Somatic Pharmacogenomics in Cancer

Somatic pharmacogenomics in cancer

ON Ikediobi Many of the initial examples of the clinical utility of pharmacogenetics were elucidated in the field of oncology. Those examples were largely based on Department of Clinical Pharmacy, School of the existence of germline genetic variation that influences the metabolism of Pharmacy, University of California, San Francisco, cytotoxic drugs. However, with the development of kinase inhibitors, drugs CA, USA designed to preferentially target altered proteins driving oncogenesis, pharmacogenetics in cancer has shifted to understanding the somatic Correspondence: Dr ON Ikediobi, Department of Clinical differences that determine response to these targeted agents. It is becoming Pharmacy, School of Pharmacy, University of increasingly clear that understanding the molecular genetics of cancer will California, 3333 California Street, Box 0613, lead to the further development of targeted therapeutics. Therefore, it is San Francisco, CA 94118, USA. imperative that pharmacogenomics researchers understand the motivations E-mail: [email protected] and challenges of developing targeted therapies to treat cancer as a paradigm for personalized medicine. However, much of the discussion in the pharmacogenomics community in cancer is still largely focused on the germline variants as predictors of drug toxicity. In light of that fact, this review presents a detailed discussion of the development of commonly used targeted therapies for the treatment of hematological and solid tumors, the somatic mutations that determine response to those therapies, and the mechanisms of drug resistance. The Pharmacogenomics Journal (2008) 8, 305–314; doi:10.1038/tpj.2008.8; published online 5 August 2008

Keywords: somatic; mutation; cancer; pharmacogenomics; kinase; inhibitor

Introduction

Pharmacogenomics is the study of the genetics of interindividual response to drugs and aims at molecular subsetting of patients for more effective therapy.1 The field of pharmacogenomics is especially important in oncology where most clinically used drugs have a narrow therapeutic window, that is the difference between the dose required to achieve the desired therapeutic effect and that causing toxicity, is small.2 Therefore, knowledge of genetic variations, inherited or acquired, that may predict differential response to cancer chemotherapy is key to personalized therapy. Genetic variations in drug effect are classified into two groups: those due to either pharmacokinetic or pharmacodynamic factors. The pharmacokinetic factors that influence drug effect involve the drug’s absorption, distribution, metabolism and excretion.3 In contrast, the pharmacodynamic factors that influence drug effect involve the transport of the drug into the cell and the interaction of the drug (ligand) and its target(s).3 Many of the initial examples from pharmacogenomics in cancer chemotherapy center on understanding the inherited (germline) interindividual differences involved in drug metabolism (Table 1).2,4–7 However, there are also examples where acquired (somatic) mutations within the tumor DNA are Received 6 March 2008; revised 17 June 2008; accepted 3 July 2008; published online predictive of response to cancer chemotherapy. The elucidation of the signal- 5 August 2008 transduction networks that drive neoplastic transformation has led to rationally Somatic pharmacogenomics ON Ikediobi 306

Table 1 Standard chemotherapeutics and germline variants reported to modulate their action

Drug Tumor type Allelic polymorphism Drug action (references)

6-Mercaptopurine ALL TPMT*3A Enhanced toxicity 2,4 Irinotecan Colorectal UGT1A1*28 Enhanced toxicity 5 Tamoxifen Breast CYP2D6*4, *10 Decreased efficacy 6 5-Fluorouracil Colorectal DPYD*2A Enhanced toxicity 7

Abbreviations: ALL, acute lymphoblastic leukemia; CYP2D6, cytochrome P450 2D6; DPYD, dihydropyrimidine dehydrogenase; TPMT, thiopurine methyltransferase; UGT1A1, uridine 50-diphosphate-glucuronosyl-transferase. Numbers in brackets indicate cited references.

