Influence of Fludarabine on Pharmacokinetics and Pharmacodynamics of Cytarabine: Implications for a Continuous Infusion Schedule1

Vol. 2, 653-658, April 1996 Clinical Cancer Research 653

John F. Seymour,2 Peng Huang, are achieved during a continuous infusion schedule. Given William Plunkett, and Varsha Gandhi3 the important role intracellular ara-CTP concentrations and ara-CMP incorporation into DNA have on the ultimate Department of Clinical Investigation, University of Texas M. D. cytotoxic capacity of ara-C against acute myelogenous leu- Anderson Cancer Center, Houston, Texas 77030 kemia blasts, these studies suggest a promising pharmaco- logical model for improving the efficacy of the continuous ABSTRACT infusion ara-C regimen. Arabinosylcytosine (ara-C) is a cytotoxic agent with major activity against acute leukemias. To exert this effect, INTRODUCTION it must first be phosphorylated to its active 5’-triphosphate, The clinical efficacy of ara-C4 in the therapy of patients ara-CTP, which is incorporated into DNA. Our previous with AML was first demonstrated more than 25 years ago ( 1). studies demonstrated that preincubation with arabinosyl-2- Although many drugs and biologicals have been evaluated since fluoroadenine (F-ara-A) increased the rate of ara-CTP ac- that time, it remains one of the most effective chemotherapeutic cumulation in leukemia cells when incubated with 10 aM agents in use against hematological malignancies (2-5). For this ara-C. Such concentrations of ara-C are readily obtained reason, novel strategies are still sought to optimize the use of during intermittent bolus infusions of ara-C, and clinical cytarabine, particularly in combinations with other effective trials were conducted using fludarabine in combination with agents. For ara-C to exert its cytotoxicity, it must first be 2-h infusions of intermediate-dose ara-C. During continuous phosphorylated intracellularly by the rate-limiting enzyme dCyd infusion of ara-C, however, serum ara-C levels are < 10 tM. kinase and subsequently to its 5’-triphosphate, ara-CTP (6, 7). Because the effectiveness of ara-C depends on the levels of Ara-CTP then competes with dCTP for incorporation into DNA intracellular ara-CTP and its incorporation into DNA, we (8). Numerous in vitro studies have demonstrated that the sought to investigate the influence of fludarabine on phar- amount of ara-CMP incorporated into cellular DNA is a predic- macodynamics of ara-C at concentrations of ara-C achieved tor of loss of clonogenicity in human leukemia cells (9-I 1). during continuous infusion. Using the K562 human leuke- Although technically possible (12), it is extremely difficult to mic cell line, we established that incubation with 30 M reliably and reproducibly measure the incorporation of ara-CMP F-ara-A was able to modulate intracellular dNTP pools and into the DNA of circulating human leukemic myeloblasts during achieve maximum enhancement of ara-CTP levels at all therapy. Thus, alternative predictors of the clinical cytotoxic concentrations of ara-C tested (0.3-10.0 FM). The relative effect of ara-C have been sought. enhancement of ara-CTP concentrations ranged from 2.2- to In the in vitro setting, the major determinant of ara-CMP 2.8-fold. Combination of F-ara-A with 1.0 and 3.0 piM ara-C incorporation into DNA was found to be the product of the intra- also increased the incorporation of ara-CTP into DNA. To cellular ara-CTP concentration and time ( I I ). This observation has model the influence of F-ara-A on continuous infusion been confirmed in patients in vivo where statistically significant ara-C, cells were incubated with 1 LM ara-C alone or in correlations have been demonstrated between the intracellular phar- combination with F-am-A. The F-ara-A-incubated cells ac- macokinetics of ana-CTP and clinical response to single-agent cumulated effective intracellular concentrations of F-ara- high-dose am-C therapy given either on an intermittent schedule ATP, which resulted in greatly increased intracellular ara- (13, 14) or by continuous infusion (15). Similarly, very low incor- CTP levels. These studies demonstrate the capacity of poration of ara-C into DNA in vitro was predictive of an adverse clinically attainable concentrations of F-ara-ATP to enhance outcome with subsequent ann-C-based therapy in vivo (16). the formation of ara-CTP at concentrations of ara-C that Based on these studies demonstrating the importance of ara-CTP formation and retention on treatment outcome, our laboratory ( 17, 18), and others ( 19) have investigated potential means to favorably modulate ara-CTP metabolism. One of the Received 9/22195; revised 1 1120195; accepted 12/6195. most promising methods of biochemical modulation identified I Supported in part by Grants CA32839, CAS5 164, and CA57629 from was the capacity of pretreatment with F-ara-A to enhance the the National Cancer Institute, Department of Health and Human Ser- vices. 2 pesent address: Ludwig Institute for Cancer Research, Melbourne Tumor Biology Branch, do Post Office Royal Melbourne Hospital, Grattan Street, Parkville 3050, Australia. 3 To whom requests for reprints should be addressed, at Department of 4 The abbreviations used are: ara-C, l-3-D-arabinofuranosylcytosine or Clinical Investigation, University of Texas M. D. Anderson Cancer cytarabine; AML, acute myelogenous leukemia; dCyd, deoxycytidine: Center, Box 71, 1515 Holcombe Boulevard, Houston, TX 77030. dNTP, deoxynucleoside triphosphate; F-ara-A, 943-D-arabinofuranosyl- Phone: (713) 792-2989; Fax: (713) 794-4316. 2-fluoroadenine.

