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Stephen A Bustin
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General Information.
INTRODUCTION Colorectal cancer is a disease of the genome, which is altered at multiple sites in cancer cells by cumulative epigenetic and genetic changes. The progression of a tumour from normal to pre-cancerous cells, to cancer and then on to local invasion and finally metastasis is the result of the clonal expansion of cells that have acquired a selective growth advantage and allows them to outnumber neighbouring cells. This advantage is the result of alterations in genes that control cellular proliferation and cell death and involve specific target genes that are generally divided into three major categories: 1. Alterations that activate proto-oncogenes (e.g. K-ras and H-ras) result in cellular proliferation or inhibition of cell death. 2. Alterations that inactivate tumour suppressor genes (e.g. APC and TP53) result in the inhibition of cell proliferation or promotion of cell death. 3. Alterations in DNA mismatch repair genes result in microsatellite instability and accelerated tumour development. There are good reasons why colorectal cancer has served as a paradigm for the genetic description of tumourigenesis. Firstly, colorectal cancer is an extremely common tumour and its evolution is staged through a relatively consistent series of pathological precursors (the adenoma-carcinoma sequence). Secondly, the cancer and its precursors can be sampled easily by colonoscopy, yielding material for molecular analysis. Finally, there are distinct syndromes in which individuals display a marked predisposition to the development of colorectal cancer. The genetic lesions in FAP (familial adenomatous polyposis) and HNPCC (hereditary non-polyposis colorectal cancer) are now well delineated and their study has contributed enormously to our understanding of the molecular pathology of sporadic colorectal cancer. More recently, mutations in the STK11 (LKB1) gene were identified as the genetic basis of familial Peutz-Jeghers syndrome (PJS), an autosomal dominant hereditary syndrome conferring an increased risk of cancer within the intestinal tract as well as in other areas of the body. STK11 is a serine/threonine kinase and the majority of the mutations lead to a truncated protein product. It has been described as a multitasking kinase that plays a role in chromatin remodelling, cell cycle arrest, cell polarity, and energy metabolism, all of which may require the tumour suppressor function of this kinase and/or its catalytic activity. Intriguingly mutated STK11 is unable to activate the GSK-3b kinase, which phosphorylates beta-catenin and inhibits the wnt signalling pathway. Consequently, mutations in the STK11 gene can result in the inappropriate activation of the wnt/beta-catenin pathway.
Sporadic colorectal cancer is initated by epigenetic changes in the epithelial stem cells located towards the bottom of the crypts. These populate the crypt and acquire alterations that result in the cells no longer responding appropriately to signalling. Precursor lesions form and their exposure to environmental carcinogens eventually results in the acquisition of a malignant phenotype. 
Cell cycle
Cyclin dependent kinases (CDK) drive the cell through the cell cycle by phosphorylation of other proteins. CDK are G1- (Cdk4), S- (Cdk2) or M-phase (Cdk1) specific. Cyclins, which are degraded periodically, bind to CDK and regulate their function by selecting the proteins to be phosphorylated. There are G1-specific cyclins (D), S-phase-specific cyclins (E and A) and mitotic cyclins (B and A).The CDK-molecules can be compared with an engine and the cyclins with a gear box controlling whether the engine will run in the idling state or drive the cell forward in the cell cycle. Genes for CDK-molecules and cyclins can function as oncogenes and CDK and cyclin expression is often upregulated in colorectal cancers. Mitogenic signals, particularly those acting through the K-ras pathway, stimulate transcription of cyclin D isoforms, resulting in increased complex formation between CDK4 and CDK6 and cyclin D. The retinoblastoma gene product, pRB, in its unphosphorylated state, normally binds to and sequesters the E2F family of transcription factors. Successive phosphorylation of pRB by CDK4 and CDK6 (bound to cyclin D) and CDK2 (bound to cyclin E) inhibits its ability to bind and sequester E2F. Upon its release from pRB, E2F becomes transcriptionally active and switches on the transcription of multiple genes important for DNA synthesis and progression into the S phase, including cyclins E and A. Members of both the INK4 and Cip/Kip families of CDK inhibitors inhibit the function of cyclin D/ CDK4/6 complexes, whereas members of the Cip/Kip family also inhibit cyclin E/CDK2 and cyclin A/CDK2 complexes. The products of P53 and PTEN (mutated in some juvenile polyposis families) can strongly induce the expression of certain CDK inhibitors as shown. Loss of P53 function in many colorectal cancers and of PTEN function result in decreased expression of the CDK inhibitors p21/WAF1 and p27/Kip1, respectively, as shown. The CDK inhibitors also function in other phases of the cell cycle not shown here. 