Table 2 Kinase inhibitors and somatic mutations reported to modulate their drug action

Kinase inhibitor Tumor type Gene product Somatic alterations

Imatinib CML, ALL, GIST BCR-ABL, KIT, PDGFRA Mutation, deletion, translocation Nilotinib CML BCR-ABL Mutation Dasatinib CML, ALL BCR-ABL Mutation Sunitinib GIST KIT, PDGFRA, FLT3, VEGFR2 Mutation Trastuzumab Breast ERBB2 Amplification, overexpression Pertuzumab Breast ERBB2, EGFR, ERBB3 PTEN deletion, Akt overexpression Lapatinib Breast ERBB2, EGFR ERBB2 amplification Gefitinib NSCLC EGFR Mutation, amplification Erlotinib NSCLC EGFR Mutation, amplification

Abbreviations: Akt, serine/threonine protein kinase Akt; ALL, acute lymphoblastic leukemia; BCR-ABL, breakpoint cluster region-ABL; CML, chronic myeloid leukemia; ERBB2, v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma-derived oncogene homolog (avian); ERBB3, v-erb-b2 erythroblastic leukemia viral oncogene homolog 3; FLT3, fms-related tyrosine kinase 3; GIST, gastrointestinal stromal tumor; KIT, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homologue; NSCLC, non-small cell lung cancer; PDGFRA, platelet-derived growth factor receptor, a-polypeptide; PTEN, phosphatase and tensin homolog; VEGFR2, vascular endothelial growth factor receptor 2.

designed cancer therapeutics that target specific molecular within the tumor DNA but absent within the germline. In abnormalities.8 These targeted therapeutics, unlike tradi- this review, I have highlighted the most common examples tional cytotoxic cancer chemotherapeutics, do not have whereby somatic mutations within tumor DNA determine narrow therapeutic indices and therefore have less severe clinical response to kinase inhibitors (Table 2). side effects. The less severe side effects experienced by taking targeted therapies is as a result of their greater affinity for inhibiting the activity of somatic alterations of the target BCR-ABL, KIT, PDGFR kinase inhibitors kinase(s) that are present within the tumor DNA but absent within the germline. In addition, targeted therapeutics, The first example of the success of small-molecule targeted unlike traditional cytotoxic chemotherapy are available in therapies in treatment of cancer is demonstrated with oral dosage forms for better ease of administration. imatinib, the first small-molecule kinase inhibitor approved Many of the currently known targeted therapeutic agents for clinical use that targets the breakpoint cluster region are protein kinase inhibitors. Kinases are key components of (BCR)-ABL protein tyrosine kinase for the treatment of cell signaling pathways involved in neoplastic transforma- chronic myeloid leukemia (CML). The BCR-ABL protein tion. Genes that encode protein kinases are often over- results from the fusion of the BCR and nonreceptor protein expressed or activated as a result of mutations or tyrosine kinase ABL resulting from a reciprocal chromoso- chromosomal rearrangements in human tumors. Kinase mal translocation t(9;22) producing a shortened chromo- inhibitors are designed as adenosine triphosphate (ATP) some 22, called the Philadelphia (Ph) chromosome. The mimetics and therefore compete with ATP for binding at the resultant fusion protein has constitutive tyrosine kinase active site.8 When bound to the active site of the putative activity enabling the activation of several signaling path- kinase(s), kinase inhibitors reduce the activity of the ways such as Ras, PI3K-Akt and Jak-STAT leading to cell activated protein kinase(s), reducing the cellular oncogenic proliferation and survival.9 The BCR-ABL protein is asso- drive and inducing tumor regression. Interestingly, only a ciated predominantly not only with CML but also with subset of patients respond to these targeted therapies and acute lymphoblastic leukemia.9 Imatinib works by binding their response is governed by the presence of specific to and stabilizing an inactive conformation of the BCR-ABL somatic alterations of the target kinase that are present protein kinase.10,11 Because imatinib also has specificity for

