Molecular alterations and biomarkers in colorectal cancer

The polyp/carcinoma progression sequence

Colorectal cancer (CRC) arises as the result of the accumulation of acquired genetic and epigenetic changes that transform normal glandular epithelial cells into invasive adenocarcinomas. Steps that transform normal epithelium to benign neoplasms (adenomas and sessile serrated polyps), followed by invasive carcinoma, and eventually metastatic cancer are described in the classic tumor progression model proposed by Fearon and Vogelstein (Figure 1).(Vogelstein et al. 1988) Since this model was originally proposed our understanding of the molecular pathogenesis of CRC has advanced considerably and led to numerous revisions of the Vogelstein and Fearon model. For instance, the original model proposed that only tubular and tubulovillous adenomas had the potential to progress to invasive adenocarcinoma. It is now recognized that serrated polyps including sessile serrated adenomas/polyps (SSA/P) and traditional serrated adenomas (TSA) also have the potential for malignant transformation.(Goldstein 2006; Jass 2004) These polyps are an alternative pathway to malignancy whereby a subset of hyperplastic polyps progress to serrated neoplasms (SSP or TSA) and a fraction of these serrated neoplasms progress to CRC. Premalignant serrated polyps more frequently arise in the proximal colon (Baker et al. 2004) and are associated with microsatellite instability and aberrant DNA methylation at CpG islands, whereas conventional tubular adenomas appear to arise most commonly via biallelic inactivation of the APC tumor-suppressor gene and display chromosome instability.(Noffsinger 2009) Furthermore, other molecular alterations, such as BRAF V600E mutations, are characteristically found more often in tumors arising via the serrated neoplasia pathway.(Noffsinger 2009)

A Fundamental Feature of Colorectal Cancer: Genomic and Epigenomic Instability

Genomic and epigenomic instability distinguishes neoplastic from normal colonic epithelium and is a hallmark feature of colorectal carcinogenesis.(Hanahan and Weinberg 2000; Little et al. 2008) At least four kinds of genomic or epigenetic instability have been described in colorectal cancers: (1) chromosomal instability (CIN), (2) microsatellite instability (MSI), (3) CpG island methylator phenotype (CIMP), and (4) global DNA hypomethylation. Overlap between these categories and imprecise use of these terms has led to confusion and confounds interpretation of the literature. (Walther et al. 2009) Thus, in this section we will first define the different types of genomic and epigenetic instability in CRC and then will delineate in general terms how these mechanisms are clinically relevant.

Chromosomal Instability (CIN)

The most common form of genomic instability is chromosome instability, which is found in as many as 85% of CRCs.(Grady and Carethers 2008) Chromosome instability, which can be recognized by the presence of aneuploidy, is defined as the presence of numerical chromosome changes or multiple structural aberrations and is can be assessed by DNA flow cytometry.(Walther et al. 2008) Despite the frequent occurrence of CIN in colorectal cancer, the mechanisms that give rise to this form of genomic stability and the role of aneuploidy in tumor progression remain poorly understood. However, there is some evidence that CIN promotes cancer progression by increasing clonal diversity (Grady 2004; Hermsen et al. 2002; Maley et al. 2006). Importantly, from a clinical perspective, large meta-analyses have demonstrated that CIN is a marker of poor prognosis in colorectal cancers, but technical aspects of assays that can determine the CIN status of CRCs have limited its use in clinical laboratories.(Popat et al. 2005; Walther et al. 2008)

Microsatellite Instability (MSI)

Microsatellite unstable (MSI) tumors, which account for approximately 15% of CRCs, are generally regarded as being mutually exclusive of CIN tumors because they display a normal karyotype and exhibit unique gene mutations compared to CIN CRCs, although there does appear to be a subset of tumors that show both CIN and MSI.(Walther et al. 2009) MSI CRC has been defined by the presence of at least 30% unstable loci in a panel of 5-10 loci consisting of mono- and di-nucleotide tracts selected at a National Cancer Institute consensus conference.(Boland et al. 1998) Currently, many clinical laboratories assess MSI using a panel of 5 mononucleotide markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27) that were selected for high sensitivity and specificity.(Bacher et al. 2004) A subset of tumors with only 10-29% unstable loci has been designated as a form of microsatellite tumors designated “MSI-low”. Although there is evidence that MSI-low cancers have distinct features compared with MSI (also referred to as “MSI-High”, or “MSI-H”) and microsatellite stable tumors, there is considerable controversy regarding whether MSI-low is a unique molecular subclass of CRC.(Baron et al. ; Grady and Carethers 2008; Walther et al. 2009) CRC patients with MSI tumors have been shown to have a better prognosis compared to patients with CIN cancers(Popat et al. 2005; Walther et al. 2008), and probably respond differently to adjuvant chemotherapy compared to patients with microsatellite stable (MSS, also called CIN) CRCs.(Fallik et al. 2003; Jo and Carethers 2006; Sargent 2008)

