Pharmacogenetics and pharmacogenomics: role of mutational analysis in anti-cancer targeted therapy


The goal of cancer pharmacogenomics is to obtain benefit from personalized approaches of cancer treatment and prevention. Recent advances in genomic research have shed light on the crucial role of genetic variants, mainly involving genes encoding drug-metabolizing enzymes, drug transporters and targets, in driving different treatment responses among individuals, in terms of therapeutic efficacy and safety. Although a considerable amount of new targeted agents have been designed based on a finely understanding of molecular alterations in cancer, a wide gap between pharmacogenomic knowledge and clinical application still persists. This review focuses on the relevance of mutational analyses in predicting individual response to antitumor therapy, in order to improve the translational impact of genetic information on clinical practice.


Pharmacogenetics was first designated as a scientific matter in 19591 to investigate the unequal response to the same treatments by different patients in terms of beneficial effects, clinical efficacy and adverse reactions. Recently, in line with the increasing use of functional genomics in the clinical medicine during the post-genomic era, ‘pharmacogenetics’ was changed into ‘pharmacogenomics’, but both terms can be interchangeably used.2

The purpose of this emerging field is to understand how genetic variants of drug-metabolizing enzymes, transporters and proteins involved in mechanisms of action of specific drugs, may influence their therapeutic effect. On the other hand, prediction of clinical response and potential drug-related toxicities is essential in managing the relevant costs of treating cancer, in particular with biomolecular drugs, and accounts for the great interest in studying genetic variants of specific genes.3

The genomic sequencing of several human cancers has recently provided defined series of somatic gene mutations,4, 5 including single-nucleotide polymorphisms, namely single DNA base changes in a defined position, haplotypes as alleles at different loci together inherited, microsatellites as segments of DNA containing repeated sequences up to 6 bp, insertions and/or deletions as mutations resulting from addition/loss of DNA segments, copy number variations as gain and loss of large chunks of DNA producing an abnormal number of copies of one or more sections of DNA, aneuploidy as an irregular number of chromosomes with structural abnormality, and loss of heterozygosity that includes the defective function of a gene for which the other allele was already inactivated. Among these, single-nucleotide polymorphisms appear the most frequent and can be ordered in a single-nucleotide polymorphism map to be used in single patients to obtain genetic informations suggesting appropriate treatments producing expected clinical response.

Here, we report the most relevant applications of mutational analysis in selecting tailored anti-cancer treatments for solid tumors, also based on our own experience in specific tumors.

Pharmacogenomics and cancer

Tumor progression is strongly associated with the activity of cell membrane receptors and their intracellular signal transduction pathways, which regulate several cell functions including proliferation, apoptosis, motility, adhesion and angiogenesis. Recent studies have emphasized the role of single-nucleotide polymorphisms,6 mutations6 and copy number variations7 in different genes involved in the abnormal growth of cancer cells. In this context, based on the major role of the epidermal growth factor receptor (EGFR) in the pathogenesis of a number of solid tumors,8 the transcriptional factors of EGFR pathway are regarded as putative targets of anti-cancer treatments.

EGFR is a 170 kDa transmembrane glycoprotein belonging to the ErbB family, which includes four members: the EGFR, also known as ErbB-1/HER1, ErbB-2/Neu/HER2, ErbB-3/HER3 and ErbB-4/HER4.8 All these proteins show a similar less conserved ligand-binding extracellular domain, a single transmembrane domain, and a highly conserved intracellular domain with tyrosine kinase (TK) activity.9

The key event leading to ErbB receptor activation includes the binding of a growth factor to the extracellular domain, resulting in the formation of receptor homo- or hetero-dimers. Subsequent molecular events mediate activation of the intrinsic TK domain, which in turn triggers several downstream pathways, such as ras/raf/mitogen-activated ERK kinase (MEK)/mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways10 (Figure 1), driving many biological processes such as cell differentiation, cell cycle progression, cell invasivity, apoptosis and angiogenesis.9, 11 Thus, overexpression of EGFR is believed to have a critical role in tumor progression,8, 9, 11 and the molecular approaches inhibiting this pathway are apparently successful in treating several cancers.11

Figure 1

EGFR signaling pathway. The EGFR is activated by its appropriate ligand with autophoshorylation of SH2 binding domains that recruit small adaptor proteins SOS and Grb2. The GrB2/SOS complex stimulates activation of K-ras by binding to guanosine triphosphate (GTP), thus transducing the activation signal to the nucleus. Guanosine triphosphatase hydrolysis of guanosine triphosphate to guanosine diphosphate (GDP) leads to inactivation of Ras. In particular, guanosine triphosphate-bound Ras activates RAF/mitogen-activated ERK kinase/ERK pathway, which regulates cell proliferation and motility, and PI3K/PTEN/AKT/mTOR cascade, involved in anti-apoptotic responses. Mutational change of K-ras leads to a permanent activated status that triggers the signal transduction pathway regardless upstream EGFR activation or inhibition through anti-EGFR mAbs. AKT, protein kinase B-alpha; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; GRB2, growth factor receptor-bound protein 2; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog; RAS, Kirsten murine sarcoma virus 2; RAF, V-Raf murine sarcoma viral oncogene homolog B1; SOS, GTPases son of sevenless; TK, tyrosine kinase.

PowerPoint slide

Inhibitors of proteins encoded by the majority of overexpressed or mutated genes along the EGFR signaling pathway have been recently reviewed by McDermott et al.3 For instance, EGFR mutations are reported in non-small cell lung cancer (NSCLC) whereas the gene overexpression occurs in glioblastoma.12 Furthermore, ErbB-2/Neu/HER2 is overexpressed in breast13 and gastric cancer,14 K-ras (Kirsten rat sarcoma viral oncogene homolog) and BRAF mutations in colorectal cancer,15 specific BRAF mutations recur in melanoma,16 phosphatidylinositol 3-kinase mutations in colorectal, breast, gastric cancers and glioblastoma.17 Finally, increased activation of mammalian target of rapamycin has been detected in renal cell carcinoma.18

However, despite the wide number of mutations occurring in cancer genetics, only a few have been approved by the Food and Drug Administration (FDA) as molecular targets and predictive biomarkers for new tailored therapeutics (Table 1). They include mutation of EGFR (OMIM #131550) and echinoderm microtubule-associated protein-like 4 (EML4)–ALK in NSCLC,19, 20 c-KIT (OMIM #164920) and platelet-derived growth factor receptor alpha (PDGFRα, OMIM #173490) in gastrointestinal stromal tumors (GISTs),21 K-ras (OMIM #190070) in metastatic colorectal cancer (mCRC),22, 23 and BRAF (OMIM #164757) in melanoma,24 whereas the clinical usefulness of other promising genetic markers is currently under investigation (

Table 1 Most common pharmacogenomic biomarkers for selection of cancer therapy (

Our institutions (Department of Internal Medicine and Clinical Oncology, University of Bari, Italy, and Department of Laboratory Medicine and Advanced Biotechnologies, IRCCS San Raffaele Pisana, Rome, Italy) routinely perform pharmacogenetic screenings for K-ras, BRAF, c-KIT, PDGFRα and EGFR. Table 2 shows mutation frequency of the above-mentioned genes obtained, up to date, from overall studies performed in our laboratory.

Table 2 Mutation frequency of K-ras, BRAF, EGFR, c-KIT and PDGFRα genes obtained from overall pharmacogenetic studies in our laboratory

However, despite accumulating data on pharmacogenomics in oncology, there are very few conclusive studies providing specific clinical recommendations. Indeed, several clinicians are still unfamiliar with pharmacogenetic tests because of the lack of standardized guidelines filling the gap between pharmacogenomic knowledge and its clinical application. Only recently, a specific program aimed at conciliating pharmacogenomic informations to daily practice has been adopted by the Clinical Pharmacogenomics Implementation Consortium of the National Institutes of Health's Pharmacogenomics Research Network (

Pharmacogenomic-based studies of anti-cancer agents are also complicated by the genetic heterogeneity of a cancer population and the occurrence of additional somatic mutations within tumor, thus emphasizing the importance of microdissection from tissue samples as these populations need to be individually procured for analysis. These observations underline the need to develop and apply high-throughput molecular-profiling techniques to analyze and validate potential biomarkers.