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the platelet-derived growth factor receptor, a-polypeptide malignancies harboring mutant forms of those proteins.24 (PDGFRA) and KIT protein kinases, it is also used in Another second generation ABL kinase inhibitor that treatment of malignancies associated with dysregulated demonstrates activity against imatinib resistance is dasati- forms of those proteins.11 nib. Dasatinib, although not specifically designed to target Although the initial response to imatinib is dramatic, imatinib-resistant forms of BCR-ABL, is a potent inhibitor of treatment failure quickly ensues. As is the case with the BCR-ABL, Src-family kinases, KIT and PDGFR.25,26 In con- treatment of CML patients with imatinib, patients initially trast to imatinib and nilotinib, dasatinib binds to the active experience complete cytogenetic and hematological remis- conformation of the ABL kinase.27 On the basis of promising sion. However because imatinib fails to deplete leukemic data from phase I and II trials of dasatinib, in patients with stem cells that harbor the BCR-ABL fusion protein,12 some imatinib-resistant CML and Ph-positive ALL patients, it was patients develop resistance to imatinib, particularly in the recently approved in the United States and Europe for the advanced phases of CML and Ph-positive acute lympho- treatment of adults in all phases of CML with imatinib blastic leukemia (ALL).13 Mechanisms of imatinib resistance resistance or intolerance.28 Dasatinib was also approved for include BCR-ABL amplification and overexpression of the treatment of patients with Ph-positive ALL with mRNA and protein.14,15 However, the most common imatinib resistance or intolerance.29 mechanism of resistance is the acquisition of somatic point Because imatinib also has specificity for KIT and PDGFRA mutations within the kinase domain of the ABL gene.16 The proteins, it is used to treat gastrointestinal tumors (GISTs) first point mutation identified as associated with imatinib that harbor mutant forms of those proteins. Most GISTs resistance was T315I.16 The crystal structure of an analogue harbor oncogenic KIT or PDGFRA receptor tyrosine kinase of imatinib bound to the ABL kinase domain revealed that the mutations.30 The KIT or PDGFRA gain-of-function muta- T315 residue was crucial for the interaction between imatinib tions are early events in GIST oncogenesis and lead to the and ABL.17 It was also found that a different mutation at the activation of the mitogen-activated protein kinase (MAPK) T315 residue (T315V) conferred constitutive kinase activity to and signal transducer and activator of transcription (STAT) ABL and was less sensitive to imatinib compared with wild- downstream signaling pathways involved in cell proliferation type ABL.18 The T315I point mutation impairs imatinib and survival, respectively.31 Imatinib, a potent inhibitor of binding thereby reducing its inhibition of ABL. KIT signaling, has recently become first-line treatment of There are more than 50 different point mutations in ABL metastatic GIST following in vitro studies suggesting a that are associated with imatinib resistance.13 However, therapeutic potential for imatinib in a human GIST cell line.32 most are rare and six amino-acid residues (Gly250, Tyr253, Before treatment with imatinib, surgical resection of primary Glu255, Thr315, Met351 and Phe359) thus far account for localized GIST was the only chance for cure.33 GISTs proved 60–70% of imatinib-resistant mutations.13 Two of the more refractory to standard chemotherapy and radiation. Recur- frequently detected ABL mutants, Y253F and E255K, have rence of disease was certain with a predicted 5-year survival of been shown to have high in vitro transforming potential.13 30%.34 Prospective trials of imatinib in metastatic GISTs have This in vitro finding is consistent with clinical findings that shown that approximately 80% of patients will respond to show P-loop mutations such as Y253F and E255K are imatinib and will achieve stable disease.35,36 In addition, 70% associated with a greater likelihood of progression to blast of metastatic GIST patients will have at least a 2-year disease- crisis and shorter overall survival in imatinib-treated free survival and 50% will be free of disease progression.37 patients.19 Another ABL mutant, T315I, is generally found As is the case with CML and imatinib response, specific in patients with advanced CML, and predicts a worse overall mutations of KIT and PDGFRA in GISTs predict response to survival compared with other ABL mutations in patients on imatinib. KIT mutations occur in up to 90% of GISTs and imatinib therapy.20 clinical response to imatinib is dependent, at least in part, Those drug-resistant BCR-ABL point mutations can be on the presence of specific KIT mutations.38 KIT exon 11 found in imatinib-naı¨ve CML or can be acquired during mutations are found in 75% of GISTs and result in the imatinib treatment. Acquired imatinib resistance involves abrogation of the juxtamembrane autoinhibition of the KIT the reemergence of BCR-ABL tyrosine kinase activity.21 This kinase.39 Patients with exon 11 KIT mutations have a higher suggests that the mutant BCR-ABL protein is still a viable response rate to imatinib treatment and longer time to target for inhibition by small-molecule inhibitors.21,22 To treatment failure compared with other KIT or PDGFRA this end, second generation ABL kinase inhibitors have been mutations.40 Patients without detectable KIT or PDGFRA designed and have been shown to inhibit the growth of mutation respond less frequently to imatinib treatment most mutant forms of BCR-ABL resistant to imatinib. One of compared with exon 11 KIT mutants.38 However, up to 39% those drugs, nilotinib, was specifically designed to target of those patients without KIT or PDGFRA mutations do imatinib-resistant forms of BCR-ABL, and is approximately respond to imatinib.41 As well, patients with the rare exon 30-fold more potent than imatinib as an ABL inhibitor.13 13 KIT mutation or exon 17 PDGFRA mutation may respond Phase I and II trials of nilotinib in patients with imatinib- to imatinib.40 These data suggest that imatinib treatment resistant CML in all phases of disease and patients with Ph- ought to be considered for all GIST patients, regardless of positive ALL demonstrate significant clinical response to KIT or PDGFRA mutation status.40 The only exception may nilotinib.23 Nilotinib also inhibits KIT and PDGFRB protein be patients with primary imatinib-resistant mutation of kinases and may therefore be used for the treatment of PDGFRA (D842V).40

