[CANCER RESEARCH 55, 1670-1674, April 15, 1995]

Quantitation of 6-Thioguanine Residues in Peripheral Blood Leukocyte DNAObtained from Patients Receiving 6-Mercaptopurine-basedMaintenance Therapy1

David J. Warren,2 Anders Andersen, and Lars S10rdal

Department of Clinical Pharmacology, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway

ABSTRACT

The antimetabolite 6-mercaptopurine is widely utilized in maintenance

therapy for childhood acute lymphoblastic leukemia. Following p.o. administration, this prodrug undergoes extensive biotransformation, resulting in the generation of a plethora of metabolites including 2'-deoxy-6-

thioguanosine triphosphate. Incorporation of 6-thioguanine (6-TG) basesinto DNA is generally considered to be central to thiopurine-mediated

cytotoxicity. We have developed a novel precolumn derivatization HPLCtechnique for quantifying 6-TG base accumulation into leukocyte DNAobtained from acute lymphoblastic leukemia patients receiving 6-mercap

topurine maintenance therapy. The method is based on enzymatic degradation of DNA to 2'-deoxyribonucleosides and the derivatization of released 2'-deoxy-6-thioguanosine with a thiol-reactive reagent containing a7-amino-4-methylcoumarin-3-acetic acid fluorophore. The 2'-deoxy-6-

thioguanosine-7-amino-4-methylcoumarin-3-acetic acid adduct is resolvedby reversed-phase HPLC and quantified fluorometrically. Assay responseis linear from 15 pmol to 60 fmol 6-TG bases/fig DNA with a limit ofquantitation corresponding to the incorporation of 1 6-TG residue per

50,000 bases. In a small cohort of acute lymphoblastic leukemia patientsreceiving p.o. 6-mercaptopurine-based maintenance therapy, significantinterindividual variation in the accumulation of 6-TG bases into leukocyte

DNA was revealed. The determined levels of drug base incorporationranged from 95 to 710 fmol 6-TG bases/un DNA (6-TG base:nucleotide

ratio 1:32,000 to 1:4,000). The assay may provide a novel methodology forpharmacological monitoring of thiopurine therapy either in the routineclinical setting or during studies of alternative routes of drug delivery.

INTRODUCTION

The introduction of aggressive multimodal therapy for childhoodALL3 has resulted in a high proportion of individuals achievinglong-term DPS. The potential permanence of these "cures" is amply

demonstrated by 56% of the cohort treated in the St. Jude study V(1967-1968) being alive and in complete remission 20 years after

cessation of therapy (1). Initial remission induction therapy for lowand intermediate risk ALL is successful in >95% of cases, but withoutadequate maintenance and consolidation therapy the majority of individuals experience relapse within 1-2 months (2). Even when ap

propriate secondary therapy is provided approximately 30% of patients relapse, usually while receiving maintenance treatment (3). Inthose individuals who suffer relapses during therapy a subsequentremission is usually of short duration. Thus, although remissioninduction in ALL is relatively straightforward, maintenance of remission remains a major problem and many patients succumb to thisotherwise treatable disease. Recent observations indicate that, at least

Received 12/21/94; accepted 3/6/95.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by grants from the Norwegian Cancer Society and the Kate

Forsberg Legacy. D. J. W. is a Norwegian Cancer Society Senior Researcher.2 To whom requests for reprints should be addressed.3 The abbreviations used are: ALL, acute lymphoblastic leukemia; DPS, disease-free

survival; AMCA-HPDP, W-[6-(7-amino-4-methylcoumarin-3-acetamido)hexyl]-3'-(2'-pyridyldithio)propionamide; 6-TG, 6-thioguanine; 6-MP, 6-mercaptopurine; S6dGMP,2'-deoxy-6-thioguanosine 5'-monophosphate; S6dGuo, 2'-deoxy-6-thioguanosine; AUC,

area under the curve; TON, thioguanine nucleotide; CV, coefficient of variation.

in the low- to intermediate-risk groups, a substantial number of

relapses may be related to inadequate dosing of the chemotherapeuticagents used in remission maintenance therapy (4). These studiesindicate that the instigation of individual dose tailoring, based onpharmacokinetic evaluation at the commencement of maintenancetreatment, together with routine monitoring during the period oftherapy, may lead to significant increases in the number of patientsmaintaining long-term DPS.

