PHARMACOGENETICS OF THE

NEURODEVELOPMENTAL IMPACT OF

ANTICANCER CHEMOTHERAPY

Philippe Robaey,1,2* Maja Krajinovic,1 Sophie Marcoux,1 and Albert Moghrabi31Centre de Recherche de l’Hopital Sainte-Justine, Universit�e de Montr�eal, Montr�eal, Qu�ebec

2Children’s Hospital of Eastern Ontario, University of Ottawa, Ottawa, Ontario3Service d’H�ematologie-Oncologie, Hopital de Verdun, Universit�e de Montr�eal, Montr�eal, Qu�ebec

Pharmacogenetics holds the promise of minimizing adverse neuro-developmental outcomes of cancer patients by identifying patients atrisk, enabling the individualization of treatment and the planning of closefollow-up and early remediation. This review focuses first on methotrex-ate, a drug often implicated in neurotoxicity, especially when used incombination with brain irradiation. The second focus is on glucocorti-coids that have been found to be linked to adverse developmentaleffects in relation with the psychosocial environment. For both examples,we review how polymorphisms of genes encoding enzymes involved inspecific mechanisms of action could moderate adverse neurodevelop-mental consequences, eventually through common final pathways suchas oxidative stress. We discuss a multiple hit model and possible strat-egies required to rise to the challenge of this integrative research.

'2008 Wiley-Liss, Inc.Dev Disabil Res Rev 2008;14:211–220.

Key Words: methotrexate; glucocorticoids; polymorphisms; outcome;

irradiation

Neurotoxic complications of cancer chemotherapy arecommon. Chemotherapy may cause both peripheralneurotoxicity, consisting mainly of peripheral neurop-

athy, and central neurotoxicity, ranging from encephalopathyto neurodevelopmental cognitive deficits. Chemotherapy-induced deficits may occur long after treatment and pro-foundly affect the quality of life. Neurodevelopmental effectsof cancer treatment have long been recognized in children[Mulhern, 1994], but they are also increasingly recognized inadults who describe their state as ‘‘chemobrain’’ or ‘‘chemofog’’[Weiss, 2008]. In adults as in children, larger effects are seenfor executive functions and verbal memory [Anderson-Hanleyet al., 2003]. Neuropsychological sequelae of cancer therapyare quite variable [Mulhern, 1994]. In many cases, this vari-ability can be explained by differences in treatment (e.g., typeof chemotherapy), differences in assessment procedures (e.g.,different or incomplete test data, questionable reliability, or va-lidity of some instruments) and subject variations (e.g., differ-ences in diagnosis, in the duration of the follow-up or in thetime elapsed between treatment and evaluation, failure tocontrol for age, gender, demographic, and other confounding

variables such as school attendance). Recent research hasshown that genetic differences are likely to contribute to thisvariability as well.

Some examples of the contribution of genetic variationto neurotoxicity can be found for acute adverse effects on thebrain. Adenosine triphosphate-binding cassette (ABC) trans-porters prevent the brain entry of toxic compounds underphysiological conditions but complicate pharmacotherapies incancer [Hermann et al., 2006]. Encephalopathy episodes weremore frequent among children with acute lymphoblastic leu-kemia with the ABCB1 3435TT genotype than in the3435CC/CT group [Erdelyi et al., 2007], creating a basis foraltering therapy based on genotype. Following this approachfor long-term neurodevelopmental toxicity, a pharmacogeneticmodel could lead to a more personalized treatment throughidentification of significant genes, integration of multiple genesinto a genetic profile, and use of the profile to improve out-come in practice. Presently, the field is nascent, as there is onlyone publication that shows increased susceptibility for adverseneurodevelopmental outcome following treatment of acutelymphoblastic leukemia of children with specific gene variants[Krajinovic et al., 2005]. We will thus focus on methotrexate(MTX), the drug most often implicated in neurotoxicity,which is a mainstay in the treatment of cancer and especiallyin the treatment of acute lymphoblastic leukemia. We willthen focus on glucocorticoids (GCs), another common cancertreatment (and primarily leukemia) in children with more spe-cific adverse developmental effects. For both examples, we willreview how polymorphisms in genes encoding enzymesinvolved in their mechanisms of action could moderate theiradverse neurodevelopmental consequences. On this basis, wewill discuss the challenges and the possible strategies to under-

*Correspondence to: Philippe Robaey, Children’s Hospital of Eastern Ontario,University of Ottawa, 401 Smyth Road, Ottawa, Ontario, Canada K1H 8L1.E-mail: philippe.robaey@umontreal.caReceived 22 September 2008; Accepted 22 September 2008Published online in Wiley InterScience (www.interscience.wiley.com).DOI: 10.1002/ddrr.29

DEVELOPMENTAL DISABILITIESRESEARCH REVIEWS 14: 211 – 220 (2008)

' 2008Wiley -Liss, Inc.

stand the pharmacogenetic of neurode-velopmental impact of chemotherapy.

