Pharmacogenomics || Pharmacogenomics of Chemotherapeutic Agents in Cancer Treatment

Abstract

This chapter describes how genetic differences among patients may change thera-peutic outcome in cancer chemotherapy. The therapeutic window of anticanceragents is narrow and, in most cases, patient are treated at dose levels that areclose to those maximally tolerated. Inter-patient genetic differences altering phar-macokinetics and/or pharmacodynamics might result in unpredictable outcome.Severe toxicity in genetically predisposed patients is predominantly associatedwith mutations in drug metabolism enzyme genes. Intolerance to chemotherapyis clearly demonstrated in subsets of patients receiving 6-mercaptopurine (inacti-vated by thiopurine methyltransferase, TPMT), 5-fluorouracil (inactivated by dihy-dropyrimidine dehydrogenase, DPD), irinotecan [the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) is inactivated by UDP-glucuronosyltransferase 1A1,UGT1A1], amonafide (metabolized by N-acetyltransferase 2, NAT2). Moreover,cancer patients carrying a mutation in the methylenetetrahydrofolate reductase(MTHFR) gene are highly susceptible to myelosuppressive effects of CMF regi-men (cyclophosphamide+methotrexate+5-fluorouracil). It is emerging that notonly toxicity, but also response to chemotherapy could be influenced by pharmaco-genetic determinants. As a matter of fact, recent studies highlighted the correla-tion between mutations in glutathione-S-transferase (GST) and thymidylate syn-thase (TS) genes and patients’ response to chemotherapy.

14.1Pharmacological Treatment of Cancer and Importance of Pharmacogenomics

Chemotherapy of cancer is part of a multimodal treatment including surgery andradiation therapy. In drug-sensitive tumors, the major obstacle to successful che-motherapy is the occurrence of resistance, and is related to the impossibility to ad-minister curative doses of drugs due to the occurrence of toxicity. Reduced inten-sity of treatment allows the emergence of cell clones with a resistant phenotype asa result of somatic mutations occurring in surviving cells. Maximizing tumorexposure while reducing the risk of intolerable toxicity is a mandatory task, in par-

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Pharmacogenomics of Chemotherapeutic Agentsin Cancer TreatmentFederico Innocenti, Lalitha Iyer and Mark J. Ratain

Pharmacogenomics: The Search for Individualized Therapies.Edited by J. Licinio and M.-L. Wong

Copyright © 2002 Wiley-VCH Verlag GmbH & Co. KGaAISBNs: 3-527-30380-4 (Paper); 3-527-60075-2 (Electronic)

ticular because chemotherapy is the only alternative available in cases of inoper-able and metastatic disease.

Cancer patients are treated at doses close to those maximally tolerated, makinganticancer agents a class of drugs with a very narrow therapeutic window, definedas the interval between the dose required to produce a therapeutic effect and thatresponsible for toxicity. Current modes to administer anticancer drugs do not takeinto account differences among individuals. Cancer patients receive fixed doses“normalized” by body surface area, a very imprecise approach of dose individual-ization. Existing differences between individuals in pharmacokinetics and pharma-codynamics imply that some patients might benefit from chemotherapy, butothers might experience adverse reactions without any therapeutic advantage. Theexplosion of genetic investigation of human molecular biology is increasingly de-monstrating that unpredictability in patient outcome could be due to genetic dif-ferences in the way drugs are handled and react with targets of action.

Multiple steps occur from drug administration to pharmacological effect on nor-mal and neoplastic tissues. Processes mediated by membrane transporters andmetabolizing enzymes are critical for achieving effective intracellular drug concen-trations. At intracellular level, killing action of cytotoxic drugs is dependent upondrug activation/inactivation pathways, levels of the molecular target of action,mechanisms of DNA repair, and balance between pro- and anti-apoptotic path-ways. Theoretically, genetic mutations can lead to reduced or increased efficiencyin each of these processes. So far, germ line mutations in drug metabolizing en-zymes have been demonstrated to be major determinants of severe toxicity in ge-netically predisposed patients. The field of cancer pharmacogenomics is extraordi-narily expanding, and recent findings also pointed out that the detection of bothgerm line and somatic genetic polymorphisms in detoxifying enzymes and in mo-lecular targets of action can be used as predictors of response and outcome of che-motherapy. Somatic mutations in the tumor can be also used to select appropriatechemotherapy treatment after surgery in order to overcome drug resistance.

In cancer patients with normal liver/kidney function who receive single agentchemotherapy, the possible presence of genetic determinants of toxicity/responsecould be argued by the following observations:

(1) high interpatient variability in pharmacokinetic parameters of active drug,(2) bimodal distribution of area under the concentration versus time curve (AUC)

metabolic ratios of inactive metabolite to active drug,(3) occurrence of severe toxicity after the first cycle of treatment, and re-occur-

rence of toxicity in following cycles, even at reduced doses.

Once a candidate gene has been identified, anticancer drug therapy can be ratio-nalized by means of genetic principles. The current application of pharmacoge-netics in cancer chemotherapy suggests that pharmacogenetic differences amongpatients can be identified, and the therapeutic window of new and old anticanceragents can be enlarged. Patient predisposition to severe toxicity and genetic mark-ers of response should be characterized prospectively, in order to eliminate toxicity

14 Pharmacogenomics of Chemotherapeutic Agents in Cancer Treatment284

of ineffective therapy and allow more rational search for new therapies in patientswho cannot benefit from conventional chemotherapy.

14.2Pharmacogenetic Determinants of Toxicity after Cancer Chemotherapy

Among the cancer patient population, a subgroup of patients is genetically predis-posed to develop more prolonged and severe toxicity. Mutations in genes codingdrug inactivating/activating enzymes, as well as enzymes involved in reduced fo-late metabolism, can be responsible for intolerance to standard doses of severaldrugs currently used in cancer treatment.

