Colorectal Cancer
Ramesh A. Shivdasani
The cumulative lifetime risk of developing colorectal cancer (CRC) in the United States is about 6%, and this increases about fourfold in persons with a history of CRC in first- or second-degree relatives. Although fewer than 5% of cases occur in patients with uncommon inherited predisposition syndromes and most CRCs are accordingly considered to be sporadic, 20% to 30% of cases might have a familial basis despite absence of a known germline defect. Characteristic somatic mutations, epigenetic alterations, and defects in DNA repair or chromosomal stability promote disease progression and malignant behaviors. Well-characterized predisposing conditions and somatic mutations profoundly inform the molecular understanding of CRC and serve as a paradigm for the genetic basis of cancer.
THE ADENOMA-CARCINOMA SEQUENCE AND MULTISTEP MODELS OF COLORECTAL TUMORIGENESIS
The genetic basis of CRC is best appreciated in light of the adenoma-carcinoma sequence and the premise that CRCs arise from benign precursor polyps. Most hyperplastic polyps harbor little potential for invasive cancer, although those of the sessile serrated variety may represent precursors of cancers with microsatellite instability.1,2 By contrast, adenomatous polyps are known to be the important precursor lesions, with those larger than 1 cm carrying an estimated 15% risk of progression to adenocarcinoma over 10 years; endoscopic removal of such adenomas reduces CRC incidence and mortality.3 Adenomas are marked by epithelial overgrowth, dysplasia, and abnormal differentiation, sometimes with small foci of invasive cells; residual areas of benign adenomatous tissue are frequently identified in surgical CRC specimens.
The prevalence of adenomas in the United States, estimated at 25% by age 50 and up to 50% by age 70,4 dwarfs the 6% cumulative lifetime risk of CRC. This is because few adenomas progresses to invasive cancer, in a process that unfolds over one to three decades.5 Alterations in three classes of genes drive tumors: oncogenes, tumor suppressor genes, and genes that prevent DNA damage. Although oncogenic events may have a genetic (mutational) or epigenetic basis, at present more is known about the somatic mutations that fuel step-wise increases in malignant potential.6 The order of mutations can vary and most tumors do not carry every known genetic alteration. Nevertheless, certain mutations appear at appreciable frequency in different tumor stages, allowing assignment of a typical sequence (Fig. 19.1). Considered in light of the adenoma-carcinoma sequence, these mutations support the idea of cancer as a multifaceted disease that breaches natural checks on cell survival, growth, and invasion.7 Individual mutations rarely correlate precisely with a particular feature, such as survival or angiogenesis, and common mutations often impinge on multiple cell functions. Nevertheless, the combination of somatic mutations defines cancer subtypes, their unique properties, and sensitivity to certain therapies. Specific mutations illuminate the normal controls on colonic epithelium, reveal key cellular pathways as rational targets for therapy, and may guide future prevention strategies. Furthermore, knowledge of the mutations classifies CRC into subgroups with distinctive features. For example, KRAS or BRAF gene mutations (together accounting for nearly half of all U.S. cases) predict for lack of response to epidermal growth factor receptor (EGFR) antibodies.8,9 KRAS mutation status is now used to dictate EGFR antibody therapy in CRC, a practice likely to expand as the prognostic and predictive value of additional molecular features becomes apparent.
Global Events in Colorectal Cancer
In light of the central importance of somatic mutations, cellular conditions that elevate mutation rates might enable or accelerate tumor progression. Over 80% of CRCs display widespread
chromosomal gains and losses, phenomena that favor amplification of oncogenes and loss of tumor suppressors.10 Chromosomal segregation defects may account for this background of chromosomal instability (CIN), as illustrated by the segregation factor Bub1 in mice,11 but few specific gene defects are implicated with confidence. The remaining fraction of CRCs appears euploid at the level of whole chromosomes but may carry thousands of point mutations, small deletions, and insertions near nucleotide repeat tracts, a defect known as microsatellite instability (MSI).12 Molecular determinants of progression in MSI+ adenomas differ from those associated with CIN; for example, BRAF V600E mutations occur more commonly in MSI+ serrated adenomas than in other subtypes.13 Hypermutability with CIN or MSI results in many inconsequential or detrimental “passenger” mutations, an important consideration that focuses attention on “driver” changes. Such changes are distinguished by their appearance in a significant proportion of tumor specimens and, ideally, by laboratory demonstration of their contribution toward malignancy.
