Pharmacogenetics and biomarkers in colorectal cancer - Nature

The Pharmacogenomics Journal (2009) 9, 147–160 & 2009 Nature Publishing Group All rights reserved 1470-269X/09 $32.00 www.nature.com/tpj

REVIEW

Pharmacogenetics and biomarkers in colorectal cancer AS Strimpakos1, KN Syrigos2 and MW Saif2 1 Department of Medicine, Royal Marsden Hospital, Sutton, UK and 2Section of Medical Oncology, Yale Cancer Center, Yale University School of Medicine, New Haven, CT, USA

Correspondence: Dr AS Strimpakos, Department of Medicine, Royal Marsden Hospital, Downs Road, Sutton, SM2 5PT, UK. E-mail: [email protected]

The prognosis of patients with colorectal cancer (CRC) is affected by various factors at the time of diagnosis, including location of the tumor, gender, age and overall performance status of the patient. Predicting response and limiting drug-induced toxicity for patients with CRC are also critical. Interpatient differences in tumor response and drug toxicity are common during chemotherapy. Genomic variability of key metabolic enzyme complexes, drug targets and drug transport molecules are important contributing factors. At present, there is inconsistent and rather low use of pharmacogenetic testing in the clinical setting because of a lack of robust evidence or of resources. Patients’ selection and tailored treatments by the introduction of genetic testing will hopefully allow better response prediction and limit drug-induced toxicity leading to improved patient outcomes in the most cost-effective way. Here, we review the main genetic alterations observed in familial and sporadic CRC and their associations with the metabolism, efficacy and toxicities of drugs used in this disease. The Pharmacogenomics Journal (2009) 9, 147–160; doi:10.1038/tpj.2009.8; published online 21 April 2009 Keywords: colorectal cancer; pharmacogenetics; individualized treatment

Introduction

Received 8 December 2008; revised 2 March 2009; accepted 16 March 2009; published online 21 April 2009

Colorectal cancer (CRC) is the third most common cancer among men and women, following breast, lung and prostate, and constitutes the second most common cause of cancer-related death among patients in the United Kingdom and the United States.1 The lifetime probability of developing CRC has been estimated to be 1 in 18 in men and 1 in 19 in women.1 Our understanding of the etiology of CRC and of the progress in its treatment has shown a slow but steady improvement over the past 30 years. From the era of colon-specific antigens and the use of carcinoembryonic antigen, yet a useful tool in diagnosis and treatment monitoring, as the main screening, diagnostic and predictive markers in bowel cancer, we have come to the beginning of individualized tailored therapies and selection of the population subgroups for appropriate management as directed by genomic intraindividual variations. This has only been possible in the last few years after the revolution in genomic analysis supported by incredible technological and laboratory progress able to produce consistent and validated results. Across a variety of solid cancers and hematological malignancies, many genetic alterations have been identified as being responsible for carcinogenesis and tumor growth. Genetic variations may well predict therapeutic success or failure. The therapeutic outcome, that is, the sensitivity or resistance of cancer cells to various cytotoxic and biological agents, is related to their pharmacokinetic and

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pharmacodynamic properties. These properties are generally subject to genetic regulation and control of metabolic pathways and molecular targets. Numerous genes, nucleotides, antigens and enzymes are implicated in the metabolism and efficacy of drugs used in the treatment of CRC. Many of them are unanimously similar in patients, but often show small variations/polymorphisms that may result in a significant effect in clinical practice. This is exactly the field now recognized as pharmacogenetics and pharmacogenomics, associating the incidence, variations and effects of genomic abnormalities across the whole spectrum of disease, from etiology and pathogenesis to treatment tolerance, efficacy and clinical outcome. In this review, we will summarize the hitherto identified alterations in genes and molecules involved in CRC pathophysiology and treatment with regard to their incidence and clinical effect.

Genetic abnormalities in CRC Germline genetic alterations Approximately 80% of CRC cases are sporadic. The remaining 20% are considered as familial or related to specific genetic syndromes, for example, familiar adenomatous polyposis and hereditary non-polyposis colorectal carcinoma, which account for about 1 and 5% of all CRC cases, respectively. Familiar adenomatous polyposis is associated with mutations of the adenomatous polyposis coli (APC) gene, whereas hereditary non-polyposis colorectal carcinoma is associated with germline mutations of mismatch repair (MMR) genes (mainly hMSH2, hMSH6 and hMLH1).2,3 In contrast to single genetic abnormalities seen in inherited syndromes, the majority of sporadic CRC cases are associated with multiple genetic abnormalities (Table 1). Some of these genetic changes have prognostic value and some may serve as predictive markers. Quite central in the process of carcinogenesis is genomic instability, which incorporates chromosomal instability

Table 1

(CIN) and microsatellite instability (MSI).4 Microsatellites are short repeated DNA sequences present normally in human genome. In mutated genes, microsatellites may become abnormally shorter or longer rendering the DNA unstable. In MSI, which is caused by mutations in MMR genes, the abnormal microsatellites replicate and pass to daughter cells uncorrected. MSI is found in about 15% of sporadic colon cancer cases; however, it is the main genetic abnormality (495%) in Lynch’s syndrome (hereditary nonpolyposis colorectal carcinoma).5 There is growing evidence of the prognostic and predictive value of MSI status in stage II CRC. There are published studies suggesting that sporadic primary CRC stage II with deficient MMR genes (MSI þ ) treated with surgery has a better prognosis than do the proficient MMR (MSI) tumors, and furthermore, MSI þ tumors do not benefit from adjuvant chemotherapy with fluoropyrimidines; therefore, MSI may serve as a useful tool in selecting those patients who may benefit the most from adjuvant treatment.6–9 On the contrary, other studies have failed to come to the same conclusion and have suggested that MSI þ tumors may even have worse outcome or that they do not bear a prognostic value.10–13 Possibly, the most reliable data are provided from a recent pooled molecular analysis on a randomized trial in patients with mainly stage II (and a few III) CRC who received either 5-fluorouracil (5-FU) adjuvant chemotherapy or surgery alone. It was found in that analysis that only MSI tumors benefit from the addition of adjuvant 5-FU, and that administration of chemotherapy in stage II MSI þ patients may even be harmful.14 Therefore, the authors underlined the value of assessing the MMR status of tumors before offering adjuvant treatment, although no prospective testing has been conducted yet.14 It is possible, though, that other underlying or coexistent genetic change may influence the outcome, which may explain the contradictory results observed in some studies. For example, French et al.15 reported a favorable 5-year survival in the group of patients with MSI þ tumors and wild-type (WT) BRAF WT gene (a gene involved in the