Since the late 1960s, daily p.o. 6-MP for a period of 2-3 years,

combined with weekly p.o. methotrexate treatment, has been the"backbone" of maintenance regimens for childhood ALL. In these

protocols 6-MP is administered in standardized doses which are

titrated on the basis of routine peripheral WBC determinations. However, the relationship between bone marrow 6-MP exposure and

subsequent alterations in peripheral blood cell counts is poorly understood and open to misinterpretation (5). The comparatively recentuse of HPLC methods for the determination of plasma 6-MP concen

trations has revealed highly variable drug levels following p.o. administration (6-8). This wide variation in plasma 6-MP levels indi

cates that it is impossible to deduce the degree of systemic drugexposure from standardizing the dose by body weight or area. Indeed,in a study of 19 children, Sulh et al. (7) noted 6-MP AUCs (normalized to a drug dose of 1 mg/m2) of between 75 and 815 ng X min/ml

and identified an individual who failed to achieve detectable plasma6-MP concentrations after two separate drug challenges. These studiesindicate that the current practice of 6-MP dosing may result in

suboptimal therapy and that inadequate systemic exposure to thisagent may be a causal factor in relapse during maintenance therapy.The importance of optimalizing 6-MP therapy is supported by recent

studies that demonstrate a correlation between systemic drug exposure(AUC) and risk of relapse in children receiving 6-MP for remission

maintenance in ALL (9, 10).The routine use of plasma AUC determinations as a basis for 6-MP

dosing is complicated by a requirement for serially timed bloodsampling, the wide intraindividual variation in plasma AUC (8), andthe influence of concomitant methotrexate therapy on AUC determinations (11). A more fundamental problem is that 6-MP is an inactive

prodrug and hence its plasma concentration may poorly reflect thelevels of its active metabolites at their intracellular sites of action. Inan attempt to overcome these problems a number of investigators haveinstigated the pharmacological monitoring of a population of long-lived 6-TGN metabolites which accumulate in patient erythrocytesduring 6-MP therapy (4). These studies have revealed significantinterindividual variations in erythrocyte 6-TGN concentrations and a

possible correlation between metabolite levels and risk of relapse. Itshould be remembered, however, that erythrocytes possess quantitatively different purine metabolic pathways when compared to nucleated cell populations. Thus, both the relative concentrations and rangeof metabolites observed in erythrocytes may not adequately reflecttheir levels in the nucleated leukemic cell population.

6-MP is metabolically activated to 6-thioinosinate (6-thioinosine5'-monophosphate) by the purine salvage pathway enzyme hypoxan-

thine-guanine phosphoribosyltransferase. Subsequent biotransforma-

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DETERMINATION OF 6-THIOGUAN1NE IN LEUKOCYTE DNA

tion results in the generation of a plethora of nucleotide metabolitesincluding 6-thiodeoxyguanosine 5'-triphosphate and 6-thioguanosine5'-triphosphate which can be incorporated into nucleic acids as 6-TG

bases. Several observations suggest that incorporation of 6-TG

residues into DMA is a major determinant of thiopurine toxicity.These include the demonstration of single-strand damage, double-strand breaks and interstrand cross-links in DNA from thiopurine-exposed cells (12-14). 6-Thiodeoxyguanosine 5'-triphosphate is

an efficient in vitro substrate for mammalian DNA polymeraseenzymes (15). In contrast, DNA containing incorporated 6-TG

bases has been found to be a poor substrate for the ligase enzymesinvolved in DNA replication (16). These observations suggest thatperturbations in DNA-protein interactions resulting from 6-TG

incorporation may be responsible for the DNA damage associatedwith thiopurine exposure (16, 17).