chromosomal gains and losses, phenomena that favor amplification of oncogenes and loss of tumor suppressors.10 Chromosomal segregation defects may account for this background of chromosomal instability (CIN), as illustrated by the segregation factor Bub1 in mice,11 but few specific gene defects are implicated with confidence. The remaining fraction of CRCs appears euploid at the level of whole chromosomes but may carry thousands of point mutations, small deletions, and insertions near nucleotide repeat tracts, a defect known as microsatellite instability (MSI).12 Molecular determinants of progression in MSI+ adenomas differ from those associated with CIN; for example, BRAF V600E mutations occur more commonly in MSI+ serrated adenomas than in other subtypes.13 Hypermutability with CIN or MSI results in many inconsequential or detrimental “passenger” mutations, an important consideration that focuses attention on “driver” changes. Such changes are distinguished by their appearance in a significant proportion of tumor specimens and, ideally, by laboratory demonstration of their contribution toward malignancy.
 FIGURE 19.1 Genetic pathways to colorectal carcinoma. All colorectal cancers (CRCs) arise within benign adenomatous precursors, fueled by mutations that serially enhance malignant behavior. Mutations that activate the Wnt signaling pathway seem to be necessary initiating events, following which two possible courses contribute to accumulation of additional mutations. A: Chromosomal instability is a feature of up to 80% of CRCs and is commonly associated with activating KRAS point mutations and loss of regions that encompass P53 and other tumor suppressors on 18q and 17p, often but not necessarily in that order. B: About 20% of CRCs are euploid but defective in DNA mismatch repair (MMR), resulting in high microsatellite instability (MSI-Hi). MMR defects may develop sporadically, associated with CpG island methylation (CIMP), or as a result of familial predisposition in hereditary nonpolyposis colorectal cancer (HNPCC). Mutations accumulate in the KRAS or BRAF oncogenes, p53 tumor suppressor, and in microsatellite-containing genes vulnerable to MMR defects, such as TGFβIIR. Epigenetic inactivation of the MMR gene MLH1 and activating BRAF point mutations are especially common in serrated adenomas, which progress in part through silencing of tumor suppressor genes by promoter hypermethylation. Progression from adenoma to CRC takes years to decades, a process that accelerates in the presence of MMR defects. |
Epigenetic mechanisms are probably as significant as mutations in cancer but also less well understood. Various covalent histone modifications and methylation of cytosine residues in DNA represent the principal means for gene regulation, the latter far better characterized in CRC than the former. The 5′-CpG-3′ dinucleotide pairs are particular targets for methylation in localized areas of high CpG content in promoters, where abundant methylation silences adjacent genes. Compared to normal tissue, CRCs show 8% to 15% lower total DNA methylation,14 even in colorectal
adenomas.15 Reduced pericentromeric methylation might decrease the fidelity of chromosomal segregation, and altered methylation and loss of imprinting at the IGF2 locus are associated with increased CRC risk,16 suggesting broad effects of global hypomethylation on cell growth. However, because some animal models show increased tumor susceptibility with global hypomethylation,17 whereas ApcMin mice that lack or overexpress the de novo DNA methyltransferase DNMT3B show reduced or increased progression of small adenomas, respectively,18,19 its precise significance is unclear. Against the background of genomewide hypomethylation, a subset of CRCs show coordinate hypermethylation of characteristic CpG-rich promoter islands, conferring the CpG island methylator phenotype (CIMP), with transcriptional attenuation of associated genes, including tumor suppressors such as HIC1 and the secreted Wnt-inhibiting secreted Frizzled-related proteins (sFRPs).20,21 Adenomatous precursors of CIMP cancers show the distinctive histology of sessile serrated adenomas, with dysplasia within an architectural pattern typical of hyperplastic polyps.
adenomas.15 Reduced pericentromeric methylation might decrease the fidelity of chromosomal segregation, and altered methylation and loss of imprinting at the IGF2 locus are associated with increased CRC risk,16 suggesting broad effects of global hypomethylation on cell growth. However, because some animal models show increased tumor susceptibility with global hypomethylation,17 whereas ApcMin mice that lack or overexpress the de novo DNA methyltransferase DNMT3B show reduced or increased progression of small adenomas, respectively,18,19 its precise significance is unclear. Against the background of genomewide hypomethylation, a subset of CRCs show coordinate hypermethylation of characteristic CpG-rich promoter islands, conferring the CpG island methylator phenotype (CIMP), with transcriptional attenuation of associated genes, including tumor suppressors such as HIC1 and the secreted Wnt-inhibiting secreted Frizzled-related proteins (sFRPs).20,21 Adenomatous precursors of CIMP cancers show the distinctive histology of sessile serrated adenomas, with dysplasia within an architectural pattern typical of hyperplastic polyps.