In contrast to CIN, the mechanisms underlying MSI are relatively well understood and involve inactivation of genes in the DNA Mismatch Repair (MMR) family either by aberrant DNA methylation or by somatic mutation.(Grady 2004) Furthermore, individuals with the hereditary cancer syndrome, Lynch syndrome (also called hereditary non-polyposis colorectal cancer, HNPCC), almost exclusively develop MSI colorectal cancers because they have germline mutations in one of the MMR genes, which include MLH1, MSH2, MSH6, and PMS2. In contrast, sporadic MSI colorectal cancer most often have loss of MMR activity as the result of silencing of MLH1 by aberrant DNA methylation.(Grady 2004; Kane et al. 1997) It is also now recognized that sporadic MSI tumors are associated with the serrated neoplasia pathway and frequently carry BRAF V600E mutations, while cancers resulting from germline mutations in MMR genes (Lynch syndrome) do not have mutated BRAF.(Domingo et al. 2004; Wang et al. 2003) Thus, the presence of a BRAF mutation in an MSI CRC effectively excludes the possibility that the tumor arose as the consequence of Lynch syndrome (Figure 2).

CpG Island Methylator Phenotype (CIMP)

Epigenetic instability in CRC is manifested as both hypermethylation of gene promoters that contain CpG islands and global DNA hypomethylation. Aberrant DNA methylation is present in essentially all CRC's, however, there is a subset of CRCs (approximately 20%) that have an extremely high proportion of aberrantly methylated CpG loci. This class of CRCs has been characterized as having a CpG Island Methylator Phenotype (CIMP). The mechanism(s) that give rise to CIMP are still unknown, although the strong association between BRAF V600E mutations and CIMP colorectal cancer suggests a role for activated BRAF in the pathogenesis of CIMP CRCs and a link between sporadic MSI and CIMP.(Barault et al. 2008; Weisenberger et al. 2006) However, in vitro studies of mutant BRAF in colorectal cancer cell lines have not demonstrated a direct cause and effect relationship between BRAF and CIMP.(Hinoue et al. 2009) Furthermore, although CIMP tumors do appear to represent a distinct subset of CRC, the clinical utility of this designation is hindered by lack of a universally accepted criteria for determining CIMP. CIMP is usually defined as methylation of at least three loci from a selected panel of five gene associated CpG islands. Because this panel is not always the same across studies, attempts are being made to facilitate standardization of CIMP markers for clinical use.(Nosho et al. 2008; Weisenberger et al. 2006) Some authors have proposed two classes of CIMP, CIMP-low, and CIMP-high, depending on the number of methylated marker loci detected.(Barault et al. 2008) Another group suggested that CIMP colorectal cancers be divided into two distinct classes (called CIMP1 and CIMP2) based on the results of unsupervised cluster analysis of a large panel of methylation markers.(Shen et al. 2007) Finally, considerable overlap between CIMP and sporadic MSI tumors adds to the challenge of incorporating CIMP-status into clinical trials and clinical decision making.(Weisenberger et al. 2006) Retrospective studies suggest CIMP will ultimately be shown to be a predictive marker for CRC, but the data is not adequate at this time to recommend its clinical use.(Iacopetta et al. 2008; Shen et al. 2007) Thus, the discovery and classification of CIMP tumors has advanced our understanding of the molecular pathology of CRC but has not yet impacted clinical care.

In addition to aberrant gene methylation, a global decrease in methylation has also been identified in many colorectal cancers and is tightly associated with CIN CRCs.(Matsuzaki et al. 2005; Rodriguez et al. 2006) Further research is needed to determine if the measurement of global DNA hypomethylation in CRC has any clinical utility.

Gene Mutations and Epigenetic Alterations: Impact on signaling pathways

Just as important as genomic and epigenomic instability for the pathogenesis of CRC is the accumulation of mutations in specific genes and the resulting deregulation of specific signaling pathways that control the hallmark behaviors of cancer: cell proliferation, differentiation, apoptosis, immortalization, angiogenesis, and invasion.(Hanahan and Weinberg, 2000) The best-studied pathways that are deregulated in colorectal cancer are the WNT-β-catenin signaling pathway, the transforming growth factor β (TGFβ) signaling pathway, the epidermal growth factor receptor (EGFR)-MAPK pathway, and the phosphatidylinositol 3-kinase (PI3K) pathway.(Siena et al. 2009; Walther et al. 2009) Selected deregulated pathways in CRC and targeted therapies in clinical use or in clinical trials are summarized in Table 2.

Key tumor suppressor genes that do not necessarily mediate their effects through signal pathway deregulation, such as TP53, and recurrent cytogenetic aberrations such as 18q loss of heterozygosity (LOH) are also well-studied in CRC and affect the malignant transformation of colon epithelial cells through specific effects on the behavior of the cells (Figure 1). The use of these molecular alterations in the management of patients with CRC is discussed in more detail below.