In view of the apparent relationship between 6-TG incorporationinto nucleic acids and cytotoxicity the determination of 6-TG bases in

leukemic cell DNA would most probably provide a useful system formonitoring patients receiving thiopurine therapy. However, the difficulty in obtaining sufficient quantities of purified leukemic cells frompatients in remission precludes this approach. As mature circulatingblood cells are lost through aging or injury they are replaced by theextensive proliferation and differentiation of a population of hemato-

poietic progenitor cells present in the bone marrow. Thus, in an ALLpatient receiving thiopurine therapy significant 6-TG base incorpora

tion must occur during the extensive DNA synthesis associated withhematopoiesis. As the mature end-stage differentiated blood cells

leave the bone marrow and enter the circulation, they will carry the6-TG bases accumulated during their production in the hematopoietictissues. Thus the determination of DNA-associated 6-TG bases in

circulating nucleated blood cells should provide an indication of bonemarrow, and hence leukemic cell, drug exposure. In view of theextensive literature on thiopurine drugs accumulated over the last 40years it is surprising that few methods are available to measurethiopurine uptake into DNA. A HPLC assay based on DNA hydrolysisand quantitation of a fluorescent oxidation product of 6-TG by anion-

exchange HPLC has been described (18). This method is poorly suitedfor therapeutic drug monitoring due to its low sensitivity and hencethe large quantities of pure DNA needed for analysis.

In this report we describe a novel method for the determination of6-TG residues in leukocyte DNA obtained from ALL patients on6-MP maintenance therapy. The procedure involves enzymaticdigestion of DNA containing 6-TG bases to nucleosides, derivatiza-tion of the released S6dGuo with the thiol-reactive fluorophore

AMCA-HPDP, and adduci quantitation by reversed-phase HPLC with

fluorescence detection.

MATERIALS AND METHODS

Reagents. P, nuclease was obtained from Boehringer Mannheim GmbH,Mannheim, Germany. Acid phosphatase and general laboratory reagents werepurchased from Sigma Chemical Co., St. Louis, MO. HPLC grade methanolwas from Rathburn Chemicals, Ltd., Walkerburn, United Kingdom. AMCA-

HPDP was supplied by Pierce Chemical Company, Rockford, IL. Aqueousreagents and mobile phases were made up in water purified by reversedosmosis followed by polishing with a Milli-Q UF-PLUS system (Millipore

Corp., Bedford, MA). All pH determinations were undertaken at roomtemperature.

Apparatus. Chromatographie equipment was produced by ShimadzuCorp., Tokyo, Japan. The solvent delivery system consisted of a DGU-3Aon-line degasser coupled to a LC-9A quaternary pump. Column temperaturewas maintained using a CTO-6A column oven and on-line solvent preheater.Samples were injected with a SIL-9A autoinjector maintained at ambient

temperature. An RF-551 scanning fluorescence detector was used. Plotting andintegration were performed by a Chromatopac C-R6A intergrator.

Chromatography. Chromatography was performed on a Supelcosil LC-8

column (4.6 x 150 mm; particle size, 3 jxm; Supelco, Bellefonte, PA) equippedwith a 20-mm Supelguard precolumn. The mobile phase consisted of 0.2 M

sodium formate buffer (pH 4.0):methanol (63:37, v/v). The formate buffer wasmade by pH adjustment of a solution of formic acid with 10 M sodiumhydroxide. The mobile phase was delivered at a rate of 1 ml/min and thecolumn temperature was maintained at 45°C.The fluorescence detector was

operated at excitation and emission wavelengths of 345 and 450 nm, respectively. Twenty-five /j.1of derivatized sample were injected. Between analyses

the column was washed for 3 min with 0.2 M sodium formate buffer (pH4.0):methanol (20:80, v/v). The working and rinsing fluid for the autoinjectorwas a 60% (v/v) solution of methanol in water.