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The majority of metastatic GIST patients will develop acquired) have not been fully elucidated, however there are resistance to imatinib. The most common resistance numerous proposed mechanisms. Elucidating the molecular mechanisms involve the acquisition of secondary exon 13, mechanisms underlying primary or acquired trastuzumab 14 or 17 KIT mutations that prohibit imatinib binding.40 resistance is critical to improving the survival of metastatic Some of these secondary mutations are intrinsically breast cancer patients whose tumors overexpress ERBB2.55 imatinib-resistant, such as the frequently occurring V654A Altered downstream signaling from ERBB2 has been substitution.42 However, other mutations such as those shown to confer primary resistance to trastuzumab. ERBB2 involving the N822 residue are intrinsically imatinib signaling activates the PI3K signaling pathway. Constitutive sensitive but are associated with clinical imatinib resistance PI3K/AKT activity has been shown to inhibit trastuzumab- when coincident with an exon 11 KIT mutation.42 mediated cell-cycle arrest and apoptosis.56 An ERBB2 over- It is therefore crucial to develop more potent inhibitors of expressing breast cancer cell line, BT474, resistant to KIT and PDGFRA to potentially overcome imatinib resis- trastuzumab had elevated levels of phosphorylated AKT tance. Sunitinib, an inhibitor of KIT, PDGFRA, fms-related compared to the parent line.57 The resistant cells were tyrosine kinase 3 (FLT3) and vascular endothelial growth sensitive to a small-molecule inhibitor of PI3K.57 Patients factor receptor 2 (VEGFR2), has recently been approved for with phosphatase and tensin homolog (PTEN)-deficient, the treatment of imatinib-resistant GIST and patients unable ERBB2 overexpressing breast tumors have a poorer response to tolerate treatment with imatinib.43 In a randomized to trastuzumab-based therapy.58 Subsequently, it was shown phase III trial of sunitinib in patients who had progressed on that in phosphatase and tensin homolog (PTEN)-deficient imatinib therapy, sunitinib was found to prolong median cells, PI3K inhibitors rescued trastuzumab resistance in vitro time to tumor progression compared with placebo.44 and in vivo.58 Therefore, PTEN loss may serve as a predictor of However, sunitinib provides only a temporary benefit for trastuzumab resistance and PI3K inhibitors may be potential imatinib-resistant GIST patients, therefore more therapeutic therapies in PTEN-null trastuzumab-resistant tumors.49 options are needed. Preclinical studies in GIST cell lines have Novel therapeutic strategies are being employed to over- shown that treatment with heat-shock protein 90 inhibitors come resistance to trastuzumab. Pertuzumab is a mono- resulted in degradation of the KIT oncoprotein and may clonal ERBB2 antibody that represents a new class of drugs therefore be of benefit in imatinib-resistant GIST.45 called dimerization inhibitors.49 Pertuzumab can block signaling by other EGFR family receptors, as well as inhibit signaling in cells expressing normal ERBB2 levels. Pertuzu- ERBB2 kinase inhibitors mab sterically blocks dimerization of ERBB2 with EGFR and