EGFR mutational status and treatment of NSCLC

NSCLC is the most common type of lung cancer and a major cause of death from cancer.27

For early-stage disease, surgery, even when followed by adjuvant chemotherapy,28 offers the best opportunity for long-term survival. In patients with advanced NSCLC, the use of cytotoxic chemotherapy is associated with a response rate of 20–35% and a median survival time of 10–12 months.29 Despite the improvement of the overall survival provided by the use of multilinear therapeutic regimens, advanced NSCLC is currently considered incurable and the only new options available are based on mutational studies of EGFR. In fact, inhibition of EGFR with either Gefitinib or Erlotinib appears effective in restraining the tumor growth and both of drugs were approved as first-line treatment of NSCLC.30

These small molecules directly inhibit TK phosphorylation by interfering with either the adenosine triphosphate and/or the enzyme substrate-binding sites, thus suppressing the EGFR pathway.11 Several trials have confirmed their clinical efficacy31, 32 as second- or third-line therapy in advanced NSCLC and, based on longer progression-free survival (PFS) and lower toxicity obtained with these drugs as compared with standard chemotherapy, recent studies support their use also as first-line treatment.33, 34

However, clinical responses to both of TK Inhibitors (TKIs) differ among NSCLC patients. Increasing evidence indicates that therapeutic response to TKIs is associated to aberrant EGFR TK signaling because of activating mutations in the TK domain (exons 18 through 21).35, 36, 37, 38, 39 Major oncogenic mutations include deletion of a leucine–arginine–glutamate–alanine motif (E746_A750del) within exon 19 and thymine-to-guanine transversion in exon 21 leading to an arginine-for-leucine substitution at amino acid 858 (L858R).38 Figure 2 shows location and frequency of somatic mutations in the TK domain of EGFR (Genbank accession no. NC-000007) reported in NSCLC,38 and an electropherogram, obtained in our laboratory, showing the most recurrent somatic change L858R.

Figure 2

Graphic representation of location and frequency of somatic mutations in the tyrosine kinase (TK) domain of epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC). Exons 18–21 in the TK domain are expanded. The frequencies of mutations are reported by the Somatic Mutations in EGFR Database—SM-EGFR-DB ( An example of the recurrent somatic change of Leucine to Arginine at position 858 (L858R) in exon 21, which comprises approximately 45% of EGFR mutations, is showed. Sequence analysis was performed in our laboratory.

PowerPoint slide

After TKI-based treatment, NSCLC patients harboring such mutations show higher response rate and improved survival times compared with EGFR wild-type (WT) patients.40 Clinical studies indicated that the higher response rates achieved in Asiatic never-smoker female patients with lung adenocarcinoma treated with EGFR TKIs were related to the high incidence in these populations of mutations in the TK domain of EGFR.31, 41, 42 Thus, clustering of mutations within specific regions of the EGFR TK domain makes feasible the development of rapid diagnostic tests to guide the clinical use of both Gefitinib and Erlotinib.

A number of patients treated with EGFR TKIs, however, develop resistance to both of drugs. It has been reported that about half of these patients may express an additional EGFR mutation, namely the substitution of threonine with methionine at codon 790 in the TK domain (T790M)43, 44 leading to a steric interference with the binding of TKIs that restores the adenosine triphosphate affinity.44 This mutation mainly occurs in relapsed tumors after initial response to the TKI-based therapy, whereas its recurrence, as germ-line mutation, in a family with inherited susceptibility to lung cancer suggests the pivotal role of the mutation in tumor growth.45 Moreover, the identification of the T790M mutation in circulating cancer cells of patients showing tumor progression during treatment with TKIs further supports the use of non-invasive serial monitoring of emerging EGFR mutations during TKI-based therapy.46

These findings suggest that prospective validation of EGFR TK domain mutations as predictors of responsiveness to TKIs improves benefit from either Gefitinib or Erlotinib in terms of PFS and overall response rate, although no study has shown so far a real advantage in overall survival.41 The use of Gefitinib in patients with NSCLC harboring EGFR mutations was thus approved in July 2009 by European Medicines Agency, while in 2010, the Italian Association of Medical Oncology, in collaboration with the Italian Society of Anatomic Pathology and Diagnostic Cytopathology, published the recommendations for mutational analysis of EGFR in lung carcinoma.47 This is crucial for a correct clinical use of TKIs because it helps in anticipating both responsiveness and occurrence of resistance, although this latter could be mediated by other mechanisms including c-MET amplification48 and HER2, HER3, mitogen-activated protein kinase and AKT1 activation.49

EML4–ALK rearrangements in NSCLC

The ALK gene (OMIM #105590) encodes a TK receptor and is involved in formation of many fusion proteins.50, 51 The fusion of ALK with EML4 (OMIM #607442), on the short arm of chromosome 2, was reported in approximately 4% of never- or light-smoker patients with NSCLC52 and studies involving East Asian patients reported that up to 13% of lung tumors may harbor EML4–ALK fusions.53, 54, 55, 56, 57, 58, 59

The EML4–ALK encodes a transforming fusion protein that mediates the ligand-independent oligomerization of ALK resulting in constitutive ALK kinase activation.56, 60, 61 Apart from rare exceptions, EML4–ALK rearrangements are mutually exclusive of EGFR and K-ras mutations,54, 55, 58, 62 as patients with EML4–ALK do not benefit from EGFR TKIs’ therapy, similarly to those lacking EGFR mutations.58 Nevertheless, cancers holding ALK fusions are sensitive to specific ALK inhibitors. Most of them are currently under investigation63 whereas only Crizotinib (PF-02341066) has been approved for clinical use.62, 64 Early results from a phase I study emphasized the therapeutic efficacy of Crizotinib, because an objective response rate of 56% and a median PFS of 9.2 months were reached in a cohort of 105 EML4–ALK-positive NSCLC patients.63, 64 These data led to open a phase-3 registration trial ( and, in August 2011, the FDA approved Crizotinib for the treatment of patients with locally advanced or metastatic NSCLC with ALK mutation. Recently, two studies reported that patients who relapsed after Crizotinib may hold two secondary resistance mutations within the ALK TK domain,65, 66 namely the C1156Y substitution and the gatekeeper mutation L1196M.65, 66

To date, more than nine different variants of the EML4–ALK fusion gene have been identified.67 This supports the importance of using highly sensitive rapid amplification of cDNA ends-coupled PCR sequencing methods for identifying patients for targeted therapies.68

K-ras mutational analysis and treatment of mCRC

Although a number of therapeutic approaches have been introduced in the clinical practice over the last few years, mCRC represents a major challenge for clinical oncology. EGFR-directed monoclonal antibodies (anti-EGFR MoAbs), namely Cetuximab and Panitumumab have entered in clinic based on the predominant expression (up to 80%) of EGFR in colorectal tumors.69 Cetuximab is an immunoglobulin G1 (IgG1) chimeric mouse/human MoAb that, after binding to EGFR, promotes its internalization resulting in a selective inhibition of downstream signal transduction.70