Main genetic abnormalities of CRC

Altered genes

Frequency

Normal function

APC DCC (SMAD2/4)

70–80% B10%

Tumor suppressor Tumor suppressor

Clinical significance

Mutations linked to FAP and sporadic CRC ? Relation to advanced disease and poor prognosis. SMAD4 is linked to juvenile polyposis syndrome Src B80% Oncogene Overexpression/mutations lead to Akt/PI3K pathway overactivation MSH2, MSH6 MLH1 B15% sporadic DNA MMR, Mutations linked to HNPCC (Lynch syndrome). Microsatellite and other MMRs cases, 495% HNPCC microsatellite stability unstable tumors (MSI) related to poor response to adjuvant CT but better prognosis TP53 (p53) B50–60% Tumor suppressor Mutations associated with poor prognosis KRAS B40–50% Oncogene Overexpressed mutations lead to resistance to EGFR mAbs BRAF B5–12% Oncogene Mutations lead to resistance to EGFR mAbs PTEN 18–40% Tumor suppressor Loss of activity related to poor response to EGFR mAbs is linked to Cowden’s syndrome Abbreviations: APC, adenomatous polyposis coli; CRC, colorectal cancer; CT, chemotherapy; EGFR, epidermal growth factor receptor; FAP, familiar adenomatous polyposis; HNPCC, hereditary non-polyposis colorectal carcinoma; mAbs, monoclonal antibodies; MMR, mismatch repair; MSI, microsatellite instability.

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epidermic growth factor (EGF) pathway) compared with that in the other groups, MSI þ /BRAF V600E, MSI/BRAF WT and MSI/BRAF V600E, where V600E was the observed BRAF mutation.15 Undoubtedly, more research and more information of the exact significance of any of these genetic variants are needed and hopefully will be available in the near future. The majority (85%) of CRCs show CIN. CIN has long been reported to be a genetic abnormality having a dominant effect in colorectal carcinogenesis.4 In contrast to MSI, which involves short repeats or simple nucleotide repeats (usually CAs), CIN is associated with gross chromosomal abnormalities, such as gene deletions and insertions, activation of proto-oncogenes, inactivation of suppressor genes, as well as aneuploidy or polyploidy of chromosomes.16 A battery of genes is involved in bowel carcinogenesis and contribute toward CIN, such as APC, TP53, KRAS, BRAF, PTEN, Src, TGF-b, SMAD 2 and 4, as well as thymosin b-4; other genes, such as DCC, bcl-2, those coding for molecules of sonic hedgehog signaling pathway and matrix metalloproteinases have been found to be altered in preclinical studies on CRC models. Abnormal hypermethylation of DNA promoter sequences (CpG) occurs frequently in CRC, causing inhibition of transcription factor binding and leading to the silencing of tumor-suppressor genes and DNA repair genes.5,17,18 Normally, methylation of cytosine–guanosine dinucleotides (CpG) occurs throughout DNA genome, whereas gene promoters contain mainly unmethylated CpG islands, events that are reversed in carcinogenesis (that is, global genomic hypomethylation with promoter CpG islands hypermethylation).18 There are numerous published preclinical studies suggesting a strong association of CpG islands methylation phenotype with BRAF mutations and also to a lesser degree with MSI.19–21 Finally, changes of genes regulating telomerase and telomerase reactivation may contribute toward carcinogenesis and cancer cells immortality.22 APC tumor-suppressor gene mutation/inactivation is the main genetic culprit in familiar adenomatous polyposis, but APC is also found inactive in about 80% of sporadic cases, occurring early during the transformation of a normal epithelium to aberrant crypt foci then to adenoma and finally to carcinoma. The normal APC protein regulates the Wtn/b-catenin pathway, finally effecting the degradation of b-catenin by proteasomes and preventing b-catenin migration to the nucleus where it acts as a transcription factor for proliferating genes. APC also promotes cell adhesion and migration by binding to microtubule bundles. Thus, APC gene inactivation and/or mutation of b-catenin (found in 10% of sporadic CRC) results in an uncontrolled activity of b-catenin to the nucleus and in excessive cell proliferation.23 Genetic alterations in tumor tissues Mutation and overexpression of the Src gene is a genetic event, taking place early in colon carcinogenesis and is

seen frequently (in about 80%) in CRC cell lines. Src overexpression activates, by phosphorylation, the Akt/PI3K pathway, leading to tumor progression, invasion and metastases.23,24 Irby et al.25 reported an infrequent Src mutation (in 12% of CRC) at codon 531, which was associated with tumorigenesis, progression and metastasis. The KRAS gene plays a very important role in colon carcinogenesis and also in the recently adopted therapeutic strategy. Proto-oncogene KRAS (Kirsten rat sarcoma virus oncogene) is positioned in chromosome 12p12.1. The EGFR/RAS/RAF pathway is significant in growth-promoting signal transduction from the cell surface receptors to the nucleus, affecting the production and regulation of other key proteins. The three proto-oncogenes belonging to the RAS family (HRAS, KRAS and NRAS) are located in the inner plasma membrane, bind guanosine diphosphate (GDP) and guanosine triphosphate (GTP) and own an intrinsic GTPase activity that cleaves the GTP to GDP (switch-off position). The KRAS protein is active and transmits signals by binding to GTP (turn on), but it is inactive (turn off) when GTP is converted to GDP. Mutations of KRAS are a rather late event in colorectal carcinogenesis, found in less than 10% in early adenomas, in up to 60% in large (41 cm) adenomas and in approximately 40–50% of carcinomas, according to various published studies.23,26 As many as 70% of CRC tumors will eventually show mutations of one of the genes encoding for proteins of the EGFR/RAS/RAF pathway.23 The clinical importance of KRAS lies in its predictive value of treatment with EGFR monoclonal antibodies, as we will present later in this review. Other molecules of value in the same signaling pathways with KRAS are BRAF, PI3K/Akt, PTEN, MEK, MAP, mTOR and so on. Not surprisingly, sporadic mutations and genetic derangements have been reported for most of these molecules/genes in CRC and are currently investigated as potential therapeutic targets in drug development. Further genetic abnormality seen later in carcinogenesis of CRC, as also in most solid tumors, is mutations on the apoptotic gene, TP53 (in 50% of cases) or mutations of the p53-related pathway (90%). In the remaining of cancers (without TP53 mutation), WT p53 is inhibited by binding with protein mdm2, which is a p53 usual regulator in normal cells, but found overexpressed in malignancies.27 This p53–mdm2 tie prevents p53 action and leads to p53 binding to ubiquitin and to degradation by proteasome (process known as ubiquitination). In abnormal cells with damaged DNA (as seen in aging and ionizing radiation), p53 blocks the cell cycle at the G1 to S-phase transition, inhibits proliferation and growth, leads to cell cycle arrest and finally induces a programed cell death or repair. Hence, the significance of TP53 mutations lies in the loss of the aforementioned crucial functions allowing uncontrolled cell growth and proliferation, increased cellular survival and chromosomal instability, leading eventually to carcinogenesis. Many studies on CRC patients have shown evidence of the prognostic value of TP53 mutations/loss of function and its association with worse survival.28–30 No robust