ERBB3, inhibiting signaling from ERBB2/EGFR and ERBB2/ Another example of the success of targeted therapies in the ERBB3 heterodimers.59 Pertuzumab is also able to disrupt treatment of cancer is that of trastuzumab, a monoclonal the interaction between ERBB2 and insulin-like growth antibody against v-erb-b2 erythroblastic leukemia viral factor-I receptor in trastuzumab-resistant cells.60 Trastuzu- oncogene homolog 2 (ERBB2) that was developed to treat mab and pertuzumab bind to different epitopes in the breast cancer patients whose tumors overexpress the extracellular domain of ERBB2.61,62 The combination of protein. ERBB2 protein tyrosine kinase amplification and trastuzumab and pertuzumab produced synergistic apopto- overexpression occurs in approximately 30% of metastatic sis in ERBB2 overexpressing trastuzumab-naı¨ve breast cancer breast cancers and leads to aberrant signaling through the cells,63 without any significant effect on the viability of PI3K and MAPK pathways, resulting in cell proliferation and trastuzumab-resistant breast cancer cells.60 evasion of apoptosis.46 Amplified ERBB2 in breast cancer Another alternative therapeutic agent against trastuzu- shows clinical correlates with earlier relapse and shorter mab-resistant tumors is lapatinib, a dual tyrosine kinase overall survival.47 Therefore, a drug, trastuzumab that could inhibitor that targets both EGFR and ERBB2. Binding of target ERBB2 protein in those cancers could increase lapatinib to EGFR and ERBB2 is reversible but its dissociation survival. Trastuzumab was the first genetic-based drug is much slower allowing for prolonged downregulation of approved for cancer therapy.48 Unlike the small-molecule receptor tyrosine phosphorylation.49 ERBB2 status and not kinase inhibitors, which are administered orally, trastuzu- EGFR status is a determinant of lapatinib activity.49 It has mab, a monoclonal antibody, is administered intravenously. been shown that the combination of lapatinib with Trastuzumab is active as a single agent after failure of other trastuzumab enhanced apoptosis of ERBB2 overexpressing chemotherapy regimens.49 However, primary resistance to breast cancer cells.64 Resistance to lapatinib seems to be trastuzumab monotherapy occurs in approximately 66–88% mediated by increased signaling from the estrogen receptor of ERBB2 overexpressing metastatic breast tumors.50,51 in estrogen receptor-positive ERBB2 overexpressing breast Trastuzumab with adjuvant chemotherapy (paclitaxel or cancers.65 This suggests that targeting of both the estrogen docetaxel) significantly improves disease-free and overall receptor and ERBB2 may be beneficial in that subset of survival in patients with early stage ERBB2 overexpressing cancer patients.65 A recent phase III trial of trastuzumab- breast cancers compared with trastuzumab monotherapy.52–54 resistant ERBB2 overexpressing breast cancer patients Invariably, the majority of patients who achieve an initial demonstrated that the combination of lapatinib and response to trastuzumab-based regimens develop resistance capecitabine resulted in longer median progression-free within 1 year.49 The mechanisms of resistance (primary or survival compared with capecitabine alone.66