The Tumour microenvironment The microenvironment plays an essential role in the development of colorectal cancer. Inflammatory cells influence cancer initiation/promotion by secreting cytokines, growth factors and chemokines and generate reactive oxygen species. mutations in stromal fibroblasts can precede carcinoma development. 
Genetic testing for hereditary colorectal cancer
Clearly, the development of a genetic technique for making a firm diagnosis of a hereditary colorectal cancer syndrome is of great importance to affected patients and their families. Despite the identification of some of the causative genes, genetic testing in this context is not in routine clinical use. The genes are either very large (as in APC) or multiple (DNA mismatch repair genes). Moreover, in both FAP and HNPCC, there are no mutational hot-spot regions. Several techniques have been employed to detect alterations in these genes, including direct DNA sequencing, heteroduplex analysis and SSCP or single-stranded chain polymorphism. However, each of these techniques has its specific limitations; in particular, they are either quite insensitive or are appropriate only for examining small segments of DNA.
Notwithstanding these limitations, the use of in vitro transcription translation assays is able to detect mutations in approximately 85% of FAP and 50-60% of HNPCC kindreds. The development of this technique is aided by the knowledge that virtually all APC mutations, and at least 50% of those occurring in HNPCC, result in the production of a truncated protein. The gene transcript is amplified and subsequently transcribed and translated in an in vitro system. In the presence of a mutation, protein truncation will then be revealed by altered electrophoretic mobility of the reaction products, with the truncated protein migrating faster than the full-length gene product. For example, an individual who is heterozygous for such a mutation will show two bands, a shortened polypeptide generated from the mutated allele and a full-length protein from the wild-type allele. Such tests have now been developed for FAP and HNPCC.
There are still considerable problems that need to be overcome before these tests can be offered routinely to patients. Firstly, they do not detect all FAP and HNPCC kindreds. Secondly, the validity of a negative result cannot be guaranteed as this may be due either to the relative insensitivity of the technique or to the presence of a mutation that simply is not detected by the test. Finally, genetic testing for these conditions, which with appropriate management are essentially non-lethal, has significant ethical considerations. For instance, should prenatal testing be offered? These issues underscore the importance of conducting any such testing under the auspices of a family cancer clinic with significant input from trained genetic counsellors who can address the medical and psychological questions with patients and their families. Nonetheless, the potential value of these techniques should not be underestimated as they may obviate the need for repeated examination of family members who do not harbour a kindred-specific mutation.
Early detection of colorectal cancerCurative interventions for colorectal cancer depend upon early detection of the tumour. In colorectal cancer various genetic targets have been exploited for early detection strategies.
1. As stated previously, the presence of a mutational hot-spot at codon 12 in the K-ras gene allows detection of allelic alteration with relative ease, compared with analysis of the APC gene which would be a daunting task. The initial strategy used in colorectal cancer patients examined paired primary tumours and stool samples. Stool testing has several valuable features:it is non-invasive,does not require bowel preparation,might enable screening of the entire length ofthe colon and rectum, and specimens are transportable. DNA was extracted and purified from the stool samples and then examined for the presence of ras mutations by using PCR to amplify the small amounts of target DNA followed by a ras plaque hybridization assay. This study revealed that eight of nine patients with a K-ras mutation in their primary tumours also exhibited similar ras mutations in stool DNA from exfoliated tumour cells. Patients without a ras mutation in their cancers and those without cancer demonstrated a negative test. Since then other investigators have confirmed these findings in patients with colorectal cancer targeting mutated K-ras as well as p53 genes, although in the latter case sensitivity was very low (28%). Furthermore, as with K-ras mutations, there are reports of p53 mutations in the stool of patients with pancreatic disease,which would again reduce the specificity of CRC detection.