METHOTREXATE

Metabolism of Folate, Methionine,and Transsulfuration Pathway

Methylation is a key biochemicalprocess involved in the regulation ofgene expression, protein function, andRNA metabolism. The major donor formost methyltransferase reactions is S-adenosylmethionine (SAM), which issynthesized by the methionine adeno-syltransferase from methionine and ATP.Dietary supply of methionine does notprovide enough methyl groups for themetabolism, and methionine has to berecycled. The product of the demethy-lation of SAM is S-adenosylhomocys-teine (SAH), which is subsequentlyhydrolyzed by the SAH hydrolase inadenosine and homocysteine (Hcy).Hcy is remethylated in the liver by themethionine synthase (MTR), whichaccounts for about half of the Hcyremethylation capacity [Finkelstein andMartin, 1984]. MTR catalyzes thetransfer of a methyl group from 5-methyltetrahydrofolate (5-methyl THF)to Hcy to produce methionine andTHF. This reaction requires cob(I)ala-min as cofactor. Over time, the cob(I)alamin cofactor of MTR becomes oxi-dized to cob(II)alamin, rendering theenzyme inactive. Regeneration of func-tional enzyme necessitates reductivemethylation using SAM as a methyldonor, which is supported in humansby the methionine synthase reductase(MTRR) in presence of NADPH aselectron donor [Olteanu and Banerjee,

2001]. Alernatively, the betaine-Hcymethyltransferase (BHMT) catalyzes thetransfer of a methyl group from betaineto Hcy, resulting in the formation ofdimethylglycine and methionine.

The THF produced by the MTRis reloaded with a carbon to form 5,10-methylene-THF by the serine hydroxy-methyltransferase (SHMT) that catalysesthe conversion of serine to glycine. Themethylene tetrahydrofolate reductase(MTHFR) catalyzes the conversion ofthe 5,10-methylene-THF to 5-methylTHF, which reenters the cycle of reme-thylation of Hcy by the MTR. Theproduction of 5-methyl THF by theMTHFR is nonreversible under physio-logical conditions, and all the dietaryfolates of the one-carbon pool [Arinze,2005] can end up as 5,10-methylene-THF. Alternatively, the 5,10-methylene-THF is used by the thymidylate(dTMP) synthase to catalyze the reduc-tive methylation of deoxyuridylate toproduce dTMP and dihydrofolate(DHF). Using NADPH, the dihydrofo-late reductase (DHFR) reduces DHF toTHF that can be converted back to5,10-methylene-THF by the SHMT.dTMP, the other product of dTMPsynthase, is subsequently phosphorylatedto thymidine triphosphate for usein DNA synthesis and repair. MTHFRis thus pivotal, balancing the homeosta-sis between DNA methylation andsynthesis.

About half of the Hcy formed isconserved by remethylation to methio-nine in the methionine cycle. Theother half is irreversibly converted bycystathionine b-synthase (CBS). CBS isa lyase that catalyzes the condensation

of serine with Hcy, generating cysta-thionine in a rate-limiting reaction ofthe transsulfuration pathway. Then, thehydrolysis of cystathionine yields a-ketobutyrate and cysteine, a precursorof glutathione. Thus, CBS is directlyinvolved in the removal of Hcy fromthe cycle and in the biosynthesis of cys-teine, a precursor of glutathione, themajor metabolite regulating the redoxstatus of the cell. In vitro studies haveindicated that SAM functions as aswitch between the methionine cycleand the transsulfuration pathway [Fin-kelstein and Martin, 1984]. At highconcentration, SAM activates CBSwhile limiting Hcy remethylation byinhibiting MTHFR and inactivatingBHMT. At low concentration, the Hcyremethylation is unimpaired (Fig. 1).

MTX: Mechanisms of TherapeuticActions and Adverse Effects

MTX inhibits DHFR. The affin-ity of MTX for DHFR is about 1,000-fold that of the normal substrate DHFfor DHFR. Moreover, MTX is metab-olized in target cells into MTX poly-glutamates (MTX-[Glu]n) that inhibitssome enzymes involved in de novo pu-rine and pyrimidine synthesis, especiallydTMP synthase. As THF can no longerbe regenerated, 5,10-methylene-THF,5-methyl THF, methionine, and SAMare depleted. As a consequence, dTMPsynthesis and the methylation of cyto-sine in DNA decreases, which in turnenhances gene transcription and DNAstrand breakage, and impairs DNArepair, resulting in genetic mutationsand apoptosis. SAM also providesmethyl group in the methylation ofproteins, neurotransmitters, and phos-pholipids, which extends the effects offolate deficiency beyond DNA alone.These mechanisms account for the ther-apeutic effects of MTX [Fotoohi andAlbertioni, 2008].

Folate deficiency also producesaccumulation of Hcy, the product ofthe demethylation of methionine thatcan no longer be remethylated. WhenHcy accumulates, it has different adverseeffects on the brain processes. Acuteand subacute toxic neurological effectshave been observed after intrathecal orparenteral, low or high doses of MTX[Kay et al., 1972; Allen et al., 1980;Jaffe et al., 1985; Poskitt et al., 1988;Rubnitz et al., 1998]. Because of thereversibility of the action of SAHhydrolase, increased level of Hcy leadsto accumulation of SAH, the precursorof Hcy. SAH has much greater affinityfor methyltransferases than SAM and is

Fig. 1. Simplified schema depicting the main steps involved in homocysteine toxicity followingMTX treatment. SAM functions as a switch between the methionine cycle and the transsulfura-tion pathway. MTHFR is balancing the homeostasis between methylation and DNA synthesisand repair, while eNOS activity is buffering the endothelial HCy toxicity. See text for details.