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evidence or consensus about predictive role of p53 in relation to treatment is yet available, and therefore p53 is not routinely used in clinical practice. A rather late finding in bowel carcinogenesis is the role of the insulin growth factor-1R system, which has been found overexpressed in CRC, preventing apoptosis, enhancing proliferation and inducing angiogenesis by upregulation of the vascular endothelial growth factor (VEGF).31–33 Insulin growth factor-1R is currently being tested as a new therapeutic target in clinical trials, in combination with other targeted agents and cytotoxics. Testing for a germline mutation is often technically easier and more reliable than obtaining and testing tumor samples for genetic or protein alterations. Germline mutations can be detected in blood, feces or other tissues and are unlikely to be altered after antineoplastic treatments. Tumor tissues, although are not always easily obtained, are often improperly preserved, jeopardizing subsequent molecular analyses, and may be affected by the various treatments; therefore, testing for tumor markers may be unreliable or suboptimal. Different methodologies and variable laboratory conditions may contribute toward the commonly discrepant results in various preclinical and clinical studies. Furthermore, genetic tests on heterogeneous populations (for example, different stages, ages, sites of disease and so on) of different ethnicities and of different environmental influences may produce less reliable and often confounding results, as we often observe in published literature. Cytotoxics: pharmacogenetics and molecular markers Drugs used in early (adjuvant) or advanced CRC are subject to various steps of metabolism and transformation to active or inactive compounds, until finally excreted out of the host through the hepatobiliary or renal route. Drug metabolism is regulated by various enzymatic and metabolic pathways prone to genetic variations. The main enzymatic pathways involved in the metabolism of these agents are shown in Table 2. The standard agents licensed for use in CRC include conventional cytotoxics, that is, fluoropyrimidines (5fluorouracil (5-FU), capecitabine, tegafur (UFT) and S1), oxaliplatin, irinotecan, mitomycin C, raltitrexed and targeted agents, such as, cetuximab, panitumumab and bevacizumab. Fluoropyrimidines 5-FU. 5-Fluorouracil is a fluoropyrimidine analog that inhibits the target enzyme, thymidylate synthase (TS) through its metabolite, fluorodeoxyuridylate (FdUMP). 5-FU is converted to the active metabolite, FdUMP, by the enzymes, thymidine phosphorylase (TP) and thymidine kinase intracellularly. FdUMP binds and inhibits TS in the presence of 5,10-methylenetetrahydrofolate (MTHF). Inhibition of TS results in the accumulation of deoxyuridylate (dUMP), which then gets misincorporated into DNA in the form of deoxyuridine triphosphate (dUTP), resulting finally in inhibition of DNA synthesis and

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function. Similarly, integration of fluoro-deoxyuridine triphosphate (FdUTP) into DNA leads to inhibition of DNA formation and function, and incorporation of the fluorouridine triphosphate (FUTP) into RNA results in alterations in RNA processing and translation. The vast majority (approximately 80–85%) of administered 5-FU is catabolized and inactivated by the enzyme, dihydropyrimidine dehydrogenase (DPD), in the liver into the inactive form, dihydrofluorouracil (FDHU) and excreted as a-fluoro-b-alanine (FBAL). Thymidylate synthase has a predictive value in terms of predicting response to fluoropyrimidine-based chemotherapy and potential toxicities. Located in chromosome 18, TYMS, the gene encoding for TS, is found in three different genotypic forms on the basis of the number of 28-base tandem repeats (R) in the promoter 50 -untranslated region (UTR) of the TS gene. Thus, TS forms are seen as 2R/2R (double repeats), 2R/3R (double/triple repeats) and 3R/3R (triple repeats). Studies on tumors from CRC patients have shown that homozygous 3R/3R allelic status is associated with higher levels of TYMS mRNA, higher levels of TS and inferior response to 5-FU-based chemotherapy.34,35 In contrast, 2R/ 2R genotype has been associated with low levels of TS expression and better clinical outcome to therapy.35,36 In addition to the above genotypic groups, a single-nucleotide polymorphism (SNP; G-C), observed at the 12th position of the second repeat of the 3R allele, at its upstream stimulatory factor family E-box elements, reduces its translational activity and causes low TS expression and therefore an altered outcome similar to 2R.37 This result has not been reproduced yet in subsequent studies. Mandola et al.38 provided evidence of a 6bp nucleotide deletion polymorphism at the 30 -UTR end related to lower intratumoral TS expression and mRNA instability in individuals with homozygous 6 bp deletion (6 bp/6 bp), especially among Singaporean, Chinese and African-American patients with colorectal cancer. There are few published studies correlating the level of TS expression at the various tumor sites to treatment response. CRC patients with lung or liver metastases expressing higher levels of TS showed poor response to 5-FU chemotherapy compared with those with metastatic tumors expressing lower TS levels.39,40 In a retrospective study on 862 patients with Duke’s B and C colorectal adenocarcinoma, Edler et al.41 reported that higher intratumoral TS expression of the primary tumor was associated with better response to adjuvant fluoropyrimidine chemotherapy and prognosis.41 In contrast, Soong et al.42 reported that CRC patients (Duke’s B and C) with high TS expression showed a better survival when treated with surgery alone than those who received additional adjuvant 5-FU chemotherapy.42 In a further study on 94 CRC patients undergoing adjuvant chemotherapy, the researchers reported no relation between TS expression and chemotherapy efficacy.43 In general, most published data support that patients with metastatic disease and low TS expression showed a trend toward better response to chemotherapy and longer

