Thymidylate synthase gene polymorphism determines response and toxicity of 5-FU chemotherapy

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1 The Phrmogenomis Journl (2001) 1, Nture Pulishing Group All rights reserved X/01 $ ORIGINAL ARTICLE Thymidylte synthse gene polymorphism determines response nd toxiity of 5-FU hemotherpy ST Pullrkt 1 J Stoehlmher 1 V Ghderi 1 Y-P Xiong 1 SA Ingles 1 A Sherrod 1 R Wrren 2 D Tso-Wei 1 S Groshen 1 H-J Lenz 1 1 University of Southern Cliforni/Norris Comprehensive Cner Center, Los Angeles, Cliforni; 2 Deprtment of Surgery, University of Cliforni Sn Frniso, Sn Frniso, CA, USA Correspondene: H-J Lenz, MD, Diretor, GI Onology, USC/Norris Comprehensive Cner Center, 1441 Estlke Avenue, Suite 3456, Los Angeles, CA 90033, USA Telephone: Fx: E-mil: lenz hs.us.edu Reeived: 16 Ferury 2001 Aepted: 12 Mrh 2001 ABSTRACT Thymidylte synthse (TS) tlyses the onversion of deoxy-uridylte to deoxy-thymidylte nd is essentil for DNA synthesis. The humn TS gene promoter is polymorphi, hving either doule or triple tndem repets of 28-p sequene. Here we determined the signifine of this polymorphism in humns nd its predition for linil outome of ptients with metstti oloretl ner treted with 5-fluorouril. The TS mrna level ws nlyzed using RT-PCR. Individuls homozygous for the triple repet vrint (L/L) hd 3.6 times higher TS mrna levels ompred to those homozygous for the doule repet vrint (S/S) in tumor tissue (P = 0.004). We tested 50 ptients with disseminted oloretl ner who reeived 5-FU tretment to determine whether this TS polymorphism will predit linil outome. We found individuls with S/S genotype hd response rte of 50% (4/8) when ompred to 9% (2/22) in those with L/L nd 15% (3/20) in those with S/L genotype (P = 0.041). Ptients with L/L hd less severe side effets to 5-FU (P = 0.008). The dt suggest tht genotyping for the TS polymorphism my hve the potentil to identify ptients more likely to respond to 5-FU sed hemotherpy. The Phrmogenomis Journl (2001) 1, Keywords: TS-polymorphism; TS-gene expression; oloretl ner; 5-FU resistne; fluoropyrimidine hemotherpy INTRODUCTION Coloretl ner is leding use of moridity nd mortlity with out new ses nd deths in Europe nd USA eh yer. 1 Sine its introdution 4 dedes go, 5-fluorouril (5-FU) hs remined the minsty of hemotherpeuti regimens for oloretl ner, oth in the metstti nd djuvnt settings. 2 The mehnism of tion of 5-FU is through the inhiition of thymidylte synthse (TS). This enzyme tlyses the intrellulr onversion of deoxyuridylte to deoxythymidylte whih is the sole de novo soure of thymidylte, n essentil preursor for DNA synthesis. 3 The tive metolite of 5-FU, 5-fluorodeoxyuridylte (5FdUMP), inds to TS nd inhiits it y forming stle ternry omplex. 4 The humn thymidylte synthse gene (hts) is polymorphi with either doule or triple tndem repets of 28 se-pir sequene downstrem of the p-site in the 5 terminl regultory region. 5 In in vitro studies, the tivity of reporter gene linked to the 5 terminl frgment of the hts gene with triple tndem repets ws 2.6 times higher thn tht with doule tndem repets. 6 Thus this polymorphi region ppers to e funtionl nd my modulte TS gene expression. In ddition, we hve previously shown tht the intrtumorl TS mrna level is determinnt of response to 5-FU hemotherpy nd survivl in ptients with olon nd gstri ner. 7,8 Hene if this polymorphism modultes the TS gene expression, then knowledge of the TS genotype ould predit response to fluoropyrimidine hemotherpy nd survivl. Bsed on these previous findings, we onduted pilot retrospetive study to investigte the