2. Unlike the small number of specific mutations in K-ras, mutations in APC can occur at almost any site in the first 1,600 codons of the gene, with the type of mutation also varying widely. Some groups have focused on specific regions in which mutations occur frequently. A digital protein-truncation assay, which relies on the amplification of a small number of APC templates and the detection of truncated polypeptides generated by in vitro transcription and translation of the PCR products has a sensitivity of 57% for the stool-based detection of colorectal neoplasia, with no false positives.
3. Colorectal neoplasms characteristically exfoliate non-apoptotic colonocytes, unlike normal colonic mucosa,which typically sheds apoptotic colonocytes. The presence of intact DNA as a marker of non-apoptotic cells presumed to be colonocytes, as compared with the fragmented DNA in apoptotic cells shed from normal mucosa, might therefore allow identification of patients with CRC. Indeed, the presence of long DNA fragments in stool has been shown to be associated with CRC.
4. An important early defect in oncogenesis is an imbalance in cytosine methylation, as manifested by hypermethylation of CpG islands and genome hypomethylation. Detection of hypermethylated DNA markers might therefore help identify patients with CRC and precursors using stool. The gene encoding secreted frizzled-related protein 2 (SFRP2), an antagonist of the wnt signalling pathway that is commonly methylated in CRC, has been identified as a potential stool-based marker of CRC. Using a fluorescence-based real-time PCR assay for the faecal detection of SFRP2, a sensitivity of 90% and a specificity of 77 % for CRC (49 samples total) was achieved, including successful detection of three of five proximal cancers. Detection of DNA methylation holds promise as a key component of multitarget assays, not least in view of its potential contribution to detecting proximal cancers.
5. Since colorectal neoplasms are genetically heterogeneous, however, no one mutation has been identified that is universally expressed. Mutant K-ras, for example, is present in less than half of all colorectal neoplasms; this would restrict the maximum sensitivity of this test for colorectal cancer to less than 50% if it was used as the sole marker for screening in a stool assay. Also, since mutant K-ras may arise from non-neoplastic sources, such as pancreatic hyperplasia, this marker may lack specificity. This has resulted in the targeting of multiple DNA alterations to increase overall sensitivity. Reported sensitivities using this approach in initial small studies ranged from 63% to 100%. As well as detection of distal cancers, the method has enabled successful detection of proximal cancers and might also detect some cancers of sites proximal to the colon, such as the stomach and pancreas.
So far, most studies have used a single stool sample for analysis, and recent data indicate that there might be no additional advantage from performing multitarget DNA assays on more than one stool specimen per patient. Clearly, there are prospects of developing tests based on analysis of stool, which promise improved accuracy, safety, affordability and patient compliance. However, as with all of the molecular markers, no DNA test has yet demonstrated a reduction in the incidence or mortality of CRC and the reliability of DNA multitarget testing remains uncertain. Further prospectively designed studies are clearly indicated to corroborate these early outcomes. In the meantime, significant technological development is required before these methodologies can be translated into large screening protocols recruiting asymptomatic patients.
Staging and prognostic stratification
Conventional histopathological staging provides a static description of the anatomical extent of tumour spread within a surgical specimen. Currently, such tumour staging, when performed accurately, gives the best guide to the prognosis of patients who have undergone putatively curative colorectal cancer resection. The limitations of conventional staging are highlighted by the considerable prognostic heterogeneity of patients within a given tumour stage. For instance, not all patients with lymph node-negative cancers are cured of their disease. Conversely, treatment failure is not a uniform outcome in patients with Dukes' stage C tumours. More precise stratification of prognostic groups assumes importance with the emerging evidence of the clinical value of adjuvant chemotherapy regimens.
The urgent need for new prognostic strategies has engendered the search for alternative techniques that allow rapid, accurate and personalized detection of clinically significant occult disease. A huge effort has been made to detect tumour-associated antigens (immunohistochemistry), genetic markers (PCR) and tissue-specific messenger RNA (mRNA; reverse transcription [RT]-PCR) in lymph nodes, bone marrow or peripheral blood and associate their presence with more accurate prognosis for the individual patient. Most recently, global tumour genomic or transcriptome profiling has become feasible. In the future, proteome and even metabolome analysis may be applied to the understanding of colorectal cancer biology. Can the knowledge of the molecular alterations during colorectal tumourigenesis aid in this problem? Although numerous alterations in the tumour progression cascade have been identified to date, no single human cancer has been completely described with a clear delineation of all the genes altered in that cancer. In addition, even a complete profile of the mutations in a given cancer represents only a single point in time. As cancers evolve, mutations accumulate, and cellular heterogeneity develops, complicating the static analysis of tissue samples. Cancers may also develop in the setting of field defects, where surrounding tissue, while appearing histologically normal, contain abnormalities that not only give rise to the tumour but may affect its behavior. It is now recognized that completely normal stroma plays a role in tumour invasion and metastases. This effect may vary depending on the molecular alterations in a cancer cell, adding yet another layer of complexity to defining the changes that result in cancer.