There are few variations to the adenoma-carcinoma sequence. A tenfold elevated risk of CRC in patients with long-standing inflammatory bowel disease, especially ulcerative colitis (UC),22 probably reflects heightened mutation and tumorigenesis with repeated cycles of mucosal injury and repair. Such cancers arise not only from typical polyps but also within flat adenomatous plaques and nonadenomatous areas of dysplasia. A p53 gene mutation tends to occur earlier in the cancer sequence,23 and APC gene inactivation is less common than in sporadic CRC. Conversely, even in the absence of CIMP, methylation of the p16INK4a tumor suppressor gene, which is rare in sporadic CRC, is common in UC-associated cancers.24
EARLY EVENTS AND CRITICAL PATHWAYS IN COLORECTAL TUMORIGENESIS HIGHLIGHTED BY INHERITED SYNDROMES OF INCREASED CANCER RISK
Two uncommon but highly penetrant inherited syndromes, familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC), together account for about 5% of all CRC cases. Other rare syndromes, familial juvenile polyposis (FJP), Peutz-Jeghers syndrome (PJS), and Cowden disease, each occurring in fewer than 1 in 200,000 births, also elevate the risk of CRC, and some genes responsible for these autosomal dominant disorders have been identified (Table 19.1). Elucidation of the corresponding molecular defects serves not only in accurate molecular diagnosis, risk assessment, and disease prevention in affected families but also informs understanding of the considerably larger proportion of sporadic cases.
Familial Adenomatous Polyposis and the Central Importance of Wnt Signaling
In FAP, an autosomal dominant monogenic disorder that underlies about 0.5% of all CRCs, individuals develop hundreds to thousands of colonic polyps by their teens or early 20s, and the lifetime risk of progression to invasive cancer approaches 100%, with cancer diagnosed at a median age of 39. Extraintestinal manifestations include duodenal and gastric adenomas; congenital hypertrophy of the retinal pigmented epithelium; osteomas and mesenteric desmoid tumors in the Gardner syndrome variant25; and less commonly, brain tumors in the Turcot syndrome variant,26 cutaneous cysts, thyroid tumors, and adrenal adenomas. Although most features are benign, rare patients develop hepatoblastoma or thyroid cancer. Reflecting the similar regulation of small bowel and colonic epithelia, patients have a 5% to 10% risk of developing duodenal or ampullary adenocarcinoma, mandating close endoscopic monitoring of the upper intestine after prophylactic colectomy.27
The gene affected by mutations in this disorder, adenomatous polyposis coli (APC) on chromosome 5q21, encodes a 2842-residue protein. Germline mutations occur throughout the locus but cluster in the 5′ half and exon 15,28 mostly introducing stop codons or frame shifts that truncate the protein. Although a few mutations correlate with phenotypic severity or specific extraintestinal manifestations, identical mutations can produce different clinical features. In the attenuated APC (AAPC) variant, disease onset is delayed, individuals develop fewer colonic polyps or cancers, and mutations cluster in the extreme 5′ or 3′ ends of APC exons.29 The I1307K allele, present in the Ashkenazi Jewish population, barely doubles the lifetime risk of CRC and does not affect APC protein function but replaces an (A)3T(A)4 coding sequence with an extended (A)8 tract that is occasionally targeted for nearby truncating mutations.30 Identification of an APC mutation in a proband allows reliable testing of family members. Carriers should have screening colonoscopy annually after age 10, gastroduodenoscopy after age 25, and treatment with non-steroidal anti-inflammatory drugs to reduce the risk of progression to cancer.31 Prophylactic colectomy is highly recommended, with subsequent monitoring of the rectal stump and other at-risk tissues.