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Table 2

Drugs, enzymes and pharmacogenomics in CRC

Cytotoxic, biological agent 5-FU

Enzyme involved TS DPD MTHFR MMR TS/DPD and TP/DPD

Capecitabine

Genetic alteration

Clinical importance and response to treatment

Polymorphisms—altered expression of TYMS DPD SNPs

m Expression/activity - k response

SNPs (118 T) SNPs (156A,312Asn,751Gln) GST polymorphisms AGXT polymorphism

k k m k

k Expression - m toxicity and ? m response MTHFR SNPs (mainly 1298C & 677 T) k Activity -kTS - m toxicity/response Deficiency of MMR genes (MSI+) Stage II (?III) : MSI+ tumors - better prognosis without CT and worse with adjuvant CT m Ratio - m response

Likely similar to 5-FU Activity - m response Activity - m response Expression/activity - k response Activity - m oxalate levels - m neurotoxicity

Oxaliplatin

ERCC1 ERCC2 (XPD) GST (P1,M1,T1) AGXT

Irinotecan

UGT1A1 ABC family Topo-I

UGT1A1*28 allelic variant SNPs Altered expression

k Activity - mtoxicities, ?mresponse k Activity - mtoxicities, ? response m Expression - m response to FOLFIRI or FOLFOX but k response to 5-FU alone

KRAS BRAF PTEN

KRAS codon 12 mutation BRAF V600E mutation PTEN loss of expression

KRAS mutant- k response, k OS and k toxicities k Response k Response

EGFR mAbs (cetuximab, panitumumab)

Abbreviations: ABC, ATP-binding cassette; 5-FU, 5-fluorouracil; CRC, colorectal cancer; CT, chemotherapy; DPD, dihydropyrimidine dehydrogenase; EGFR, epidermal growth factor receptor; ERCC, excision repair cross complementation; GST, glutathione S-tranferase; mAbs, monoclonal antibodies; MMR, mismatch repair; MSI, microsatellite instability; MTHFR, methylenetetrahydrofolate reductase; OS, overall survival; SNPs, single-nucleotide polymorphisms; TP, thymidine phosphorylase; TS, thymidylate synthase.

survival, but possibly more severe toxicities, than did the high TS expression counterpart genotypes (for example, 3R/ 3R, þ 6 bp/ þ 6 bp), both in the adjuvant and advanced settings.34,44,45 This trend was confirmed in a meta-analysis reviewing the association of TS expression and prognosis in advanced and localized CRC, but there was evidence of heterogeneity and possible publication bias.46 Therefore, TS was not recommended as a prognostic or predictive tumor marker by the American Society of Clinical Oncology in the 2006 Update of Recommendations for the Use of Tumor Markers in Gastrointestinal Cancer.47 Additional studies with consistent and standardized methodology are required to clarify the prognostic significance of TS. The prognostic role of TP in patients with CRC is less well investigated. The few conducted studies have suggested that low expression of TP is associated with better response to adjuvant fluoropyrimidines and better overall survival, whereas a high expression of TP is associated with more advanced histopathological stage and therefore worse outcome.42,48–50 Similar to TS, there is no high-level evidence to support the routine use of TP in clinical practice. Orotate phosphoribosyltransferase (OPRT) is an enzyme that, along with TP and uridine phosphorylase (UP), contributes to phosphorylation and activation of 5-FU.

Preclinical and clinical studies have shown polymorphisms of the OPRT gene in colorectal tumors. Overexpression and high levels of OPRT mRNA, as well as a high OPRT/DPD ratio, have been associated with improved response to fluoropyrimidine chemotherapy (in both adjuvant and mCRC), suggesting that this enzyme may serve as a predictive tool.51–53 In a retrospective PCR amplification study on genomic DNA extracted from blood of CRC patients treated with adjuvant 5-FU, TYMS and OPRT genotypes were investigated and correlated to treatmentinduced toxicity. It was found that co-expression of the OPRT Gly213Ala polymorphism of the Ala allele with the 2R/ 2R TYMS genotype was associated with grade 3/4 neutropenia and diarrhea.54 The catabolic enzyme, DPD, plays the most important role in 5-FU excretion. Expression of DPD is related to tolerance and response to 5-FU therapy. In particular, high expression of DPD was found to be associated with increased catabolism and clearance of 5-FU. Low levels or absence of DPD was associated with accumulation of 5-FU, leading frequently to significant toxicities and adverse events, even death.55 There are studies supporting that increasing expression of DPD mRNA is associated with poor response to 5-FU and higher resistance, possibly because of a high catabolic rate.42,56 Although low levels of DPD expose individuals to higher risk