212 Dev Disabil Res Rev � Pharmagogenetics of Brain Chemotoxicity � Robaey et al.

therefore a potent inhibitor of nearly allmethyltransferases [Hoffman et al.,1979]. Methyltransferases are crucial forbrain functions (e.g., catechol-O-meth-yltransferase catabolizes catecholamineneurotransmitters, phenylethanolamine-N-methyltransferase synthesizes epi-nephrine, etc.). Elevated SAH in Alz-heimer brain inhibited different methyl-transferases and was related to markersof disease progression [Kennedy et al.,2004]. Similarly, reduced availability ofmethionine as a methyl donor affects,among other processes, neurotransmitterformation. In addition, Hcy and itsmetabolic product (excitatory aminoacids, such as homocysteic acid and cys-teine sulfinic acid) can be especiallytoxic to neurons, as they stimulateNMDA receptors directly or indirectlyvia an effect on Naþ/Kþ-pumps, lead-ing to massive increases in calciuminflux, which eventually leads to oxida-tive damage and cell death.

However, the main toxic effect ofHcy accumulation is cerebrovascular is-chemia through oxidative stress [Stamleret al., 1993; Loscalzo, 1996]. As freeamino acid, Hcy exists in either areduced (a thiol RSH) or oxidized (adisulfide RSSR) form. In healthy indi-viduals, the free reduced form is about2% of total plasma Hcy, while themixed disulfide forms (Hcy-SS-cysteine,and Hcy-SS-glutathione-mixed disul-fide, R-SS-R0) accounts for about 30%and the protein crosslinked form (Hcy-SS-protein) for about 70%. The con-centration of free Hcy increases as theplasma total Hcy rises [Mudd and Levy,1995]. In the presence of oxygen andmetal ions, Hcy can autooxidize, gener-ating disulfide forms and highly reactiveoxygen species (ROS) [Misra, 1974;Starkebaum and Harlan, 1986]. ROSgenerated during autooxidation of Hcy-initiated lipid peroxidation at the endo-thelial cell surface. The endotheliumplays a dynamic role in counteractingthe adverse effect of Hcy by secretingnitric oxide (NO), a reaction catalyzedby the endothelial nitric oxide synthe-tase (eNOS) that converts L-arginineand O2 to L-citrulline and NO. NO isan important regulator of vascular tone.Once produced by the normal endothe-lial cell, it diffuses to the smooth musclecells, where it induces relaxation[Ignarro et al., 1987]. NO confers animportant antithrombotic property onthe endothelial surface by inhibiting theadhesion, activation, and aggregation ofplatelets [Azuma et al., 1986; Radomskiet al., 1987]. It also limits the prolifera-tion of smooth muscle cells, but stimu-

lates the migration and proliferation ofendothelial cells, facilitating a remodel-ing of the vasculature after an injury[Fukuo et al., 1995].

NO reacts readily with Hcy andforms S-nitroso-homocysteine. S-ni-troso-homocysteine is one of theS-nitrosothiols that serve as a pool ofmolecules active for vasodilatation andplatelet inhibition. However, the coun-terregulatory mechanisms are eventuallyovercome by chronic exposure of theendothelial cell to high level of Hcy. Asthe production of NO is increasinglycompromised, ROS produced by theautooxidation of Hcy increases the oxi-dative injury of the endothelium, whichfurther decreases the production of en-dothelial NO [Stamler et al., 1993].The relation between total Hcy leveland vascular disease has been demon-strated in Caucasian population throughlong-term prospective studies [Folsomet al., 1998; Stehouwer et al., 1998;Whincup et al., 1999; Wald et al.,2002; Zylberstein et al., 2004] andmeta-analysis [Wald et al., 2002], as wellas in Asian population [Cui et al.,2008].

Interactions between genevariants implicated indifferent anticancertreatment components(e.g., chemotherapyagents, irradiation) areenhancing brain toxicity

Adverse effects may accumulateover time. An increased plasma Hcyconcentration was related to worse cog-nitive performance over a 6-year periodin the normal aging population [Teu-nissen et al., 2005] and was a strong,independent risk factor for the develop-ment of dementia and Alzheimer’s dis-ease [Seshadri et al., 2002]. During thecourse of cancer treatment, high-doseMTX transiently increased Hcy con-centration in the plasma [Kishi et al.,2003], as well as excitatory amino acidderived from Hcy in the cerebrospinalfluid [Quinn et al., 1997; Becker et al.,2007]. Hcy whole-body accumulationalso increased over the courses (3–4days every 2 weeks) of systemic high-dose MTX, and this increase was im-mediately reversed by administration of

folinic acid. Plasma folate concentrationat the start of each cycle (originatingfrom the folinic acid rescue adminis-tered during previous therapeuticcycles) was the principal determinant ofthe extent of whole-body Hcy accumu-lation in response to MTX administra-tion [Sterba et al., 2006]. Hcy accumu-lation has been associated in some casestudies with severe neurotoxic effects[Kishi et al., 2003; Quinn et al., 2004;Valik et al., 2005].

Functional Polymorphismsand Cognition

All enzymes involved in Hcymetabolism are essential for maintainingadequate intracellular folate pools andensuring that Hcy concentrations donot reach toxic levels. Homozygousmutations in genes encoding nonfunc-tional forms of these enzymes are wellknown and lead to severe inborn errorsof metabolism characterized by hyper-homocysteinemia and homocysteinuria[Selhub, 1999]. However, variations inenzyme activity exist in healthy popula-tion. These interindividual variationsare due to the existence of commonfunctional polymorphisms in genesencoding these enzymes. We will focusmore specifically on the polymorphismsof the enzymes that directly control thepathways utilizing Hcy (MTR andMTRR for Hcy methylation and CBSfor the transsulfuration pathway) and its‘‘buffering’’ (eNOS). Given the impor-tance of the balance between DNAmethylation and synthesis, MTHFRvariants have also been considered. Suchpolymorphisms are more plausible can-didates for moderating MTX long-termneurotoxicity if they also constitute arisk for brain dysfunctions or diseasesindependent of exposure to MTX. Thegene polymorphism would also be moreplausible as accounting for long-termneurological adverse effects if thegenetic variant had been showed re-sponsible for modifying the risk foracute brain side effects of MTX. Wewill thus review the polymorphismsassociated with brain adverse effects.