2 66 Thymidylte synthse polymorphism nd hemotherpy ST Pullrkt et l Tle 1 TS genotype nd TS mrna levels in tumor tissue Tissue TS genotype n % TS men 95% CI Comprison of TS mens P-vlue Metstti tumor tissue (n = 52) L/L 15 29% 9.42 (5.51, 16.12) L/L vs S/S S/L 26 50% 5.53 (3.68, 8.31) L/L vs S/L 0.10 S/S 11 21% 2.60 (1.39, 4.87) S/L vs S/S Overll TS men = geometri men of mrna expression of TS reltive to tin mrna. 95% onfidene intervl. P-vlue for the overll omprison is sed on the F-test, ll other P-vlues re sed on the Student Newmn Keuls method for multiple omprisons. signifine of this polymorphism on TS mrna levels nd on linil outome of ptients with oloretl ner. RESULTS TS mrna Level nd TS We found tht mong 52 ptients with metstti oloretl ner (3C-92-2 nd CALGB tril) nlyzed for TS mrna expression, 15 (29%) were homozygous for the triple repet, 26 (50%) were heterozygous, nd 11 (21%) were homozygous for the doule repet vrint within the humn TS promoter region. Tle 1 summrizes the men intrtumorl TS mrna levels in tumor tissue ording to the three different genotypes. There ws signifint ssoition etween TS mrna expression nd inresing numer of the 28-p repets within the 5 -untrnslted region of the humn TS gene. Sujets with the S/S genotype hd signifintly lower TS mrna levels (2.60 (1.39, 4.87)) thn oth S/L sujets (5.53 (3.68, 8.31); P = 0.050) nd L/L sujets (9.42 (5.51, 16.12); P = 0.004) y pir-wise omprison. The TS mrna level in norml liver tissue of 26 ptients (CALBG) ws lso exmined. We found seven ptients (27%) with the L/L genotype, 14 ptients (54%) with the S/L genotype, nd five ptients (19%) with the S/S genotype. A similr ssoition ws oserved etween TS mrna expression nd genotype: L/L group 8.18 (4.90, 13.67), the S/L group 4.41 (3.07, 6.33), nd the S/S group 3.19 ((1.74, 5.85), P = 0.051, Tle 2). The TS mrna expression level in tumor tissue ws fold inresed ompred to the djent norml liver tissue. The rtio of TS expression in tumor tissue to TS expression in norml tissue ws inverse ssoited with the numer of 28-p tndem repets in the 5 -untrnslted region of the TS gene (Tle 3, P 0.001). TS nd Clinil Outome Under Fluoropyrimidine Chemotherpy We evluted 50 ptients with disseminted oloretl ner (SWOG 9420 nd 3C-92-2) for response nd survivl treted with protrted infusion of 5-FU. Tle 4 summrizes the demogrphi informtion of these ptients. Ptients onfirmed for the S/S genotype showed higher response rte (50% (4/8)) to 5-FU ompred to 15% (3/20) in the S/L group nd 9% (2/22) in the L/L group (P = 0.041, Fisher s Ext test, Tle 5). Forty-nine out of the ove 50 ptients were evlule for toxiity. A signifint inverse ssoition ws seen etween the numer of 28-p tndem repets in the 5 -untrnslted region of the TS gene nd the severity of toxiity (P = 0.008, Jonkheere Terpstr ext test, Tle 5). In 63% (5/8) of ptients with S/S genotypes toxiity grde 3 ( severe ) ws oserved ompred to 27% (6/22) in the L/L group nd 32% (6/19) in the S/L group. A mild toxiity (grde 1) ws found in 41% (9/22) of ptients homozygous for the L llele ompred to 5% (1/19) in the heterozygous group nd 0% (0/8) in the S/S group. Toxiitygrde 0 ( none ) s well s grde 4 ( life-thretening ) ws not seen in ny of those 49 ptients. Ptients with the S/S Tle 2 TS genotype in norml tissue Tissue TS genotype n % TS men 95% CI Comprison of TS mens P-vlue Norml liver tissue (n = 26) L/L 7 27% 8.18 (4.90, 13.67) L/L vs S/S S/L 14 54% 4.41 (3.07, 6.33) L/L vs S/L S/S 5 19% 3.19 (1.74, 5.85) S/L vs S/S 0.35 Overll TS men = geometri men of mrna expression of TS reltive to tin mrna. 95% onfidene intervl. P-vlue for the overll omprison is sed on the F-test, ll other P-vlues re sed on the Student Newmn Keuls method for multiple omprisons. The Phrmogenomis Journl