Thiopurine Standard Solution. Chemically synthesized S6dGMP (gener

ously provided by Dr. J. Arly Nelson, M. D. Anderson Cancer Center,Houston, TX) was repurified by anion-exchange HPLC and used to prepare a

50 JIM stock solution in degassed water.Cell Culture. The acute lymphoblastic leukemia cell line MOLT-4 was

cultured as described previously (19). Cells were maintained in logarithmicphase and diluted to 5 X IO5 cells/ml in prewarmed medium prior to drug

exposure.DNA Extraction. Whole blood (2 ml) was collected using EDTA as

anticoagulant and stored at -20°C. Specimens were obtained during routine

phlebotomies following institutional approval and informed consent of thepatient's guardian. Frozen samples were thawed at room temperature, diluted

with an equal volume of PBS, and centrifuged at 3,000 X g for 15 min atambient temperature. The nucleated cell pellet was resuspended in 2(H) ;ul ofPBS. DNA was then extracted using a commercially available spin columnmethod (QIAamp Blood Kit; Diagen GmbH, Hilden, Germany). In brief, thedigestion mix was treated with proteinase K for 10 min at 70°C.The mixture

was then applied to a disposable spin column by centrifugation (1 min) in amicrofuge. The column was washed (twice for 1 min each) with buffer and thepurified DNA eluted with 200 /il of 10 mM Tris-HCl-0.1 mM EDTA (pH 9.0).DNA was quantitated by absorption at 260 nm using a UV 1201 spectropho-

tometer (Shimadzu Corp.). Prior to derivatization, DNA samples weredenatured at 100°Cfor 5 min followed by rapid chilling on ice.

Derivatization of Thiopurine-containing DNA. To 100 /.il of spin columneffluent (1-5 /ng DNA) 10 fil of digestion buffer (500 mM sodium acetatebuffer-10 mM MgCl2, pH 4.5) were added prior to addition of 20 p,l of enzyme

stock solution (25 fig/ml P, nuclease, 12.5 units/ml acid phosphatase in 50 mMsodium acetate buffer-1 HIMMgCl2 , pH 4.5). Enzyme digests were incubatedat 42°Cfor l h followed by the addition of 10 /¿I400 mM formic acid and 60

fi\ methanol. Samples were derivatized overnight at room temperaturefollowing the addition of 1 ¿ilAMCA-HPDP (5 mM stock in DMF).

RESULTS

Chromatograms obtained after the enzymatic digestion and precolumn derivatization of spin column-purified DNA from thetissue culture cell line MOLT-4 are shown in Fig. 1. Each chro-

matogram was obtained by injecting 25 /nl of the derivatizednucleoside solution corresponding to 600 ng of starting DNA. Peakexcitation and emission wavelengths of the S6dGuo-AMCA adduci

were established by prior scanning of an adduci preparation dissolved in mobile phase (data noi shown). When DNA obtainedfrom non-drug-exposed cultures (control DNA) was spiked withchemically synthesized S6dGMP before sample processing theappearance of a novel peak corresponding to the S6dGuo-AMCA

adduci was observed. This component eluled as a symmelricalpeak with a relenlion lime of 11.5 min with minimal interferencefrom reagent peaks or endogenous components present in Ihe DNAsample. Similarly, DNA from MOLT-4 cells previously exposedfor 18 h lo 10 nM concenlralions of eilher 6-TG or 6-MP was found

to form a chromatographically idenlical adduci on derivalizalionwilh AMCA-HPDP. A peak eluling al 11.5 min, associaled withthe S6dGuo-AMCA adduci, is observed only when DNA is

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DETERMINATION OF 6-THIOGUANINE IN LEUKOCYTE DNA

Untreated S6dGMP spiked 6-TG 6-MP

lp

l"öS

13 0 13 0 13 0

MinutesFig. l. Chromatograms of derivatized nucleosides obtained using DNA from untreated

MOLT-4 cells, untreated cells with DNA spiked with chemically synthesized S6dGMP

prior to derivatization, and cells previously exposed in vitro to 10 nM concentrations ofeither 6-TG or 6-MP for 18 h. Each chromatogram was obtained by injecting 25 (il ofderivatized nucleoside sample corresponding to 600 ng of starting DNA. *, peaksassociated with the S6dGuo-AMCA adduci.

obtained from cells preexposed to thiopurine drugs. A consistentobservation is that the levels of 6-TG base incorporation intocellular DNA following treatment of cell lines with 6-MP are lower

than those seen in cells treated with equimolar concentrations of6-TG.