Since 2004, Cetuximab has been approved, in combination with Irinotecan, for the treatment of Irinotecan-refractory EGFR-expressing (EGFR+) patients, or as monotherapy in patients intolerant to Irinotecan.71 Panitumumab, a fully human IgG2 MoAb, also directed to EGFR, was introduced in 2006 for treatment of EGFR+- metastatic-mCRCs in clinical progression.72 Although both of these MoAbs bind EGFR, their mechanism of action slightly differs. In fact, unlike IgG2 MoAbs, those belonging to the IgG1 isotype can activate the complement and induce antibody-dependent cellular cytotoxicity, although this has not been proven for Cetuximab.73

The response variability shown by mCRC to EGFR-targeting agents gained attention on the putative mechanisms underlying primary resistance to these agents.74 Although the high EGFR expression is a reliable predictor of response,75 other genetic factors such as K-ras mutations, may drive unresponsiveness of patients76 with poor prognosis.77

K-ras belongs to a proto-oncogene family comprising other two genes, namely H-ras (Harvey rat sarcoma viral oncogene homolog), and N-ras (neuroblastoma RAS viral (v-ras) oncogene homolog), positioned on unrelated chromosomes.78 When bound by guanosine triphosphate, K-ras protein is active, whereas it becomes inactive as guanosine triphosphate is converted to guanosine diphosphate. Active K-ras interferes with several kinase effectors such as RAF, extracellular signal-regulated kinase and phosphatidylinositol 3-kinase, thus regulating a variety of cellular processes.78 As K-ras is a downstream effector of EGFR, in case of K-ras-activating mutations, EGFR signaling is maintained activated in a ligand-independent manner and the upstream inhibition of EGFR by MoAbs results ineffective (Figure 2).

Activating mutations of K-ras are observed in approximately 40% of sporadic colorectal cancers,78 and up to 90% include G-to-A transitions79 and G-to-T transversions80 in codons 12 and 13. Less frequently, mutations occur in codons 61, 63 and 146, and, rarely, in the presence of concomitant mutations.81, 82, 83 Figure 3 shows the frequency of K-ras (GenBank accession no. NC_000012) mutations, represented as electropherograms obtained from mCRC samples in our laboratory.84, 85, 86, 87, 88

Figure 3

Frequency of K-ras mutations in metastatic colorectal cancer patients. Data extracted from Normanno et al.,84 Rascal II study85 and Neumann et al.86 Other rare mutations are available on the Catalogue Of Somatic Mutation In Cancer87 (COSMIC, Sanger Institute) ( All sequence analyses were performed in our laboratory.

PowerPoint slide

Therefore, a correct assessment of K-ras mutational status is required for the selection of mCRC patients who can benefit from anti-EGFR MoAb-based treatment, as pointed by a number of clinical trials.76, 88, 89 The phase III CRYSTAL study compared efficacy of FOLFIRI (5-fluorouracil, folinic acid/Irinotecan) plus Cetuximab protocol to FOLFIRI alone as first-line treatment in 540 patients with EGFR+- mCRC stratificated for K-ras mutational status WT.89 Approximately 35% of patients were found to hold K-ras mutations and, retrospectively, the addition of Cetuximab to FOLFIRI significantly improved PFS in K-ras WT patients with respect to the K-ras mutants. Similarly, Cetuximab also leads to significant increase in overall response rate in K-ras WT patients (43% vs 59%, P=0.0025), whereas no improvement was observed in the mutant population (40% vs 36%, P=0.46).88

In the phase II OPUS study, 233 tumor specimens were assessed for K-ras mutations, in order to evaluate the efficacy of adding Cetuximab to the FOLFOX protocol in the first-line treatment of mCRC.89 Again, addition of the MoAb prolongated PFS only in the subset of WT patients (58%), as compared with mutants (42%).89

In the multicenter randomized phase III trial evaluating efficacy of Panitumumab in combination with chemotherapy (PRIME), mCRC patients with no prior chemotherapy, Eastern Cooperative Oncology Group performance status of 0–2, and available tissue for biomarker testing were randomly assigned 1:1 to receive Panitumumab plus infusional Fluorouracil, Leucovorin, and Oxaliplatin (FOLFOX4) vs FOLFOX4 alone, in order to evaluate the efficacy and safety of Panitumumab as initial treatment for mCRC.90 This study demonstrated that Panitumumab—FOLFOX4 was well tolerated and significantly improved PFS in patients with WT K-ras tumors, emphasizing the importance of K-ras testing for patients with mCRC.90

However, a minor subset of patients with WT K-ras do not benefit from anti-EGFR MoAb treatment, because of either alteration of other genes enrolled in the EGFR signaling pathway, such as BRAF91 and PIK3CA92 or overexpression of phosphatase and tensin homolog protein93 and EGFR ligands.94

c-KIT/PDGFRα mutational analysis in GIST

GISTs are tumors of the gastrointestinal tract originating from the interstitial cells of Cajal of the myenteric plexus95 and characterized by coexpression of the CD34 and the type III TK receptor KIT (CD117).96 Approximately 60–70% of these tumors develop in stomach, followed by small intestine (30%), esophagus or large intestine and rectum96 (<5%), whereas they rarely occur outside the gastrointestinal tract.96

It is estimated that >80% of patients with GIST carry a gain-of-function somatic mutation in c-KIT,97 whereas 5–7% hold mutation in PDGFRα.98 c-KIT and PDGFRα mutations are mutually exclusive and drive the tumorigenesis of GISTs, as they underlay structural changes of the relative receptors, resulting in a constitutive persistent autoactivation.97, 98

Four hot spots have been identified in c-KIT sequence,97 as represented in Figure 4. Up to 65% of c-KIT mutations affect the juxtamembraneous domain (exon 11), an autoinhibitory region that maintains the inactivated conformation in the absence of the KIT ligand,97 whereas other mutations rarely occur in the extracellular region (exon 9, 10–15%)99 or in the TK domain (exons 13 and 17, <1%).100

Figure 4

KIT and platelet-derived growth factor receptor alpha (PDGFRα) receptors with locations of most common activating mutations in gastrointestinal stromal tumors (GISTs). Ligand bindings to extracellular domains activate signal transduction with consequent modulation of cell proliferation, chemotaxis and apoptosis. Mutations frequencies are available in GIST Support International (

PowerPoint slide

PDGFRα mutations are prevalently found within the TK domain (exon 18, up to 90%) and, at a lesser extent, in the juxtamembraneous domain (exon 12) or in the first part of the TK domain (exon 14) (Figure 4).98

Discovery of these mutations fueled the development and clinical use of selective TKIs. Imatinib, a TKI successfully adopted in chronic myeloid leukemia,101 was approved in 2002 for treatment of unresectable and/or metastatic GIST102 and several studies have confirmed its efficacy also as adjuvant therapy of these tumors, because of the high homology between the adenosine triphosphate-binding pocket of c-kit and Bcr–Abl. The pivotal trial Z9001,103 indeed, showed that patients receiving the TKI in adjuvant (400 mg per day × 12 months) had a significant improvement in PFS with respect to the placebo group.103 Therefore, in 2008 the FDA approved Imatinib for adjuvant therapy of resected adult GIST, whereas additional ongoing trials are aimed at defining the therapeutic value of Imatinib in the neoadjuvant setting.104

Binding of Imatinib to the adenosine triphosphate-binding pocket of c-KIT and PDGFRα mediates a competitive inhibition of the enzymatic activity of these receptors,102 thus driving anti-proliferative and pro-apoptotic effects on GISTs cells. Affinity of Imatinib depends, in part, on the type of mutation, and on the codons affected, thus emphasizing the role of mutational analysis in identifying GIST genotypes that are likely to be scarcely responsive to Imatinib.105, 106 Several studies suggest that tumors with mutations in the TK domain (exons 13 and 17) are not effectively inhibited by Imatinib, but these types of mutations occur in <1%. Fortunately, most of GISTs carry mutations in the juxtamembraneous domain (exon 11), so that they are intrinsically higher sensitive to Imatinib than those with an exon 9 mutation or without c-KIT mutations at all.105 In addition, based on recent studies demonstrating that another TKI, namely Sunitinib, is effective in GIST with exon 9 mutation,107 the FDA approved Sunitinib malate for the treatment of patients with GIST in clinical progression or intolerant to Imatinib.