14.2.16-Mercaptopurine and TPMT Pharmacogenetics

14.2.1.1 Clinical Use and Toxicity of 6-MP in Childhood Acute LymphoblasticLeukemia

6-mercaptopurine (6-MP) is a purine analog used in the cure of childhood acutelymphoblastic leukemia (ALL), the most common malignancy in children. Leuke-mic clones are eradicated from the bone marrow after an intense poly-chemother-apeutic regimen. To maintain clinical remission, patients receive daily oral 6-MPin combination with methotrexate (MTX) for a period of two-three years, duringwhich children are under a permanent condition of intentionally “controlled” mye-lotoxicity, a surrogate endpoint to monitor treatment efficacy. Most ALL protocolsinclude individual tailoring of 6-MP dose depending on white blood cell count.Hematological toxicity is not related to 6-MP dosage but to the conversion of 6-MP into active metabolites [1, 2]. At least 2/3 of children with ALL are diseasefree for five years and appear cured after the termination of chemotherapy. In thepast two decades, it became evident that 6-MP is the cornerstone of the mainte-nance chemotherapy and patient outcome could be improved by understandingthe complex pharmacology of 6-MP. Childhood ALL can be regarded as a curabledisease, but about 1/3 of children will not be cured. About 80% of first relapsesin ALL occur in the hematopoietic tissues, and adequate bone marrow exposure isof considerable importance.

14.2.1.2 Metabolism of 6-MP – Activating and Inactivating Pathwaysand Their Clinical Relevance

At cellular level, 6-MP is transformed in a number of active and inactive metabo-lites, and in the bone marrow, the balance between activation and inactivation of6-MP is the main determinant of its antiproliferative effect. Similar to other anti-metabolites, 6-MP is a prodrug lacking any cytotoxic activity and needs to be acti-vated [3] (Figure 14.1). The first step is 6-MP transformation into 6-thioinosinemonophosphate (6-TIMP), which is subsequently converted to 6-thioguanine tri-

14.2 Pharmacogenetic Determinants of Toxicity after Cancer Chemotherapy 285

phosphate nucleotides (6-TGN). DNA incorporation of 6-TGN mediates 6-MP an-tileukemic activity, interfering with DNA ligase, endonuclease, and polymerasefunctions [4]. The amount of 6-MP that can be activated in the bone marrowdepends upon the extent of 6-MP methylation by thiopurine methyltransferase(TPMT) [5, 6]. Although methylated 6-MP and 6-TIMP are inhibitors of the purinesalvage pathway, their contribution to the overall cytotoxicity of 6-MP is not as re-levant as 6-TGN production. Adequate activation of 6-MP to 6-TGN at bone mar-row level is required for increasing the probability of better outcome [7, 8]. By in-directly regulating the size of 6-TGN production, TPMT is the prime determinantof 6-MP antileukemic effect. TPMT is genetically polymorphic, introducing a ma-jor factor of variability in outcome of ALL patients.

14.2.1.3 Reduced Tolerance to 6-MP in Patients with Genetic Impairmentof TPMT Activity

Several case reports evidenced that ALL patients with reduced TPMT activity areintolerant to standard doses of 6-MP. Similar findings were reported also in pa-tients with skin or autoimmune disorders, as well as in transplantation patientsreceiving the 6-MP analog azathioprine. ALL patients with genetic deficiency inTPMT accumulate 6-TGN to toxic concentrations, leading to severe and prolongedmyelosuppression associated with bone marrow hypoplasia. Due to the latency of6-MP cytotoxicity, these effects are not evident after the first daily administrationsof 6-MP, becoming manifest generally after two-three weeks. In the presence ofexcessive hematological toxicity, TPMT-deficient patients can be exposed to life-threatening opportunistic infections, and drug treatment is discontinued until thebone marrow has recovered. 6-MP total dose delivered at the end of the mainte-nance is a critical factor for outcome of ALL patients [9, 10]. As a consequence,when ALL patients are not able to complete the scheduled treatment, they aremore susceptible to drug resistance and disease recurrence. In one patient withTPMT deficit, no therapy could be administered for half of the maintenance peri-od [11]. In another case report, 6-MP standard dosage was reduced by 15-folds toavoid intolerable myelosuppression persisting for 3 weeks [12]. Along with myelo-suppression, these patients experienced severe gastrointestinal toxicity, permanent


Fig. 14.1 6-MP metabolism inbone marrow cells. 6-TIMP,6-thioinosine monophosphate;6-mMP, 6-methylmercapto-purine; 6-mTIMP, 6-methyl-thioinosine monophosphate.

alopecia, and less severe mucositis [11, 12], and support therapy including erythro-cytes and platelet transfusion and antibiotics is required.

The impact of TPMT genetic make up on patient outcome has been establishedin two clinical trials. In one study, TPMT-deficient heterozygotes and homozy-gotes received full doses of 6-MP only for 65% and 7% of the maintenance peri-od, respectively. On the contrary, wild-type patients tolerated full doses of 6-MPfor 84% of the maintenance period [13]. In another study, no clear distinction in6-MP tolerance was noted between wild-type and heterozygous patients, probablydue to differences in the intensity of previous treatment protocols affecting bonemarrow sensitivity. However, the only patient who was mutant homozygous couldnot receive 6-MP for half of the maintenance period [11]. Finally, the first case oflife-threatening myelosuppression in a TPMT-deficient patient receiving 6-thiogua-nine (a 6-MP analog and TPMT substrate) was recently observed during the con-solidation phase of ALL [14].

14.2.1.4 Prediction of TPMT DeficiencyIn order to identify patients at higher risk of toxicity after 6-MP, either phenotyp-ing or genotyping procedures are successfully used. The choice between phenotyp-ing or genotyping is dependent upon the availability of such techniques in thelaboratory. TPMT activity is already routinely measured in some centers (some-times coupled to the measurement of 6-TGN production in erythrocytes) andabout 90–95% of the TPMT-deficient phenotypes are concordant with their geno-type. ALL patients receive blood transfusions when they experience severe ane-mia, and the use of TPMT activity measurement in erythrocytes is not reliable. Inthese cases, genotyping should be indicated. Ideally, genotyping associated withphenotyping should be used, since there are still unknown mutations accountingfor at least 10% of cases of TPMT phenotypic deficiency.