The most recent and definitive immunohistochemical study concludes that this technique reveals no significant prognostic information for node-negative colorectal cancer patients, and hence is unreliable as a clinical assay. DNA-targeted PCR has been used to enhance conventional tumour staging by examining lymph nodes for the presence of genetically detected metastases. However, no firm conclusions can be drawn from these studies which have investigated the prognostic significance of many of the common genetic alterations observed in colorectal cancers. Careful prospective studies with appropriate statistical analysis are needed to determine if the presence or absence of any of these genetic alterations are independent prognostic indicators. Some limited data are available on the genetic staging of colorectal cancer. The presence of mutations in the K-ras and p53 genes has been used in this context. One study demonstrated that the presence of these specific genetic alterations in the lymph nodes of colorectal cancers assessed as tumour-free by conventional histopathology is associated with reduced survival. The obvious implication of this is that these mutations are contained in occult lymph node micrometastases not detected by histology. However, K-ras mutations are present in only 40-50% of colorectal cancers, and mutations in the p53 gene, while a more frequent finding in colorectal carcinoma, are not confined to a single site. Therefore, genetic diagnosis of lymph node metastasis in colorectal cancer will require analysis of a panel of genes and/or several sites within target genes.
Amplification of tissue-specific mRNA has also been evaluated extensively for the molecular assessment of tumour stage and disease recurrence. However, there are significant technical and conceptual limitations that hold back its adaptation into clinical practice and it continues to constitute a "proof of principle" rather than robust and reliable clinical assay. The introduction of significant variability by the RT-step makes it problematic to delineate universal, biologically relevant quantitative cut-off points and the lack of standardised protocols results in lack of reproducibility between laboratories. However, most crucially, there are no disease-specific markers uniquely associated with colorectal cancer. Hence the use of tissue-specific markers, which are presumed to detect the presence of cancer cells in patients' blood, bone marrow or lymph nodes. Unfortunately, it is unclear just how tissue-specific these markers are.
There have been numerous studies reporting the detection of mRNA markers such as carcinoembryonic antigen (CEA), cytokeratins (ck), mucins, CD44 and guanylyl cyclase C (GCC) in different tissue compartments and attempting to assess their prognostic significance. One report suggests that it is possible to distinguish histologically positive lymph nodes from histologically negative ones by counting the number of CEA-expressing cells. However, there was significant overlap between the two groups and cell numbers were calculated relative to a CEA-expressing cell line, ignoring inter-sample or inter-patient variation of CEA mRNA levels. A second study calculated CEA copy numbers in lymph nodes relative to 18S RNA levels, and used cut-off levels to suggest that high CEA mRNA levels might be predictive of distant recurrence. A third study also concluded that quantification of CEA mRNA levels in the lymph nodes from patients with advanced colorectal cancer yielded prognostic information. Unfortunately, quantitating the amount of an mRNA does not allow the calculation of the number of circulating tumour cells since the expression of most genes varies by several orders of magnitude between tumours in different individuals and often varies in the tumour of the same individual. Also, none of these authors discussed how to implement a relative quantification assay in practice.