On the other hand, PDGFRα mutations, and particularly the most frequent point mutation D842V in exon 18, may lead to a primary resistance to Imatinib.108 Moreover, with longer duration of TKI-based treatment, the risk of resistance to therapy increases, also because of the potential acquisition of secondary mutations in the kinase domain.109

These evidences underline that molecular subclassification of GISTs, even at the time of progression during the first-line treatment, provides valuable information for patient management.110

BRAF mutational analysis in melanoma

Malignant skin melanoma is one of the most chemoresistant human neoplasias and its incidence has risen faster than other solid tumors over the past 60 years ( Early-stage patients can be successfully treated with surgical resection, whereas those in advanced stage have <5% of survival rate within 5 years.111

Approximately 70–80% of acquired melanocytic nevi112 and 40–60% of malignant melanoma113 express somatic mutations of BRAF, particularly the oncogenic-activating V600E mutation.113, 114, 115, 116, 117, 118, 119, 120, 121 Thus, many selective inhibitors of mutant BRAF were developed and are currently under preclinical and clinical investigation (

Vemurafenib, a potent inhibitor of the V600E BRAF kinase activity, was the first agent showing clinical advantages in treating melanoma.120 In a single phase I trial, 11 out of 16 BRAF V600E mutation-bearing patients showed considerable tumor response to Vemurafenib, whereas no clinical response was observed in patients with WT BRAF tumor.120

In the phase II trial, BRIM-2 enrolling 132 patients with advanced melanoma who had received one prior therapy, the overall response rate was 53%, with a complete response rate equal to 5% and a PFS of 6.7 months.122 In tumors expressing the BRAF V600E mutation, Vemurafenib lowered levels of phosphorylated extracellular signal-regulated kinase, which is associated with clinical response.123

Furthermore, Vemurafenib was recently tested in a phase III randomized trial against Dacarbazine in 675 never-treated melanoma patients harboring the V600E mutation.119 It showed a significant overall survival advantage as single agent against Dacarbazine119 (48% vs 5%, respectively), thus prompting the recent FDA approval for the treatment of advanced melanoma with mutant BRAF.

As in other cancers, however, mechanisms of resistance must be considered. Resistance to Vemurafenib may arise within 8 months119, 120 and depend on the reactivation of the mitogen-activated protein kinase pathway124, 125 or, alternatively, on the acquisition of additional activating mutation in the mitogen-activated ERK kinase protein.126 Once again, this emphasizes the clinical impact of genotyping tumors to select the best patients for the most appropriate treatment.

Conclusions and future perspective

Genomic heterogeneity among tumors inevitably causes differences between unrelated patients in terms of responsiveness to the same treatment. Pharmacogenomics has made significant progress in recent years and traced a new face of cancer therapy.

In the light of the close link between drug mechanism of action, genomics and biology, the goal of cancer pharmacogenomics is to obtain the highest benefits from personalized anti-cancer approaches in both cancer treatment and prevention. Moreover, this may also be relevant in cost–benefit analysis in applying the genetic diagnostic tests. A number of genetic markers have been identified so far, and even if many of them are not candidate for routinary use, the analysis of tumor-biopsy samples for a subgroup of key mutations has been introduced as a mandatory test in the clinical practice.

The FDA has recently addressed the integration of pharmacogenomic studies into drug development and medical practice, and approved the adoption of novel drugs, including Gefitinib or Erlotinib as first-line therapy of NSCLC, Cetuximab for the treatment of EGFR-expressing mCRC and Imatinib as adjuvant therapy of GIST.

To optimize pharmacogenomic studies, clinicians must routinely collect biospecimens by using standardized protocols, and obtain high-quality samples for any subsequent genomic analyses. The importance of pharmacogenomics, indeed, is also related to the identification of mutations accounting for the drug resistance acquired by some patients during treatment.

At present, this effect may be explained as product of secondary mutations acquired by cancer cells during the treatment. Recent findings, however, emphasize the existence within tumors of a subset of stem cell-like cells, namely cancer stem cells, endowed with high tumor-regenerative capacity and drug resistance.127, 128, 129 The cancer stem cell hypothesis may thus provide an alternative explanation for the failure of targeted approaches, because these treatments are able to ablate proliferating cancer cells whereas favoring emergence of a small pool of quiescent cancer stem cells that hold resistant mutations responsible for tumor relapse.

The application of gene sequencing strategies to predict the efficacy of novel anti-cancer drugs recently benefits from a new tool allowing high-throughput DNA sequencing. This method, called next-generation sequencing, uses high number of parallel sequence reactions to read millions of bases in a single run.130 Next-generation sequencing has the potential to rapidly detect, at single base pair resolution, all somatic mutations, rearrangements, small insertion/deletions and copy number variations in an entire genome.131 Another important application of next-generation sequencing is represented by the so-called ‘deep sequencing’, an approach allowing an extensive reading coverage of targeted genomic regions during the same sequencing process.132, 133 This methodology holds many considerable advantages, including (i) extension of pharmacogenomics studies to entire genetic pathways instead of a single gene;133, 134, 135 (ii) identification of secondary mutations responsible for drug resistance;136, 137, 138 (iii) selection of somatic mutations in small cell subclones within a large component of normal cells;131 and (iv) recovery of short DNA fragments from formalin-fixed, and paraffin-embedded samples.130, 139

Based on increasingly available scientific data, the ‘next-generation sequencing’ techniques, although at the beginning of their development, can represent the definitive tools for introducing pharmacogenetic markers in the common clinical practice, enabling us to look forward to an exciting future of this developing science.

Accession codes




  1. 1

    Meyer UA . Pharmacogenetics 5 decades of therapeutic lessons from genetic diversity. Nat Rev Genet 2004; 6: 669–676.

  2. 2

    Evans WE, Relling MV . Pharmacogenomics: translating functional genomics into rational therapeutics. Science 1999; 286: 487–491.

  3. 3

    McDermott U, Downing JT, Stratton M . Genomics and the continuum of cancer care. N Engl J Med 2011; 36: 340–350.

  4. 4

    Stratton MR, Campbell PJ, Futreal PA . The cancer genome. Nature 2009; 458: 719–724.

  5. 5

    Pleasance ED, Cheetham RK, Stephens PJ, McBride DJ, Humphray SJ, Greenman CD et al. A comprehensive catalogue of somatic mutations from a human cancer genome. Nature 2010; 463: 191–196.

  6. 6

    Huang YT, Heist RS, Chirieac LR, Lin X, Skaug V, Zienolddiny S et al. Genome-wide analysis of survival in early-stage non-small- cell lung cancer. J Clin Oncol 2009; 27: 2660–2667.

  7. 7

    Zhang Y, Martens JW, Yu JX, Jiang J, Sieuwerts AM, Smid M et al. Copy number alterations that predict metastatic capability of human breast cancer. Cancer Res 2009; 69: 3795–3801.

  8. 8

    Normanno N, De Luca A, Bianco C, Strizzi L, Mancino M, Maiello MR et al. Epidermal growth factor receptor (EGFR) signaling in cancer. Gene 2006; 366: 2–16.

  9. 9

    Raymond E, Faivre S, Armand JP . Epidermal growth factor receptor tyrosine kinase as a target for anticancer therapy. Drugs 2000; 60 (suppl 1): 15–23.

  10. 10

    Jorissen RN, Walker F, Pouliot N, Garrett TP, Ward CW, Burgess AW . Epidermal growth factor receptor: mechanisms of activation and signalling. Exp Cell Res 2003; 284: 31–53.