TABLE 19.1 GENETICS OF INHERITED COLORECTAL TUMOR SYNDROMES | ||||||||||||||||||||||||||||||||||
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The larger significance of the APC gene derives from its somatic inactivation in about 80% of sporadic CRCs and early colorectal adenomas.32 Indeed, somatic APC mutations are found in tiny adenomas, containing few dysplastic glands. Attesting to the tumor suppressor function, tumors arising sporadically or in FAP patients show biallelic APC gene inactivation and loss of heterozygosity, with one copy usually lost by deletion. Except for the small bowel, APC mutations are rare in other cancers, including those in other digestive organs. APC gene inactivation is a ratelimiting step for development of adenomas, and its designation as a gatekeeper gene in CRC is now well supported by knowledge of its cellular functions. APC encodes several functional domains and proteins truncated by mutation that could in principle interfere with a wide range of cellular activities. Disruption of its known role in chromosome segregation might, for example, contribute to CIN.33 However, attention on APC centers rightfully on its control of the Wnt signaling pathway. About half the sporadic CRCs with intact APC function carry activating point mutations in the CTNNB1 gene,34,35 which encodes β-catenin, a transcriptional effector of Wnt signaling. Moreover, acute loss of APC function in mice produces intestinal defects identical to those observed upon Wnt pathway activation.36
The Wnt glycoproteins are secreted morphogens with diverse functions in development and homeostasis. In the absence of Wnt signaling, cells use a complex containing APC, Axin2, and other cytoplasmic proteins to promote phosphorylation, by casein kinase I and glycogen synthase kinase (GSK)-3β, of several conserved serine and threonine residues in the β-catenin N-terminus, thereby targeting β-catenin for ubiquitin-mediated proteasomal degradation.37 When Wnt ligands bind a surface protein complex that contains a member of the Frizzled protein family and the obligate coreceptor LRP5/6, they antagonize APC/Axin2 activity and thereby stabilize β-catenin. CTNNB1 mutations in CRC alter consensus residues for N-terminal phosphorylation and render the mutant protein resistant to
degradation. Thus, inactivating APC or activating CTNNB1 mutations, two alternative lesions in CRC, have the same effect: constitutive, Wnt-independent stabilization of β-catenin (Fig. 19.2). Accumulated β-catenin translocates to the cell nucleus, where it acts as a transcriptional coactivator for the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors. Nuclear β-catenin provides TCF/LEF proteins with an activating partner, resulting in transcription of target genes.38 Of the four known TCF/LEF proteins, TCF4 is the most important in normal bowel epithelium and CRC.34,39 Among the many components of the Wnt signaling cascade, rare AXIN2 mutations of uncertain significance are reported in MSI+ cases,40 but mutations in CRC are otherwise found only in APC and CTNNB1.
degradation. Thus, inactivating APC or activating CTNNB1 mutations, two alternative lesions in CRC, have the same effect: constitutive, Wnt-independent stabilization of β-catenin (Fig. 19.2). Accumulated β-catenin translocates to the cell nucleus, where it acts as a transcriptional coactivator for the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors. Nuclear β-catenin provides TCF/LEF proteins with an activating partner, resulting in transcription of target genes.38 Of the four known TCF/LEF proteins, TCF4 is the most important in normal bowel epithelium and CRC.34,39 Among the many components of the Wnt signaling cascade, rare AXIN2 mutations of uncertain significance are reported in MSI+ cases,40 but mutations in CRC are otherwise found only in APC and CTNNB1.
 FIGURE 19.2 Outline of Wnt signaling, the key driver pathway in colorectal cancer. Members of the Wnt family of glycoprotein morphogens bind the cell surface coreceptors Frizzled and LRP5/6. In the absence of Wnt binding, normal cells use a complex containing adenomatous polyposis coli (APC), Axin, and other cytoplasmic proteins to promote glycogen synthase kinase (GSK)-3β-mediated phosphorylation of the β-catenin N-terminus, which targets β-catenin for proteasomal degradation (from ref. 37.). Binding of a Wnt ligand to Frizzled and its obligate coreceptor LRP5/6 antagonizes the APC/Axin destruction complex, stabilizing β-catenin (CTNNB1), which moves into the nucleus and coactivates genes through T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors. Either of the two principal gatekeeper events in colorectal cancer, inactivating APC or activating CTNNB1 mutations, results in constitutive, Wnt-independent stabilization of β-catenin and unregulated activation of the cognate transcriptional program. Wnt signaling in the intestine is normally confined to crypt progenitors, and its aberrant activation by APC or CTNNB1 mutations confers a permanent cryptlike state that favors cell replication. |
Although Wnt ligands signal in many tissues, intestinal homeostasis is particularly dependent on this pathway. Wnt signaling in the intestine is confined to proliferative crypt stem and progenitor cells. In mice, cycling crypt cells that express the surface marker LGR5 are far more susceptible to Wnt-induced transformation than their differentiated progeny, implying that CRC arises in a primitive stem or progenitor cell and not in mature descendants.41 Moreover, the Wntdependent transcriptional program in CRC cell lines overlaps materially with that in intestinal crypts.42 Wnt signaling is hence necessary for intestinal epithelial self-renewal, and constitutive, ligand-independent activation imposed by APC or CTNNB1 mutations induces and sustains adenomas. Among the diverse transcriptional targets of the TCF-β-catenin complex identified to date, CMYC seems especially important because its absence in mice abrogates the effects of acute APC loss in the intestine.43,44 Mice that lack CD44, another prominent Wnt-pathway target, develop fewer adenomas in an APC-deficient back-ground.45 Gene expression profiling offers an expanded list of over 100 candidate transcriptional targets,42,46 but the individual significance of each will take many years to investigate.