3 Thymidylte synthse polymorphism nd hemotherpy ST Pullrkt et l 67 Tle 3 Differene of TS expression in norml nd tumor tissues y TS genes TS genes n Norml tissue Tumor tissue Rtio of TS 95% P-vlue TS geometry men TS geometry men Confidene intervl L/L (0.80, 4.19) 0.13 S/L (1.30, 4.66) S/S (1.59, 8.12) Overll (1.64, 3.65) Tumor nd norml tissue in pir from eh ptient. Rtio of TS expression = TS (tumor)/ts (norml). Bsed on pired t-test. Tle 4 Bsi demogrphi informtion P-vlue L/L (n = 22) S/L (n = 20) S/S (n = 8) n % n % n % Age % 5 25% 1 13% % 12 60% 3 38% % 3 15% 4 50% Medin (rnge) 62 (39 77) 59 (34 77) 66 (53 80) Ethniity 0.36 Asin 8 36% 2 10% 1 13% Blk 2 9% 1 5% 0 0% Hispni 5 23% 8 40% 2 25% White 7 32% 9 45% 5 63% Sex 0.74 Femle 9 41% 7 35% 4 50% Mle 13 59% 13 65% 4 50% All P-vlues re sed on Fisher s Ext test. Tle 5 TS genotype nd linil outome for ptients treted with 5-FU P-vlue L/L (n = 22) S/L (n = 20) S/S (n = 8) n % n % n % Response to 5-FU Response 2 9% 3 15% 4 50% Non-response 20 91% 17 85% 4 50% Toxiity 0.008* Grde % 1 5% 0 0% Grde % 12 63% 3 37% Grde % 6 32% 5 63% Missing 1 Overll survivl 0.37** No. of deths Medin survivl (months) Reltive risk of dying Bsed on Fisher s Ext test exept *sed on Jonkheere Terpstr ext test, **sed on the Logrnk test. Response: 50% dispperne of ll tumors. Referene group.

4 68 Thymidylte synthse polymorphism nd hemotherpy ST Pullrkt et l genotype hd longer medin survivl of 16.2 months when ompred to 8.3 months nd 8.5 months in those with the S/L genotype nd L/L genotype, however it did not reh sttistil signifine (P = 0.37, Log-rnk test, dt not shown). The medin follow-up ws 2.9 yers. DISCUSSION 5-fluorouril-sed therpy of metstti oloretl ner hs n overll response rte of 26% in our ptient popultion. 7 However there re inter-individul differenes in response, survivl nd toxiity in ptients treted with 5- FU. Reently, the mesurement of intrtumorl TS mrna expression hs een estlished s preditor of response nd survivl to 5-fluorouril hemotherpy. It hs een shown tht low expression levels of TS mrna were ssoited with higher proility of response to 5-FU-sed tretment nd longer survivl. Ptients with TS mrna level higher thn 4.1 did not respond to 5-FU-sed hemotherpy. 7 Similr results were seen in gstri ner ptients treted with 5 FU-sed regimen. 8 Regultion of intrtumorl TS expression is not very well understood. Reent studies hve demonstrted tht mutnt p53 is ssoited with higher TS protein nd gene expression. 9,10 Further it hs een reported tht 28-p tndem repet polymorphism is ssoited with TS tivity in vitro. 6 This study is demonstrting for the first time signifint ssoition etween TS mrna level in oth metstsized oloretl tumors nd norml tissue nd 28-p tndem repet polymorphism in the 5 -untrnslted region of the TS gene. A higher intr-tumorl TS mrna expression ws oserved with n inresing numer of the 28-p tndem repets. Reent dt showed tht TS protein expression in gstrointestinl tumors is ssoited with this TS polymorphism in the 5 -untrnslted region. 11 These dt suggest tht this TS polymorphism my e signifint preditor of TS gene nd protein expression. In our study we lso found n ssoition etween the TS genotype nd the TS mrna expression in norml liver tissue suggesting the signifine of this polymorphism on TS gene regultion s it hs een onluded from in vitro studies. 6 Compring the TS mrna expression in metstsized tumor tissue nd in djent norml liver tissue, we found signifint higher TS levels in the tumor. These dt suggest tht the TS mrna expression in tumor tissue my e due to trnsriptionl or post-trnsriptionl proess. Reently 6- p deletion in the 3 -untrnslted region of the TS gene ws reveled nd suggested tht this new polymorphism my lter mrna instility nd trnsltion. 12 We were le to show tht the presene of triple repets (L) predited not only intrtumorl TS mrna expression, ut lso response to 5-FU sed hemotherpy. Ptients with the S/S genotype hd the most fvorle response to 5-FU therpy, euse the intrtumorl TS mrna expression is signifintly lower in this group. The lower numer of ptients homozygous for the S/S genotype might e due to the ft tht they omprise only out 10 20% of the popultion. 