rate of ara-CTP accumulation in a human leukemia cell line three volumes of ice-cold PBS. After centrifugation, the cell (20). Mechanistic studies suggest that the major mechanism by pellet was extracted with HC1O4 as previously described (32). which F-ara-A augments the intracellular anabolism of ara-C to Ara-CTP and F-ara-ATP were separated from nibonucleoside ara-CTP is through an enhanced rate of ara-CTP accumulation triphosphates by high-pressure liquid chromatography on an (20, 21) due to both a direct stimulatory effect of F-ara-ATP on anion exchange Partisil-lO SAX column (Waters Associates, dCyd kinase and indirectly through reduced concentrations of Milford, MA) as previously described (33). Nonradioactive ara- dCTP, which acts as a feedback inhibitor of dCyd kinase activ- CTP and F-ana-ATP were quantified at 262 nm by electronic ity. Subsequent clinical studies have demonstrated the effective- integration with reference to external standards. The [3H]ara- ness of this strategy in circulating AML blasts (21), chronic CTP peak was collected, and radioactivity was quantitated with lymphocytic leukemia lymphocytes (22), or leukemic lympho- a liquid scintillation counter. The intracellular concentrations of blasts (23) where F-ara-A is administered as a more soluble nucleotides were calculated and expressed as the quantity of form, fludarabine (the monophosphate of F-ara-A). nucleotides contained in the extract from a given number of These studies of nra-C modulation by F-nra-A or fludarabine cells of a determined mean volume. This calculation assumes were carried out at concentrations of ann-C (10 p.M or above) that that nucleotides are uniformly distributed in total cell water. saturate the rate of ara-CTP formation in human leukemia cells in Determination of Deoxynucleotides. K562 cells (5 X vitro and during therapy (24). This concentration of ara-C can 106) were obtained before and 3 h after incubation with the easily be attained or exceeded in vivo with intermittent high-dose or different concentrations of F-ara-A. These cells were extracted intermediate dose ara-C therapy (24, 25). However, serum con- by 60% methanol for determination of dNTPs. The DNA poly- centrations of ara-C are generally less than 10 p.M when maxi- merase assay as modified by Sherman and Fyfe (34) was used to mally tolerated continuous infusion schedules are administered quantitate dNTPs in the cell extracts. DNA polymerase I (U. S. (26). Continuous infusion schedules of single-agent ara-C have Biochemical Corporation, Cleveland, OH) was used to start a been extensively used for treatment of acute leukemias (27-29) reaction in a mixture that contained 100 nmi HEPES buffer (pH