APC/ß-catenin/WNT Pathway

Mutations in the adenomatous polyposis coli (APC) gene occur in up to 70% of sporadic colorectal cancers and are the cause of the familial adenomatous polyposis (FAP) cancer predisposition syndrome. APC mutations can be found at the earliest stages of neoplasia and are predominantly associated with the classic tubular adenoma pathway and CIN cancers (Figure 1).(Chung 2000; Miyaki et al. 1999; Vogelstein et al. 1988) The APC protein negatively regulates WNT signaling by facilitating the targeting of the transcription factor β-catenin for ubiquitin-mediated proteasomal degradation. Disruption of the APC protein results in increased WNT signaling through stabilization of nuclear β-catenin, which increases the transcription of WNT target genes. Activating mutations in the β-catenin gene (CTNNB1) that inhibit APC-mediated degradation are also observed in colorectal neoplasia, although they are found more frequently in adenomas (12.5%) than invasive cancer (1.4%), suggesting that CTNNB1-mutant neoplasms do not frequently progress to carcinoma.(Samowitz et al. 1999) Despite the critical and nearly universal role of WNT pathway activation in colorectal carcinogenesis, there is currently no clinical use for APC or CTNNB1 mutations for treatment selection, prognosis, or early cancer detection. There has been intense effort to develop small molecule inhibitors of this pathway, but these efforts are still confined to the preclinical arena. (Anastas and Moon) If these agents eventually reach the clinic, the assessment of APC mutations, activated ß-catenin (by the detection of nuclear localization of ß-catenin by immunostaining), and epigenetic alterations that can affect WNT ligand availability (e.g. methylation of SFRP2, etc.) is likely to have a role in directing the selection of patients who will respond to these agents.

Transforming Growth Factor Beta (TGF-β) Pathway

Deregulation of TGF-β signaling, which is generally considered a tumor-suppressor pathway in the colon, occurs in the majority of CRCs.(Chittenden et al. 2008) Inactivating mutations have been observed in receptor genes (TGFBR2 and TGFBR1), post-receptor signaling pathway genes (SMAD2, SMAD4), and TGF-β superfamily members (ACVR2).(Deacu et al. 2004; Eppert et al. 1996; Grady et al. 1999; Grady and Markowitz 2008) Functionally significant mutations in TGFBR2 have been detected in as many as 30% of all CRC and are associated with the malignant transformation of late adenomas. TGFBR2 mutations are most common in MSI tumors, but also occur in approximately 15% of microsatellite stable (MSS) tumors (Figure 1).(Grady et al. 1999; Grady et al. 1998; Markowitz et al. 1995) SMAD4 is located on 18q in a region commonly deleted in CRCs, and is associated with adenoma formation and adenoma-carcinoma progression in mouse models, supporting a role for SMAD4 as a tumor suppressor gene.(Takaku et al. 1998) Furthermore, loss of SMAD4 expression as detected by immunostaining has been reported in >50% of CRCs and is associated with lymph node metastases.(Tanaka et al. 2008) There is still not any definite clinical role for any genetic markers in the TGF-β signaling pathway, however there is some evidence that SMAD4 expression levels may be associated with prognosis and response to 5-Fluorouracil (5-FU) and there is ongoing investigation of 18qLOH as a predictive marker, which is discussed further in the next section.(Alazzouzi et al. 2005; Boulay et al. 2002)

Loss of the long arm of chromosome 18 (18q loss of heterozygosity; LOH) is the most frequent cytogenetic alteration in colorectal cancer and is observed in up to 70% of CRCs, and it may influence CRC behavior by deregulating TGF-ß signaling.(Popat et al. 2005; Vogelstein et al. 1988) Two genes implicated as tumor suppressor genes that are affected by this cytogenetic alteration are deleted in colorectal carcinoma (DCC) and SMAD4. Additional mediators of the TGF-β pathway, SMAD2 and SMAD7, are also in the 18qLOH region, suggesting that 18qLOH may promote tumorigenesis at least in part through deregulation of TGF-β signaling. It appears that deletion at 18q is associated with a worse prognosis, however efforts to definitively link 18qLOH to prognosis are limited by a lack of consistent results across studies and by heterogeneous detection methods.(Popat et al. 2005) Ongoing clinical trials (e.g. NCT00217737, also designated ECOG 5202) are assessing the utility of 18qLOH for directing the use of specific adjuvant therapies.

TP53

Mutations in the tumor-suppressor gene TP53 occur in about half of all colorectal cancers and promote the malignant transformation of adenomas (Figure 1).(Vogelstein et al. 1988) Like APC, TP53 is a key tumor suppressor that has been extensively studied in colorectal cancer but currently has no predictive or prognostic role in the clinical setting.(Walther et al. 2009)

EGFR/RAS/RAF/RAF/MAPK signaling

KRAS, a member of the RAS family of proto-oncogenes, is the most frequently mutated gene in all of human cancer and an important oncogene in CRC. The KRAS protein is a downstream effector of EGFR that signals through BRAF to activate the mitogen activated kinase (MAPK) pathway and promote cell growth and survival (Figure 3). Mutations in KRAS codons 12 or 13 occur in approximately 40% of colorectal cancers and lead to constitutive signaling by impairing the ability of GTPase activating proteins to hydrolyze KRAS-bound GTP.(Downward 2003) KRAS mutations occur after APC mutations in the adenoma-to-carcinoma progression sequence, but are still a relatively early event in tumorigenesis (Figure 1).(Vogelstein et al. 1988) Acquired KRAS mutations are maintained throughout carcinogenesis, as evidenced by the nearly perfect concordance of KRAS-mutation status in primary and metastatic colorectal cancer.(Artale et al. 2008; Zauber et al. 2003) This fact is critical to the utility of KRAS mutational analysis on archived primary tumor specimens in patients with metastatic disease and usually eliminates the need for additional biopsy tissue from a metastatic lesion.