The stability of the sulfhydryl group of S6dGMP during the nucle-

ase/phosphatase digestion step was determined by processing controlDNA (5 jag) which had been spiked with S6dGuo either before

enzymatic digestion or immediately prior to AMCA-HPDP addition.Observed recoveries were 85.9 ±2.9% (SD) when S6dGuo was added

to the DNA solution to a concentration of 100 nM and 86.4 ±7.0%(n = 3) when it was added to 10 nM. Within-run and between-day CVs

were determined using two samples obtained by diluting DNA fromMOLT-4 cells following exposure to 6-TG in control DNA. On assay,these samples were found to contain 2 and 0.1 pmol 6-TG bases//xgDNA. Within-run CVs of independently digested and derivatizedsamples (5 jag DNA/replicate) were 2.6 and 6.5% (n = 6) for samplescontaining 2 and 0.1 pmol 6-TG bases//ag DNA, respectively.Between-day CVs, determined by daily (n = 6) digestion andderivatization of DNA aliquots stored at -20°C, were 6.6% at 2 pmol

and 8.2% at 0.1 pmol 6-TG bases//o.g DNA.To determine assay linearity, DNA from 6-TG-exposed MOLT-4

cells containing 30 pmol 6-TG bases//j,g DNA (assayed as described

in Ref. 19) was serially diluted in control DNA. The samples (5 /u,gDNA/replicate, n = 3) were then individually heat denatured, nucle-

ase/phosphatase treated, and derivatized. As shown in Fig. 2, the assaydisplayed a linear response from 15 pmol 6-TG bases/jug DNA downto the lowest concentration tested, i.e., 59 fmol 6-TG bases//u.g DNA.

In addition, the assay displayed good precision with SDs of the means<10% for each of the concentrations tested.

Fig. 3 shows four chromatograms produced by derivatizing humanperipheral blood leukocyte DNA from one non-drug-treated individual

and three ALL patients receiving maintenance therapy. Each of thepatients had been receiving 6-MP-based therapy (50-75 mg/m2) for at

least 1 year. In each case derivatized nucleosides equivalent to 625 ng ofstarting DNA were injected per run. The unique peak associated with theS6dGuo-AMCA adduci was present after derivatization of DNA samples

from the three ALL patients but absent in the sample from the nontreatedindividual. We have consistently failed to observe the characteristic peak

at 11.5 min on derivatization of leukocyte DNA from individuals whohave not been exposed to thiopurine drugs.

The assay was utilized to quantitate 6-TG base accumulation intoleukocyte DNA obtained from nine ALL patients receiving 6-MP-

based maintenance therapy (Table 1). All patients had been receivingmaintenance therapy for at least 12 months before blood samples weredrawn for DNA preparation. With the exception of one patient, eachof the subjects had been treated with a daily p.o. dose of 6-MPaveraging 50-75 mg/m2. In these individuals significant variations in

the accumulation of 6-TG bases into leukocyte DNA were observedwith values ranging from 130-710 fmol 6-TG bases//ag leukocyte

DNA. Since 1 /ag DNA contains 3 nmol nucleotide bases, these levelsof drug base accumulation correspond approximately to the incorporation of 1 6-TG base for every 4,000-23,000 nonthiolated bases. Thelowest level of 6-TG base accumulation (95 fmol 6-TG bases/jug

leukocyte DNA) was seen in the patient who had also been receiving

I8

a

Io>

I13

y = -2.1 + 18.8x, r2 = 0.998

IO1 IO2 IO3 IO4 IO5

6-TG base content (fmol///g DNA)Fig. 2. Titration of DNA obtained from MOLT-4 cells following exposure to 6-TG.