13,14 The differenes in the frequeny of these tndem repets in Chinese nd Cusins my explin differenes in linil outome to 5-FU hemotherpy in those ethni groups. Tking these oservtions together, our dt suggest tht sreening of ptients for this TS polymorphism my hve the potentil to selet ptients of whom 50% will respond to 5-FU-sed tretment. Reently two novel lleles in the enhner region of the TS gene ontining four nd nine opies of the tndem repet hve een found in Afrin popultions. The frequeny of those lleles in Cusins ppers to e very low, however it might e interesting to evlute whether n ssoition etween these high opy numers nd the TS mrna expression n e found. 15 TS is not the only enzyme ritil in the 5-FU metolism. It hs een shown tht high levels of thymidine phosphorylse (TP) nd dihydropyrimidine dehydrogense (DPD) gene expression re ssoited with low sensitivity to 5-FU tretment in gstrointestinl tumors. Reently our group demonstrted tht using ll three genes, TS, TP nd DPD, we were le to predit response to 5-FU in ll ptients. 16,17 Using TS mrna expression lone, ptients with TS expression levels 3.5 hd response rte of 52%. In this study ptients with the S/S genotype showed response rte of 50% inditing the linil signifine of this TS polymorphism. Another mehnism of 5-FU resistne ws shown to e ssoited with instility of the TS polypeptide, used y n mino id sustitution. 18 Bsed on our dt, tht the TS genotype predits not only TS mrna expression in metstsized olon tumors ut lso in norml liver tissue, we tested whether genotyping of TS would not only predit for response ut lso for toxiity to 5-FU. Ptients with the L/L genotype hd signifintly less toxiity, when ompred to the S/L nd S/S genotype under 5-FU-sed hemotherpy. This my e due to TS mrna expression levels in norml tissue. The inresed TS mrna expression in oth norml nd tumor tissue of ptients with the L/L genotype protets the ells ginst dmge y 5-FU tretment due to the low effiy of TS inhiition. The resulting deresed ell deth rte leds to resistne (tumor tissue) nd low toxiity (norml ells). On the other hnd the dt suggest tht the lower TS mrna level in the norml tissue of ptients with S/S or S/L genotype my enhne the ytotoxi effets of 5-fluorouril leding to more severe side effets in these ptients. The survivl enefit of the S/S group with medin survivl of 16.2 months vs 8.3 nd 8.5 months in the S/L nd L/L group did not reh sttistil signifine. However, tking ll dt together these findings suggest tht genotyping for this TS polymorphism my e helpful in seleting ptients who most likely enefit from 5-FU hemotherpy. The simple method of PCR mplifition of genomi DNA from peripherl lood prior to ommening hemotherpy provides tool to llow individul hemotherpy sed on genomi profiling. Ptients unlikely to respond to 5 FU, those with the L/L genotype, my e treted with other gents suh s irinoten or oxlipltin whose mehnisms of tion re independent of TS inhiition. In ddition genotyping for the TS polymorphism my eome n importnt tool to identify ptients, who require lose monitoring or The Phrmogenomis Journl