The BRAF gene, mutated in ∼10-15% of colorectal cancers, encodes a protein kinase that is the direct downstream effector of KRAS in the Ras/Raf/MAPK signaling pathway. The majority of BRAF mutations are a single base change resulting in the substitution of glutamic acid for valine at codon 600 (V600E; sometimes referred to as “V599E”).(Siena et al. 2009) KRAS and BRAF mutations are mutually exclusive, supporting the hypothesis that an activating mutation in either gene is sufficient to promote tumorigenesis via increased MAPK signaling.(Rajagopalan et al. 2002) As discussed above, BRAF mutations are much more frequent in MSI tumors (∼35%) compared to MSS tumors (∼5%) and are very tightly linked to CIMP cancers and the serrated neoplasia pathway.(Lubomierski et al. 2005; Rajagopalan et al. 2002) Emerging evidence supports a role for BRAF as a genetic marker for prognosis and possibly for predicting response to therapy.

Alterations in EGFR ligands and the EGFR gene itself are also observed in a subset of colorectal cancers. There is some data to support that upregulation of the EGFR ligands epigregulin and amphiregulin are associated with an anti-EGFR drug response.(Jacobs et al. 2009; Lievre et al.)

PI3K/AKT Signaling Pathway

Mutations in phosphatidylinositol 3-kinase (PI3K) pathway genes are observed in up to 40% of colorectal cancer and are nearly mutually exclusive of one another.(Parsons et al. 2005) The most frequent mutations of the PI3K pathway occur in the p110α catalytic subunit PIK3CA, which are reported in up to 32% of colorectal cancers and may promote the transition from adenoma to carcinoma (Figure 1).(Samuels et al. 2004) Mutations are also observed in PTEN, a tumor suppressor gene that negatively regulates PI3K signaling in as many as 30% of MSI tumors and 9% of CIN tumors.(Danielsen et al. 2008) In addition, loss of PTEN expression (as assessed by immunostaining) occurs in 30-40% of CRCs.(Yu et al. 2013) The PI3K pathway is modulated by EGFR signaling in part via KRAS activation, and there is a plausible role for both PIK3CA and PTEN mutations as predictive markers of anti-EGFR therapy (Figure 3).(Razis et al. 2008; Sartore-Bianchi et al. 2009a) Recent evidence suggests that mutations in PIK3CA are predictive biomarkers of response to aspirin therapy (Liao et al.). Mutations of genes in this pathway also have the potential to be used a predictive biomarkers for therapies that target the PI3K pathway, mTORC pathway, as well as the MAPK pathway.

Lynch Syndrome (aka Hereditary NonPolyposis Colon Cancer; HNPCC) syndrome

Identifying individuals with Lynch syndrome (also known as HNPCC) dramatically alters their clinical management, and can lead to effective colorectal cancer prevention programs for these individuals and their family members. However, currently the definitive molecular diagnosis of Lynch Syndrome requires expensive germline DNA mutation analysis of multiple DNA Mismatch Repair (MMR) genes (MLH1, MSH2, MSH6, EPCAM, and PMS2). To facilitate the most cost-effective strategies for identifying patients at high risk for Lynch syndrome who are candidates for genetic testing, the evaluation of molecular features of CRCs that have occurred in these individuals or other family members can be used to predict the likelihood of identifying a germline mutation in one of the MMR genes. It is now common practice for molecular diagnostics labs to offer a step-wise series of molecular tests that are used to identify CRCs that arose secondary to Lynch syndrome. These tests are based on the molecular pathology of CRC (Figure 2).(Baudhuin et al. 2005) A common approach is to initially test the tumors for loss of MMR gene products (MLH1, MSH2, MSH6, PMS2) by immuohistochemistry (IHC) and for MSI by polymerase chain reaction (PCR) as the first-tier screening test (see for example http://www.mayomedicallaboratories.com/test-catalog/Clinical+and+Interpretive/17073), although there is support for the use of IHC alone as a first-line test.(Shia 2008) Tumors that display MSI and loss of MLH1 protein expression by IHC are then subjected to reflex testing for BRAF V600E mutation status and MLH1 promoter hypermethylation to help distinguish sporadic MSI tumors (∼35% BRAF-mutant and 99% MLH1-methylated) from Lynch syndrome MSI tumors (BRAF-WT, infrequent MLH1-methylation) (Figure 2, Table 3).(Bettstetter et al. 2007; Domingo et al. 2004; Zhang 2008) This strategy is most effective in excluding individuals who are unlikely to have a MMR gene mutation from undergoing germline mutation testing. It is notable that in those tumors that have MSI and loss of MSH2, MSH6, or PMS2 the likelihood of having a germline mutation is extremely high. Also of interest, it is now recognized that a strategy that relies on clinical criteria alone for the diagnosis of individuals at risk for Lynch Syndrome underdiagnose this syndrome (Hampel et al. 2005). In light of the substantial effect of a missed diagnosis on an individual's likelihood of developing cancer in the future, a strategy that employs universal testing of all colorectal cancer is being advocated by some experts in this area.(South et al. 2009)[NCT01850654]. It remains to be determined if this strategy is cost -effective and if the benefits outweigh the risks.