and have demonstrated clinical efficacy in previously untreated 7.3), 10 ifiM MgC12, 7.5 p.g BSA, and synthetic oligonucleotides AML (30). Thus, the present study aimed to explore the capacity of defined sequences as templates annealed to a primer, [2,8- of F-nra-A to modulate the metabolism of ara-C at concentra- 3H]dATP (16.7 Cilmmol; Moravek Biochemicals) or [methyl- tions below 10 p.M and to quantify the influence of F-ara-A on 3H]d’rrp (47 Ci/mmol; ICN Radiochemicals, Irvine, CA), and the incorporation of ara-CMP into DNA. The results provide a either standard dNTP or the extract from 1 to 3 X lO cells. rational basis for the clinical application of intermittent bolus Reactants were incubated for 1 h and applied to filter discs; after fludarabine along with continuous infusion nra-C in the treat- washing, the radioactivity on the discs was determined by liquid ment of patients with relapsed and refractory AML. scintillation counting. nra-C Incorporation into DNA. ara-C incorporation MATERIALS AND METHODS into cellular DNA was determined following incubations with Cells. The K562 cell line (3 1) was obtained from the varying concentrations of [3H}ara-C with and without prior American Type Culture Collection (Rockville, MD). Mainte- incubation with F-ara-A. The nra-C stock solution used for these nance procedures for the cultures, Mycoplasma analysis, and experiments was at a concentration of 1 .0 nmi, with a specific growth characteristics of this cell line have been published activity of 500 p.Ci/ml. At the completion of incubations, cells were washed twice in three volumes of ice-cold PBS and lysed previously (20). Cells were maintained in exponential growth in RPMI 1640 medium supplemented with 10% heat-inactivated with digestion buffer [100 mri NaCl, 10 mrsi Tris-HCI (pH 8.0), fetal bovine serum in 5% CO2 in fully humidified room air. Cell 25 nmi EDTA (pH 8.0), 0. 1 mg/ml proteinase K, and 0.5% SDS]. Following a 12-h incubation at 50#{176}C,DNA was extracted density and size were determined with a Coulter counter (Coulter Electronics, Hialeah, FL). using a standard phenol/chloroform/isoamylalcohol method Chemicals. ara-C and ara-CTP were purchased from (35). DNA was quantified by determining absorption at 260 nm Sigma Chemical Co. (St. Louis, MO). [5-3H]ara-C (11.7 Cii and [3H]ara-C content measured using a liquid scintillation mmol) was obtained from Moravek Biochemicals (Brea, CA). counter. The amount of ara-C incorporated into DNA was quan- F-nra-A was produced by alkaline phosphatase treatment of titated and expressed as pmol ara-C per mg of cellular DNA. fludarabine, which was obtained from Berlex Laboratories (Richmond, CA). F-ara-ATP was synthesized by Sierra Biore- RESULTS search (Tucson, AZ). All other chemicals were reagent grade. Influence of F-ara-A Concentration on ara-CTP Accu- Cellular Pharmacology. Cells were incubated with ali- mulation. Previous work has established that F-ara-ATP ac- quots of standard solutions of ara-C or F-ara-A to yield desired cumulation is saturated following incubation of cells with F- drug concentrations. Incubations were maintained for 3 to 4 h, ara-A at concentrations of 300 p.M and that this pretreatment and aliquots were removed at hourly intervals. In studies of enhances subsequent ara-CTP accumulation in cells incubated sequential drug exposure, the cells were washed three times in with 10 p.M nra-C (17). To establish the minimum concentration

three volumes of ice-cold PBS (8. 1 g NaC1, 0.22 g KC1, 1 . 14 g of F-nra-A needed to achieve this potentiation, we incubated Na,HPO4, and 0.27 g KH,PO4fliter H20, pH 7.4), and then cells with 10-100 p.M F-nra-A for 3 h, followed by incubation resuspended in fresh medium, to which the appropriate concen- with 10 p.M ara-C. Fig. 1 demonstrates that K562 cells prein- tration of the second drug was added. At the completion of the cubated with F-ara-A accumulated ara-CTP at an increased rate final incubation, the reaction was stopped by the addition of compared to that achieved with ara-C alone. At 10 p.M F-ara-A,

completion of the 3-h incubation with 30 pM F-ara-A was 75 (± 1 1 JIM), confirming that an F-ara-ATP concentration adequate to achieve optimal potentiation of ara-CTP metabolism was present. These data suggest that, in addition to the established capacity of F-ara-A to modulate ara-CTP accumulation at ara-C concentrations that saturate the rate of ara-CTP accumulation, F-ara-A can also modulate the accumulation of ara-CTP when ara-C is present at lower concentrations (0.3-3 p.M). Influence of F-ara-A on ara-CMP Incorporation into DNA. Cells were incubated with 30 p.M F-ara-A for 3 h, washed, then incubated with varying concentrations of [3H]ara- C for 3 h. At the completion of the incubation, the cell suspen- sion was divided into separate aliquots for DNA isolation and Hours nucleoside triphosphate quantitation as described. In spite of the Fig. 1 Influence of different concentrations of F-ara-A preincubations ability of F-nra-A pretreatment to enhance intracellular ara-CTP on ara-CTP accumulation. Cells were incubated with the indicated accumulation at all concentrations of ara-C studied, there was a concentrations of F-ara-A for 3 h, washed, and then incubated with 10 dose-dependent effect on ara-CMP incorporation into DNA p.M ara-C. Samples were taken every hour up to 4 h to analyze the (Fig. 3). At intermediate concentrations of ara-C (I .0 and 3.0 concentrations of ara-CTP, as described in ‘ ‘Materials and Methods.” Data points, means of duplicate determinations from two to four exper- 1i.M), F-nra-A pretreatment resulted in a mean 1 .8- and 1 .9-fold iments; the SE was <15%. increase in the quantity of ara-CMP incorporated into cellular