DNA from 6-TG-exposed cells (containing 30 pmol 6-TG bases//ig DNA) was serially

diluted in DNA obtained from nonexposed cells. Each dilution was divided into aliquots(5 ^g DNA each) and independently digested and derivatized. SDs were <6.5% (n = 3)

for all concentrations examined.

Untreated Patient 1 Patient 2 Patient 3

O

8M

§

T3Oí

13 O

MinutesFig. 3. Chromatograms obtained by derivatizing leukocyte DNA from one untreated

individual and three ALL patients who had been receiving 6-MP-based maintenancetherapy (50-75 mg/m2) for at least 1 year. Derivatized nucleosides corresponding to 625ng of starting DNA were injected. *, peaks associated with the S6dGuo-AMCA adduci.

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DETERMINATION OF 6-THIOGUANINE IN LEUKOCYTE DNA

Table l 6-TG base accumulation into leukocyte DNA of ALL patients receiving 6-MP

maintenance therapyThe concentration of 6-TG residues in peripheral blood leukocyte DNA from nine

patients receiving 6-MP-base maintenance treatment for ALL was determined. Eachindividual had been receiving 6-MP, 50-75 mg/m2 p.o., except patient I (25 mg/m2), for

at least 1 year. In three patients (numbers in parentheses) an additional determination wasmade on blood drawn 2-3 months after the first determination had been made. Data are

means of triplicate Chromatographie runs.

PatientABCDEF0HI6-TG(fmol/ng DNA) 6-TG:nonthiolated baseratio710

1:4,000700(670)1:4,000500(610)480410400270130

(150)95:6,000:6,000:

7,000:7,000:11,000:23,000:32,000

the lowest average dose of 6-MP (25 mg/m2). In three of the nine

patients we have determined DNA-associated 6-TG bases in peripheral leukocyte DNA obtained from blood drawn between 2 and 3months after the first determination was made. The values of 6-TGbase accumulation obtained on analysis of these repeat samples agreewell with those found in the initial determinations. However, the smallpatient cohort studied to date precludes an assessment as to whether asteady state occurs in individuals on continuous therapy.

DISCUSSION

The development of intensive chemotherapy regimens has dramatically improved the prognosis of children with low to intermediaterisk ALL. Recently, a number of aggressive induction-consolidationprotocols which utilize repeated "reinduction" phases with maximumtolerated doses of multiple presumably non-cross-resistant agentshave been introduced. These protocols have resulted in >90% 2-yearDPS in individuals displaying poor prognostic features at presentation(20, 21). The improvement in the prognosis of high-risk individualstreated with these therapies will almost inevitably result in theirapplication to lower-risk individuals in an attempt to increase theproportion of patients maintaining long-term DPS. However, the useof these strategies is associated with serious toxicities including fatalopportunistic infections and the spectre of long-term side effectsassociated with aggressive combination chemotherapy. In particular,considering the high proportion of standard-risk individuals expectedto maintain long-term DPS, the extensive use of anthracyclines isassociated with significant cardiotoxicity which may manifest itselfup to 20 years after therapy (22). Thus, at least in the low- tointermediate-risk individuals, we are most probably approaching thelimits of treatment intensity and alternative strategies must be used toincrease the proportion of patients maintaining long-term DPS. In thisregard, the highly variable pharmacokinetics during 6-MP maintenance therapy indicates that dose adjustment based on pharmacological evaluation may result in improved numbers of standard-riskpatients maintaining long-term remission. Furthermore, by using thisapproach, additional therapeutic stategies, associated with a higherrisk of acute and long-term toxicities, could be reserved for thoseindividuals who consistently display unfavorable 6-MP pharmacokinetics. Since the accumulation of 6-TG bases into nucleic acid appearsto mediate thiopurine cytotoxicity, quantitation of thiolated bases inDNA may provide an informative parameter for pharmacologicalmonitoring of patients receiving 6-MP therapy.