  11. 11

    Krause DS, Van Etten RA . Tyrosine kinases as targets for cancer therapy. N Engl J Med 2005; 353: 172–187.

  12. 12

    De Witt Hamer PC . Small molecule kinase inhibitors in glioblastoma: a systematic review of clinical studies. Neuro Oncol 2010; 12: 304–316.

  13. 13

    Campone M, Berton-Rigaud D, Bourbouloux E, Sophie S, Zanetti A, Frenel JS . Her2 positive breast cancer: practices. Bull Cancer 2011; 98: 154–163.

  14. 14

    De Vita F, Giuliani F, Silvestris N, Catalano G, Ciardiello F, Orditura M . Human epidermal growth factor receptor 2 (HER2) in gastric cancer: a new therapeutic target. Cancer Treat Rev 2010; 36 (Suppl 3): 11–15.

  15. 15

    Bass A . Impact of KRAS and BRAF gene mutations on targeted therapies in colorectal cancer. J Clin Oncol 2011; 29: 2728–2729.

  16. 16

    Sigalotti L, Fratta E, Parisi G, Coral S, Maio M . Stability of BRAF V600E mutation in metastatic melanoma: new insights for therapeutic success? Br J Cancer 2011; 105: 327–328.

  17. 17

    Cleary JM, Shapiro GI . Development of phosphoinositide-3 kinase pathway inhibitors for advanced cancer. Curr Oncol Rep 2010; 12: 87–94.

  18. 18

    Klümpen HJ, Beijnen JH, Gurney H, Schellens JH . Inhibitors of mTOR. Oncologist 2010; 15: 1262–1269.

  19. 19

    Dahabreh IJ, Linardou H, Siannis F, Kosmidis P, Bafaloukos D, Murray S . Somatic EGFR mutation and gene copy gain as predictive biomarkers for response to tyrosine kinase inhibitors in non-small cell lung cancer. Clin Cancer Res 2010; 16: 291–303.

  20. 20

    Pillai RN, Ramalingam SS . The biology and clinical features of non-small cell lung cancers with EML4-ALK translocation. Curr Oncol Rep 2012; 14: 105–110.

  21. 21

    Wardelmann E, Büttner R, Merkelbach-Bruse S, Schildhaus HU . Mutational analysis of gastrointestinal stromal tumors: increasing significance for risk assessment and effective targeted therapy. Virchows Arch 2007; 451: 743–749.

  22. 22

    Plesac TP, Hunt JL . KRAS mutation testing in colorectal cancer. Adv Anat Pathol 2009; 16: 196–203.

  23. 23

    Allegra CJ, Jessup JM, Somerfield MR, Hamilton SR, Hammond EH, Hayes DF et al. American Society of Clinical Oncology provisional clinical opinion: testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy. J Clin Oncol 2009; 27: 2091–2096.

  24. 24

    Vultur A, Villanueva J, Herlyn M . Targeting BRAF in advanced melanoma: a first step toward manageable disease. Clin Cancer Res 2011; 17: 1658–1663.

  25. 25

    Sim SC, Ingelman-Sundberg M . Pharmacogenomic biomarkers: new tools in current and future drug therapy. Trends Pharmacol Sci 2011; 32: 72–81.

  26. 26

    Relling MV, Altman RB, Goetz MP, Evans WE . Clinical implementation of pharmacogenomics: overcoming genetic exceptionalism. Lancet Oncol 2010; 11: 507–509.

  27. 27

    Goldstraw P, Ball D, Jett JR, Le Chevalier T, Lim E, Nicholson AG et al. Non-small-cell lung cancer. Lancet 2011; 378: 1727–1740.

  28. 28

    Sangha R, Price J, Buttes CA . Adjuvant therapy in non-small cell lung cancer: current and future directions. Oncologist 2010; 15: 862–872.

  29. 29

    Ohe Y, Ohashi Y, Kubota K, Tamura T, Nakagawa K, Negoro S et al. Randomized phase III study of cisplatin plus irinotecan versus carboplatin plus paclitaxel, cisplatin plus gemcitabine, and cisplatin plus vinorelbine for ad- vanced non-small-cell lung cancer: Four- Arm Cooperative Study in Japan. Ann Oncol 2007; 18: 317–323.

  30. 30

    Cataldo VD, Gibbons DL, Pérez-Soler R, Quintás-Cardama A . Treatment of non-small-cell lung cancer with erlotinib or gefitinib. N Engl J Med 2011; 364: 947–955.

  31. 31

    Shepherd FA, Rodrigues Pereira J, Ciuleanu T, Tan EH, Hirsh V, Thongprasert S et al. Erlotinib in previously treated non–small-cell lung cancer. N Engl J Med 2005; 353: 123–132.

  32. 32

    Kim ES, Hirsh V, Mok T, Socinski MA, Gervais R, Wu YL et al. Gefitinib versus docetaxel in previously treated non-small-cell lung cancer (INTEREST): a randomised phase III trial. Lancet 2008; 372: 1809–1818.

  33. 33

    Oxnard GR, Miller VA . Use of erlotinib or gefitinib as initial therapy in advanced NSCLC. Oncology 2010; 24: 392–399.

  34. 34

    Rosell R, Gervais R, Vergnenegre A, Massuti B, Felip E, Cardenal F et al. Erlotinib versus chemotherapy (CT) in advanced non-small cell lung cancer (NSCLC) patients (p) with epidermal growth factor receptor (EGFR) mutations: Interim results of the European Erlotinib Versus Chemotherapy (EURTAC) phase III randomized trial. J Clin Oncol 2011; 29, abstr 7503. Presented at: 47th Annual Meeting of the American Society of Clinical Oncology. Chicago, IL, USA. 3–7 June 2011.

  35. 35

    Pao W, Miller V, Zakowski M, Doherty J, Politi K, Sarkaria I et al. EGF receptor gene mutations are common in lung cancers from ‘never smokers’ and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci USA 2004; 101: 13306–13311.

  36. 36

    Paez JG, Jänne PA, Lee JC, Tracy S, Greulich H, Gabriel S et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004; 304: 1497–1500.

  37. 37

    Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004; 350: 2129–2139.

  38. 38

    Riely GJ, Politi KA, Miller VA, Pao W . Update on EGFR mutations in non-small cell lung cancer. Clin Cancer Res 2006; 12: 7232–7241.

  39. 39

    Toschi L, Cappuzzo F . Understanding the new genetics of responsiveness to epidermal growth factor receptor tyrosine kinase inhibitors. Oncologist 2007; 12: 211–220.

  40. 40

    Linardou H, Dahabreh IJ, Bafaloukos D, Kosmidis P, Murray S . Somatic EGFR mutations and efficacy of tyrosine kinase inhibitors in NSCLC. Nat Rev Clin Oncol 2009; 6: 352–366.

  41. 41

    Thatcher N, Chang A, Parikh P, Rodrigues Pereira J, Ciuleanu T, von Pawel J et al. Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet 2005; 366: 1527–1537.

  42. 42

    Bell DW, Brannigan BW, Matsuo K, Finkelstein DM, Sordella R, Settleman J et al. Increased prevalence of EGFR-mutant lung cancer in women and in East Asian populations: analysis of estrogen-related polymorphisms. Clin Cancer Res 2008; 14: 4079–4084.

  43. 43

    Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski MF et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2005; 2: e73.

  44. 44

    Yun CH, Mengwasser KE, Toms AV, Woo MS, Greulich H, Wong KK et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci USA 2008; 105: 2071.

  45. 45

    Bell DW, Gore I, Okimoto RA, Godin-Heymann N, Sordella R, Mulloy R et al. Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR. Nat Genet 2005; 37: 1315–1316.