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of toxicities, there may be an association with better response to chemotherapy and longer survival.42,57 The DPD gene is subject to various SNPs, and roughly 3–5% of the population is partially or completely deficient in this important enzyme. As a consequence, patients lacking the DPD enzyme may experience severe to lethal toxicities when treated with a fluoropyrimidine, mainly myelosuppression, diarrhea and mucositis.58,59 The most known DPD SNPs related to grade 3 to 4 toxicities are found on chromosome 1p. They mainly consist of the polymorphisms IVS14 þ 1G-A, 2846A-T, 1679T-G and 85T-C.60 Recently, a new SNP mutation was found at exon 5 of the DPD gene (464T-A) and was associated with a life-threatening toxicity.44,61 It has also been reported that the TS/DPD and TP/DPD ratios are of prognostic significance in CRC. Higher TS/DPD and TP/DPD ratios were related to better outcome and longer disease and progression-free survival,62 because of increasing 5-FU efficacy (likely a result of a reduced catabolism and increased active bioform), although this association had been better described in other gastrointestinal malignancies, such as gastric and pancreatic. Another important enzyme that is fundamental in fluoropyrimidine activity is MTHF reductase (MTHFR), which converts 5,10-MTHF to 5,10-methyltetrahydrofolate. Satisfactory levels and activity of methyltetrahydrofolate allow folate to facilitate 5-FU binding to TS and in the inhibition of DNA synthesis. MTHFR is regulated by the gene, MTHFR, which is subject to genetic alterations, such as mutations and SNPs occurring also on chromosome 1p. The two most common polymorphisms of clinical significance are MTHFR 1298 A-C and 677C-T. These mutations are related to lower enzyme activity, accumulation of 5,10MTHF, increased inhibition of TS and higher efficacy, but also higher toxicity risk, from 5-FU chemotherapy (mainly referring to genotypes 1298 A/A and 677 C/C). Even though the mutations do not bear prognostic value on their own, it has been suggested that the combination of wild-type MTHFR genotype (1298A or 677C) with the 3R/3R TS genotype shows significantly lower overall survival.63 The clinical importance of the MMR status of tumors with regard to prognosis and response to adjuvant 5-FU chemotherapy has been increasingly studied the last few years. Patients diagnosed with stage II colorectal cancer, whose tumors were found to have unstable microsatellites (MMR deficient or MSI þ ), carry a better prognosis than do the microsatellite stable (MMR proficient or MSI) ones. Although it seems paradoxical, MSI þ patients treated with adjuvant 5-FU chemotherapy showed worse prognosis than did MSI patients who derived benefit from adjuvant treatment.14 Therefore, testing tumors for the expression of specific MMR proteins (for example, MLH1, MSH2 and MSH6) may provide an additional tool in our clinical practice in selecting patients and individualizing treatments. Capecitabine. Capecitabine is an oral fluoropyrimidine (5-FU prodrug) broadly used in colorectal cancer as monotherapy, or in combination with other drugs in adjuvant and advanced treatment. Capecitabine undergoes

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metabolism by carboxylesterase 2 , cytidine deaminase and TP to be transformed to 5-FU. Various polymorphisms of these two enzymes have been reported, but no convincing data regarding their predictive or prognostic power are yet available. Low DPD expression and low DPD enzymatic activity have been linked to better response but possibly higher toxicity to capecitabine in a manner similar to that by 5-FU chemotherapy.64,65 Other predictive parameters of capecitabine toxicity include the aforementioned mutations of MTHFR 1298 A-C and 677C-T, as reported in a recent study by Sharma et al.66 MTHF 677C-T mutation was associated with a better response to capecitabine treatment. Meropol et al.67 studied the correlation of TP expression in advanced CRC patients treated with capecitabine and irinotecan, and reported a positive association between high expression of TP in primary tumors and response to chemotherapy. A study from Japan on CRC patients treated with adjuvant 50 -deoxy-5-fluorouridine (50 -DFUR, doxifluridine), a capecitabine metabolite, showed that high TP levels, low DPD levels and a higher TP/DPD ratio were all associated with better disease-free and overall survival.68 The upregulation of TP by capecitabine, in combination with other cytotoxics or radiation therapy, has been proposed as a predictor of response and survival in various malignancies. In particular, TP was found upregulated in patients with locally advanced pancreatic cancer or rectal cancer after treatment with concomitant capecitabine and radiation, and was associated with tumor response.69,70 Overexpression of TP after treatment with capecitabine or 5FU and docetaxel in breast cancer patients, and after association with clinical benefit, has been proposed in a few studies, although others failed to find such an association, suggesting that differences in studied patients, clinical scenarios, methodologies and other unknown factors may influence the results.71–73 Oxaliplatin Oxaliplatin, a novel third-generation platinum analog, was first tested in drug development about 20 years ago and has shown particular activity in CRC treatment in combination with fluoropyrimidines.74.Oxaliplatin bears a 1,2-diaminocyclohexane carrier ligand through which it is linked with DNA, forming a DACH-platinum-DNA adduct preventing repair and further DNA replication.75 Although no specific enzymatic activity directly affects oxaliplatin’s efficacy or metabolism, there are enzymes involved in DNA repair that influence oxaliplatin’s activity and contribute to drug resistance. Excision repair cross-complementation proteins (ERCC), regulated by genes ERCC1 and 2, are involved in the nucleotide repair systems. The hypothesis that polymorphisms and altered activity of DNA repair systems, such as ERCC and XPD (xeroderma pigmentosum group D), may influence oxaliplatin efficacy has been evaluated in a few studies. A polymorphism (SNP) at the codon 118 (C-T), which resulted in a less active ERCC1 genotype (T/T) compared with the WT (C/C), and a better response to FOLFOX

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chemotherapy, but no association with 5-FU or irinotecan treatment, was found to be responsible for the reduced activity of ERCC1.76 Likewise, another study showed that metastatic colorectal cancer (mCRC) patients carrying the ERCC (118 T/T) and XPD (Gly751Gly) polymorphisms showed better survival and response to FOLFOX chemotherapy.77 Contradictory results were published by others who suggested that ERCC1 polymorphism, T/T, at codon 118, was associated with poorer response to FOLFOX and shorter progression-free survival (PFS).78–80 Other researchers have shown that low levels of ERCC mRNA expression are correlated to better response to oxaliplatin combination therapy and survival.81,82 In any case, inspite of the contrasting results about the specific codon genotypes, it seems that a change of function and expression of the DNA-repairing enzymes does affect the chemotherapy efficacy. With regard to the repairing enzyme, XPD (also known as ERCC2), Park et al.79,83,84 reported three common polymorphisms of the XPD gene (that is, C156A, Asp312Asn and Lys751Gln). Of these three, the researchers showed a positive predictive and prognostic value for the Lys/Lys genotype of Lys751Gln compared with that for Lys/Gln and Gln/Gln genotypes, which did not respond to oxaliplatinbased chemotherapy. Overexpression of x-ray cross-complementation (XRCC) genes is related to resistance to oxaliplatin-based therapy. A polymorphism at exon 10 of the XRCC gene (genotype Gln/Gln) was associated with poor response to oxaliplatinbased chemotherapy in patients with mCRC, as opposed to that in patients with the normal Arg/Arg genotype.85 The importance of EGFR inhibitors in KRAS wild-type tumors has been well established. A hypothesis that has emerged recently is the role of the EGFR inhibitor, cetuximab, in downregulation of the ERCC1 protein and in amplification of oxaliplatin efficacy in colon cancer cell lines, earlier resistant to oxaliplatin.86,87 These data have been provided from preclinical studies and have not been prospectively validated. Apart from the role of DNA repair systems in oxaliplatin efficacy, there are various metabolizing enzymes belonging to the glutathione S-tranferase (GST) group regulated by the relevant GST genes, P1 (p), T1(y) and M1(m). These enzymes are involved in oxaliplatin metabolism and detoxification, and understandably their altered function and expression result ultimately in variations in oxaliplatin efficacy. This was mostly shown for GSTP1, but not for the other two enzymes.88 For example, polymorphism of the GSTP1 gene (allele 105 Ile/Ile) was associated with increasing risk of progression and mortality in patients treated with oxaliplatin-based chemotherapy in contrast to the favorable genotype, GSTP1-105 Val/Val.80 Overexpression of GSTP1 has been shown in CRC tumors, especially of the mucinous type, and may be responsible for diminished oxaliplatin activity and response.89 Though there are polymorphisms of GSTT1 and M1 genes reported to be related to clinical response in breast cancer and childhood leukemia, no similar association has been found in colorectal cancer. It