Molecular markers and colorectal cancer early detection

Colonoscopy is the most accurate test currently used for colorectal cancer screening, however, it is expensive and associated with procedure-related complications and poor patient compliance.(Ahlquist et al. 1993) In contrast, another commonly used colorectal cancer screening test , fecal occult blood testing (FOBT) is inexpensive and simple to perform, but has a relatively low sensitivity and specificity.(Smith et al.) Advances in our understanding of the molecular pathology of colorectal cancer, has led to the identification of promising early detection molecular markers for use in non-invasive colorectal cancer screening assays.(Ahlquist et al. ; Osborn and Ahlquist 2005) Stool-based methylated VIMENTIN (mVim) is a clinically validated marker for early colorectal cancer detection that was previously available as for clinical use in the United States (Table 1).(Itzkowitz et al. 2007) The test relies on the fact that a majority of colorectal cancers (53-84%) carry an aberrantly methylated vimentin (VIM) gene. A PCR-based assay that simultaneously measured mVim and DNA integrity reported a sensitivity of 83% and a specificity of 82%, with approximately equal sensitivity in Stage I-III colorectal cancer patients. (Itzkowitz et al. 2008) At this time, methods are under development to enhance the performance of stool- and plasma-based methylation assays for clinical purposes.(Li et al. 2009; Zou et al. 2009) The use of molecular assays, such as the fecal-methylated VIM assay, in the clinical care of patients is an area that is likely to undergo rapid advances in the near future.

Genomic Instability and Prognosis: MSI vs. CIN

Meta-analyses across a diverse range of patients have firmly established that MSI CRC have a better prognosis, which is independent of stage, and that CIN tumors have an unfavorable prognosis (Table 4).(Popat et al. 2005; Walther et al. 2008) The combined hazard ratio for MSI colorectal cancers for overall survival was estimated to be 0.65 (0.59-0.71, 95% CI) with only one of thirty-two included studies reporting an HR >1.0.(Popat et al. 2005) Conversely, the overall HR associated with CIN colorectal cancer was determined to be 1.45 (1.27-1.45, 95% CI) based on 63 eligible studies and over 10,000 patients.(Walther et al. 2008) Despite the clear association of MSI and CIN with prognosis, these markers have not yet been adopted into routine clinical decision making. It is most likely that MSI testing will be adopted into clinical practice before CIN testing because of the availability of a reliable assay for assessing MSI status.

18qLOH and Prognosis

Colorectal cancer patients with 18qLOH appear to have a worse prognosis compared to patients with tumors that do not carry 18qLOH. A meta-analysis of seventeen independent studies that was limited by evidence of publication bias found an overall HR of 2.00 (1.49-2.69 95% CI) for 18qLOH across all patients, and an HR of 1.69 (1.13-2.54 95% CI) in the adjuvant setting.(Popat et al. 2005) Candidate genes in the 18q region, including DCC and SMAD4, have been studied individually for prognostic roles, with inconsistent results.(Carethers et al. 1998) The independent prognostic contribution of 18q deletion in CRC has been called into question due to the tight association between 18qLOH and CIN, and the inverse association of 18qLOH and MSI.(Walther et al. 2009) This assertion is supported by a recent study that found no difference in prognosis attributable to 18qLOH in a prospectively collected cohort of 555 non-MSI tumors Stage I-IV.(Ogino et al. 2009a) Thus, it is unclear at this time if 18qLOH represents an independent prognostic marker, or is merely a surrogate marker for CIN/MSS colorectal cancers.

Mediators of EGFR signaling and prognosis

Several recent studies have assessed the prognostic significance of KRAS, BRAF, and PIK3CA mutations in colorectal cancer.(Fuchs 2009; Ogino et al. 2009b; Richman et al. 2009; Roth et al. ; Tol et al. 2009; Zlobec et al.) In the vast majority of studies, mutant KRAS has not been independently associated with differences in relapse-free or overall survival in Stage II or III colorectal cancer, but mutant BRAF has been shown to be prognostic for overall survival (OS) in this group of patients.(Fuchs 2009; Roth et al. ; Yokota) In contrast, mutant KRAS and BRAF have been reported as markers of poor prognosis in advanced colorectal cancer. In the largest study that has addressed the prognostic role of KRAS mutations in advanced colorectal cancer to date, patients with mutant KRAS cancers had a worse overall survival (HR= 1.40; 1.20-1.65 95%CI) but similar PFS compared to patients with tumors bearing wild-type KRAS.(Richman et al. 2009) The potential prognostic value of KRAS mutations is of particular interest in advanced colorectal cancers because the KRAS mutational status of tumors is now being routinely collected in this setting in order to assess for eligibility for treatment with cetuximab or panitumumab. At this time, the use of KRAS mutation status for prognosis in colorectal cancer is still premature but appears to have significant potential to be adopted into clinical use in the near future. Similarly for mutant PIK3CA, most studies to date have not demonstrated a role for PIK3CA mutations in either exon 9 (helical domain) or exon 20 (kinase domain) as prognostic markers, although one observational cohort study did find that patients with concurrent exon 9 and 20 had a worse prognosis.(Liao et al.)