  46. 46

    Maheswaran S, Sequist LV, Nagrath S, Ulkus L, Brannigan B, Collura CV et al. Detection of mutations in EGFR in circulating lung-cancer cells. N Engl J Med 2008; 359: 366–377.

  47. 47

    Marchetti A, Normanno N, Pinto C, Taddei GL, Adamo V, Ardizzoni A et al. Recommendations for mutational analysis of EGFR in lung carcinoma. Pathologica 2010; 102: 119–126.

  48. 48

    Bean J, Brennan C, Shih JY, Riely G, Viale A, Wang L et al. MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc Natl Acad Sci USA 2007; 104: 20932–20937.

  49. 49

    Giovannetti E, Zucali PA, Peters GJ, Cortesi F, D’Incecco A, Smit EF et al. Association of polymorphisms in AKT1 and EGFR with clinical outcome and toxicity in non-small cell lung cancer patients treated with gefitinib. Mol Cancer Ther 2010; 9: 581–593.

  50. 50

    Morris SW, Kirstein MN, Valentine MB, Dittmer KG, Shapiro DN, Saltman DL et al. Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science 1994; 263: 1281–1284.

  51. 51

    Palmer RH, Vernersson E, Grabbe C, Hallberg B . Anaplastic lymphoma kinase: signalling in development and disease. Biochem J 2009; 420: 345–361.

  52. 52

    Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S et al. Identification of the transforming EML4-ALK fusion gene in nonsmall-cell lung cancer. Nature 2007; 448: 561–566.

  53. 53

    Inamura K, Takeuchi K, Togashi Y, Nomura K, Ninomiya H, Okui M et al. EML4-ALK fusion is linked to histological characteristics in a subset of lung cancers. J Thorac Oncol 2008; 3: 13–17.

  54. 54

    Inamura K, Takeuchi K, Togashi Y, Hatano S, Ninomiya H, Motoi N et al. EML4-ALK lung cancers are characterized by rare other mutations, a TTF-1 cell lineage, an acinar histology, and young onset. Mod Pathol 2009; 22: 508–515.

  55. 55

    Koivunen JP, Mermel C, Zejnullahu K, Murphy C, Lifshits E, Holmes AJ et al. EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. Clin Cancer Res 2008; 14: 4275–4283.

  56. 56

    Shinmura K, Kageyama S, Tao H, Bunai T, Suzuki M, Kamo T et al. EML4-ALK fusion transcripts, but no NPM-, TPM3-, CLTC-,ATIC-, or TFG-ALK fusion transcripts, in non-small cell lung carcinomas. Lung Cancer 2008; 61: 163–169.

  57. 57

    Martelli MP, Sozzi G, Hernandez L, Pettirossi V, Navarro A, Conte D et al. EML4-ALK rearrangement in non-small cell lung cancer and non-tumor lung tissues. Am J Pathol 2009; 174: 661–670.

  58. 58

    Shaw AT, Yeap BY, Mino-Kenudson M, Digumarthy SR, Costa DB, Heist RS et al. Clinical features and outcome of patients with nonsmall-cell lung cancer who harbor EML4-ALK. J Clin Oncol 2009; 27: 4247–4253.

  59. 59

    Wong DW, Leung EL, So KK, Tam IY, Sihoe AD, Cheng LC et al. The EML4-ALK fusion gene is involved in various histologic types of lung cancers from nonsmokers with wild-type EGFR and KRAS. Cancer 2009; 115: 1723–1733.

  60. 60

    Mano H . Non-solid oncogenes in solid tumors: EML4-ALK fusion genes in lung cancer. Cancer Sci 2008; 99: 2349–2355.

  61. 61

    Takeuchi K, Choi YL, Togashi Y, Soda M, Hatano S, Inamura K et al. KIF5B-ALK, a novel fusion oncokinase identified by an immunohistochemistry-based diagnostic system for ALK-positive lung cancer. Clin Cancer Res 2009; 15: 3143–3149.

  62. 62

    Soda M, Takada S, Takeuchi K, Choi YL, Enomoto M, Ueno T et al. A mouse model for EML4-ALK-positive lung cancer. Proc Natl Acad Sci USA 2008; 105: 19893–19897.

  63. 63

    Kwak EL, Bang YJ, Camidge DR, Shaw AT, Solomon B, Maki RG et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010; 363: 1693–1703.

  64. 64

    Shaw AT, Yeap BY, Solomon BJ, Riely GJ, Gainor J, Engelman JA et al. Effect of crizotinib on overall survival in patients with advanced non-small-cell lung cancer harbouring ALK gene rearrangement: a retrospective analysis. Lancet Oncol 2011; 12: 1004–1012.

  65. 65

    Choi YL, Soda M, Yamashita Y, Ueno T, Takashima J, Nakajima T et al. ALK Lung Cancer Study Group EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors. N Engl J Med 2010; 363: 1734–1739.

  66. 66

    Sasaki T, Okuda K, Zheng W, Butrynski J, Capelletti M, Wang L et al. The neuroblastoma associated F1174L ALK mutation causes resistance to an ALK kinase inhibitor in ALK translocated cancers. Cancer Res 2010; 70: 10038–10043.

  67. 67

    Choi YL, Takeuchi K, Soda M, Inamura K, Togashi Y, Hatano S et al. Identification of novel isoforms of the EML4-ALK transforming gene in non-small cell lung cancer. Cancer Res 2008; 68: 4971–4976.

  68. 68

    Zhang X, Zhang S, Yang X, Yang J, Zhou Q, Yin L et al. Fusion of EML4 and ALK is associated with development of lung adenocarcinomas lacking EGFR and KRAS mutations and is correlated with ALK expression. Mol Cancer 2010; 9: 188.

  69. 69

    Goldstein NS, Armin M . Epidermal growth factor receptor immunohistochemical reactivity in patients with American Joint Committee on Cancer Stage IV colon adenocarcinoma: implications for a standardized scoring system. Cancer 2001; 92: 1331–1346.

  70. 70

    Graham J, Muhsin M, Kirkpatrick P . Cetuximab. Nat Rev Drug Discov 2004; 3: 549–550.

  71. 71

    Cunningham D, Humblet Y, Siena S, Khayat D, Bleiberg H, Santoro A et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. N Engl J Med 2004; 351: 337–345.

  72. 72

    Saltz L, Easley C, Kirkpatrick P . Panitumumab. Nat Rev Drug Discov 2006; 5: 987–988.

  73. 73

    Mendelsohn J, Baselga J . Status of epidermal growth factor receptor anatgonists in the biology and treatment of cancer. J Clin Oncol 2003; 21: 2787–2799.

  74. 74

    Camp ER, Summy J, Bauer TW, Liu W, Gallick GE, Ellis LM . Molecular mechanisms of resistance to therapies targeting the epidermal growth factor receptor. Clin Cancer Res 2005; 11: 397–405.

  75. 75

    Moroni M, Veronese S, Benvenuti S, Marrapese G, Sartore-Bianchi A, Di Nicolantonio F et al. Gene copy number for epidermal growth factor receptor (EGFR) and clinical response to antiEGFR treatment in colorectal cancer: a cohort study. Lancet Oncol 2005; 6: 279–286.

  76. 76

    Amado RG, Wolf M, Peeters M, Van Cutsem E, Siena S, Freeman DJ et al. Wild-type KRAS is required for panitumumab efficacy in patients with metastatic colorectal cancer. J Clin Oncol 2008; 26: 1626–1634.

  77. 77

    Lièvre A, Bachet JB, Boige V, Cayre A, Le Corre D, Buc E et al. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab. J Clin Oncol 2008; 26: 374–379.

  78. 78

    Malumbres M, Barbacid M . RAS oncogenes: the first 30 years. Nat Rev Cancer 2003; 3: 459–465.