was suggested by a published study that, not only is the GSTP1-105 Ile/Ile genotype associated with adverse outcome after oxaliplatin treatment but it is also related to a significant oxaliplatin neurotoxicity predisposition.90 An important role in the prediction and pathogenesis of oxaliplatin-related neurotoxicity lies with polymorphisms of the gene, AGXT, which codes for the AGXT enzyme responsible for oxalate (which increases drastically after oxaliplatin infusion) metabolism. Patients with the minor haplotype (in contrast to the normal major haplotype) AGXT showed reduced enzymatic activity, higher levels of oxalate and much higher risk of neurotoxicity, both acute and chronic, as was well reported by Gamelin et al.91 Irinotecan Irinotecan (CPT-11 or Campto) is a commonly used cytotoxic drug in colorectal cancer. Irinotecan is a hemisynthetic analog of the natural product, camptothecin, able to inhibit topoisomerase I, a DNA helicase (cutting enzyme). Campto has gained license of use in combination with fluoropyrimidines not only in the first-line mCRC but also as a single second-line agent in patients who failed treatment with fluoropyrimidines and oxaliplatin.92,93 After entering the systemic circulation, irinotecan is metabolized by carboxylesterase to the active metabolite, SN-38, which in turn inhibits the topoisomerase I enzyme and subsequently DNA replication and transcription. SN-38 is inactivated to the compound SN-38 glucuronide (SN-38G) by glucuronidation mediated by the liver enzyme, uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1). Various important polymorphisms of the UGT1A1 enzyme are currently known and associated with a reduced enzymatic activity, such as the ones found in the known metabolic syndromes, Gilbert’s and Crigler–Najjar.94 In these syndromes, the leading genetic event is the addition of a TA dinucleotide to the TATA sequence at the promoter region, creating a variant that is associated with a decreased expression and activity of the UGT1A1 enzyme. The activity of this glucuronidation enzyme is even lower in patients with the homozygous variant allele (known as UGT1A1*28 or 7/7 variant). Approximately 6—12% of the general population carries the UGT1A1*28 variant.94–97 The incidence of this polymorphism is much lower in patients of Asian origin.97,98 Treatment of UGT1A1*28 patients with irinotecan results in reduced metabolism and increased accumulation of the active SN-38 compound and subsequently to higher risk of myelosuppression and diarrhea, as shown in a few prospective studies.96,99 On the basis of this evidence, the Food and Drug Administration of the United States approved the use of the UGT1A1* 28 genotype test in 2005 and recommended a lower starting dose of irinotecan in patients with the homozygous variant. In Europe, UGT1A1*28 testing is not yet routinely applied. In clinical practice, a starting dose reduced by approximately 25–50% is often introduced in patients with the known UGT1A1*28 genotype or with elevated bilirubin (41.5–3.0 times of the upper limit of normal), to minimize the risk of adverse effects. Despite the increased risk of toxicity, patients with

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lower UGT1A1 enzyme activity showed similar response rates and survival than did patients with normal enzymatic activity.95 Carboxylesterase enzymes facilitate irinotecan metabolism to the active SN-38 compound. Although various SNPs of carboxylesterase regulatory genes have been reported, there is no solid evidence and consensus regarding their significance up to now. Other important pathways in irinotecan metabolism include the ATP-binding cassette family, which contains subfamilies/members, such as member 1 (glycoprotein-P, ABCB1), member 2 (multiresistanceassociated protein, ABCC2), breast cancer-resistant protein (BCRP, ABCG2) and many others. Their role is to act as transmembrane pumps and facilitate excretion of noxious metabolic products of drugs and xenobiotics. Polymorphisms (SNPs) of encoding genes for these ATP-binding cassette proteins have been identified and linked to altered irinotecan renal clearance and systemic drug exposure.100,101 It seems that race and ethnicity variation may play a role in these SNPs and may be related to their clinical importance. A recent interesting genetic study led by McLeod et al.102, on mCRC Caucasian patients on FOLFOX versus FOLFIRI chemotherapy, suggested that an SNP (34 A-G) of the ABCG2 gene was associated with higher response to FOLFOX and resistance to FOLFIRI treatment compared with that of WT gene (Po0.013). Enzymes of the cytochrome P450 family (in particular CYP3A) play a role in irinotecan oxidation and catabolism. It was reported that mutations of these enzymes may result in decreased metabolism of irinotecan, but not in increased toxicities.103 The DNA-repair enzymes, XRCC1, ERCC1 and GST-P1, and the expression of the EGF receptor (EGFR), may also be of predictive value in treatment with irinotecan. A molecular study on tumors from CRC patients treated with first-line irinotecan and 5-FU combination reported that intratumoral overexpression of EGFR, ERCC1 and GST-P1 mRNA was associated with better response to treatment. Furthermore, high levels of EGFR and ERCC1 mRNA were correlated with improved PFS.104 The authors suggested that high levels of ERCC1 were likely to be indicative of increased DNA repair and instability, rendering it vulnerable to topoisomerase I inhibitors. With regard to the mechanism through which EGFR is associated with irinotecan activity, no convincing theory is currently available. A particular polymorphism of the XRCC1 gene (the haplotype GGCC-G) was found to be predictive of tumor response to irinotecan in a small patients cohort study (n ¼ 107). Patients homozygous for the GGCC-G haplotype showed the best response to treatment compared with those with other haplotypes probably because of lower XRCC1 activity, and thus reduced DNA repair and higher sensitivity to chemotherapy.105 Expression of topoisomerase I (topo I), the therapeutic target of irinotecan/SN-38, has been lately studied as a prognostic marker in CRC. It was shown in the large prospective FOCUS trial that topo I expression in tumors yields prognostic and predictive significance. Patients with high topo I expression had in general poor prognosis and poor outcome when treated with 5-FU monotherapy, but