We have previously described a HPLC-based assay for the determination of 6-TG residues in DNA obtained from tissue culture celllines exposed to thiopurines (19). Herein, we report a variation of thistechnique that permits the determination of 6-TG bases in leukocyte

DNA obtained from ALL patients receiving 6-MP maintenance therapy. Substantial modifications to the original method have been madeto speed up sample handling and enhance assay sensitivity. Theseinclude the use of a commercially available spin column DNApurification technique and the application of a one step nuclease/phosphatase digestion method. Additionally, the monobromobimanefluorophore used in the original method has been replaced with anAMCA derivative which has a significantly higher absorbance andfluorescence yield resulting in an approximately 50-fold increase inassay sensitivity.

We have used a number of techniques for preparing leukocyte DNAfor derivatization including traditional phenol/chloroform extraction(23), salt precipitation (24), or silica-based (25) methods, each withsatisfactory results. The spin column DNA extraction procedure described can rapidly handle large numbers of samples and providesmaterial of high purity (A2haIA2m> 1.8). A convenient feature of thespin column method is that DNA can be subjected to enzymaticdigestion in the column elution buffer without prior ethanol precipitation. Additionally, we have noted that DNA can be stored in columnelution buffer for at least 9 months at —20°Cwithout influencingassay results. Incorporated 6-TG residues are released from the purified DNA in the form of S6dGuo by enzymatic digestion. The use of

a combination of Pt nuclease and acid phosphatase permits the simultaneous digestion and dephosphorylation of samples at low pH allowing a degree of protection of the drug sulfhydryl from oxidation. Thisfeature may be partially responsible for the high (>85%) analyticalrecovery of S6dGuo noted even at analyte concentrations as low as 10

nM.Although the derivatization process requires multiple steps, it ishighly reproducible and relies on the sequential addition of reagents.Optimal reaction conditions were established in preliminary studiesand confirmed by the failure of increases in individual reagent concentrations to influence adduci yield (data not shown). Subsequent tothis optimalization procedure the method has displayed consistentlyhigh precision revealed by low within-run and between-day CVs.

The Chromatographie system used provides good resolution of theS6dGuo-AMCA adduci from derivatizalion biproducts using a staight-

forward isocralic elulion methodology. Evidence for correct peakidentification includes its presence only when DNA from thiopurine-exposed cells is used and the identical Chromatographiebehavior of anadduci formed on derivatizing chemically synthesized S''dGMP. Additionally, concomilanl treatment of MOLT-4 cells with mycophe-nolic acid, an inhibitor of inosinate dehydrogenase, totally abrogatesthe detection of DNA-associated thionucleolide in cultures exposed to6-MP but not those treated with 6-TG (data not shown).

The titration of 6-TG-containing DNA in this assay demonstratedgood linearity and sensitivity (Fig. 2). A detection limit of 60 fmol6-TG bases//j.g DNA indicates that the method can quantify 6-TGbase incorporation as low as approximately 1 thiolated base in every50,000 nucleotides. Although we have found it unnecessary, sensitivity can be further increased by derivatization of larger quantities ofstarting DNA or by the injection of larger sample volumes, bothwithout compromizing resolution.

The assay was used to determine 6-TG base accumulation intoleukocyte DNA in a small cohort of individuals on 6-MP therapy forALL (Table 1). In eight of the patients, each of whom had beenreceiving p.o. 6-MP averaging 50-75 mg/m2 for >1 year, significantinterindividual variation in DNA-associated 6-TG bases was noted.The levels of 6-TG residues incorporated were found to vary over a5-fold range from 130 to 710 fmol 6-TG bases//xg DNA corresponding to 1 thiolated base for every 4,000 to 23,000 nucleotides. Thesevalues represent, to our knowledge, the first quantitation of 6-TGaccumulation into the DNA of patients receiving 6-MP asmaintenance therapy.