  79. 79

    Martinez-Garza SG, Núñez-Salazar A, Calderon-Garcidueñas AL, Bosques-Padilla FJ, Niderhauser-García A, Barrera-Saldaña HA . Frequency and clinicopathology associations of K-ras mutations in colorectal cancer in a Northeast Mexican population. Digest Dis 1999; 17: 225–229.

  80. 80

    Urosević N, Krtolica K, Skaro-Milić A, Knezević-Usaj S, Dujić A . Prevalence of G-to-T transversions among K-ras oncogene mutations in human colorectal tumors in Yugoslavia. Int J Cancer 1993; 54: 249–254.

  81. 81

    Palmirotta R, Savonarola A, Formica V, Ludovici G, Del Monte G, Roselli M et al. A novel K-ras mutation in colorectal cancer. A case report and literature review. Anticancer Res 2009; 29: 3369–3374.

  82. 82

    Palmirotta R, Savonarola A, Ludovici G, Marchis ML, Covello R, Ettorre GM et al. Concurrent mutation in exons 1 and 2 of K-ras oncogene in colorectal cancer. Folia Histochemica Et Cytobiologica 2011; 49: 729–733.

  83. 83

    Edkins S, O’Meara S, Parker A, Stevens C, Reis M, Jones S et al. Recurrent KRAS codon 146 mutations in human colorectal cancer. Cancer Biol Ther 2006; 5: 928–932.

  84. 84

    Normanno N, Tejpar S, Morgillo F, De Luca A, Van Cutsem E, Ciardiello F . Implications for KRAS status and EGFR-targeted therapies in metastatic CRC. Nat Rev Clin Oncol 2009; 6: 519–527.

  85. 85

    Andreyev HJ, Norman AR, Cunningham D, Oates J, Dix BR, Iacopetta BJ et al. Kirsten ras mutations in patients with colorectal cancer: the ‘RASCAL II’ study. Br J Cancer 2001; 85: 692–696.

  86. 86

    Neumann J, Zeindl-Eberhart E, Kirchner T, Jung A . Frequency and type of KRAS mutations in routine diagnostic analysis of metastatic colorectal cancer. Pathol Res Pract 2009; 205: 858–862.

  87. 87

    Forbes S, Clements J, Dawson E, Bamford S, Webb T, Dogan A et al. Cosmic 2005. Br J Cancer 2006; 94: 318–322.

  88. 88

    Van Cutsem E, Köhne CH, Láng I, Folprecht G, Nowacki MP, Cascinu S et al. Cetuximab plus irinotecan, fluorouracil, and leucovorin as first-line treatment for metastatic colorectal cancer: updated analysis of overall survival according to tumor KRAS and BRAF mutation status. J Clin Oncol 2011; 29: 2011–2019.

  89. 89

    Bokemeyer C, Bondarenko I, Hartmann JT, de Braud F, Schuch G, Zubel A et al. Efficacy according to biomarker status of cetuximab plus FOLFOX-4 as first-line treatment for metastatic colorectal cancer: the OPUS study. Ann Oncol 2011; 22: 1535–1546.

  90. 90

    Douillard JY, Siena S, Cassidy J, Tabernero J, Burkes R, Barugel M et al. Randomized, phase III trial of panitumumab with infusional fluorouracil, leucovorin, and oxaliplatin (FOLFOX4) versus FOLFOX4 alone as first-line treatment in patients with previously untreated metastatic colorectal cancer: the PRIME study. J Clin Oncol 2010; 28: 4697–4705.

  91. 91

    Di Nicolantonio F, Martini M, Molinari F, Sartore-Bianchi A, Arena S, Saletti P et al. Wild-type BRAF is required for response to panitumumab or cetuximab in metastatic colorectal cancer. J Clin Oncol 2008; 26: 5705–5712.

  92. 92

    Sartore-Bianchi A, Martini M, Molinari F, Veronese S, Nichelatti M, Artale S et al. PIK3CA mutations in colorectal cancer are associated with clinical resistance to EGFR-targeted monoclonal antibodies. Cancer Res 2009; 69: 1851–1857.

  93. 93

    Loupakis F, Pollina L, Stasi I, Ruzzo A, Scartozzi M, Santini D et al. PTEN expression and KRAS mutations on primary tumors and metastases in the prediction of benefit from cetuximab plus irinotecan for patients with metastatic colorectal cancer. J Clin Oncol 2009; 27: 2622–2629.

  94. 94

    Jacobs B, De Roock W, Piessevaux H, Van Oirbeek R, Biesmans B, De Schutter J et al. Amphiregulin and epiregulin mRNA expression in primary tumors predicts outcome in metastatic colorectal cancer treated with cetuximab. J Clin Oncol 2009; 27: 5068–5074.

  95. 95

    Rubin BP, Heinrich MC, Corless CL . Gastrointestinal stromal tumor. Lancet 2007; 369: 1731–1741.

  96. 96

    Miettinen M, Lasota J . Gastrointestinal stromal tumors: definition, clinical, histological, immunohistochemical, and molecular genetic features and differential diagnosis. Virchows Arch 2001; 438: 1–12.

  97. 97

    Rubin BP, Singer S, Tsao C, Duensing A, Lux ML, Ruiz R et al. KIT activation is a ubiquitous feature of gastrointestinal stromal tumors. Cancer Res 2001; 61: 8118–8121.

  98. 98

    Heinrich MC, Corless CL, Duensing A, McGreevey L, Chen CJ, Joseph N et al. PDGFRA activating mutations in gastrointestinal stromal tumors. Science 2003; 299: 708–710.

  99. 99

    Antonescu C, Sommer G, Sarran L, Tschernyavsky S, Riedel E, Woodruff J et al. Association of KIT exon 9 mutations with nongastric primary site and aggressive behavior: KIT mutation analysis and clinical correlates of 120 gastrointes- tinal stromal tumors. Clin Cancer Res 2003; 9: 3329–3337.

  100. 100

    Kinoshita K, Isozaki K, Hirota S, Nishida T, Chen H, Nakahara M et al. c-Kit gene mutation at exon 17 or 13 is very rare in sporadic gastrointestinal stromal tumors. J Gastroenterol Hepatol 2003; 18: 147.

  101. 101

    Gafter-Gvili A, Leader A, Gurion R, Vidal L, Ram R, Shacham-Abulafia A et al. High-dose Imatinib for newly diagnosed chronic phase chronic myeloid leukemia patients-systematic review and meta-analysis. Am J Hematol 2011; 86: 657–662.

  102. 102

    Joensuu H, Roberts PJ, Sarlomo-Rikala M, Andersson LC, Tervahartiala P, Tuveson D et al. Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N Engl J Med 2001; 344: 1052–1056.

  103. 103

    Dematteo RP, Ballman KV, Antonescu CR, Maki RG, Pisters PW, Demetri GD et al. Adjuvant imatinib mesylate after resection of localised, primary gastrointestinal stromal tumor: a randomised, double-blind, placebo-controlled trial. Lancet 2009; 373: 1097–1104.

  104. 104

    Eisenberg BL, Trent JC . Adjuvant and neoadjuvant imatinib therapy: current role in the management of GIST. Int J Cancer 2011; 129: 2533–2542.

  105. 105

    Debiec-Rychter M, Sciot R, Le Cesne A, Schlemmer M, Hohenberger P, van Oosterom AT et al. KIT mutations and dose selection for imatinib in patients with advanced gastrointestinal stromal tumors. Eur J Cancer 2006; 42: 1093–1103.

  106. 106

    Gastrointestinal Stromal Tumor Meta-Analysis Group (Meta-GIST). Comparison of two doses of imatinib for the treatment of unresectable or metastatic gastrointestinal stromal tumors: a meta-analysis of 1,640 patients. J Clin Oncol 2010; 28: 1247–1253.