Predictors of Response to anti-EGFR mAb Therapies

EGFR-targeted monoclonal antibodies cetuximab (Erbitux®), and the fully humanized mAb panitumumab (Vectibix®), have proven to be effective in patients with metastatic colorectal cancer both as single agents and in combination with traditional chemotherapy.(Ciardiello and Tortora 2008; Cunningham et al. 2004; Saltz et al. 2004) However, while these therapies improve both progression free survival (PFS) and overall survival (OS), they are effective in only a minority of metastatic colorectal cancer patients.(Bardelli et al.) These drugs are generally well-tolerated, but are still associated with treatment-related morbidity, including skin rash, diarrhea, and nausea, and are also expensive. To better target anti-EGFR mAb therapy to patients most likely to benefit, KRAS mutation status and additional molecular markers of cetuximab and panitumumab resistance have been extensively evaluated.(Siena et al. 2009)

KRAS

Results of four large phase III randomized have established unequivocally that metastatic colorectal cancer patients with KRAS mutations in codon 12 or 13 do not benefit from cetuximab or panitumumab therapy.(Amado et al. 2008; Bokemeyer et al. 2009; Karapetis et al. 2008; Van Cutsem et al. 2007) Prior to the publication of these pivotal trials, the link between KRAS mutation status and anti-EGFR mAb response was already firmly supported by several smaller studies(Benvenuti et al. 2007; Di Fiore et al. 2007; Lievre et al. 2006), but the data was not sufficient to warrant routine clinical testing. The recently published randomized trials have established the use of KRAS mutational analysis as a predictive marker for anti-EGFR mAb resistance in patients with metastatic colorectal cancer in most of the relevant clinical settings. These settings include the use of cetuximab or pantumimab in combination with conventional cytotoxic chemotherapy (e.g. 5-FU, FOLFOX, FOLFIRI) as first line treatment of metastatic disease(Bokemeyer et al. 2009; Douillard 2009; Van Cutsem et al. 2009), and as monotherapy in relapsed/refractory patients.(Amado et al. 2008; Karapetis et al. 2008; Van Cutsem et al. 2007)

A second relevant question related to anti-EGFR mAb therapy is whether mutant KRAS predicts an adverse outcome in the setting of these treatments. The reported hazard ratios (HR) were almost exactly 1.0 in a total of 348 KRAS-mutant chemotherapy-resistant or refractory cancers treated with either panitumumab(Amado et al. 2008) or cetuximab(Karapetis et al. 2008) as monotherapy, confirming lack of benefit, but also suggesting no harm from anti-EGFR mAb treatment related to PFS or OS in this population. In contrast, the reported HRs were usually greater than 1.0 in studies of cetuximab or panitumumab as first line treatment in combination with FOLFOX4 (fluorouracil, leucovorin, and oxaliplatin) or FOLFIRI (fluorouracil, leucovorin, and irinotecan) chemotherapy.(Bardelli et al.) The results of the OPUS trial (Oxaliplatin and Cetuximab in First-Line Treatment of Metastatic Colorectal Cancer) in particular suggest it may be harmful to add anti-EGFR mAb treatments to 5FU, leukovorin, and oxaliplatin in patients with KRAS-mutant metastatic colorectal cancer.(Bokemeyer et al. 2009)

Based on the evidence from large trials, European and United States practice guidelines either recommend or require KRAS mutational analysis on colorectal cancer tumor tissue prior to the initiation of cetuximab or panitumumab treatment.(2008; 2010; Allegra et al. 2009) The European health authority confines use of panitumumab monotherapy, and cetuximab as mono- or combination therapy, to metastatic colorectal cancer patients who are found to carry non-mutated (wild-type, WT) KRAS in the primary tumors.(2008) The American Society for Clinical Oncology recently published a provisional opinion stating that “All patients with metastatic colorectal carcinoma who are candidates for anti-EGFR antibody therapy should have their tumors tested for KRAS [codon 12 and 13] mutations…and [KRAS-mutant] patients should not receive anti-EGFR antibody therapy”.(Allegra et al. 2009) Similarly, the National Comprehensive Cancer Network (NCCN) guidelines require evidence of wild-type KRAS prior to cetuximab or panitumumab therapy in all metastatic colorectal cancer settings.(2010)

Despite the nearly perfect negative predictive value of mutant KRAS, it is still only a minority (∼30%) of KRAS codon 12/13 wild-type patients who respond to anti-EGFR mAb therapy.(Bardelli et al.) This has led to research into additional biomarkers that might predict lack of benefit in those individuals with tumors that have wild-type KRAS. There is evidence that rare KRAS mutations in codons 61 or 146 (∼2% of colorectal cancer) behave similarly to codon 12/13 mutations(Loupakis et al. 2009), but incorporating these mutations into routine clinical practice will require analysis of a larger group of patients. Other promising markers of anti-EGFR mAb resistance are BRAF V600E mutations, PIK3CA mutations, and loss of PTEN protein expression.(Siena et al. 2009)