5 Thymidylte synthse polymorphism nd hemotherpy ST Pullrkt et l 69 djustment of the 5-FU dose (S/S group). These findings my lso pply to the newer fluoropyrimidine gents like peitine nd UFT, whih re inresingly eing used for the tretment of vriety of ners. Lrger prospetive linil studies re neessry to vlidte our dt from this retrospetive pilot study. PATIENTS AND METHODS Ptients Seletion Ptients inluded in this study hd disseminted oloretl ner nd were enrolled in the following protools: Southwest Onology Group protool 9420 (24 ptients) opened for rul in My 1995 nd losed in My 1999; University of Southern Cliforni protool 3C-92-2 (26 ptients) opened for rul in Septemer 1992 nd losed in June 1995; nd Cner nd Leukemi Group B protool 9481 (26 ptients). All ptients signed n informed onsent to prtiipte in the linil tril nd for evlution of the TS polymorphism. Genotyping for the TS polymorphism ws performed on fresh-frozen or prffin-emedded tissues in ll ptients. All speimens were tken from metstsized liver tumors. The reltion etween genotype nd intrtumorl TS mrna ws determined for 52 ptients enrolled in the 3C-92-2 nd CALGB 9481 protools s fresh frozen speimens were not ville for TS mrna quntittion from ptients enrolled in the SWOG 9420 protool. Quntittion for mrna levels in norml tissues ws performed on 26 CALGB (protool 9481) ptients. Anlysis of the genotype nd linil outome ws done on 50 ptients enrolled in the SWOG 9420 nd 3C-92-2, s linil dt re not yet ville for the CALGB ptients. All ptients were previously untreted, hd stge IV disese nd reeived 5-FU s definitive therpy (Tle 6). All ptients hd i-dimensionlly mesurle disese t the time of protool entry. Responders to 5-FU therpy were lssified s those ptients whose tumor urden ws deresed y 50% or more (prtil response) or ompletely disppered (omplete response) for t lest 6 weeks. Non-responders inluded those with stle disese or ner progression. Toxiity ws grded ording to the NCI riteri version 2 onsidering grde 0 none, grde 1 mild, grde 2 moderte, grde 3 severe, nd grde 4 life-thretening. Survivl ws omputed s the numer of months from the initition of hemotherpy with 5-FU to deth of ny use. Ptients who were live t the lst follow-up evlution were ensored t tht time. The doule tndem repet vrint of the TS gene ws designted s the S llele wheres the triple repet ws designted s the L llele. TS Gene Polymorphism DNA ws extrted from frozen or prffin-emedded tissues using the QiAmp kit (Qigen, Vleni, CA, USA). The promoter region of the hts gene ws mplified y polymerse hin retion (PCR) using the following primers: Primer 1 (sense): 5 GTGGCTCCTGCGTTTCCCCC-3, nd Primer 2 (ntisense): 5 -GCTCCGAGCCGGCCACAGGCATGGCGCGG- 3 s previously desried. 14 Briefly, 25 l retion mixture ontining 1.25 mm MgCl 2 ws trnsferred to therml yler (PTC-100 TM, MJ Reserh Lortories, Wtertown, MA, USA) nd mplified for 35 yles. Eh yle onsisted of 1 min t 96 C, 30 s t 60 C nd 1 min t 72 C with finl extension phse t 72 C for 5 min. The PCR produt ws nlyzed y eletrophoresis on 4% grose gel. Homozygotes for the triple repet vrint L/L hd 250-p produt, homozygotes for the doule repet vrint S/S hd 220-p produt nd heterozygotes S/L hd 220 nd 250-p produts (Figure 1). TS mrna Quntittion Messenger RNA ws isolted from fresh frozen tissue smples using QuikPrep miro mrna isoltion kit (Phrmi Bioteh), sed on the method desried y Chomzynski nd Shi. 19 TS mrna ws quntitted using quntittive RT-PCR method s previously desried. Briefly, TS mrna ws trnsried to DNA using reverse trnsriptse nd rndom hexmers. The DNA ws PCR mplified using TS primers TS 61: T7- GGGAGA GAGTTGACCA ACTGCAAAGAGTG (ses of the TS oding sequene) nd TS 60: GATGTGCGCA Tle 6 Summry of ptients inluded in study Study Totl Tumor Norml Norml Clin. RNA RNA DNA dt CALGB ND USC 3C ND SWOG ND ND Totl FU doses used: 450 mg dy 1 d1 d5 vs i.. infusion of 5-FUDR. 5-FU doses used: CI 200 mg m 2 dy 1 for 3 weeks followed y 1 week rest. 5-FU doses used: CI 300 mg m 2 dy 1 vs CI 2600 mg m 2 dy 1 every week. ND, not determined. CI, ontinuous infusion. Figure 1 Eletrophoresis of PCR produts on 4% grose gel showing single 220-p nd 250-p nd in homozygotes for S/S nd L/L respetively. Doule nds re seen in heterozygotes (S/L).