TPMT PhenotypingTMPT activity in human erythrocytes is transmitted as an autosomic codominanttrait [15] and is trimodally distributed, with 89–94% of the individuals havinghigh, 6–11% intermediate, and 0.3% low activity [7, 15–17] (Figure 14.2). The mea-surement of TPMT activity in erythrocytes closely reflects the ability of bone mar-row to inactivate 6-MP. TPMT activity is inversely related to erythrocyte 6-TGNlevels [7, 13, 18, 19], and children with low TPMT activity and very high 6-TGNlevels experienced profound myelotoxicity [20, 21]. Moreover, TPMT phenotype inerythrocyte reflects that in leukemic blasts [22]. Patients with intermediate TPMTactivity had a 5-fold greater cumulative incidence of dose reductions than subjectswith high activity [13], and TPMT activity has been inversely related to the time oftreatment withdrawal due to cytopenia [21].

TPMT GenotypingTen TPMT variants associated with low enzyme activity have been described, andTPMT*2, TPMT*3A, and TPMT*3C account for about 80–95% of the TPMT-defi-


cient phenotype [16, 23–27]. TPMT genotype and phenotype are highly concor-dant. Wild-type individuals have high TPMT activity, while heterozygotes andhomozygotes for one variant allele have intermediate and low activity, respectively[24] (Figure 14.2). TPMT*3A allele comprises two non-synonymous single nucleo-tide polymorphisms in exons 7 (G460A) and 10 (A719G) and is the most commonvariant (frequency of 3.2–5.7% in Caucasians). TPMT*3A represents 55–86% ofall defective variants, and has been found in about 55% of deficient phenotypes.The frequency of TPMT*2 (G238C in exon 5) and TPMT*3C (A719G in exon 10)is about 0.2–0.8% in Caucasians [16, 24, 28, 29].

14.2.1.5 6-MP Dose Adjustment in ALL PatientsClinical experience in TPMT-deficient patients suggests that they should receive 5-10% of the planned 6-MP dose. With regard to wild-type patients with highTPMT activity (about 90% of ALL children), the molecular basis of the interpatientdifferences in TPMT activity is still unclear. Up to 5-fold variability in TPMT activ-ity has been found in wild-type patients [24, 30], suggesting the possible contribu-tion of differences in TPMT gene expression. A polymorphism in the variable tan-dem repeat region of the TPMT promoter has been proposed to modulate TPMTactivity, however, the magnitude of this modulation is probably not relevantenough to explain such differences in TPMT activity in wild-type individuals[31–34]. It would be scientifically reasonable to treat this subgroup with higher6-MP doses to avoid underdosing, in particular because patients with high TPMTactivity and low 6-TGN are more at risk for relapse [7]. However, 6-MP dose esca-lation in the absence of toxicity paradoxically reduces dose intensity (i.e., theamount of drug delivered per unit of time) because of increased toxicity, and fulldoses of 6-MP are still recommended in wild-type patients [9, 35]. The future chal-lenge in 6-MP pharmacogenetics is to identify the genetic basis of TPMT variabil-ity in wild-type patients.


Fig. 14.2 Trimodal distribution ofhuman TPMT activity in erythrocytes.High (TPMTH/TPMTH), intermediate(TPMTL/TPMTH), and low (TPMTL/TPMTL) metabolizer genotypes are in-dicated.

14.2.25-Fluorouracil and DPD Pharmacogenetics

14.2.2.1 Clinical Use and Toxicity of 5-Fluorouracil5-Fluorouracil (5-FU) is a pyrimidine analog widely used in the treatment of colo-rectal, breast, and head and neck cancers. In combination with leucovorin (folinicacid), 5-FU represents the standard adjuvant treatment of non-metastatic coloncancer, one of the most frequent tumors in developed countries. In metastatic dis-ease, combinations of 5-FU/leucovorin with either irinotecan or oxaliplatinshowed better efficacy compared to 5-FU/leucovorin alone [36, 37]. The mainschedules for 5-FU administration are intravenous bolus given daily for 5 daysevery 3–4 weeks or weekly bolus. 5-FU is generally well tolerated, and highly repli-cating epithelial tissues are targets of its toxic action. Dose-limiting toxicity ofbolus 5-FU includes nausea/vomiting, myelotoxicity, oral mucositis, diarrhea, des-quamation of the palms and soles, and rarely, cardiac and neurological toxiceffects.

14.2.2.2 Metabolism of 5-FU – Activating and Inactivating Pathwaysand Their Clinical Relevance

After intravenous administration, about 80–90% of the dose is catabolized in theliver by dihydropyrimidine dehydrogenase (DPD) [38] (Figure 14.3). The formationof the inactive 5-fluoro-5,6-dihydrouracil (5-FUH2) by DPD is the rate-limitingstep of 5-FU catabolism [39]. DPD is widely distributed among tissues, with thehighest levels found in the liver. Once 5-FU entered tumor cells, its antitumor ef-fect is mainly dependent on the extent of 5-FU anabolism. After two sequentialanabolic steps involving thymidine phosphorylase (TP) and thymidine kinase


Fig. 14.3 5-FU catabolism, anabolism and mechanism of action.5-FUH2, 5-fluoro-5,6-dihydrouracil; 5-FdUMP, 5-fluorodeoxyuridinemonophosphate; TP, thymidine phosphorylase; TK, thymidine kinase;TS, thymidylate synthase; CH2THF, 5,10-methylenetetrahydrofolate.

(TK), 5-FU is activated to 5-fluorodeoxyuridine monophosphate (5-FdUMP). Potentinhibition of thymidylate synthase (TS) by 5-FdUMP is considered critical for 5-FU cytotoxicity. TS catalyzes the rate-limiting step of DNA synthesis, such as theconversion of dUMP into dTMP. Optimal TS function requires the formation of acovalent ternary complex consisting of TS, the folate cofactor 5,10-methylenetetra-hydrofolate (CH2THF), and 5-FdUMP. Inadequate cellular levels of 5,10-methyle-netetrahydrofolate reduce the stability of the ternary complex and consequentlythe inhibition of TS by 5-FdUMP. For this reason, 5-FU is administered in asso-ciation with folinic acid, a precursor of 5,10-methylenetetrahydrofolate [40].