DNA, respectively. At the extreme low (0.3 ELM) and high (10 i.M) concentrations of ara-C, F-ara-A pretreatment did not discernibly influence ara-CMP incorporation into DNA. The mean there was a marginal increase in ara-CTP accumulation. Pretreat- ratios of ara-CMP incorporation for F-am-A pretreated to control ment with 30-100 p.M F-ara-A potentiated ara-CTP at similar cells were 1.09 and 0.94 at 0.3 and 10 p.M am-C, respectively. levels. This suggests that a 3-h incubation with 30 p.M F-am-A Modeling of the Influence of F-ara-A on Continuous results in optimal potentiation of ara-CTP formation. Incubation Infusion am-C. The preceding studies demonstrated the ca- with greater concentrations of F-am-A do not further enhance pacity of F-ara-A to enhance both intracellular ara-CTP accu- ara-CTP formation. All subsequent combination experiments used mulation and ara-CMP incorporation into cellular DNA at con- a 3-h incubation with 30 liM F-nra-A. Preliminary experiments centrations (1 and 3 .LM) of ara-C, which are attained in plasma demonstrated that washing and resuspension of cells alone did not significantly alter the accumulation of ara-CTP (data not shown). during therapy with well tolerated and efficacious dose of 1500 Dose-dependent Accumulation of F-ara-ATP and Effect mgim2iday continuous infusion of ara-C (26, 30). Additionally, on dNTP Pools. As reported previously, the potentiation of the F-ara-ATP concentration (SO .tM) required for this biochem- the rate of ara-CTP accumulation by F-ara-ATP was due to a ical modulation is achieved in circulating leukemia blasts during direct effect of F-ara-ATP on dCyd kinase and an indirect therapy with a 30-mgim2 infusion of fludarabine, the dose used mechanism (through alterations in the dNTP pool) on this en- in the clinic (21). To model the influence of intermittent bolus zyme. Hence, F-ara-ATP levels and its effect on endogenous F-ara-A administration on continuous infusion ara-C, aliquots of deoxynucleotides were determined in cells treated with mdi- K562 cells were incubated for 10 h with 1 iM ara-C along with cated concentrations of F-ara-A for 3 h (Table I). Incubation SO J.M tetrahydrouridine to minimize loss of ara-C through with F-ara-A resulted in a dose-dependent increase in F-ara- deamination. Following attainment of steady-state intracellular ATP formation. Levels of F-ara-ATP were 20 .M or less at ara-CTP concentrations at 3 h, half of the cells were coincubated with 30 iM F-am-A. Portions of the cell mixtures from each flask exogenous F-nra-A levels of 10 pM or lower. At 30 p.M or higher were collected at hourly intervals for measurement of intracellular concentrations of F-ara-A, >50 p.M F-ara-ATP was achieved after 3 h of incubation. Purine deoxynucleotides were affected at am-CTP concentrations. Accumulation of am-CTP reached a pla- 10 pM level of intracellular F-ara-ATP; dATP was 51% of teau 3 h after incubation with 1 i.M am-C (Fig. 4), and continued control and dGTP was 70% of the pretreatment value. At higher incubation with am-C in the absence of F-am-A demonstrated a concentrations of F-ara-ATP, all dNTPs except dTFP were true steady-state level of ara-CTP. However, addition of F-am-A to lowered. Concentrations of dATP and dCTP were lowered most the parallel culture resulted in a continuous increase in ara-CTP significantly: a 60-70% reduction after a 3-h incubation with accumulation up to 10 h. Compared to the concentration of ara- 100 iM F-ara-A which accumulated 175 p.M F-ara-ATP. CTP in cells incubated with ara-C alone, the level of ara-CTP at 10 Ability of F-ara-A to Modulate ara-CTP Formation at h was 2.3-fold higher in cells coincubated with F-am-A. Similar Low Concentrations of ara-C. Cells were incubated with experiments conducted using 3 i.M F-am-A in combination with 1 30 p.M F-ara-A for 3 h, washed, and then exposed to 0.3-10.0 LM am-C resulted in a 1 .5-fold increase in the accumulation of p.M ara-C for 3 h. The resulting intracellular levels of ara-CTP am-CTP at 10 h (data not shown). were between 2.2- and 2.8-fold higher than those attained with the corresponding concentration of ara-C alone, i.e. , without DISCUSSION prior F-ara-A exposure (Fig. 2). In these experiments, the mean Ara-C remains the basis of standard cytotoxic treatments (±SE) intracellular F-ara-ATP concentration attained at the for most patients with AML. Despite more than 25 years of