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ERBB2, a member of the EGFR family, is also over- cancers.88–93 Mutations outside of the kinase domain expressed, to a lesser degree, in lung cancers, specifically (extracellular domain) are rare in NSCLC,81–83 but are adenocarcinomas and large-cell carcinomas and is predictive common in gliomas94 and squamous cell lung cancers.95 of poorer outcomes.67–69 In addition, intragenic mutations Therefore, the use of EGFR inhibitors in subsets of those have also been found in the conserved kinase domain of the tumors may be a viable therapeutic option. ERBB2 gene in some lung cancers.70,71 The ERBB2 mutations In NSCLC, somatic mutations of EGFR are sometimes seem to occur exclusively in non-small cell lung cancer associated with amplification.96 Studies have shown that (NSCLC) of adenocarcinoma histology and are more patients with EGFR amplification were more likely to common in female patients and never smokers,71 similar respond to gefitinib or erlotinib and had longer median to the clinicophathologic manifestation of EGFR mutations. time to disease progression and overall survival compared to It was then thought that trastuzumab might be of clinical patients with normal EGFR copy number.97 However, it benefit for the treatment of ERBB2 mutant NSCLC. How- remains to be established whether amplification of EGFR ever, thus far, there has not been demonstrated the benefit (wildtype or mutant) contributes to lung cancer develop- of trastuzumab monotherapy or in combination with cancer ment and response to gefitinib or erlotinib.97 chemotherapeutics for the treatment of NSCLC with over- Thus far there are five mutations known to confer expressed ERBB2.72–75 sensitivity to EGFR tyrosine kinase inhibitors. The drug- sensitive mutations are: point mutations in exon 18 (G719A or G719C), point mutations of exon 21 (L858R and L861Q) EGFR kinase inhibitors and in-frame deletions of exon 19 that eliminates four amino acids (LREA).98 The most common of these drug- To further illustrate the importance of the EGFR family of sensitive mutations are the exon 19 in-frame deletion and proteins in cancer progression and treatment, I discuss the exon 21 missense amino-acid substitution (L858R) account- timely development of EGFR inhibitors and its subsequent ing for up to 90% of EGFR mutations in NSCLC.98 In clinical use. EGFR protein tyrosine kinase overexpression retrospective studies, the association between the presence has been implicated in numerous cancer types, is associated of EGFR mutation and sensitivity to gefitinib and erlotinib is with poor prognosis, and leads to enhanced signaling quite consistent showing 75% response rate for patients through antiapoptotic proteins, Akt and STAT.76–78 The fact with EGFR mutations compared to 10% response rate for that EGFR is overexpressed in many cancer types and patients with wild-type EGFR.99–103 Prospective trials have associated with poor prognosis made it a good candidate confirmed the association between EGFR mutation and for the development of targeted therapeutics. Therefore, two sensitivity to gefitinib and erlotinib. Those studies showed small-molecule inhibitors of EGFR, gefitinib and erlotinib, that 78% of patients with somatic exon 19 deletion or exon received speedy approval from the US Food and Drug 21 L858R mutation had radiographic responses to gefitinib Administration (FDA) in May 2003 and November 2004, and erlotinib.104–106 respectively. Gefitinib and erlotinib were approved as single- However, there is controversy around what prolongs drug therapy for the treatment of patients with advanced survival of NSCLC patients; treatment with EGFR inhibitors chemotherapy-refractory NSCLC.79,80 It was later observed, or presence of EGFR mutation? Large phase III retrospective in the clinic, that only a subset of NSCLC patients achieved trials have been conducted where NSCLC patients were dramatic response to these drugs. By molecular genetic randomized to receive either standard cytotoxic chemother- analyses, it was found that specific somatic mutations in the apy alone or standard chemotherapy in combination with conserved kinase domain of the EGFR gene are associated gefitinib or erlotinib.99,107 Those studies have reported that with sensitivity to gefitinib81,82 and erlotinib.83 Recently, patients with EGFR mutations have prolonged survival however, a large clinical trial comparing gefitinib with compared with patients with wild-type EGFR treated with placebo in NSCLC patients who had failed other courses of gefitinib or erlotinib. Interestingly, the studies have also chemotherapy showed no survival benefit from taking found that the prolonged survival may occur in the absence gefitinib.84 Following this report in 2005 the FDA restricted of treatment with gefitinib, erlotinib, surgery or standard use of gefitinib only to patients who had previously cancer chemotherapy.97 In the standard cytotoxic che- responded to the drug.85 In contrast, clinical trials have motherapy alone treatment arm, patients with EGFR muta- demonstrated that erlotinib improves survival in NSCLC tion had prolonged progression-free and overall survival patients whose cancers have progressed on other courses of compared with patients with wild-type EGFR.99,107 chemotherapy.86 Therefore, erlotinib is FDA approved for Even within the group of NSCLC patients with EGFR use in new NSCLC patients with chemotherapy-refractive mutations, there are reported differences in the clinical tumors. course between patients with exon 19 in-frame deletions Somatic mutations of EGFR kinase domain are most and patients with exon 21 L858R missense substitution. One common in NSCLC and have a distinct clinicophathologic study has shown that NSCLC patients with the L858R manifestation common in adenocarcinomas, East Asians, mutation treated with surgery alone have a prolonged women and never smokers.87 Rare mutations of EGFR are overall survival compared with NSCLC patients with exon found in head and neck cancers, cholangiosarcomas, colon 19 deletions.87 Another study has shown that after treat- cancers, ovarian cancers, esophageal cancers and pancreatic ment with gefitinib or erlotinib, NSCLC patients with EGFR