Methylene terahydrofolate reductaseTwo common polymorphisms

have been identified in MTHFR gene,C677T [Frosst et al., 1995] andA1298C [van der Put et al., 1998;Weisberg et al., 1998] base changes,leading to amino-acid substitutions atcodons 222 (Ala-to-Val) and 429 (Glu-to-Ala), respectively. Both variants resultin reduced enzyme activity. Accord-ingly, in homozygotes for the C677T

Dev Disabil Res Rev � Pharmagogenetics of Brain Chemotoxicity � Robaey et al. 213

mutation, Hcy level significantlyincreased when folate status was low,and reached intermediate values in het-erozygotes [Jacques et al., 1996; Girelliet al., 1998]. A sequence homologywith DHFR suggests that the region inMTHFR that contains the Ala residueis involved in folate stabilization ofMTHFR [Goyette et al., 1995; Rozen,1996]. Because of its pivotal role inDNA methylation and synthesis, andthe role of methylation changes in car-cinogenesis [Das and Singal, 2004], thehypothesis that polymorphic variationof MTHFR influences the risk of pri-mary tumors, especially in the brain,has been tested. Increased risk formeningioma and glioma has been foundfor both MTHFR variants [Dutta et al.,2008].

It has also been suspected thatpolymorphisms in genes resulting in adeficit of DNA methylation could alterthe meiotic recombination and segrega-tion, particularly of chromosome 21.However, the association between thesame polymorphisms and Down syn-drome (DS) could also exist through aneffect on fetal survival up to birth [Mar-tinez-Frias et al., 2006]. The allele C ofMTHFR A1298C polymorphism wasassociated with increased risk of havingan offspring with DS, especially formothers above 34 years of age at thetime of conception [Scala et al., 2006],although MTHFR C677 T allele alonewas not associated with the risk of DS[Chango et al., 2005]. In humans,hydrolyzed folates are absorbed in theproximal small intestine by specializedcarriers (reduced folate carrier, RFC1).A common polymorphism at position80 in exon 2 of RFC-1 changes a gua-nine (G) to an adenine (A). Double-homozygous subjects for MHTFR677TT and RFC-1 80GG had thehighest level of Hcy [Chango et al.,2000]. The same gene–gene interactionbetween the MTHFR C677TT andRCF-1 80GG was also suspected inyoung Italian women to increase therisk of having a child with DS [Cop-pede et al., 2006]. In children with DS,IQ correlated with Hcy level, especiallyin carriers of MTHFR C677 T allele[Gueant et al., 2005], but no associationwas found between MTHFR variantsand idiopathic mental retardation [Duttaet al., 2008].

In Turkish patients, an associationwas found between migraine and theMTHFR 677TT genotype [Kara et al.,2003]. This association appeared specificto migraine with aura in Caucasian [Leaet al., 2004; Oterino et al., 2004; Scher

et al., 2006] and Japanese populations[Kowa et al., 2000]. Migraine sufferersare at increased risk of vascular brainlesions, and the MTHFR C677T poly-morphism has been associated with theoccurrence of ischemic stroke [Kawa-moto et al., 2005]. However, the rela-tion between the C677T mutation andcardiovascular disease remains contro-versial [Brattstrom et al., 1998; Changoet al., 2000; Trabetti, 2008].

Cystationine beta-synthaseSeveral polymorphisms have been

described for the CBS gene [Krauset al., 1998]. An insertion of 68 bp inexon 8 (844ins68) was shown to occurin about 5% of Caucasian alleles, butwas not associated with hyperhomocys-teinemia [Sebastio et al., 1995; Kluijt-mans et al., 1997]. However, the pres-ence of the 68 bp insertion abolishedthe high plasma Hcy level observed inhomozygotes for the MTHFR C677T[De Stefano et al., 1998]. In healthymiddle-aged men, MTHFR C677T andthe MTR A2756G alleles increased theHcy plasma level in an additive manner.On the contrary, carrying a CBS844ins68 allele lowered Hcy level. Thislowering effect was seen most stronglyin homozygotes for the MTHFR andMTR variants [Dekou et al., 2001].The carriers of the 844ins68 allele seemto have a higher CBS activity, and abetter control of the balance betweenthe methionine cycle and the transsulfu-ration pathway. In agreement with thisview is the finding that the CBS844ins68 may be a protective factoragainst vascular thromboembolic diseasein the Chinese population [Zhang andDai, 2001]. The fact that the CBS844ins68 allele could play a role in cog-nitive development was suggested bythe fact that this allele was significantlyunderrepresented in children with veryhigh IQ (above percentile 99) as com-pared to normal IQ (percentile 58)[Barbaux et al., 2000].