BRAF

The biological rationale for BRAF V600E mutations as an additional biomarker of anti-EGFR mAb resistance is strong:(1) BRAF is the immediate downstream effector of KRAS in the Ras/Raf/MAPK signaling pathway (Figure 3), and (2) BRAF V600E activating mutations are 100% mutually exclusive of KRAS mutations in colorectal cancer, implying that activation of either protein is sufficient for colon tumorigenesis. Prior studies support the use of BRAF V600E as a negative predictor of response to anti-EGFR mAb therapy, leading to the evolving use of BRAF mutation testing in KRAS-WT patients prior to treatment as a means to further stratify patients into responders and nonresponders. A retrospective analysis showed that 0/11 tumors with mutant BRAF responded to cetuximab or panitumumab compared to 22/68 (32%) of BRAF-WT/KRAS-WT patients.(Di Nicolantonio et al. 2008) Similar results were observed for patients treated with cetuximab plus irinotecan. None of the patients with tumors with mutant BRAF (N=13) responded compared to 24/74 (32%) patients with tumors with BRAF-WT/KRAS-WT.(Loupakis et al. 2009) although not all studies have found as robust a relationship between BRAF V600E mutation status and anti-EGFR antibody response.(Laurent-Puig et al. 2009; Tol et al. 2009) BRAF mutations also appear to be associated with worse prognosis independent of treatment, which can confound the assessment of its role as a predictive marker for response to EGFR directed therapies.(Laurent-Puig et al. 2009; Tol et al. 2009) Despite the currently limited data, and lack of complete consensus, it is likely that BRAF mutation status has a role in anti-EGFR mAb treatment decisions and soon will be adopted into the planning for treatment with cetuximab and panitumumab.

PI3K Pathway members

Molecular lesions in the PI3K pathway, which in colorectal cancer are primarily mutations in PIK3CA and loss of PTEN protein expression(Yu and Grady), have been proposed as additional anti-EGFR mAb resistance markers because EGFR mediates its effects through the PI3K pathway aas well as the MAPK pathway.(Bardelli et al.) However, the relationship of oncogenic alterations in PI3K signaling with cetuximab or panitumumab response is much less clear than that of KRAS and BRAF mutations. In several small studies published to date, PIK3CA mutations or PTEN loss have been associated with lack of response to cetuximab.(Jhawer et al. 2008; Mao et al. ; Prenen et al. 2009; Sartore-Bianchi et al. 2009a; Sartore-Bianchi et al. 2009b) Both PIK3CA mutations and PTEN loss may coexist with KRAS or BRAF mutations, which weakens the biological rationale of the activation of this pathway as an absolute predictor of anti-EGFR mAb therapeutic response. Nonetheless, the balance of evidence points towards a probable predictive role of molecular events that activate the PI3K pathway for being negative predictive markers for EGFR monoclonal antibody based therapy. In fact, there is modest data demonstrating that when PIK3CA mutations and PTEN loss of expression are combined with KRAS and BRAF mutational analysis, up to 70% of patients unlikely to respond to cetuximab or panitumumab may be identified.(Bardelli and Siena ; Sartore-Bianchi et al. 2009a) This observation has led to the idea that colon cancer may be able to be classified like breast cancers (e.g. triple negative breast cancers), and these cancers have been termed “quadruple-negative” for patients who do not have alterations in any of these four biomarkers.(Bardelli and Siena ; Sartore-Bianchi et al. 2009a) However, at this time, further studies are needed to determine if mutant PIK3CA or PTEN loss should be incorporated into clinical practice.(Bittoni et al.)

EGFR Mutations and amplification

The most obvious candidate biomarker for resistance to antibodies that target EGFR is the EGFR gene itself. Early studies that focused on EGFR overexpression failed to show a consistent relationship with treatment response because of lack of standardization of the assays used to assess EGFR expression, which were based on immunostaining, fluorescent in-situ hybridization (FISH) or quantitative RT-PCR, and because of inter-observer variability inherent in some of these techniques.(Shia et al. 2005) EGFR gene amplification appears to be a more promising predictive biomarker, but it has also been fraught with technical challenges that have limited its use in the clinic (e.g. false negative results secondary to dilution of tumor DNA with wild-type DNA in PCR-based assays, and lack of consistent tissue processing and scoring systems in FISH assays).(Siena et al. 2009) Activating mutations in the EGFR catalytic domain are seen frequently in lung cancer and are associated with sensitivity to anti-EGFR tyrosine kinase inhibitors, but these mutations are quite rare in colorectal cancer.(Siena et al. 2009) Thus, EGFR is unlikely to be a clinically useful predictive marker for anti-EGFR monoclonal antibody therapy. Furthermore, although preliminary studies have shown that the EGFR ligands amphiregulin and epiregulin are overexpressed in colorectal cancer and may predict response to cetuximab, lack of standardization of the assays and studies that reproducibly demonstrate the same effect have prevented amphiregulin and epiregulin expression levels from being used as clinical biomarkers for directing therapy with EGFR monoclonal antibodies.(Jacobs et al. 2009)

Predictive molecular markers for cytotoxic chemotherapy: 5-FU, irinotecan, and oxaliplatin

Currently, the tumor biomarkers that demonstrate the greatest promise for guiding adjuvant chemotherapy with conventional cytotoxic drugs in colorectal cancer patients are MSI and 18qLOH.

