Pemetrexed in Previously Treated Non–Small-Cell Lung Cancer

ABSTRACT: Several decades of chemotherapy trials in non–small-cell lung cancer(NSCLC) have clearly shown a survival benefit for chemotherapyover best supportive care. However, only short-lived responses are attained,with an average of four cycles of chemotherapy, before tumorprogression is observed. Second-line chemotherapy has been demonstratedto improve outcome, with docetaxel (Taxotere) as the predominantcytotoxic drug. A recent randomized trial in second-line NSCLCindicated that the novel drug pemetrexed (Alimta) attained the sameresponse, time to progression, and survival as docetaxel. This findingushers in a new age in second-line treatment that can be further invigoratedby the addition of targeted agents. Accumulated evidence indicatesthat overexpression of epidermal growth factor receptor andHER2/neu, which occurs frequently in NSCLC, leads to the deregulationof PI3K and MAPK, activating Akt and enhancing chemoresistance.Future clinical trials in NSCLC will include tailored andmultitargeted therapy and pemetrexed represents a significant step forSward in this direction.Second-line chemotherapy innon-small-cell lung cancer(NSCLC) is a relatively newapproach. For a long time, it was notrecognized that cisplatin-based chemotherapyimproved survival in themetastatic setting. A Canadianstudy[1] showed a median survival of4 months with best supportive careand 8 months (P = .01) with vindesine(Eldesine) at 3 mg/m² weekly* 4 and then every 2 weeks plus cisplatinat 120 mg/m², days 1 and 21and then every 6 weeks. One-yearsurvival rates were 10% and 22%,respectively.The cisplatin-based chemotherapymeta-analysis[2] of cisplatin combinedwith vinca alkaloids or etoposideshowed a chemotherapy benefitwith a hazard ratio of 0.73 and anincrease in median survival of 1.5months. The response rate with gemcitabine(Gemzar)/cisplatin was higherthan with etoposide/cisplatin (41%vs 22%; P = .02) and time to progressionwas also longer in the gemcitabinearm (6.9 vs 4.3 months; P =.01).[3] In the British study, mediansurvival was 6.7 months with mitomycin(Mutamycin)/ifosfamide (Ifex)/cisplatin (MIC) as compared to 4.8months with best supportive care (P =.03).[4]In the Southwest Oncology Group study,[5] cisplatin-based chemotherapywas a strong significant prognosticfactor for survival. Furthermore,in cisplatin-treated patients, three differentprognostic subsets were observedbased on performance status,age, hemoglobin, and lactate dehydrogenase(LDH), with 1-year survivalof 27%, 16%, and 6% (P More recently, overexpression ofgenes involved in the nucleotide excisionrepair pathway has been relatedto cisplatin response and survival.In gemcitabine/cisplatin-treated patients,overexpression of excision repaircross-complementing 1 (ERCC1)mRNA was associated with a mediansurvival of 5 months, in contrast with15 months in patients with normalERCC1 mRNA levels (P = .009).[6]In spite of the statistically demonstratedbenefit of cisplatin chemotherapy,patients often perceive arelatively small benefit, and hence arereluctant to receive chemotherapy.In scripted interviews, when patientswere asked to indicate the benefitrequired to accept the toxicityassociated with chemotherapy, only22% chose chemotherapy over bestsupportive care for a survival benefitof 3 months.[7] Moreover, in a Britishstudy, patients were randomizedto receive three or six cycles of mitomycin/vinblastine/cisplatin (MVP); nodifferences were observed in time toprogression (5 months in both arms)or in median survival (6 vs 7months).[8]No single chemotherapy combinationhas demonstrated an overall superiorityto any other in survivalbenefit[9,10] or in quality of life[10]in metastatic NSCLC. The averagetime to progression ranged from 3.4to 5.5 months, median survival from7.4 to 9.9 months, 1-year survival from31% to 43%, and 2-year survival from10% to 13%.[9,10] In these studies, afraction of patients received poststudytherapy, mainly with crossover doubletcombinations.[10] However, noconsistent second-line treatment hadbeen undertaken until recently. TheBritish randomized study[8] highlightedthe point that time to progressionoccurred early on, after three to fivecycles of chemotherapy.Customized chemotherapy canhelp to improve outcome by selectingpatients[11] and planning second-linetreatment in all clinical trials. Thereare only a few drugs that have demonstratedactivity in the second-line approach, and there is some evidenceof activity for cisplatin-based chemotherapyin patients receiving frontlinenoncisplatin combinations.[12]Phase II Second-LineTrials in NSCLCMultiple phase II studies have beenperformed with almost all availablecytotoxic drugs. The majority of thesestudies included vinorelbine (Navelbine),gemcitabine, paclitaxel, anddocetaxel (Taxotere). A complete review[13] found that with the exceptionof docetaxel, the response in someof these studies was null. Median survivalfrom the time of starting second-line treatment ranged from 3 to11 months (Table 1). Many of theseearly phase II studies included a smallnumber of patients. Moreover, fromthe inclusion criteria, it is difficult todiscern whether the patients were cisplatin-refractory (no response wasobserved) or cisplatin-resistant (a priorresponse was attained), as therewas no common agreement on theconcept of cisplatin-resistant and-refractory tumors.[13] The largestgemcitabine study, including 83 patients,attained a partial response of19%, including patients who had previouslyresponded to gemcitabine/cisplatinchemotherapy.[14]Pemetrexed (Alimta) has been testedin patients with progressive diseasewithin 3 months after first-linechemotherapy or progression whilebeing treated with first-line chemotherapy.[15] Patients were stratifiedaccording to whether the first-linetreatment included a platinum regimen.Pemetrexed was administered at500 mg/m² every 21 days. The responserate was 4.5% in the platinumpretreatedgroup and 14% in thenon-platinum-pretreated group. Mediansurvival was 6.4 and 4 months,respectively. Time to progression was2.3 and 1.6 months, respectively (Table1). In this trial, no folic acid orvitamin B₁₂ was administered. Vitaminsupplementation with daily folicacid (350 μg) and vitamin B₁₂ (1,000μg) every 9 weeks significantly loweredhomocysteine levels and reducedsevere myelosuppression and gastrointestinaltoxicity.[16]