Methionine synthaseThe A2756G polymorphism in

the MTR gene results in substitution ofaspartic acid by glycine at codon 919. Ithas been suggested that the glycine resi-due, a strong helix breaker compared toaspartatic acid, could affect the func-tional structure of the protein [Leclercet al., 1996; Ma et al., 1999]. Afteradjustment for age, Hcy level and thepresence of the MTR A2756G allelewere significant risks for having a childwith DS, especially if the mother wasalso carrying a MTRR 66G allele

[Bosco et al., 2003]. Combined allelesalso constitute a significant risk for highplasma Hcy level [Laraqui et al., 2006,2007]. Finally, the observation of anacute MTX-induced encephalopathy ina patient homozygous for the variantMTR A2756G gives some support tothis variant as influencing long-termadverse neurological effects [Linnebanket al., 2007].

Methionine synthase reductaseReplacement of the Ile by Met at

position 22 of MTRR caused by anA66G substitution in the MTRR genewas associated with an increased risk ofneural tube defects when cobalaminlevel is low or when an MTHFR 677T allele is present [Leclerc et al., 1999;Wilson et al., 1999]. A meta-analysisconfirmed that mothers with theMTRR 66GG genotype were 55%more at risk of having a child affectedby spina bifida. The risk increased inthe presence of the MTHFR 677TTgenotype, and when the vitamin B12status was low [van der Linden et al.,2006]. A significant association betweenrisk of meningioma and homozygosityfor MTRR 66G was also reported[Bethke et al., 2008].

Endothelial nitric oxide synthaseNO is synthesized by the nitric

oxide synthase family of oxidoreduc-tases. To date, three isoforms of nitricoxide synthase have been cloned andcharacterized: neuronal NOS, inducibleNOS, and endothelial NOS (eNOS). Avariant of eNOS has been identified inexon 7 of the gene (G to T substitutionat nucleotide position 894) resulting ina Glu to Asp change at codon 298[Yoshimura et al., 1998]. The eNOSG894T polymorphism was found to beassociated with asymptomatic whitematter lesions in patients with essentialhypertension, but not with ischemicstroke [Henskens et al., 2005]. Estradiolsignificantly increased platelet aggrega-tion in individuals homozygous for theeNOS G894T variant [Tanus-Santoset al., 2002]. The eNOS 894 TT geno-type interacted with low-serum folateto increase the risk for elevated Hcylevels [Brown et al., 2003]. In turn,high Hcy level interacted with the samegenotype to lead to an increased risk ofrecurrent thrombotic events [Heil et al.,2004] and of coronary artery disease[Kerkeni et al., 2006]. Compromisedbuffering of homocysteinemia bydecreased production of NO anddecreased formation of S-nitroso-homocysteine might lead to more

214 Dev Disabil Res Rev � Pharmagogenetics of Brain Chemotoxicity � Robaey et al.

severe Hcy-mediated oxidative injury ofendothelium in individuals carrying theeNOS gene variant. Another T-786Cmutation resulted in a significant reduc-tion in eNOS gene promoter activityand was associated with coronary spasms[Nakayama et al., 1999] and with severecoronary disease, adding its own contri-bution of other common risk factors forcoronary disease, such as overweight,low LDL cholesterol, and smoking[Rossi et al., 2003].

Testing the effects of polymorphisms of genescontrolling Hcy levels on IQ

In children treated for acutelymphoblastic leukemia, the effects ofdifferent polymorphisms (MTHRC677T, MTHFR A1298C, MTRA2756G, MTRR A66G, NOS3G894T, NOS T-786C, and CBS844ins68) were tested on changes in IQscores over a period of 4 years postdiag-nosis [Krajinovic et al., 2005]. Two var-iants, the CBS 844ins68 and the homo-zygotes NOS3 G894T were associatedwith changes in IQ scores 2 years afterdiagnosis. Consistent with the sugges-tion that carriers of the 844ins68 allelehave a higher CBS activity, IQ scorewas slightly increased in carriers of thisvariant, which could reflect some prac-tice effects and the overall healthimprovement over time. Carriers of twoNOS3 894T alleles showed a loss in IQscores at the end of the first year post-diagnosis that did not recover 3 or 4years after the diagnosis. When bothvariants and clinical variables (gender,age, total number of hospitalizationdays, treatment protocol, and use ofcranial radiation therapy) were enteredin a multivariate model, only the effectof the NOS3 894TT genotyperemained significant, in addition to ageat diagnosis and cranial radiation ther-apy. The IQ decrease was significantonly in the carriers of the NOS3894TT, who also received cranial radia-tion therapy. It is thus possible that thesame gene variant would be implicatedat the crossing of different brain toxicitypathways, which would enhance theircombined harmful effects. This interac-tion was already demonstrated in girlstreated for acute lymphoblastic leuke-mia, as high-dose MTX followed by 18Gy cranial radiation therapy was associ-ated with IQ decline [Waber et al.,1995]. Children treated with intrathecalMTX and 18 Gy CRT were also foundto exhibit more behavioral rigidity andslower, less fluent processing 6 yearspostdiagnosis in later cohorts [Waberet al., 2007]. In order to explore this

issue, we will briefly review the brainradiation-induced toxicity.