Table I Accumulation of intracellular F-ara-ATP after a 3-h incubation with indicated concentrations of exogenous F-ara-A and its effect on the deoxynucleotide pool

F-ara-ATP or dNTP (p.M ± SE)

F-ara-A (p.M) F-ara-ATP dATP dCTP IGTP d1TP

0 24.8 ± 0.6 13.6 ± 0.3 7.6 ± 0.3 73.6 ± 3.3 3 10.4 ± 0.5 12.7 ± 0.4 1 1.7 ± 0.6 5.3 ± 0.1 53.9 ± 2.3 10 20.8 ± 0.8 9.1 ± 0.2 12.2 ± 0.3 4.1 ± 0.2 68.7 ± 3.4 30 53.8 ± 0.9 9.1 ± 0.2 1 1.1 ± 0.4 4.9 ± 0.3 73.1 ± 3.2 50 99.6 ± 0.4 9.8 ± 0.3 9.6 ± 0.3 5.3 ± 0.4 74.3 ± 3.3 100 174.9 ± 8.1 7.3 ± 0.4 5.6 ± 0.1 5.0 ± 0.5 77.0 ± 1.9

. z

0 E 0.

Ara-C (sM) Ara-C (tM) Fig. 2 Influence of F-nra-A preincubations on ara-CTP accumulation Fig. 3 Incorporation of ara-CMP into cellular DNA. DNA was isolated at different concentrations of ara-C. Parallel cultures of cells were after 3 h of ara-C incubation with or without F-ara-A as described in Fig. incubated with no drug or 30 p.M F-ara-A for 3 h, washed, and then 2. Columns, means of duplicate determinations from two to three ex- incubated with the indicated concentrations of l3Hlara-C for an addi- periments. Bars, SE. tional 3 h. Nucleotides were extracted and separated by high-pressure liquid chromatography. Columns, means of duplicate determinations from three to six experiments. Bars, SE. At present, it is not possible to quantify the relative contributions of these components to the observed clinical efficacy of the combination (36, 37). Preliminary studies using an in vitro model preclinical and clinical evaluation in patients with AML, opti- system demonstrated that incorporation of both ara-CTP and mal application of ara-C remains elusive. Pioneering studies F-ara-ATP in a DNA primer by human DNA polymerase a established the quantitative relationship between ara-C incorpo- results in more than 99% inhibition of further extension (38). ration into cellular DNA and cytotoxicity (9-1 1), and the means The results we obtained in vitro using human leukemic of enhancing this end point are keenly sought. One of the blasts in this study suggest that the influence of F-ara-A on limiting factors has been the difficulty of measuring ara-C ara-CMP incorporation into DNA are related to the concentra- incorporation into leukemic cellular DNA during therapy in tion of ara-C. At the extremes of ara-C concentrations studied, vivo. The best predictive index readily measured in clinical 0.3 and 10.0 ELM, pretreatment with 30 p.M F-ara-A did not samples is the intracellular ara-CTP concentration, which has discernibly influence ara-CMP incorporation into DNA. This been demonstrated to be strongly correlated with clinical re- was despite the fact that ara-CTP concentrations were increased sponse to ara-C therapy (13-15). Enhancement of ara-CTP more than 2-fold. It is likely that at 0.3 p.M ara-C, the difference