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DETERMINATION OF 6-THIOOUANINE IN LEUKOCYTE DNA

The complexity of 6-MP metabolism has hampered the identification of a single 6-MP biotransformation product which may serve as

a logical target for pharmacological monitoring. Still, assays whichmeasure both the inactive parent compound (9, 10) and erythrocyte6-TGNs (4) have been shown to have predictive value and may indeedenhance the efficacy of 6-MP-based maintenance therapy. Since themajority of the peripheral blood cells are not actively dividing, 6-TG

accumulation into leukocyte DNA must principally occur during theontogeny of these cells. Thus, the data from this assay should reflect themean bone marrow drug exposure during the development of the matureblood cell population. Approximately 80% of first relapses in ALL occurin the hematopoietic tissues, indicating that maintenance of adequatebone marrow drug exposure may be of considerable importance. Themethod may also prove to be useful in limited sampling strategies forpharmacological monitoring of 6-MP therapy and in the long-term as

sessment of patient compliance. In contrast to the commonly used methodologies, the main advantage of monitoring 6-TG base incorporation

into DNA may lie in the ability to quantify a terminal cytotoxic metabolite the accumulation of which represents the net result of the interplayof a multitude of anabolic and catabolic events.

In conclusion, we describe an assay for quantitation on 6-TGresidues in leukocyte DNA obtained from individuals taking thiopu-

rine drugs. The assay is sensitive and specific and permits the analysisof large numbers of samples. This method may be useful for clarifyingthe relationship between 6-TG uptake into DNA and the maintenance

of remission in ALL. In addition, it may provide a basis for improvedclinical monitoring during thiopurine pharmacotherapy.

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2. Lonsdale, D., Gehan, E. A., Fernbach, D., Sullivan, M. P., Land, D. M., and Ragab,A. H. Interrupted vs. continued maintenance therapy in childhood acute leukemia.Cancer (Phila.), 36: 341-352, 1975.

3. Mauer, A. M. Therapy of acute lymphoblastic leukemia in childhood. Blood, 56:1-10, 1980.

4. I .aiii.inl. L. The clinical pharmacology of 6-mercaptopurine. Eur. J. Clin. Pharmacol.,43: 329-339, 1992.

5. Lafolie, P., Hayder, S., Björk,O., ahström,L., Liliemark, J., and Peterson, C. Largeinterindividual variations in the pharmacokinetics of oral 6-mercaptopurine in maintenance therapy of children with acute leukemia and non-Hodgkin lymphoma. ActaPaediatr. Scand., 75: 797-803, 1986.

6. Zimm, S., Collins, J. M., Riccardi, R., O'Neill, D., Narang, P. K., Chabner, B., and

Poplack, D. G. Variable bioavailability of oral mercaptopurine. Is maintenancechemotherapy in acute lymphoblastic leukemia being optimally delivered? N. Engl.J. Med., 308: 1005-1009, 1983.

7. Sulh, H., Koren, G., Whalen, C., Soldin, S„Zipursky, A., and Greenberg, M.Pharmacokinetic determinants of 6-mercaptopurine myelotoxicity and therapeutic

failure in children with acute lymphoblastic leukemia. Clin. Pharmacol. Ther., 40:604-609, 1986.

8. Lafolie, P., Hayder, S., Björk, O., and Peterson, C. Intraindividual variation in6-mercaptopurine pharmacokinetics during maintenance therapy of children withacute lymphoblastic leukemia. Eur. J. Clin. Pharmacol., 40: 599-601, 1991.

9. Koren, G., Ferra/ini, G., Sulh, H., Langevin, A. M., Kapelushnik, J., Klein,J., Giesbrecht, E., Soldin, S., and Greenberg, M. Systemic exposure to mercaptopurine as a prognostic factor in acute lymphocytic leukemia in children. N. Engl.J. Med., 323: 17-21, 1990.

10. Hayder, S., Lafolie, P., Björk,O., and Peterson C. 6-Mercaptopurine plasma levels in

children with acute lymphoblastic leukemia: relationship to relapse risk andmyelotoxicity. Ther. Drug Monit., 11: 617-622, 1989.

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1995;55:1670-1674. Cancer Res   David J. Warren, Anders Andersen and Lars Slørdal  6-Mercaptopurine-based Maintenance TherapyLeukocyte DNA Obtained from Patients Receiving Quantitation of 6-Thioguanine Residues in Peripheral Blood

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