Radiation EffectsIn presence of O2, ionizing radia-

tions form free radicals that includehydroxyl radicals (OH�), superoxide(O2

2), and organic radicals (R�). Imme-diately upon formation, these free radi-cals give rise to other ROS includinghydrogen peroxide (H2O2) and organichydroperoxides (ROOH). In the pres-ence of redox active metal ions (such asFe or Cu), these ROS produce more ofthem, through Fenton-type reactionand contribute to oxidative damagewithin a few milliseconds after irradia-tion. In response to irradiation, cellsand tissues increase the expression ofcellular antioxidant defenses [Shimizuet al., 1998; Guo et al., 2003], mitigat-ing the radiation-induced damages.Radiation-induced processes includeredox-sensitive signaling pathways, tran-scription factors activation, gene expres-sion, and metabolic activities that gov-ern the cellular redox state. They mayremain perturbed for minutes, hours, ordays [Spitz et al., 2004].

The main target of oxidativedamage is the cerebral vascular endothe-lium. Disruption of the blood–brainbarrier has long been recognized as theprimary adverse effects of CNS irradia-tion [Rubin et al., 1994]. Irradiationselectively impairs the NO pathway as aconsequence of oxidative stress [Solo-viev et al., 2003]. The deficit in eNOSactivity contributes to impair the endo-thelium-dependent relaxation of theirradiated vessels and to increase theirthrombotic property [Qi et al., 1998;Sugihara et al., 1999], which can bereversed by a free radical scavenger[Zhang et al., 2003]. The endothelialNO synthesis could thus be impairedby irradiation and more easily overcomeby the subsequent oxidative stress asso-ciated with Hcy autooxidation duringMTX treatment. Moreover, other che-motherapeutic agents have the potentialto generate ROS. Doxorubicin is a qui-none-containing anthracycline promot-ing oxidative stress in the brain [Joshiet al., 2005] that could be prevented byg-glutamyl cysteine ethyl ester [Joshiet al., 2007]. Some combinations ofchemotherapeutic agents could thusinteract in a nonadditive manner tocontribute to brain oxidative stress.Similarly, individuals with multiple riskalleles would be more susceptible toradiation effects than those with fewerrisk alleles [Alsbeih et al., 2007], andthe allelic architecture may underlie

interindividual differences in radiosensi-tivity [Andreassen et al., 2003].

As the additional reduction ofCNS relapses provided by irradiationwhen compared with long-term intra-thecal chemotherapy is inexistent orsmall (about 3% absolute benefit), irra-diation could be replaced without detri-ment on event-free or overall survival[Clarke et al., 2003]. With 18 Gy ofcranial irradiation, a large-scale studyestimated the full-scale IQ loss to 0.25standard deviation per year over a pe-riod of 6 years postdiagnosis, whilewithout cranial irradiation, the decreasewas insignificant [Jankovic et al., 1994]Replacing cranial radiation therapy byhigh-dose or very high-dose intrave-nous MTX was found to be associatedwith a favorable long-term neurodeve-lopmental outcome [Spiegler et al.,2006]. However, given the lack of de-finitive proof of efficacy and its toxicity,it has been suggested that high-dose in-travenous MTX should not be consid-ered the ‘‘standard of care’’ for childrenwith acute lymphoblastic leukemia[Mantadakis et al., 2005].

GLUCOCORTICOIDSGCs are used for a prolonged pe-

riod of time and at relatively high dosesin the treatment of ALL in order toreduce cell proliferation, promote cellcycle arrest, and induce cell death byapoptosis.

The theoretical possibility thatGC leukemia therapy could also con-tribute to adverse neurodevelopmentaloutcomes was first raised in 1995[Waber et al., 1995] and was later con-firmed, especially when using dexa-methasone [Waber et al., 2000]. Highdose of GCs impaired the capacity ofneurons to survive various neurologicalinsults, but exclusively or predominantlyin the hippocampus [Packan and Sapol-sky, 1990]. Evidence from a primatemodel indicated that high-dose GCsenhanced the behavioral toxicity ofMTX and irradiation [Mullenix et al.,1994]. Other drugs commonly used incancer chemotherapy have also beenshowed to trigger neurotoxicity. The 5-fluorouracil reduced BDNF and DCXlevels in the hippocampus, indicatingalterations in neurotrophin levels andneurogenesis [Mustafa et al., 2008], andcould also potentially interact withGCs.

The hippocampus plays a support-ive role in associating complexmultimodal information (especially asso-ciating external cues with an internalrepresentation of context) and laying

Dev Disabil Res Rev � Pharmagogenetics of Brain Chemotoxicity � Robaey et al. 215

down new memory traces [Redish,2001]. A meta-analysis of studies inves-tigating the effects of acute GCs admin-istration showed that memory perform-ance was impaired when GCs wereadministered before retrieval, but notwhen they were administered beforelearning [Het et al., 2005], providingpartial support for the model suggestingthat cortisol enhances memory consoli-dation of the current event and its con-text whilst compromising its retrieval inmemory [Roozendaal, 2002].

GCs exert their action by bindingto the GC receptor, which then acts asa transcriptional regulator of respon-sive genes. GC receptors are widelyexpressed in glial cells and neurons inlimbic-cortical brain structures, espe-cially in the hippocampus which plays acrucial role in controlling the hypothal-amo–pituitary–adrenocortical (HPA)axis responsiveness [Herman et al.,2003]. There is considerable variationamong patients with regard to sensitiv-ity to GC treatment and at relapse, aloss of sensitivity to GCs is common[Gaynon and Carrel, 1999]. GC recep-tors signaling can vary at several levels.First, the corticosteroid-binding globu-lin in blood regulates the availability ofcortisol. Second, the multidrug resist-ance P glycoprotein regulates the GCspenetration through the blood–brainbarrier. Third, the binding of GCs tothe multimeric GC receptor–proteincomplex changes its conformation in anactive form after dissociation from heat-shock proteins. Fourth, steroids can beinactivated or regenerated in an activeform. Fifth, the GC receptor is trans-ferred into the nucleus and can interactwith transcription factors. Sixth, therecruitment of corepressors and coacti-vators induces the repression and trans-activation of gene expression, respec-tively [de Kloet et al., 2005].