6 70 Thymidylte synthse polymorphism nd hemotherpy ST Pullrkt et l ATCATGTACGTGAG (ses of the TS oding sequene. 20 PCR onditions, T7 RNA polymerse trnsription nd the quntittion proedure re desried in detil y Horikoshi et l. 21 Sttistil Anlysis TS mrna levels were log-trnsformed prior to the nlysis. An nlysis of vrine (ANOVA) ws performed to test for differenes in TS expression y TS genotype. Anlysis ws done for the norml liver tissue nd metstti tumor tissue seprtely. The overll P-vlues were sed on the F-test from the ANOVA. The SNK (Student Newmn Keuls) method 22 ws used for the pir-wise omprisons of the S/S, S/L, nd L/L groups. For eh genotype, the geometri men (ie fter trnsformtion then using the exponentil trnsformtion to onvert k to originl sle) nd the ssoited 95% onfidene intervl were used to summrize the TS expression. Fisher s ext test nd Jonkheere Terpstr ext test were used to evlute the ssoition of genotype with the seline, nd linil outome (response to the hemotherpy of 5-FU, nd the toxiity). To evlute the ssoition etween TS genotype nd survivl, Kpln Meier plots nd logrnk test were used. 23,24 Medin survivl ws sed on Kpln Meier estimte. The reltive risks were sed on the oserved nd expeted numer of deths s lulted in the Log-rnk test sttistis. 25 ACKNOWLEDGEMENTS This work is funded y NIH grnts R01 CA 82655, R01 CA74166 nd P30 CA JS is supported y Dr Mildred Sheel Stiftung, Bonn, Germny. DUALITY OF INTEREST None delred. Arevitions 5-FU 5-Fluorouril TS Thymidylte synthse TP Thymidine phosphorylse DPD Dihydropyrimidine dehydrogense REFERENCES 1 Midgley R, Kerr D. Coloretl ner. Lnet 1999; 353: Moertel CG. Chemotherpy for oloretl ner. N Engl J Med 1994; 330: Heidelerger C, Chudhuri NK, Dnneerg P, Mooren D, Griesh L, Dushinsky R et l. Fluorinted pyrimidines: new lss of tumor inhiitory ompounds. Nture 1957; 179: Dnenerg P. Thymidylte synthse trget enzyme in ner hemotherpy. Biohem Biophys At 1977; 473: Horie N, Ai H, Ogur K, Hojo H, Tkeishi-K. Funtionl nlysis nd DNA polymorphism of the tndemly repeted sequenes in the 5 terminl regultory region of the humn gene for thymidylte synthse. Cell Strut Funt 1995; 20: Horie N, Chimoto M, Nozw R, Tkeishi K. Chrteriztion of the regultory sequenes nd nuler ftors tht funtion in oopertion with the promoter of the humn thymidylte synthse gene. Biohem Biophys At 1993; 1216: Leihmn CG, Lenz HJ, Leihmn L, Dnenerg K, Brnd J, Groshen S. Quntittion of intrtumorl thymidylte synthse expression predits for disseminted oloretl ner response nd resistne to protrted-infusion fluorouril nd weekly leuovorin. J Clin Onol 1997; 15: Lenz HJ, Leihmn CG, Dnenerg KD, Dnenerg PV, Groshen S, Cohen H et l. Thymidylte synthse mrna level in denorinom of the stomh: preditor for primry tumor response nd overll survivl. J Clin Onol 1995; 