous but marginally effective are painted with specific probes, requires new therapies. short-term cell culture but reveals consid- 4.2 erable information in terms of aneuploidy Genomic Instability as a Clinical Prognostic and chromosomal rearrangements. The Tool cost and expertise level required for this technique presently make it of rather lim- Clinical application of genomic instability ited general clinical utility. for purposes of diagnosis and prognosis is Array-based approaches comparing tu- constrained by the limitations of assaying mor DNA with normal DNA effectively a biopsy specimen in vitro, and generally at reveal amplifications and deletions, in a a single point in time. Thus, what is being higher resolution form of comparative ge- measured is more accurately genomic nomic hybridization. Such approaches can damage, but even from this, meaningful exploit conventional expression arrays, but clues often can be obtained. Tumors with nuclear DNA copies substituting for 14 Mutagenesis, Malignancy and Genome Instability

cDNAs copied from mRNA. Alternatively, adult-onset cancers. This same instability specialized arrays utilizing ordered bac- gives rise to genomic and cellular hetero- terial artificial chromosomes (Bacs) can geneity within the tumor as it presents in representatively scan the entire genome, the patient; such heterogeneity generates at a resolving power of about 150 kb. serious problems for nonsurgical thera- Future improvements in resolution, ex- peutic approaches. ploiting single nucleotide polymorphisms, Yet, all is not hopeless. Most cancer cures can be anticipated. Current analyses of ar- are currently achieved with surgery, and ray data are not yet practical for widespread there remains considerable room for im- clinical application due to cost and lim- provement in this area. Promising routes itations in data evaluation, although this for the future include improving early should be surmounted in the near future. detection, particularly through the use An alternative approach is the applica- of proteomic technologies, in turn en- tion of genomic sampling methodologies, abling more timely surgical intervention arbitrarily primed PCR and its variant, before metastasis has occurred. Identifi- inter-(simple sequence repeat) PCR. In cation of at-risk individuals with familial these approaches, small pieces of the cancer genes, and definition of tumor spe- genome are amplified by PCR, and these cific markers and tests, both fall in the genomic samples prepared from tumor area of early detection. Targeting the ge- DNA are compared with genomic samples nomically stable endothelial cells of the prepared from the patient’s normal tissue tumor vasculature has its own inherent DNA. An electrophoretic comparison of advantages over targeting the genomically the PCR products reveals what fractions of unstable and genomically heterogeneous the bands are altered, providing a quanti- tumor cell population. And basic preven- tative estimate of the degree of genomic tion measures such as eliminating tobacco damage. use must not be taken lightly. These methodologies have been applied The cancer problem will be solved nei- as a prognostic tool for breast cancer, ther quickly nor easily. But as the nature of ulcerative colitis, and colorectal cancer, the disease becomes better appreciated, re- along with Barrett’s esophagus. Genomic search efforts will hopefully come to focus instability quantitated by inter- (simple more on truly productive areas. sequence repeat) PCR has further been shown to be capable of distinguishing be- tween thyroid tumors and benign lesions, See also Epigenetic Mechanisms in which should be of substantial utility in Tumorigenesis; Intracellular Sig- dealing with relatively common thyroid naling in Cancer; Oncology, Mole- nodules of indeterminate cytology, which heretofore have been surgically removed cular. as a precautionary measure. Bibliography 5 A Difficult Future Books and Reviews

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