At the level of the GC receptor-protein, several germ line polymor-phisms have been described in the GCreceptor gene (NR3C1). One of them,the BclI RFLP polymorphism, corre-sponds to a C?G substitution in intron2 and is located 646 bp from the exon/intron boundary (NR3C1 IVS2 þ646C/G) [Fleury et al., 2003]. A reduc-tion in survival probability in childrenwith ALL was associated with beinghomozygotous for the G allele of theNR3C1 BclI RFLP polymorphism,particularly in certain patients classifiedat high risk based on common clinicalcriteria or for a certain type of thetreatment protocol [Fleury et al., 2004].However, in another study, no associa-

tion was found between in vitro and/orin vivo resistance to prednisone and thepresence of the BclI polymorphism[Tissing et al., 2005].

To summarize, among the varioussources of change in GCs action, poly-morphisms of the gene encoding theGC receptor may account for differ-ences in treatment responses. On theother hand, although it is not clear howthis intronic polymorphism would affectthe function (in vitro data are not avail-able), it was associated with HPA reac-tivity and mental health outcomes.Homozygous carriers of the BclI poly-morphism had a 30% increased risk ofdeveloping a major depressive episode[van Rossum et al., 2006]. Beyond theincreased risk for depression, BclI wasassociated with a form of depressionmore resistant to treatment. Patientswith a diagnosis of major depressive dis-order [American Psychiatric Associa-tion, 1994] and treated with a specificserotonin reuptake inhibitor showed atrend toward lower decrease in depres-sion ratings [Williams, 1988] and lower

response rates if they carried the BclIpolymorphism [Brouwer et al., 2006].Treatment response rates were especiallylower in a subgroup of BclI carrierswith high corticotrophin (ACTH)response to a challenge using cortico-trophin releasing hormone (CRH) justafter suppression by dexamethasone[Heuser et al., 1994]. In this study,ACTH levels in response to CRH chal-lenge were higher in BclI carriers, andeven more so in homozygous patientsthan in heterozygous patients [Brouweret al., 2006]. The BclI carriers with aclinical depression were thus character-ized by a hyperresponsive HPA axis.Moreover, the BclI has also been associ-ated with a specific metabolic profilecharacterized by an increased responseto the ACTH- and cortisol-suppressiveeffects of low-dose dexamethasone, anda body composition with increased ab-dominal fat suggestive of GC receptor

hypersensitivity, but only early in life[van Rossum and Lamberts, 2004]. Inline with the risk for depression, theBclI polymorphism is associated withchanges in the HPA response to a socialstressor. Compared with subjects carry-ing two wild-type alleles, the mean sali-vary cortisol response to a standardizedpsychosocial stress [The ‘‘Trier SocialStress Test’’ (TSST)] [Kirschbaum et al.,1993] was attenuated in BclI G homo-zygotes. When the responses were aver-aged across three successive stress expo-sures, no significant elevation of cortisollevels was detectable in BclI G homozy-gotes [Wust et al., 2004]. In a follow-up study, male BclI GG carriers showedrelatively diminished ACTH, serum andsalivary cortisol levels. However,women (all using ethinyl-estradiol con-taining oral contraceptives) showedhighest serum cortisol levels in responseto the TSST, which points toward sexby genotype interaction [Kumsta et al.,2007]. The cortisol response could thusvary with the BclI polymorphisms indepressed and nondepressed individuals,and among nondepressed individualswith gender, or with the nature of thestressor.

Children treated for acute lym-phoblastic leukemia with chemotherapyreceived higher rating by their parenton hyperactivity/inattention, emotionallability, sleep disturbances, listlessness,and difficulty in peer relationship dur-ing the week when they received GCswhen compared with the week they didnot receive GCs [Harris et al., 1986;Drigan et al., 1992]. One can hypothe-size that children treated with GCs havedifferent response according to GC re-ceptor gene variants, but also that theseeffects can vary according to the childand the family mechanisms of copingwith the stress of the disease and of itstreatment. The same genetic variant ofthe GC receptor gene could thusenhance or decrease the HPA responsesand hence the behavioral/affectiveproblems as a function of the psychoso-cial environment, with potentiallyadverse long-term neurobehavioral andpsychosocial consequences.

In addition to the BclI RFLPpolymorphism, other germ line poly-morphisms may moderate the risk ofdeveloping late neurodevelopmentaleffects following GC treatment. Forexample, different single nucleotidepolymorphisms (SNPs) changed in vitrothe transactivational capacity [ER22/23EK (allele frequency: 3%), N363S(4%), A3669G (15%)] or the stability ofthe mRNA of the GC receptors (GR