In complete contrast, studies using both conventional and real-time RT-PCR reported the detection of CEA mRNA in up to 85% of control lymph nodes, with significant overlap of CEA copy numbers between histologically involved and uninvolved lymph nodes. There was no correlation between CEA copy numbers and prognosis, suggesting that a CEA-based assay is unable to identify patients at risk of distant disease recurrence. At least there is a rationale for attempting to detect occult disease in lymph nodes: histological detection of occult disease during staging is an important prognostic indicator. This is not the case for blood. Nevertheless, some reports suggest that CEA mRNA levels in the blood of colorectal cancer patients is associated with disease stage and may be of prognostic value. These contrast with others that question its specificity and suggest that peripheral blood is not a suitable compartment for detection of tumour cells, or advocate analysis of yet another tissue compartment. Similar, contradictory results have been reported for other tissue-specific markers. This discordance is typical and when results are analysed in detail, there is little agreement on the specificity of the various markers and there is a significant percentage of patients that test positive for the marker in question, yet survive for five years or do not test positive for the marker yet die within five years. Characteristically, the usual conclusion is that the respective markers have not yet been evaluated sufficiently to recommend their inclusion in a clinical assay. This is not surprising, when considering that blood, bone marrow or lymph nodes sampling represents a single snapshot of a complex and dynamic process and that few of the large number of cancer cells shed from a primary tumour ever form metastatic tumours. Consequently, despite this vast effort, PCR-based techniques have still not been validated clinically in prospective studies and the presence of circulating tumour cells cannot be considered a reliable prognostic indicator.
This suggests a conceptual flaw underlying the attempts to use RT-PCR assays to allow prediction of successful distant metastasis, as it is based on a simplistic view of the biology and kinetics of tumour cell traffic through the lymphatic and systemic circulation and subsequent metastasis development. Instead RT-PCR may simply be detecting cells of no biological significance and variability in survival within each staging category probably reflects not only the inaccuracy of detecting occult residual disease but also a lack of understanding of the sequestration, release and subsequent trafficking of the tumour cell in both the lymphatic and systemic circulation. None of the qRT-PCR assays address the question of the biological relevance of detecting tumour cells in blood or lymph nodes and do not provide any information about their metastatic potential or take into account the role of patient genotype in allowing or suppressing metastasis. Animal models suggest that only 0.01% of cells circulating in the blood ultimately develop into a metastatic site and in humans the likelihood of tumour cells seeding to become metastases is also very low. Furthermore, the genotype of lymph nodes metastases differs from that of the main clone in the primary tumour in >50% of patients, with a significant minority displaying a genotype not detected in the primary tumour at all. In addition, humans themselves are genetically polymorphic, and the outcome of metastasis depends on the interplay of tumour cells with various host factors including the organ microenvironment which can influence the biology of cancer growth, angiogenesis, and metastasis. Therefore, it is not surprising that the detection of occult disease per se is unlikely to have prognostic value.
Characterization of global mRNA patterns may advance the understanding of disease pathology and increase the accuracy of staging. Transcriptome analysis is expected to lead to the identification of key markers among the complex network of gene products involved in metastasis and their association with previously undetectable features of the molecular basis of individual tumour characteristics. However, differential gene expression and prognostic value of expression patterns are not necessarily synoymous. A recent tissue microarray study was unable to show any association between the expression profiles of several cell cycle regulatory or proliferation-related markers previously correlated with prognostic relevance and disease-free survival in R0 rectal cancers treated by surgery alone. Expression profiling of highly metastatic cell lines relative to their poorly metastatic parental cell line has identified metastasis-associated expression pattern changes in 70 genes, with cell adhesion-associated markers being a recurring theme of gene lists.
A recent report addresses the issue of prognosis and suggests that expression profiling of colorectal cancers is able to distinguish clinically relevant subgroups and metastatic versus non-metastatic tumours. The authors identified a 200-odd gene set that divided patients with significantly different 5-year survival rates. Importantly, most nonmetastatic tumours that clustered with metastatic cases eventually developed metastasis, confirm ing the notion that metastatic potential can be predicted from the transcriptome of the primary tumour. Discriminator genes were associated with various cellular processes (e.g., cell cycle regulation, cell adhesion, angiogenesis), however, in common with other findings they do not point to an obvious discriminatory mechanism for metastasis. Although many of the genes identifying LN metastasis and predicting distant metastasis are the same, there are differences. This is a reflection of the clinical observation that the two are not perfectly correlated and emphasizes expression of the underlying biologic differences. A second recent study examined the expression profile from 74 patients with Stage II colorectal cancer and identified a 23 gene signature profile that predicted recurrence. The signature was validated in 36 independent patients with an overall accuracy of 78%: 13 out of 18 patients with relapse and 15 of 18 disease-free patients were correctly predicted, representing an odds ratio of 13. Taken together, these experiments are beginning to identify patterns of gene expression that correlate with metastatic potential. An understanding of metastasis requires an explana tion of the multiple ways by which cancer cells leave their site of origin, find their preferred site of ectopic residence and recreate the tumour tissue at this new site. Whether there are tumour type specific alterations or whether metastasis in different cancers follows a similar expression pattern remains to be determined. There is some evidence for the latter, with the notable exclusion of colorectal cancer. The major site for colorectal metastasis, the liver, is probably determined by the fact that its capillary network traps the vast majority of tumour cell clumps that enter the circulation through the portal vein. This does not exclude the possibility that the genotype of the primary colorectal cancer may influence the ultimate destination of successful metastases. The fact that LN and liver metastases from the same patient do not always show similar genetic aberrations suggests that there are multiple pathways through which colorectal metastases arise.