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showed significant improvement in survival when treated with combination chemotherapy.11 On the contrary, patients with low topo I expression showed better OS and better response to 5-FU monotherapy, but no benefit from combination treatment. These interesting findings are subject to independent validation, and currently tumor samples from two studies biobanks, the CAIRO trial (5-FU versus 5-FU plus irinotecan) and the FOCUS-2 study (5-FU versus 5-FU plus oxaliplatin), are evaluated for topoisomerase I expression.

Biological agents: pharmacogenetics and biomarkers EGFR inhibitors Cetuximab. Cetuximab (Erbitux, ImClone Systems, New York, NY) is a chimeric (fusion of human/mouse protein) immunoglobulin-G1 antibody, targeting the extracellular domain of the EGFR (ErbB1). This agent was initially approved by the Food and Drug Administration for use in treatment of irinotecan refractory CRC, but it has been proven recently to be effective in treatment of other solid tumors, such as head-and-neck squamous-cell carcinoma or non-small-cell lung cancer, which also express the EGFR. It is now well known from early studies of targeted agents in CRC that not everybody benefits from anti-EGFR therapy, and that there are factors predicting or influencing response. In the pivotal bowel oncology with cetuximab antibody study and in other studies, no correlation of the degree of EGFR expression with response to treatment was established.92,93 However, the grade of acneiform rash, a wellrecognized side effect of EGFR inhibitors, was noted to present a predictive marker of response. In particular, grade 3/4 rash was associated with better response to cetuximab and with better PFS.92,106,107 Later, in 2006, Lievre et al.108 tested the tumors from patients treated with chemotherapy and cetuximab for EGFR expression and for KRAS alterations. The researchers reported evidence of a specific KRAS mutation (codons 12/ 13) in 43% of patients (13/30), which was associated with no response to cetuximab, whereas no mutation of the KRAS gene was found in any of the patients who responded. The same group of researchers reported recently the results of a larger cohort of patients (89 patients of whom 24/89 or 27% were KRAS mutant), which confirmed the predictive value of KRAS mutation in patients treated with cetuximab.109 These results were validated and reproduced in subsequent subgroup analyses of large randomized trials using cetuximab with or without chemotherapy in various clinical settings (as first-line treatment, in chemoresistance mCRC or as third-line monotherapy). In the first-line CRYSTAL study, in which patients were treated with FOLFIRI±cetuximab, about 35% of patients were found to carry KRAS mutations, and this KRAS-mutant group showed no benefit from the addition of the monoclonal antibody.110 Similarly, patients treated with FOLFOX chemotherapy with or without cetuximab in the first-line

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mCRC phase II OPUS study showed benefit in response and in PFS only for the subgroup of patients with WT KRAS (58%), whereas the other 42% with KRAS mutations did not gain from the addition of cetuximab.111 Results from the phase III National Cancer Institute of Canada CTG CO.17 study (cetuximab and best supportive care (BSC) versus BSC in advanced pretreated CRC) were recently published by Karapetis et al.112 Likewise, the authors reported significant clinical and survival benefit in KRAS WT patients treated with cetuximab and BSC compared with that in KRAS mutant or BSC alone patients. KRAS mutations were observed in about 42% of studied patients.112 The above studies unanimously showed that the absence of KRAS mutations did not necessarily guarantee response to treatment, on account of the fact that as many as 40–50% of patients still remained resistant to the EGFR inhibitors. This lack of response may be in part explained by other possible genetic alterations of genes involved in the EGFR pathway; for example, mutations of BRAF or PI3K genes and loss of function of the tumor-suppressor gene, PTEN, which may be responsible for EGFR antibodies inactivity. BRAF, KRAS and PTEN alterations are genetic changes frequently found in colon carcinogenesis and are considered to be mutually exclusive.113 Mutations of the BRAF gene (most commonly studied mutation is the V600E mutation that occurs in exon 15) is another primary genetic event that happens as often as approximately 5–10% of mCRC cases. Preliminary results from studies presented in American Society of Clinical Oncology 2008 suggested that BRAF mutations can be predictive of poor response to cetuximab and of shorter overall survival.114 Similar results were produced by Di Nicolantonio et al.115 who recently published a retrospective study on patients treated with cetuximab or panitumumab. Tumors from 114 patients were tested for their KRAS status, and mutations were found in as many as 30%. The remaining 80 patients with WT KRAS were tested for their BRAF status, which showed that 11 out of the 80 patients were mutant at exon 15 (allele V600E). The authors reported that none of the BRAF-mutated patients responded to treatment, whereas none of the responders carried BRAF mutations (P ¼ 0.028). They also found that sensitivity of the BRAF-mutant CRC cells to EGFR antibodies was restored with the addition of the BRAF inhibitor, sorafenib. In conclusion, they suggested that WT BRAF is required for response to anti-EGFR monoclonal antibody treatment. Further larger prospective studies are needed to support these findings. Frattini et al.116 investigated 27 patients treated with cetuximab-based therapy for activity of PTEN, amplification of EGFR gene and KRAS mutations. The authors reported a correlation between EGFR gene amplification, presence of KRAS WT, activity of PTEN and response to therapy. Loss of PTEN protein expression was reported in 41% patients and none of them showed response to therapy, whereas 62.5% of patients with normal PTEN responded to cetuximab-based treatment.116 A rather similar conclusion was reached by Loupakis et al.117 in a retrospective study evaluating PTEN