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exon 19 deletions have a longer overall survival compared development of molecular diagnostics incorporating any with patients harboring the exon 21 L858R mutation.98 The one or combination of those biomarkers may be essential to molecular basis for such findings is yet unknown. guide the appropriate use of EGFR inhibitors. Therefore, it is no surprise that the mechanism of Adding to the complexity of predicting response to EFGR increased sensitivity of EGFR mutants to gefitinib and inhibitors, a novel drug-resistant gefitinib mutation has erlotinib is still unknown. However, it has been shown that recently been reported that does not involve mutation of the exon 19 in-frame deletions and exon 21 L858R missense EGFR. It was reported that gefitinib-resistant clones from an amino-acid substitution confer ligand-independent activa- EGFR-mutant lung cancer cell line displayed amplification tion and prolonged kinase activity after ligand stimula- of MET oncogene and maintained activation of ERBB3/PI3K/ tion,81,82 suggesting that inhibition of the aberrant EGFR AKT signaling in the presence of gefitinib.121 Following the signaling may be the predominant mechanism of action of initial observation in cell lines, a panel of 18 gefitinib or EGFR inhibitors. erlotinib-resistant primary lung tumors were assessed for As is the case with the aforementioned targeted therapies, MET amplification. MET amplification was found to occur in imatinib and trastuzumab, despite a dramatic initial re- 22% of those tumors implicating MET amplification in the sponse to gefitinib and erlotinib, NSCLC patients with EGFR development of resistance to gefitinib in a subset of lung mutations rarely achieve a complete response and resistance cancers.121 Therefore, developing targeted therapies against to treatment develops. There are thus far, three EGFR kinase amplified MET may be beneficial in the treatment of NSCLC domain mutations associated with drug resistance: an exon patients whose tumors bear the MET amplification and have 19 point mutation (D761Y), an exon 20 point mutation progressed on EGFR inhibitors. (T790M) and an exon 20 insertion (D770_N771insNPG).97 The most common of these drug-resistant mutations is the T790M reported to occur in about 50% of tumors after Discussion disease progression and has been predicted to block the binding of gefitinib or erlotinib to the kinase ATP binding It is clear from the above-mentioned examples, that somatic pocket.108,109 This mutation is analogous to the acquired mutations in cancer can be important in guiding the drug-resistant mutation, T315I, to imatinib seen in GIST and appropriate use of targeted therapies. As we continue to CML.97 Interestingly, the T790M mutation has been seen in understand the dysregulated pathways driving oncogenesis the germline and tumor DNA of family members with this will inevitably lead to the further development of hereditary bronchioloalveolar carcinoma.110 One of the targeted therapies. Elucidation of the myriad genomic family members with the T790M mutation did not respond abnormalities in cancer will be key to enhancing patient to treatment with gefitinib.110 In vitro data have suggested stratification for more accurate diagnosis and treatment. To that irreversible EGFR inhibitors may have activity in this end, a few groups are employing systematic high- patients with acquired resistance to gefitinib or erloti- throughput sequencing screens of cancer genomes in search nib.111,112 To this end, a phase II trial of HKI-272, an of other mutated kinases driving oncogenesis. This ap- irreversible EGFR kinase inhibitor, is ongoing to