14.2.2.3 Life-Threatening Toxicity in DPD-Deficient PatientsSince 1985, it was clear that reduced ability to inactivate 5-FU was heritable andcould expose patients to intolerable toxicities [41]. Further observations confirmedthis finding and clarified the biochemical determinant of this genetic defect (Fig-ure 14.4) [42]. DPD activity is completely or partially deficient in about 0.1% and3–5% of individuals [43], with at least 150 cases reported so far [40]. A neurologi-cal syndrome with thymine-uraciluria occurs in pediatric patients due to completedeficiency of DPD. In cancer patients with defective DPD, a pharmacogenetic syn-drome occurs after 5-FU dosing, and 5-FU-related toxicities are severe and life-threatening. DPD-deficient patients experience grade 4 myelosuppression, alongwith grade 3–4 neurological and gastrointestinal toxicities. The occurrence of se-vere toxicity usually requires 5-FU discontinuation and empiric dose reduction inthe following cycles of therapy, hospitalization, and, sometimes, evaluation ofalternative chemotherapy. A few cases of toxic deaths with documented DPDdefects were also reported [44–46].


Fig. 14.4 Heritability of DPD deficient phenotype. DPD activitywas measured in peripheral blood mononuclear cells from aproband, her family members, and healthy volunteers (controls).

14.2.2.4 DPD Genotype and Molecular Basis of DPD DeficiencyDPD genotype has an autosomal recessive pattern of inheritance [47]. The inactiva-tion of one allele leading to a 50% reduction in the normal DPD activity is sufficientto trigger the development of toxicities after 5-FU treatment [48]. At least 20 muta-tions in DPD gene (DPYD) coding and promoter region have been reported. AmongDPYD variant alleles, eight of them are rare polymorphisms not affecting DPDactivity [49, 50]. Correlative studies between 5-FU toxicity and mutated genotypesin cancer patients were not able to clearly identify the DPYD mutations explainingthis pharmacogenetic syndrome. Potential candidates with clinical relevance areDPYD*2A and DPYD*9A. DPYD*2A is a splice site mutation (intron 14 G1A) re-sulting in the production of a truncated mRNA. It was associated with 5-FU-relatedtoxicity and low DPD activity in three cancer patients from different studies [48, 51,52], and its allele frequency is low (1.3%). Among 14 cancer patients selected on thebasis of low DPD activity and severe toxicity, this mutation was found in six of them[53]. However, in another study, discordance was demonstrated between DPYD*2Aand DPD activity [50]. DPYD*9A is a common missense T85C mutation in exon 2resulting in a C29R amino acid change, but its association with reduced DPD activ-ity is still controversial. However, heterozygosity for DPYD*9A was found in fourand eight of 14 cancer patients with severe 5-FU toxicity in two different studies[50, 53]. DPYD*2A and DPYD*9A mutations seems to have good concordance withclinical phenotype (i.e., 5-FU toxicity), but a low concordance with biochemical phe-notype (i.e., DPD activity). Other mutations in the DPYD promoter and coding re-gion occurred in DPD-deficient patients experiencing severe toxicity but their fre-quency is unknown [50, 54, 55].

14.2.2.5 Measures to Predict DPD Deficiency in Patients Receiving 5-FUThe complexity of the genetic basis of DPD deficiency implies that the identifica-tion of patients at high risk of 5-FU toxicity is mostly based on phenotypic proce-dures. These methods are not suitable for general use and concomitant drugs,dietary intake and other environmental factors could reduce their predictive powerin cases of partial DPD deficit.

DPD Biochemical Phenotype Measured in Peripheral Blood Mononuclear Cells (PBMC)DPD activity measured in PBMC is used as a surrogate for systemic DPD activity.DPD activity is normally distributed and highly variable among individuals (coef-ficient of variation of 33.9–46.6%) [43, 56–59]. DPD activity is undetectable in to-tally deficient patients. The majority of partially deficient patients had a DPD val-ue �30% of the mean in the normal population, and this value is considered thecut-off for patients at higher risk of toxicity. Among patients experiencing severetoxicity after 5-FU, 36–59% of them were deficient in DPD activity [43, 53, 60].This suggests the involvement of other determinants in the susceptibility to 5-FUtoxicity. The concordance between liver and PBMC DPD activity is modest [61],and normal DPD activity in PBMC was found in one patient with very depressedliver DPD activity who died because of 5-FU toxicities [44].


Measurement of Natural Pyrimidines in Biological FluidsIn the majority of DPD defective patients experiencing severe 5-FU toxicity, abnor-mally high levels of natural pyrimidines are present in plasma and/or urine [62].Moreover, endogenous dihydrouracil/uracil ratio in plasma has been proposed asa measure of 5-FU catabolic deficiency in cancer patients [63], and screening ofcancer patients for these simple markers should be prospectively evaluated.

14.2.3Irinotecan and UGT1A1 Pharmacogenetics

14.2.3.1 Clinical Use and Toxicity of IrinotecanIrinotecan (CPT-11) is a semi-synthetic analog of the natural alkaloid camptothe-cin with considerable activity in colorectal cancer patients with poor prognosisdue to 5-FU resistance. Moreover, the utility of irinotecan as a component of ini-tial therapy in association with 5-FU of metastatic colorectal cancer has been re-cently demonstrated [37]. The most common administration schedule of irinote-can is a short (30–90 min) intravenous infusion, either once every three weeks orweekly for four weeks [64]. Common and dose limiting toxicities of irinotecan areneutropenia and delayed diarrhea. Both grade 3–4 neutropenia and diarrhea mayoccur in about 1/3 of patients, with variable frequency depending on the scheduleof administration. Severe nausea/vomiting is reported in less than 10% of pa-tients [65]. Increasing evidences support the correlation between toxicity and irino-tecan pharmacology.