expression on primary tumors and on metastases with regard to response on cetuximab-based therapy. Although PTEN activity on primary tumor specimens was not predictive or prognostic, loss of PTEN activity (37% of patients) on metastases was related to no response to treatment and significantly lower PFS. The frequency of the PTEN loss of expression varies among published studies from approximately 18.5 to 40%,116–118 but there is agreement regarding its predictive role. Epidermal growth factor recepter gene amplification, characterized by the presence of increased EGFR gene copy numbers (GCN), has been evaluated in CRC patients on EGFR antibodies using fluorescent in situ hybridization. Few studies reported that EGFR GCN was associated with increased response to anti-EGFR treatment (cetuximab or panitumumab) and longer PFS.119–121 In another study, fluorescent in situ hybridization failed to detect EGFR GCN in patients responding to cetuximab treatment.122 In a study on patients treated with cetuximab for resistant mCRC, the researchers evaluated EGFR GCN by PCR and reported no association of EGFR GCN to response or to PFS, but significant association to overall survival.107 Further exploration of the role of EGFR GCN and more accessible and reproducible methods are required to avoid inconsistent results.123 Panitumumab. Panitumumab is an EGFR inhibitor, a fully human immunoglobulin-G2 antibody, with longer half-life than cetuximab, allowing administration every 2 or 3 weeks. Similarly to cetuximab, panitumumab efficacy is observed only in the subgroup of CRC patients with KRAS WT. In the phase III randomized study by Amado et al.124, panitumumab was compared as a third-line treatment to BSC. KRAS status was determined in 427 (92%) of patients treated in the study and KRAS mutations were observed in 43% of them. As previously seen with cetuximab, increased response to panitumumab and longer PFS was limited to the group of patients with KRAS WT status, whereas the KRAS mutant group did not benefit from cetuximab treatment and showed similar results to the BSC group. As a result of the above data, KRAS status of tumors is required before EGFR antibodies use and strongly recommended by health authorities in the United States and Europe. Apart from selecting the subpopulation that benefits the most from anti-EGFR treatment, testing KRAS also allows the judicious use of resources and avoidance of unnecessary toxicities. In addition to KRAS mutation, it seems that BRAF status is affecting and predicting response to panitumumab, as mentioned earlier in the retrospective study presented by Di Nicolantonio et al.115 and may serve as an additional biomarker in CRC-targeted therapy, provided that these interesting results are independently validated in prospective studies. VEGFR inhibitors Bevacizumab. Bevacizumab (Avastin, Genentech, Inc., San Francisco, CA) is a humanized monoclonal antibody against VEGF-A, and therefore binds to the VEGF receptors 1 and 2.

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There is evidence from randomized clinical trials that bevacizumab is effective in various solid tumors, and particularly in advanced CRC, in conjunction with cytotoxic chemotherapy. It is well known that angiogenesis plays a crucial role in cancer progression and metastases, and it has been shown that VEGF is overexpressed in advanced CRC tumors and related to advanced stages and possibly poor survival, though may not be an independent survival prognostic factor.125–127 There is no evidence so far that the expression status of VEGF is related to bevacizumab effectiveness.128 However, not everybody responds to bevacizumab-based therapy and, unfortunately, no prognostic or predictive molecular markers have been reported so far. Given the potential disastrous complications and the high cost of targeted therapy, there is an urgent need for the development of surrogate markers of response.

Conclusion We are living in an era of vast advances in the understanding and management of solid tumors. With the rapid expansion and integration of the allied fields of genetics, biology, pharmacology, pathology and radiology, translational and medical oncology is delivering state of the art novel treatments along with conventional but still invaluable cytotoxic drugs. Up to now, we have been using observational tools, such as tumor stage, nodal status, vascular or lymphatic tumor

invasion, grade and proliferation rate of tumors, to categorize patients and predict response and survival. The identification of genetic markers of response and prognosis will aid in the development of more individualized chemotherapeutic strategies for cancer patients. Potential prognostic indicators in colorectal cancer include oncogenes, tumor suppressor genes, genes involved in angiogenic and apoptotic pathways and cell proliferation, and those encoding targets of chemotherapy. Specifically, molecular markers, such as deletion of 18q (DCC), p27 and microsatellite instability, are promising indicators of good or poor prognosis. Molecular determinants of efficacy and host toxicity of the most commonly used drugs in CRC, fluorouracil, irinotecan and oxaliplatin are being investigated. Alterations in gene expression, protein expression and polymorphic variants in genes encoding TS, DPD, dUTP nucleotide hydrolase, TP and DNA MMR proteins (for fluoropyrimidine-based chemotherapy), UGT1A1, topoisomerase I and carboxylesterase (for irinotecan therapy) and ERCC1 and ERCC2 and GST P1 (for oxaliplatin-based regimens) may be useful as markers for clinical drug response, survival and host toxicity and need further validation. In addition, KRAS status provides the base of patients’ selection that is likely to benefit from EGFR antibodies. There is growing evidence regarding the use of other biomarkers, such as BRAF and PTEN, which may allow further selection of the appropriate patients’ group likely to benefit from targeted treatments.

Figure 1 Incorporation of pharmacogenetics in the treatment of CRC. Treatment algorithm with potential PGx applications. Patients with early stage CRC may avoid unnecessary or even harmful chemotherapy by determining their MSI and/or their DPD proficiency status. Patients with advanced disease may be selected for appropriate first-, second- or third-line chemotherapy according to their tumor KRAS status, but may also avoid toxicities by determining the presence of UGT1A1*28 mutation or DPD deficiency. There is growing evidence on the role of BRAF and PTEN alterations and topoisomerase I expression before the use of EGFR antibodies and irinotecan or oxaliplatin; a validation by large studies is underway. BEV, bevacizumab; CRC, colorectal cancer; DPD, dihydropyrimidine dehydrogenase; EGFR, epidermal growth factor receptor; PGx, pharmacogenetics, MSI, microsatellite instability.

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Figure 1 illustrates a useful algorithm of pharmacogenetic applications in the management of CRC. Finally, there are still long-standing uncertain issues in the management of CRC, but new problems and conundrums also emerge as a result of new sophisticated applications. It is encouraging that we become increasingly aware of these challenges, and with the international collaboration and integration of clinical and basic research, we can hopefully offer more options and help to patients in the near future.

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