Interindividualvariability in

glucocorticoids adverseeffects may be dependenton individual andfamilial psychosocial

functioning

216 Dev Disabil Res Rev � Pharmagogenetics of Brain Chemotoxicity � Robaey et al.

exon 9b A3669G). These GC receptorSNPs (ER22/23EK, N363S, 9bA3669G) can change the dexametha-sone-induced negative feedback on theHPA axis, and several of these variantshave been found associated with depres-sion [Derijk and de Kloet, 2008]. Fur-thermore, a common SNP (rs10482605TC) is located in the promoter regionreduced transcriptional activity and wasin high-linkage disequilibrium with the9b A3669G SNP, creating a haplotypethat could increase the risk for stress-related disorders [Kumsta et al., 2008].The same is true for some mineralocor-ticoid receptor variants that are foundin limbic neurons (hippocampus, amyg-dala, septum) and show a high affinityfor cortisol in these regions. Both min-eralocorticoid and GC receptors operatein balance and genetic variants mayexplain individual differences in toxiceffects of treatment as well as in stressresponsiveness. These potential effectsmay also vary with other genetic effectsunrelated to GC receptors. For exam-ple, cytosolic glutathione S-transferases(GST) play an important role in theprotection against products of oxidativestress. They have been suggested to playa role in steroid metabolism/resistance.The GSTT1 deletion polymorphismwas associated with initial response toGCs in childhood acute lymphoblasticleukemia [Meissner et al., 2004] andmay thus also contribute to adverseeffects. Cytochrome P450 family ofenzymes polymorphisms can alsoaccount for interindividual differencesin response to various drugs. CYP3A4is highly inducible by GCs and, besidesits role in the metabolism of a variety ofdrugs and carcinogens, it catalyzes the6-b-hydroxylation of steroids [Fleuryet al., 2004]. Interindividual variabilityin CYP3A4 activity can partly beexplained by a 2290A/G polymor-phism in the CYP3A4 promoter, alter-ing RNA and protein expression[Tomlinson et al., 1997].

CONCLUSIONSDemonstrating a relationship

between a polymorphism at a singlelocus and a global neurodevelopmentaloutcome for a treatment that includesmultiple drugs exerting cytotoxicitythrough a number of different pathwaysmay appear as an insurmountable chal-lenge. The fact that over the last 25years, only two examples of pharmaco-genetic approaches have gone fromidentification of gene variants to accep-tance in practice (TPMT for 6-MP andUGT1 for irinotecan) in the prediction

of acute systemic toxicity is by itself ameasure of this challenge [Tomlinsonet al., 1997; Davies, 2006]. Pharmaco-genetic studies will also have to reportdata from studies performed as long as adecade previously, as clinical endpointssuch as IQ loss must be reached beforethe effect of a genotype can be tested.In addition, many factors can influenceglobal neurodevelopmental outcome.Alternatively, surrogate endpoints ofoutcome obtained by brain imaging orbrain event-related potential could beused as long as they provide earlier andreliable markers of the pathological pro-cess that lead to long-term negativeneurodevelopmental effects. More spe-cific and supposedly sensitive endpoints(e.g., short-term memory measure)could be used, at the cost of limitingtheir clinical significance in daily life.However, whichever the endpointsselected to measure neurodevelopmentaloutcome are, they should always beseen as complex developmental traits. Inorder to explain the interindividual dif-

ferences for such complex traits, mostlikely there will be a substantial numberof genes generating small or very smallneurodevelopmental adverse effects(much smaller than age or socioeco-nomic status for example), and few (ifany) with moderate or large effects, atleast during normal development.Adopting a genome-wide approachrather than a candidate gene approachcould allow identification of previouslyunknown genes, but at the cost oflarger sample size and of a significantrisk of false positives that will have tobe further tested using ‘‘candidate gene’’strategies. Difficulties also includeapplicability limited to specific treat-ment protocols and the challenge ofunraveling complex gene–gene or gene-environment interactions.

A promising approach to thisdaunting task could be to build models

that integrate different fields of knowl-edge, such as neurodevelopmental physi-ology, biochemistry, pharmacology, etc.[Dollery, 2007], and search for largeeffects. In order to account for the out-come of a development that has becomeatypical since the diagnosis, the bestmodel could be a multiple hit model. Ina multiple hit model, genetic influencesand early life events determine vulnera-ble phenotypes that precede the diagno-sis of cancer. This review suggests thatthe same type of pathological mecha-nisms could be at play in determiningthe precancer vulnerability and theadverse effects of cancer treatment, de-spite the very specific and unusual natureof cancer treatment. For example, oxida-tive stress affecting the cerebral endothe-lium, neurons, and glial cells appears as amajor mechanism shared by agents asdifferent as MTX and irradiation. Oxi-dative stress is also a major mechanismimplicated in the developmental conse-quences of anoxia/ischemia after a pre-mature birth for example. Free radicalsplay a central role in the relationshipbetween ischemia/reperfusion and oli-godendroglial cell death [Volpe, 1998;Blomgren et al., 2007]. HPA axis, espe-cially some of its key factors as the GCreceptors, maternal care may programepigenetically the expression of the GCreceptor gene. Epigenetic differencescould have the same consequences asgenetic polymorphisms and be influ-enced by polymorphisms [Szyf et al.,2007]. Small effects influenced by thesame gene variants and involving thesame or intricate pathways could thusaccumulate over time before and duringtreatment, and ultimately account for adetectable effect. Gene–gene interactionsand gene–environment interactionscould be more easily modeled and testedin such a multiple hit model, as theycould also be recurrent. A longitudinalapproach that allows analyzing changesover time in the same children is alsomandated by a multiple hit model. Nowthat progresses have been made indecreasing the central neurotoxicity ofcancer treatment (although muchremains to be done), innovative strat-egies could allow us to rise to the chal-lenge of addressing the more specificneeds of the children who carry a greatergenetic risk for adverse neurodevelop-mental outcome. n

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