anabolism has become a potential means of improving clinical in the concentrations of ara-CTP (10 p.M without and 25 p.M results with ara-C. One such method of modulation used clini- with F-ara-A pretreatment) was not great enough to materially cally is the combination of fludarabine with ara-C in a timed alter subsequent incorporation of ara-CMP into DNA. It is also sequential manner (20, 2 1 ). This approach enhanced intracellu- possible that the relatively small changes in ara-CMP incorpo- lar ara-CTP concentrations above that attainable using ara-C ration at low ara-C concentrations may not be detectable. alone at intermediate or high-dose infusion levels. However, Prior studies suggested that ara-C incorporation into DNA F-ara-ATP also has independent actions upon enzymes critical does not continue to increase above levels attained following for DNA synthesis, which may inhibit the subsequent incorpo- incubation with 5-10 p.M ara-C in mouse L cells, despite con- ration of the ara-CTP into DNA. Such an inhibition would tinuing increments in ara-CTP concentrations (8). This system presumably also diminish the cytotoxicity attributable to ara-C. differs from human leukemic cells, however, in which ara-CTP Of course, F-ara-ATP may also have direct cytotoxic actions itself concentrations do not continue to increase with exposure to con- on AML blasts independent of its modulation of am-C metabolism. centrations of am-C above 10 p.M but may indeed diminish due to

The current studies provide a potential means of reducing the likelihood of exposure of leukemic blasts to suboptimal ara-CTP levels during continuous infusion schedules. We have shown that clinically attainable intracellular concentrations of F-ara-ATP are able to enhance both the formation of ara-CTP, and, more important, the quantity of ara-C incorporated into 0. I- leukemic cellular DNA when exposed for prolonged periods to 1.0 p.M ara-C. This concentration approximates that attained with the clinical application of continuous infusion of ara-C at I .5 gim2iday (26). The above data along with our previous experience with fludarabine provide a rationale for combining a continuous infusion high-dose ara-C schedule (30) with bolus dose of fludarabine. Such combination has provided responses

Hours in pediatric patients with relapsed acute leukemias (43). Based on the present investigation and other studies with Fig. 4 Influence of F-ara-A addition on steady-state ara-CTP accumu- fludarabine and intermediate dose intermittent infusion of ara-C, lation. Cells were continuously exposed to L3Hlara-C. Samples were taken every hour up to 3 h, and then cultures were split into two. F-ara-A a clinical protocol has been designed using a continuous infu- at 30 p.M was added in one culture. Cells were then taken every h up to sion schedule for ara-C administration, with daily bolus doses of 10 h from each culture, and ara-CTP accumulation was quantitated. fludarabine for adults with relapsed AML. The validity of the in Data points, means of two separate determinations. Bars, SD. For some vitro studies reported here are being evaluated by measuring the data points, because of very low deviation from mean value, bars fit within the symbol and therefore are not shown. intracellular F-ara-ATP and its influence on cellular steady-state ara-CTP concentrations. We expect that those patients whose leukemic blasts synthesize sufficient quantities of F-ara-ATP will demonstrate the predicted enhancement of intracellular a direct inhibitory effect of ara-C on dCyd kinase activity (39). ara-CTP levels. This protocol will provide the opportunity to Ross et a!. (40) have also demonstrated that the relative efficiency seek correlations between clinical efficacy of this pharmacolog- of am-C incorporated into DNA diminishes with increasing ara-C ically directed treatment approach with pharmacokinetic and concentrations. This suggests that further increments in intracellu- pharmacodynamic parameters. lar am-CTP concentrations will have an increasingly smaller influence on nra-C incorporation into DNA. It is also uncertain what, if any, negative influence the intracellular F-ara-ATP would have had ACKNOWLEDGMENTS on incorporation of ara-C into DNA because F-am-AlP incorpo- We are grateful to Mm Du and Nanying Li for their expert ration results in chain termination (41). Regardless of a theoretical technical assistance and to Leslie Wildrick for editing the manuscript. inhibitory effect, the net outcome of pretreatment with 30 p.M F-am-A followed by exposure to 1.0 or 3.0 p.M am-C was a marked REFERENCES enhancement of am-C incorporation into DNA. Previous studies I. Frei. E., III, Bickers, I. N., Hewlett, i. S., Lang, M., Leary. W. V.. have demonstrated this to be the best predictor of loss of clonoge- and Talley. R. W. 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J F Seymour, P Huang, W Plunkett, et al.

Clin Cancer Res 1996;2:653-658.

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