Hereditary Nonpolyposis Colorectal Cancer and the Role of DNA Mismatch Repair
HNPCC, an autosomal dominant disorder that confers a nearly 80% lifetime risk of developing CRC, usually before age 50, is estimated to account for 2% to 4% of all CRC cases in the United States.47 Affected individuals do not lack intestinal polyps (nearly all CRCs, syndromic or sporadic, arise within adenomatous precursors) but develop many fewer colonic polyps than patients with FAP, a condition that must be excluded to satisfy criteria for diagnosis of HNPCC (Table 19.2).48 Cancers tend to develop in the ascending colon, and patients are further predisposed to develop tumors of the endometrium, small intestine, stomach, upper urothelium, ovary, biliary tract, and brain, a spectrum reflected in the revised Amsterdam II criteria (Table 19.2). The lifetime risk of endometrial cancer, in particular, is 35% to 50% and that of urologic and ovarian cancers is 7% to 8%.49 Cancers in HNPCC show pronounced variation in the lengths of microsatellite DNA sequences in tumors compared with unaffected tissues. Cancers showing such MSI at two or more among a panel of five mono- and dinucleotide tracts (BAT26, BAT25, D5S346, D2S123, and D17S250) carry the MSI-Hi designation. Most other CRCs harbor CIN and show microsatellite stability (MSS) or, in a small fraction, instability at only one of the five test tracts (MSI-Lo), a finding of uncertain significance.
TABLE 19.2 CRITERIA FOR CLINICAL DIAGNOSIS OF HEREDITARY NONPOLYPOSIS COLORECTAL CANCER | |||||||||||||||||||||||||||||||||||||||
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HNPCC results from germline mutations in any of several genes that enable DNA mismatch repair (MMR), a proofreading process that corrects base-pair mismatches and short insertions and deletions in the normal course of DNA replication. MMR in mammalian cells is mediated by homologs of bacterial and yeast repair proteins: MutS homologs (MSH) 1-6, MutL homologs (MLH) 1-3, and PMS1 and PMS2. MLH1 and PMS2 are recruited to sites of DNA mismatch as a MutLα complex and in turn recruit MSH2-MSH6 (MutSα) or MSH2-MSH3 (MutSβ) heterodimers to sites of 1-bp or 2- to 4-bp errors, respectively. These proteins excise the strand that carries the mismatch, and they resynthesize and ligate the repaired DNA. Germline mutations in MSH2, MLH1, and MSH6 together explain more than 90% of kindreds50,51; the significance of mutations in other canonical MMR pathway genes, PMS1 and PMS2, is less certain.52
MSI-Hi colorectal cancers commonly show lymphocytic infiltrates, mucinous signet-ring differentiation, and a medullary growth pattern; Bethesda guidelines (Table 19.2) combine clinical and phenotypic features to facilitate diagnosis of HNPCC.53 When these criteria are met, tumor DNA should either be tested for MSI in a simple, PCR-based assay or by immunohistochemistry for absence of the commonly implicated MLH1, MSH2, and MSH6 proteins.54 A positive result should prompt genetic testing for MLH1, MSH2, or MSH6 mutations; the personal and family history can predict the probability of identifying mutations.55 Genetic testing identifies the mutant allele and carriers, allowing targeting of a recommendation for biannual colonoscopic screening between the ages of 20 and 40, with annual screening thereafter. Women should undergo annual endometrial evaluation soon after age 25, and carriers should consider prophylactic subtotal colectomy, hysterectomy, and oophorectomy.
In incipient cancers, random events first disrupt function of the wild type allele of a mutant MMR gene, and the resulting “mutator phenotype” induces brisk accumulation of DNA replication errors.50 Consequently, adenomas progress into carcinomas over 3 to 5 years instead of two or more decades.56
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