MSI

5-FU based regimens have been shown to be ineffective, or even detrimental to patients with MSI tumors.(French et al. 2008; Ribic et al. 2003) Evidence that a functioning MMR system is required for the cytotoxic effect of fluorouracil provides a plausible biological rationale for 5-FU resistance in MSI tumors.(Grady and Carethers 2008; Jo and Carethers 2006) However, the finding of 5-FU resistance in MSI colorectal cancer is not uniform, and may vary with tumor stage and may differ between sporadic MSI cancers vs. Lynch syndrome cancers.(Kim et al. 2007; Liang et al. 2002; Sinicrope and Shi) An ongoing phase III trial enrolling stage II and III colorectal cancers (NCT00217737) will prospectively assess the role of MSI in predicting response to adjuvant chemotherapy.(Walther et al. 2009) At this time, the mixed outcomes from published studies make it unclear if MSI is a clinically useful predictive marker for 5-FU responsiveness.

MSI tumors appear to be more responsive to irinotecan-based adjuvant chemotherapy.(Bertagnolli et al. 2009; Fallik et al. 2003) Recently published results from a large randomized trial of Stage III colorectal cancer demonstrated improved outcomes (Disease-free survival; DFS) in MSI patients treated with an irinotecan-containing regimen that included 5-FU compared to 5-FU/leukovorin alone.(Bertagnolli et al. 2009) In light of the prior results of the CALGB 89803 study showing no benefit of adding irinotecan to 5FU as adjuvant therapy in unselected Stage III colorectal cancer patients, the finding that MSI is a predictive biomarker for irinotecan suggests MSI could be useful for adjusting adjuvant therapy for colorectal cancer patients.(Saltz et al. 2007) However, it should be noted that these results were not confirmed in a similar study (PETACC).(Tejpar et al.). Thus, at this time, neither the European Group on Tumour Markers nor the American Society of Clinical Oncology recommend using MSI to direct adjuvant chemotherapy for stage II or stage III colorectal cancer patients.

An important issue to consider with regards to MSI is that the majority of colorectal cancers that have MSI are sporadic colorectal cancers that have inactivated the MLH1 gene through aberrant promoter methylation. The majority of these sporadic MSI tumors also can be classified as CIMP cancers as well. It is not known whether the associations seen between 5FU and irinotecan effects in sporadic MSI tumors also apply to MSI tumors that arise in the setting of Lynch syndrome.

18q LOH

Loss of 18q has been associated with an adverse response to 5-FU based adjuvant chemotherapy.(Boulay et al. 2002; Watanabe et al. 2001) There is some evidence that this effect is due to loss of the SMAD4 gene located in the 18q21 deleted region although this remains to be determined with more definitive studies.(Alhopuro et al. 2005; Boulay et al. 2002) A number of ongoing clinical trials are assessing the predictive value of 18qLOH and MSI status for the treatment of colon cancer. These include an ECOG trial of stage II colorectal cancer patients being treated with 5-FU, oxaliplatin and bevacizumab (NCT00217737), a trial in patients being treated with olaparib for metastatic disease (NCT00912743), as well as a retrospective analysis assessing MSI and 18qLOH in patients with colorectal cancer (stage II or III) treated with 5-FU or 5-FU and irinotecan (CLB-9581 or CLB-89803).

Topoisomerase 1 expression

In a large randomized trial that compared 5-FU alone to 5-FU with irinotecan and 5-FU with oxaliplatin in advanced colorectal cancer, high expression of topoisomerase 1 (Topo1) measured by immunohistochemistry correlated with responsiveness to irinotecan.(Braun et al. 2008) Conversely, cancers with low Topo1 expression (602/1269; 47%) did not appear to benefit from the addition of irinotecan (HR 0.98; 95% CI, 0.78 to 1.22). Irinotecan is a Topo1 inhibitor, thus the level of Topo1 expression has a clear biological rationale as a biomarker for predicting irinotecan response. Replication of these initial results in multiple independent studies is required before Topo1 should be considered for use as a predictive marker.

Polymorphisms: Biomarkers for colorectal cancer

In addition to somatic molecular alterations in colorectal cancers being used as biomarkers for informing the prognosis and care of patients with this common form of cancer, germline polymorphisms that alter the pharmacokinetics and pharmacodynamics of adjuvant chemotherapy also have potential to be biomarkers for guiding treatment selection. For example, alterations in thymidylate synthetase and dehydropyrimidine dehydrogenase have been extensively studied in relation to 5-FU response and appear to have potential to identify patients that are at increased risk of adverse effects from 5-FU. However, very few of these polymorphisms have been adequately validated for clinical use, so the majority are not ready to be used to direct patient care.(Ezzeldin and Diasio 2008; Schwab et al. 2008) One exception to this generalization is a homozygous polymorphism that reduces the activity of UDP-glucuronosyltransferase (UGT1A1, an enzyme that detoxifies irinotecan), which is associated with a dose-related increased incidence of irinotecan toxicity.(Hoskins et al. 2007; Palomaki et al. 2009) This has led to a commercial UGT1A1 genotyping test that was approved by the Food and Drug Administration in 2005 to help guide irinotecan dosing.