14.2.3.2 Metabolism of Irinotecan – Activating and Inactivating Pathwaysand Their Clinical Relevance

Although irinotecan metabolism generates at least 20 metabolites, many of themare found at trace levels in patients. Clinically relevant metabolites of irinotecanare the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), inactive glucuro-nide SN-38G, and inactive aminopentane carboxylic acid (7-ethyl-10[4-N-(5-amino-pentanoic acid)-1-piperidino]carbonyloxycamptothecin, APC) (Figure 14.5).

Activating PathwayIrinotecan is a prodrug, and hydrolysis of irinotecan by the high-affinity carboxyl-esterase-2 enzyme in many normal tissues and tumors is responsible for activa-


Fig. 14.5 Inactivating and acti-vating pathways of irinotecanmetabolism.

tion of irinotecan to SN-38, a potent topoisomerase I inhibitor [66–68]. AlthoughSN-38 concentrations in plasma and urine are the lowest among all irinotecanmetabolites, SN-38 formation within the tumor is critical for irinotecan antitumoractivity.

Inactivating PathwaysInactivation pathways involve oxidation of irinotecan and glucuronidation of SN-38. Oxidation of irinotecan accounts for about 15% of irinotecan dose [69], andthe formation of inactive APC by cytochrome P450 (CYP) 3A4 reduces theavailability of irinotecan for its activation to SN-38 [70]. The final step of sequen-tial irinotecan metabolism is the inactivation of SN-38 by glucuronidation toSN-38G. Glucuronidation of SN-38 is the major elimination pathway of SN-38and protects patients from irinotecan toxicity. The severity of diarrhea is depen-dent upon the extent of inactivation of SN-38 by glucuronidation. From preclinicalexperiments in nude mice, accumulation of SN-38 in the intestine is responsiblefor the diarrhea after irinotecan [71]. When biliary excretion of SN-38 wasmeasured by the “biliary index” [which takes into account SN-38 glucuronidationrates normalized by irinotecan AUC (SN-38 AUC/SN-38G AUC�CPT-11 AUC)],patients with high biliary index are more likely exposed to the occurrence of se-vere diarrhea than patients with low biliary index (Figure 14.6). This suggests thathigher glucuronidation of SN-38 in the liver may protect against irinotecan-induced intestinal toxicity as a result of reduced elimination of SN-38 in the bile[72].


Fig. 14.6 Biliary indexes and severity of diarrhea in cancer patientsafter four different dose levels (�, �, �, �) of irinotecan. Statisti-cally significant correlation of biliary index to severity of diarrheawas shown. Cdiff+, one patient found positive for C. difficile toxin.

14.2.3.3 Increased Risk of Toxicity in Cancer Patients with Gilbert’s SyndromeSN-38 glucuronidation is catalyzed by the polymorphic UDP-glucuronosyltransfer-ase 1A1 (UGT1A1) enzyme, which is responsible for bilirubin glucuronidation[73]. Among the hyperbilirubinemic syndromes caused by genetic defects inUGT1A1 gene, a promoter polymorphism induces Gilbert’s syndrome. This is aninherited disorder, characterized by mild, chronic unconjugated hyperbilirubine-mia (serum bilirubin levels usually <3 mg dL–1). In two cancer patients withGilbert’s syndrome, grade 4 neutropenia and/or diarrhea occurred after irinotecan.Both of them had a familiar history of Gilbert’s syndrome and periodic asympto-matic increases in unconjugated bilirubin. Exaggerated toxic response to standarddoses of irinotecan was associated with abnormally elevated values of biliary index[74]. These results suggested that genetically reduced inactivation of SN-38 couldresult in a higher risk of developing irinotecan-induced toxicity.

14.2.3.4 Gilbert’s Syndrome GenotypeThe genetic defect in Gilbert’s syndrome is a TA insertion in the promoter regionof UGT1A1 gene, resulting in the variant allele (TA)7TAA (UGT1A1*28) insteadof the wild-type allele (TA)6TAA [75, 76]. The presence of an additional TA repeatresults in reduced UGT1A1 expression levels and activity, since transcriptional ac-tivity of the promoter decreases with the progressive increase in the number ofTA repeats [77]. A wide variation in the incidences of this syndrome has been re-ported, ranging from 0.5% to 23% in various groups [76–80]. In addition to the(TA)7 polymorphism, (TA)8 and (TA)5 alleles have been found in individuals fromdifferent ethnic backgrounds [77, 81, 82] and a subject with Gilbert’s syndromewas found to be heterozygous for (TA)8 [83].

While the majority of Gilbert’s syndrome patients are (TA)7 homozygotes, somepatients do not have mutations at the promoter level but are heterozygotes forG211A, T1456G and C686A missense mutations in the UGT1A1 coding region[84, 85]. G211A (G71R, UGT1A1*6) mutation results in a 30% (heterozygotes)and 60% (homozygotes) decrease in bilirubin glucuronidating activity, and ishighly prevalent in individuals of Asian origin [86, 87], being responsible forabout 60% of the Gilbert’s syndrome cases among Japanese individuals [85].G211A allele frequency of 11–13% has been reported in Asians [86, 88]. T1456G(Y486D, UGT1A1*7) mutation was found in two patients with Gilbert’s syndrome[88, 89], but its frequency in the general population is not known. Interestingly,mutations in the UGT1A1 coding region seem to be more frequent in Asian thanCaucasian populations [88].

14.2.3.5 Gilbert’s Syndrome Phenotype and SN-38 GlucuronidationInterpatient variability in SN-38 glucuronidation is considerably high in cancerpatients [72]. A 17-fold difference in SN-38 glucuronidation was found in humanlivers [90], and significant variability of UGT1A1 phenotype might account for dif-ferences in SN-38 inactivation. SN-38 glucuronidation in human livers was highly


concordant with the UGT1A1 promoter genotype, since glucuronidation rates ofSN-38 were significantly lower in homozygotes and heterozygotes for (TA)7 whencompared to wild type (Figure 14.7). Patients homozygous and heterozygous for(TA)7 might be expected to have at least a 50% and 25% decrease in SN-38glucuronidation, respectively [90].