  107. 107

    Demetri GD, van Oosterom AT, Garrett CR, Blackstein ME, Shah MH, Verweij J et al. Efficacy and safety of sunitinib in patients with advanced gastrointestinal stromal tumour after failure of imatinib: a randomised controlled trial. Lancet 2006; 368: 1329–1338.

  108. 108

    Corless C, Schroeder A, Griffith D, Town A, McGreevey L, Harrell P et al. PDGFRA mutations in gastrointestinal stromal tumors: frequency, spectrum and in vitro sensitivity to imatinib. J Clin Oncol 2005; 23: 5357–5364.

  109. 109

    Wang WL, Conley A, Reynoso D, Nolden L, Lazar AJ, George S et al. Mechanisms of resistance to imatinib and sunitinib in gastrointestinal stromal tumor. Cancer Chemother Pharmacol 2011; 67 (1s): S15–S24.

  110. 110

    Martín-Broto J, Rubio L, Alemany R, López-Guerrero JA . Clinical implications of KIT and PDGFRA genotyping in GIST. Clin Transl Oncol 2010; 12: 670–676.

  111. 111

    Mouawad R, Sebert M, Michels J, Bloch J, Spano JP, Khayat D . Treatment for metastatic malignant melanoma: old drugs and new strategies. Crit Rev Oncol/Hematol 2010; 74: 27–39.

  112. 112

    Kumar R, Angelini S, Snellman E, Hemminki K . BRAF mutations are common somatic events in melanocytic nevi. J Invest Dermatol 2004; 122: 342–348.

  113. 113

    Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S et al. Mutations of the BRAF gene in human cancer. Nature 2002; 417: 949–954.

  114. 114

    Dong J, Phelps RG, Qiao R, Yao S, Benard O, Ronai Z et al. BRAF oncogenic mutations correlate with progression rather than initiation of human melanoma. Cancer Res 2003; 63: 3883–3885.

  115. 115

    Patton EE, Widlund HR, Kutok JL, Kopani KR, Amatruda JF, Murphey RD et al. BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma. Curr Biol 2005; 15: 249–254.

  116. 116

    Dhomen N, Reis-Filho JS, da Rocha Dias S, Hayward R, Savage K, Delmas V et al. Oncogenic braf induces melanocyte senescence and melanoma in mice. Cancer Cell 2009; 15: 294–303.

  117. 117

    Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2004; 116: 855.

  118. 118

    Wellbrock C, Ogilvie L, Hedley D, Karasarides M, Martin J, Niculescu-Duvaz D et al. V599EBRAF is an oncogene in melanocytes. Cancer Res 2004; 64: 2338–2342.

  119. 119

    Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 2011; 364: 2507–2516.

  120. 120

    Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 2010; 363: 809–819.

  121. 121

    Kefford R, Arkenau H, Brown MP, Millward M, Infante JR, Long GV et al. Phase I/II study of GSK2118436, a selective inhibitor of oncogenic mutant BRAF kinase, in patients with metastatic melanoma and other solid tumors. J Clin Oncol 2010; 28, abstract 8503. Presented at 2010 ASCO Annual Meeting: Chicago, IL.

  122. 122

    Ribas A, Kim KB, Schucter LM, Gonzalez R, Pavlick AC, Weber JS et al. BRIM-2: an openlabel, multicenter phase II study of vemurafenib in previously treated patients with BRAF V600E mutation-positive metastatic melanoma. J Clin Oncol 2011; 29, abstract 8509. Presented at 2011 ASCO Annual Meeting: Chicago, IL.

  123. 123

    Joseph EW, Pratilas CA, Poulikakos PI, Tadi M, Wang W, Taylor BS et al. The RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF-selective manner. Proc Natl Acad Sci USA 2010; 107: 14903–14908.

  124. 124

    Johannessen CM, Boehm JS, Kim SY, Thomas SR, Wardwell L, Johnson LA et al. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 2010; 468: 968–972.

  125. 125

    Nazarian R, Shi H, Wang Q, Kong X, Koya RC, Lee H et al. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature 2010; 468: 973–977.

  126. 126

    Wagle N, Emery C, Berger MF, Davis MJ, Sawyer A, Pochanard P et al. Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. J Clin Oncol 2011; 29: 3085–3096.

  127. 127

    Reya T, Morrison SJ, Clarke MF, Weissman IL . Stem cells, cancer, and cancer stem cells. Nature 2001; 414: 105–111.

  128. 128

    Sánchez-García I, Vicente-Dueñas C, Cobaleda C . The theoretical basis of cancer-stem-cell-based therapeutics of cancer: can it be put into practice? Bioessays 2007; 29: 1269–1280.

  129. 129

    Crea F, Duhagon MA, Farrar WL, Danesi R . Pharmacogenomics and cancer stem cells: a changing landscape? Trends Pharmacol Sci 2011; 32: 487–494.

  130. 130

    Tzvetkov M, von Ahsen N . Pharmacogenetic screening for drug therapy: from single gene markers to decision making in the next generation sequencing era. Pathology 2012; 44: 166–180.

  131. 131

    Reis-Filho JS . Next-generation sequencing. Breast Cancer Res 2009; 11 (Suppl 3): S12.

  132. 132

    Wagle N, Berger MF, Davis MJ, Blumenstiel B, DeFelice M, Pochanard P et al. High-throughput detection of actionable genomic alterations in clinical tumor samples by targeted, massively parallel sequencing. Cancer Discovery 2011; 2: 82–93.

  133. 133

    Holbrook JD, Parker JS, Gallagher KT, Halsey WS, Hughes AM, Weigman VJ et al. Deep sequencing of gastric carcinoma reveals somatic mutations relevant to personalized medicine. J Transl Med 2011; 25: 119.

  134. 134

    Schweiger MR, Kerick M, Timmermann B, Isau M . The power of NGS technologies to delineate the genome organization in cancer: from mutations to structural variations and epigenetic alterations. Cancer Metastasis Rev 2011; 30: 199–210.

  135. 135

    Borràs E, Jurado I, Hernan I, Gamundi MJ, Dias M, Martí I et al. Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing. BMC Cancer 2011; 24: 406.

  136. 136

    Inukai M, Toyooka S, Ito S, Asano H, Ichihara S, Soh J et al. Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer. Cancer Res 2006; 66: 7854–7858.

  137. 137

    Heinrich MC, Maki RG, Corless CL, Antonescu CR, Harlow A, Griffith D et al. Primary and secondary kinase genotypes correlate with the biological and clinical activity of sunitinib in imatinib-resistant gastrointestinal stromal tumor. J Clin Oncol 2008; 26: 5352–5359.

  138. 138

    Wagle N, Emery C, Berger MF, Davis MJ, Sawyer A, Pochanard P et al. Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. J Clin Oncol 2011; 29: 3085–3096.

  139. 139

    Timmermann B, Kerick M, Roehr C, Fischer A, Isau M, Boerno ST et al. Somatic mutation profiles of MSI and MSS colorectal cancer identified by whole exome next generation sequencing and bioinformatics analysis. PLoS One 2010; 5: e15661.

Download references


We thank Dr Sabino Ciavarella for critically revising the manuscript, Dr Giorgia Ludovici for excellent technical assistance and A.R.B.O. Financial support for this work was provided by a research grant from the Italian Association of Cancer Research (AIRC IG11647) to F.S. and grant MERIT RBNE08NKH7 to F.G.

Author information

Correspondence to A Savonarola.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Savonarola, A., Palmirotta, R., Guadagni, F. et al. Pharmacogenetics and pharmacogenomics: role of mutational analysis in anti-cancer targeted therapy. Pharmacogenomics J 12, 277–286 (2012) doi:10.1038/tpj.2012.28

Download citation


  • mutational analysis
  • colon cancer
  • GIST
  • melanoma

Further reading