determine proach has been fruitful in identifying more frequently the efficacy in patients who have progressed after initial mutated oncogenes; BRAF in melanoma,122 ERBB2 in lung treatment with gefitinib or erlotinib.97 cancer123 and PIK3CA in colorectal cancer.124 This has In addition to mutations of EGFR other molecular generated interest and investigation into the development parameters involved in the EGFR signaling cascade are and clinical use of small-molecule inhibitors of those kinases associated with activity of gefitinib or erlotinib. For (BRAF and PIK3CA) or their signaling partners for the example, it has been shown that increased expression of treatment of cancers.125–128 transforming growth factor-a and heregulin, ligands for Despite the initial success of targeted therapies in produ- EGFR and ERBB3, respectively, is associated with poor cing dramatic responses and tumor regression in genetically response to gefitinib.113,114 Perhaps competitive inhibitors defined subgroups of patients, drug resistance inevitably of those ligands may be a useful therapeutic intervention. It develops. The next challenge facing drug development has also been reported that increased copy number of experts is the design of targeted therapies against those another member of the EGFR family of proteins, ERBB2, in drug-resistant tumors. One way to overcome or abate the presence of EGFR mutation in NSCLC, is associated with resistance to single-targeted therapy may be to administer response to gefitinib.115 Paradoxically, however, NSCLC multiple kinase inhibitors or multikinase inhibitors to patients with intragenic ERBB2 mutations do not respond patients whose tumors may have more than a single to gefitinib or erlotinib.116 That finding suggests there is a mutated kinase. This goal may be achieved the more we distinct genetic context of EGFR mutant NSCLC that learn about the unique somatic mutation patterns of predicates response to EGFR inhibitors. Whereas down- individual tumors and have a plethora of targeted therapies stream of EGFR signaling, it has been observed that NSCLC with which to tailor appropriate treatment regimens. patients with KRAS mutations are resistant to gefitinib or The symbiosis between molecular genetic-based disease erlotinib,117 and increased phosphorylation of AKT is classification and genetic-based therapeutic intervention predictive of response to gefitinib or erlotinib.118–120 In serves as a paradigm for the future of personalized medicine light of those observations, one can envisage that the in cancer. Ultimately, however, drug response, even with

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targeted therapies is not solely dependent on the tumor 20 Nicolini FE, Corm S, Le QH, Sorel N, Hayette S, Bories D et al. Mutation genetics. The germline genetics also plays a role, especially status and clinical outcome of 89 imatinib mesylate-resistant chronic in drug metabolism. Therefore, as we move forward with myelogenous leukemia patients: a retrospective analysis from the French intergroup of CML (Fi(phi)-LMC GROUP). Leukemia 2006; 20: implementing genetic-based tests for drug therapy in 1061–1066. oncology, it will be important to understand and consider 21 Barthe C, Gharbi MJ, Lagarde V, Chollet C, Cony-Makhoul P, Reiffers J the contribution of both somatic and germline genetics in et al. Mutation in the ATP-binding site of BCR-ABL in a patient with predicting drug response and toxicity. chronic myeloid leukaemia with increasing resistance to STI571. Br J Haematol 2002; 119: 109–111. 22 Branford S, Rudzki Z, Walsh S, Grigg A, Arthur C, Taylor K et al. 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