14.2.3.6 Possible Measures to Predict Patients at High Risk of Toxicityafter Irinotecan

Predictive measures to classify patients as low and high SN-38 glucuronidatorsand consequently identify those at higher risk of toxicity are required. Gilbert’ssyndrome remains often undiagnosed, and ratio of conjugated to unconjugated bi-lirubinemia can not be considered a predictive parameter. Recent results from twoclinical trials propose UGT1A1 genotyping as a more reliable test to predict therisk of severe toxicity after irinotecan. Preliminary findings from a phase I studyof irinotecan at two dose levels show that UGT1A1 promoter genotype correlateswith irinotecan pharmacokinetics and toxicity [91]. With irinotecan 300 mg m–2,(TA)6 wild-type patients developed grade �1 toxicity, while about 50% of (TA)7 car-riers experienced grade �2 diarrhea and neutropenia associated with reduced SN-38 glucuronidation. No significant differences were observed between homozy-gotes and heterozygotes for (TA)

7and the irinotecan dose of 300 mg m–2 has been

increased to 350 mg m–2, the dose level approved by the Food and Drug Adminis-tration. Out of five 350 mg m–2 patients, the two of them with (TA)7 allele devel-oped grade 4 neutropenia. A recent retrospective study in Japanese patients con-firmed these results [92]. Among patients with severe toxicity after irinotecan,46% of them where (TA)7 carriers. Among patients who did not experience severetoxicity, only 14% of them were (TA)7 carriers. The presence of (TA)7 allele was asignificant risk factor for irinotecan severe toxicity. Interestingly, all three patientswith a missense C686A mutation in the coding region (P229Q, UGT1A1*27) ex-perienced severe toxicity, and no statistical association was found between severetoxicity and the G211A mutation.


Fig. 14.7 In vitro glucuronidationof SN-38 in human liver microsomesgenotyped for UGT1A1 promoterpolymorphism. Each bar representsthe mean (±standard error) SN-38Gproduction in livers with 6/6 (n= 19),6/7 (n= 21), and 7/7 (n= 4) genotype.* Significantly less than 6/6, p < 0.05.

14.2.4Amonafide and NAT2 Pharmacogenetics

Amonafide is a DNA intercalating agent and topoisomerase II inhibitor whichshowed activity in breast cancer and leukemia. Highly variable and unpredictabletoxicity partly caused by interindividual differences in N-acetylation have ham-pered its clinical development. Although a dose individualization scheme was vali-dated for low and high metabolizers, amonafide is no longer in clinical develop-ment. The experience with amonafide remains an example of population pharma-cogenetics and successful phenotyping strategy in cancer chemotherapy.

14.2.4.1 Metabolism of Amonafide and NAT2 PolymorphismAmonafide is extensively metabolized, including N-acetylation by N-acetyltransfer-ase 2 (NAT2) to N-acetyl-amonafide (Figure 14.8), a metabolite approximately equi-potent in vitro with the parent drug [93]. Mutated alleles NAT2*5A, B, C,NAT2*6A, NAT2*7, NAT2*13 and NAT2*14 account for more than 99% of slowacetylators in Caucasian populations [94, 95]. Homozygosity for NAT2 mutated al-leles is required for the slow acetylator phenotype, and rapid acetylators includeboth mutant heterozygotes and wild-type individuals, the latter having signifi-cantly higher acetylation rates [96].

14.2.4.2 Dose Individualization of Amonafide Based on N-Acetylator PhenotypeDue to the polymorphic acetylation of amonafide, a phenotyping procedure foramonafide acetylation using caffeine as a probe was evaluated in cancer patients.Slow and fast acetylators of both caffeine and amonafide were identified. Fast ace-


Fig. 14.8 Amonafide metabolism. Acetylation and oxidation pathways

CY

PIA

2

tylators had significantly greater myelosuppression than slow acetylators and amo-nafide exposure was significantly greater in fast acetylators, who would be ex-pected to have a higher clearance of amonafide (Figure 14.9) [97]. This appearedto be unusual compared with most drugs metabolized by N-acetylation, whereslow acetylators are more likely to experience adverse reactions. The unexpectedbehavior of amonafide was due to the inhibition of amonafide oxidation by N-ace-tyl-amonafide, since amonafide is a substrate for CYP1A2 and amonafide oxida-tion is inhibited by its acetylated metabolite [98]. Based on acetylator phenotype, apharmacogenetic phase I study of amonafide recommended doses of 250 and375 mg m–2 for fast and slow acetylators, respectively [99]. Further investigation inphase II of studies of 300 mg m–2 amonafide demonstrated that fixed dosing wasconsidered inappropriate for all patients, as fast phenotypes would be expected toexperience severe toxicity and slow phenotypes may be significantly underdosed.Since there was still significant interpatient variability in toxicity at these doselevels, a subsequent study attempted to develop pharmacodynamic models to in-dividualize amonafide dosing, and the optimal model was defined by acetylatorphenotype, pretreatment white blood cell count and gender [100].

14.2.5MTHFR Gene Polymorphism in Breast Cancer Patients Receiving CMF Regimen

CMF regimen is a combination of cyclophosphamide, MTX, and 5-FU, and repre-sents one of the treatments of choice for women with non-metastatic breast can-cer, significantly increasing disease-free and overall survival. A recent report in a


Fig. 14.9 Degree of leukopenia in cancer patients receiving amonafide.Incidence and degree of leukopenia was higher in fast acetylatorscompared to slow acetylators.

small series of patients described an interesting association between the occur-rence of severe myelotoxicity after CMF and a single nucleotide polymorphism inthe methylenetetrahydrofolate reductase (MTHFR) gene [101]. One breast cancerpatient experienced grade 4 leukopenia after the first cycle of CMF. After a similartreatment regimen based on 5-FU and MTX, her mother affected by gastric can-cer experienced life-threatening toxicity as well. Both patients were homozygouscarriers for a single nucleotide polymorphism in the MTHFR gene. MTHFR geno-typing was extended to additional five consecutive breast cancer patients ex-periencing severe toxicity after CMF, and four of them were found to be homo-zygous for the same mutation.