14: Lenz HJ, Dnenerg KD, Leihmn CG, Florentine B, Johnston PG, Groshen S et l. p53 nd thymidylte synthse expression in untreted stge II olon ner: ssoitions with reurrene, survivl, nd site. Clin Cner Res 1998; 4: Lenz HJ, Hyshi K, Slong D, Dnenerg KD, Dnenerg PV, Metzger R et l. p53 point muttions nd thymidylte synthse messenger RNA levels in disseminted oloretl ner: n nlysis of response nd survivl. Clin Cner Res 1998; 4: Kwkmi K, Omur K, Knehir E, Wtne Y. Polymorphi tndem repet in the thymidylte synthse gene is ssoited with its protein expression in humn gstrointestinl ners. Antiner Res 1999; 19: Ulrih CM, Bigler J, Velier CM, Greene EA, Frin FM, Potter JD. Serhing expressed sequene tg dtse: disovery nd onfirmtion of ommon polymorphism in the Thymidylte synthse gene. Cner Epidemiol Biomrkers Prev 2000; 9: Pullrkt ST, Ingles SA, Xiong Y-P, Ingles SA, Sherrod A, Ghderi V et l. Anlysis of the thymidylte synthse gene polymorphism in the Ltino popultion. Pro AACR 1999; 40: Mrsh S, Collie-Duguid ESR, Li T, Liu X, MLeod HL. Ethni vrition in the thymidylte synthse enhner region polymorphism mong Cusin nd Asin popultions. Genomis 1999; 58: Mrsh S, Ameyw MM, Githng s J, Indlo A, Ofori-Adjei D, MLeod HL. Novel thymidylte synthse enhner region lleles in Afrin popultions. Hum Mut 2000; 16: Metzger R, Dnenerg K, Leihmn CG, Slong D, Shwrtz EL, Wdler S et l. High sl level gene expression of thymidine phosphorylse (pltelet-derived endothelil ell growth ftor) in oloretl tumors is ssoited with nonresponse to 5-fluorouril. Clin Cner Res 1998; 4: Ishikw Y, Kuot T, Otni Y, Wtne M, Termoto T, Kumi K et l. Dihydropyrimidine dehydrogense nd messenger RNA levels in gstri ner: possile preditor for sensitivity to 5-fluorouril. Jpn J Cner Res 2000; 91: Kithens ME, Forsthoefel AM, Brour KW, Spener HT, Berger FG. Mehnisms of quired resistne to thymidylte synthse inhiitors: the role of enzyme stility. Mol Phrmol 1999; 56: Chomzynski P, Shi N. Single-step method of RNA isoltion y id gunidium thioyte-phenol-hloroform extrtion. Ann Biohem 1987; 162: Tkeishi K, Kned S, Ayusw D, Shimizu K, Gotoh O, Seno T. Nuleotide sequene of funtionl DNA for thymidylte synthse. Nulei Aids Res 1987; 13: Horikoshi T, Dnenerg KD, Stdluer TH, Volkenndt M, She LC, Aigner K et l. Quntittion of thymidylte synthse, dihydrofolte redutse, nd DT-diphorse gene expression in humn tumors using the polymerse hin retion. Cner Res 1992; 52: Zr JH. Biosttistil Anlysis. Prentie-Hll: Englewood Cliffs, Kpln EL, Meier P. Nonprmetri estimtion for inomplete oservtions. J Am Stt Asso 1958; 53: Miller RG Jr. Survivl Anlysis. John Wiley & Sons: New York, Pike MC. Contriution to the disussion on the pper y R Peto nd J Peto, Asymptotilly effiient rnk invrint proedures. J Royl Stt So 1972; Series A 135: The Phrmogenomis Journl