These problems will limit the translation of such techniques to routine clinicopathological use for the foreseeable future. New technology based on attaching DNA oligonucleotides to a microchip for parallel hybridization will increase our ability to analyse simultaneously several mutated genes. However, by themselves these novel technologies will not translate into improved patient outcome unless corresponding advances are made in adjuvant treatment modalities for patients with colorectal cancer.
Conclusion
In the last few years we have witnessed several important breakthroughs in our understanding of the molecular basis of colorectal cancer. Clinical and molecular evidence suggests that there are several pathways leading to colorectal cancer and that many of the tumour-susceptibility genes implicated in tumour development are multifunctional. Most colorectal cancers are caused by somatic mutations that accumulate during the course of carcinogenesis. A significant minority having predisposing mutations that affect the germline, are heritable and contribute to the initiation of carcinogenesis. High-penetrance mutations confer predisposition to colorectal cancer mainly in HNPCC, involving mutations in mismatch-repair genes, and in familial adenomatous polyposis, which involves mutations in the APC tumour suppressor gene. Together, these conditions account for 5% or less of all cases of colorectal cancer. Low-penetrance mutations account for a high proportion of all the attributable risk of colorectal cancer, in both familial and sporadic cases. These mutations are more difficult to identify, hence, their value in diagnostics and prevention is limited at present. In addition, modifier alleles might have a strong influence on the penetrance and expressivity of susceptibility. However, the identification of both high- and low-penetrance mutations contributes significantly to our understanding of the molecular genetic processes occurring in cancer and facilitates the development of therapeutic drugs and preventive strategies.
Epigenetic changes, defined as clonal changes in gene expression without accompanying changes in primary DNA coding sequence, can also be a driving force in neoplastic transformation. In the colon aberrant DNA methylation arises very early, initially in normal-appearing mucosa, and may be part of the age-related field defect observed in sporadic colorectal neoplasia. Aberrant methylation also contributes to later stages of colon cancer formation and progression through a hypermethylator phenotype termed cytosine phosphoguanosine (CpG) island methylator phenotype (CIMP), which appears to be a defining event in approximately half of all sporadic tumours. In sporadic colon cancer, CIMP has distinct epidemiologic and clinical features and is responsible for most cases of microsatellite instability related to hMLH1 inactivation. The most exciting implications of research into the epigenetics of colorectal cancer lie in the potential clinical implications of the concept. Broadly, DNA methylation is being explored as a marker of disease risk and a tumour markerfor screening and prognostication. Most important, epigenetic changes ca n potentially be reversed pharmacologically, leading to novel concepts in therapy for colon cancer
Understanding the molecular mechanisms affected by these alterations will identify which proliferative, apoptotic, cell migration and differentiation functions are affected and how specific gene-expression profiles may be associated with poor clinical prognosis. There are three major challenges for the future: the first is the discovery of new genes that have a causal role in neoplasia, particularly those that initiate the process and those that drive metastasis. The second is the delineation of the pathways through which these genes act. Unfortunately, cancer biology has not kept up with cancer molecular genetics and our understanding has become clouded by the identification of too many genes, interacting proteins and potential functions. The third challenge relates to the exploitation of this knowledge for the benefit of colorectal cancer patients. This translation poses the most significant problem because of the multiple genetic abnormalities present in cancer cells, each one capable of rapidly evolving variants to combat any therapeutic intervention. Nevertheless, the fact that cancers respond to the limited therapeutics available today and that cancer development is the result of alterations in a limited number of pathways that can in principle be targeted by new generations of drugs makes it appropriate to be cautiously optimistic.
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