14.2.5.1 MTHFR Function and PolymorphismHuman MTHFR gene consists of eleven exons, and the coded enzyme converts5,10-methylenetetrahydrofolate (CH2THF) to 5-methyltetrahydrofolate (CH3THF),a methyl donor in the conversion of homocysteine to methionine during proteinsynthesis [102] (Figure 14.10). In cell folate metabolism, MTHFR regulates thepool of folates for nucleic acid synthesis. A C677T mutation in the MTHFR genecodes an enzyme variant with in vitro thermolability and reduced catalytic activity(35% compared to wild type), leading to accumulation of plasma homocysteine inhomozygous individuals [103]. In addition to this, homozygous subjects accumu-late CH2THF polyglutamates in erythrocytes at the expense of CH3THF species,the only folate form found in erythrocytes of wild-type individuals [104]. C677Tpolymorphism creates a shift in the distribution of intracellular folates, creatingretention of folates committed for purine and pyrimidine synthesis (i.e.,CH2THF). This polymorphism is common, with about 10% of homozygous indi-viduals (TT) in Caucasian population [105]. Taking into account different frequen-cies due to ethnicity, incidence of T allele ranges from 5% to 54%.


Fig. 14.10 Folate metabolism and role of MTHFR. Genetically reduced MTHFR activity affectsthe distribution between folate species required for protein and DNA synthesis. Higher availabil-ity of 5,10-methylenetetrahydrofolate (CH2THF) potentiates the TS inhibition by 5-FdUMP, theactive metabolite of 5-FU. Hcy, homocysteine; Met, methionine; CH3HF, 5-methyltetrahydrofo-late; TS, thymidylate synthase; 5-FdUMP, fluorodeoxyuridine monophosphate.

14.2.5.2 MTHFR Polymorphism as a Determinant of CMF ToxicityFive of six patients with grade 4 leukopenia after CMF were TT homozygotes[101]. Qualitatively altered distribution of intracellular folates in breast cancer pa-tients with TT genotype could have increased bone marrow sensitivity to CMF che-motherapy. When thymidylate synthase converts dUMP into dTMP, CH2THF isrequired as a donor of monocarbon groups. MTHFR deficiency induced by TTgenotype increases the availability of CH2THF, potentiating 5-FU inhibition ofthymidylate synthase mediated by 5-FdUMP, leading to severe myelosuppression.This genotype/phenotype association needs to be confirmed in a larger trial andthe postulated biochemical mechanism further investigated. These findings high-light a possible role of MTHFR polymorphism in selecting cancer patients athigher risk of toxicity after receiving the CMF regimen.

14.3Pharmacogenetic Determinants of Response after Cancer Chemotherapy

Recent studies focused on the importance of pharmacogenetic determinants ofresponse in cancer patients. Screening of patients for polymorphic mutants ofglutathione-S-transferase and thymidylate synthase has the potentiality to predictresponse and hence outcome of chemotherapy.

14.3.1Glutathione-S-Transferase Mutations in Cancer Chemotherapy

Xenobiotic detoxification in mammalian cells is efficiently mediated by conjuga-tion of the nucleophilic center of the compound with reduced glutathione (GSH)by glutathione-S-transferase (GST). GST gene mutations can lead to high pheno-typic variability. Increased GST function might arise from gene duplications(ultrarapid phenotype), increased protein level due to promoter mutations, andcoding mutations associated with increased enzyme efficiency. Reduced detoxifica-tion is generally related to gene deletions (null genotypes), as well as to conforma-tional changes induced by single amino acid changes in the coding region [106].

A broad literature exists on genetically reduced GST activity as a risk factor incarcinogenesis. Less information is available on the clinical implications of poly-morphic GSTs in cancer patients receiving chemotherapy. Conjugation with GSThas been reported for cisplatin and alkylating agents, and germ line mutations al-tering GST activity could change drug pharmacokinetics. Moreover, the GSH/GSTsystem is involved in the development of cellular resistance to cancer chemother-apy. Cancer cells protect themselves from the toxic action of chemotherapy byoverexpressing GST, and the relevance of GST mutations for patient outcome hasbeen investigated in both solid tumors and hematological malignancies.

14.3 Pharmacogenetic Determinants of Response after Cancer Chemotherapy 299

14.3.1.1 GST Pharmacogenetics and Outcome in Solid Tumor PatientsIn breast cancer patients, inherited mutations in GSTP1 gene have been shown toinfluence treatment outcome [107]. In this study, the most commonly used che-motherapeutic agents were cyclophosphamide, 5-fluorouracil, and doxorubicin. Re-active metabolites of cyclophosphamide are conjugated with GSH by GSTP1 [108],and increased GSTP expression was reported in a doxorubicin-resistant cancer cellline [109]. Single nucleotide substitutions in the GSTP1 coding region result inamino acid changes Ile105Val and Ala114Val, and Val105 variant was 2-fold lower ef-ficient than Ile105 in conjugating thiotepa [110]. In tumor biopsies, homozygosityfor the less active Val105 variant improved the survival of breast cancer patientscompared to those carrying Ile105, and the hazard of death conferred by tumorVal105/Val105 genotype was 30% of that of Ile105 patients.

Genotyping of ovarian cancer patients for null GSTM1 and GSTT1 revealed apoorer survival in patients with null genotype compared to wild type [111]. Noneof the patients with both null genotypes survived 3.5 years after diagnosis, while43% of wild-type patients survived beyond this time. In this study, 70% of pa-tients received single-agent carboplatin, the remaining of them being treated withalkylating agents. Reduced systemic detoxification of these compounds in patientswith null genotype should have led to better response rate and survival comparedto wild type, but opposite results have been observed. The most plausible explana-tion is the effect of reduced GST-mediated detoxification on the biology of ovarianepithelial cells. Ovarian tumors with loss of p53 function are characterized by lackof response to chemotherapy and poor outcome [112]. GST activity protects ovar-ian cells from chronic oxidative damage to genomic DNA potentially leading toloss of p53 function. Patients with null GST and loss of p53 function in ovariancancer cells might have experienced shorter survival caused by reduced protectionfrom oxidative damage.

14.3.1.2 GST Pharmacogenetics and Outcome in Childhood LeukemiasThe observation of 3-fold increased risk of relapse in ALL patients expressingGSTM compared to non-expressors [113] prompted to investigate the associationbetween frequency of GST variants and outcome of leukemic patients.

In ALL, null GSTT1 genotype was a major determinant of initial response toprednisone therapy in patients treated according to the Berlin–Frankfurt–Munster(BFM) protocols [114]. GSTs have been implicated in cell resistance to glucocorti-coid treatment, and initial response to prednisone is considered a strong predictorof outcome. A 6.7-fold reduced risk of poor response to prednisone was found innull GSTT1 genotype patients compared to heterozygous and wild-type patients.In another trial from the BFM study group, mutated GST genotypes were selectedfor their impact on ALL relapse [115]. Null GSTM1 and GSTT1 conferred 2-foldand 2.8-fold reduction in risk of relapse compared to wild type, respectively.Among polymorphisms in the GSTP1 gene, Val105/Val105 genotype showed 3-folddecreased risk of relapse compared to other variants at codons 105 and 114. Basedon these observations, null GSTM1, null GSTT1 and GSTP1 Val105/Val105 geno-


types were designated as “low risk” genotypes, and patients having at least two“low risk” genotypes had 3.5-fold reduced risk of relapse compared to patientswith no “low risk” genotype. A previous study did not demonstrate any impact ofnull GSTM1 and GSTT1 genotypes for event-free survival in ALL, with only a ten-dency of higher central nervous system relapse-free survival in null GSTM1 pa-tients [116]. Compared to the BFM study, these findings are more applicable tothe overall ALL population, since, in the BFM study, matching criteria led to theselection of a particular patient subgroup of the entire ALL population.

In acute myeloid leukemia (AML), intensification of both induction and post-re-mission chemotherapy improves overall survival but is associated with significantdrug-related morbidity and mortality. When AML patients receiving standard andintensive induction chemotherapy were genotyped for null GSTT1, interesting re-sults were observed [117]. In the intensive treatment arm, null GSTT1 genotypewas associated with reduced survival and increased risk of toxic death in remis-sion compared to wild-type patients.

14.3.2Thymidylate Synthase Gene Promoter Polymorphism and Response to 5-Flurouracil-Based Chemotherapy

Thymidylate synthase (TS) is the rate-limiting enzyme in the DNA synthetic path-way and the target for 5-FU and folate analogs (Figure 14.3). Compared to normaltissues, TS is often overexpressed in tumor cells, probably as a result of tumorsuppression loss of function, gene amplification or other mechanisms. Acute in-duction of TS protein as well as stable amplification of TS-specific genes may beassociated with resistance to fluoropyrimidine derivatives [118, 119], and an in-verse correlation between tumor TS expression and clinical response was found[120–122].

14.3.2.1 Regulation of TS Gene ExpressionMechanisms regulating TS gene expression are not very well understood. Tumorsuppressor elements modulate TS gene transcription. Translation of TS mRNA isnegatively regulated by direct binding of TS protein to promoter elements on itscognate mRNA [123]. A translational regulatory element within the coding regionhas also been found [124], and a 6-bp deletion in the 3�-untranslated region ofTS mRNA could affect mRNA stability and translation [125]. In addition to this,two, three, four and nine copies of 28-bp tandem repeated sequences have beendescribed in the enhancer region of the TS promoter [126–128]. Presence of a tri-ple repeat increased TS expression by 2.6-folds compared to double repeat in tran-sient expression assays [127]. In patients with gastrointestinal malignancies, TSlevels were significantly higher in tumor specimens homozygous for triple repeatscompared to those with double repeats [129].

14.3 Pharmacogenetic Determinants of Response after Cancer Chemotherapy 301

14.3.2.2 Genotyping of the TS Promoter Variable Tandem Repeatand Clinical Outcome

The chance of downstaging after radiation and 5-FU-based therapy in rectal can-cer patients was related to polymorphisms in the enhancer region of the TS genepromoter in tumor specimens [130]. The presence of downstaging is an importantprognostic factor. Based on the number of tandem repeats, 20% of patients had2/2, 38.5% 2/3, and 42.5% 3/3 genotype. The relative probability of achievingdownstaging for 2/2 and 2/3 patients was 3.7-fold higher than for 3/3 patients.Genotyping for tandem repeat promoter polymorphisms could be used to selectthe most appropriate chemotherapy in patients with 3/3 genotype, since theymight respond to irinotecan or oxaliplatin that have different mechanisms of ac-tion.

14.4Conclusion

The main focus of genetic investigation in chemotherapy over the past two de-cades has been to predict the occurrence of severe toxicity. The examples of theclinical pharmacogenetics of 6-MP, 5-FU, and CPT-11 demonstrated that about10% of cancer patient population is at high risk of severe toxicity. The challengefor the future is to use pharmacogenomics as a tool for dose individualization,since, for the vast majority of patients, it is still unclear how dosage should be se-lected on the basis of patient genotype.

Genetic investigation in cancer patients should start as early as possible duringdrug development. Candidate genes playing a significant role in the pharmacologyof new chemotherapeutic agents are often unknown before clinical trials. Geneticinvestigation in chemotherapy is complicated by the fact that multi-drug therapyis often the standard of care, confounding the results of phenotype–genotype cor-relation. For this reason, phase I–II trials of new single agents should include thesearch of genetic determinants of toxicity and response.

There is an increasing need of understanding the reasons why some cancer pa-tients respond to chemotherapy while others experience toxicity without any thera-peutic benefit. Prospective large studies with multivariate analysis will be requiredto confirm the results of retrospective studies that, so far, have demonstrated asso-ciations between genetic defects and response.

AcknowledgementsWe wish to thank Mss. Debby Stoit and Marla Scofield for their assistance in thepreparation of figures. This Pharmacogenetics of Anticancer Agents ResearchGroup (www.pharmacogenetics.org) chapter was supported by grant GM61393 fromNational Institute of Health, Bethesda, MD.


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