Randomized Second-LineTrials in NSCLCTo date, the only drug approvedfor second-line treatment of NSCLCis docetaxel, based on the results oftwo phase III randomized studies (Table2).[17,18] Among the many intriguingquestions surrounding thispoint, perhaps the most salient is whypaclitaxel has not been identified as apotential second-line treatment. Severallines of evidence indicate thatHER2/neu overexpression is relatedto paclitaxel resistance.Prez-Soler et al reported that inNSCLC heterotransplants in nudemice, lack of HER2/neu expressionwas linked to better response to paclitaxel(0% of responding tumors and48% of nonresponders expressedHER2/neu).[19] Furthermore, paclitaxelyielded a significantly lowergrowth inhibition than docetaxel inHER2/neu-overexpressing MCF/18cells (a MCF-7 human breast cancercell line transfected with HER2/neu).[20] Growth factor receptor-mediatedsignal transduction has beeninvolved in chemoresistance. TheHER2/neu/phosphatidylinositol-3 kinase(PI3K)/Akt pathway mediatesresistance to chemotherapy and EGFR inhibitors. Overexpression of Akt conferschemoresistance in NSCLC.[21]Akt regulates cell survival by phosphorylatingdownstream apoptotic targets(BAD, procaspase 9, Forkheadfamily of transcription factors, NFkappaBregulator IKK) (Figure 1).[22]The loss of phosphatase PTEN leadsto activation of Akt.[23] In breast cancercell lines, expression of bothHER2/neu and HER3 caused a PI3Kdependentactivation of Akt that waslinked to increased resistance to paclitaxel,doxorubicin, fluorouracil (5-FU), etoposide, and camptothecin.[24]

These preclinical lines of evidencehave led some investigators to designsecond-line studies of gefitinib (Iressa,an EGFR inhibitor) plus trastuzumab(Herceptin, a HER2/neuinhibitor). HER2/neu has been studiedby immunohistochemistry in resectedNSCLC tumors, and 2+ or 3+overexpression was found in 17%.[25]However, with real-time quantitativepolymerase chain reaction (RTQPCR),HER2/neu mRNA expressionwas detected in 100% of specimensanalyzed. HER2/neu expression levelsegregated patients into poor and goodprognostic subgroups. The segregationmethod used a tumor-normal tissueratio of 1.8 as a cutoff value.[26]Almost 35% of patients had highHER2/neu expression, and nearly 38%had high epidermal growth factor receptor(EGFR) expression. HighHER2/neu expression was associatedwith inferior survival in this group ofresected NSCLC patients.The Shepherd et al study[17] demonstratedthat docetaxel at 75 mg/m² every 3 weeks yielded a 6% response.When compared with best supportivecare, time to progression was 3 vs 1.7months, median survival was 7.5 vs4.6 months, and 1-year survival was37% vs 12%. These differences wereall statistically significant. Better outcomewas observed in patients with aperformance status of 1, those whohad responded to prior cisplatin treatment,those who had received onlyone prior chemotherapy regimen,those who had no weight loss, or thosewith normal LDH (Table 2). The Fossellaet al [18] study showed as wellthat docetaxel at 75 mg/m² every 3weeks achieved a 7% response rate,with a median survival of 5.7 monthsand a 1-year survival of 30%, whilevinorelbine or ifosfamide yielded a1% response rate, with a median survivalof 5.6 months and a 1-year survivalof 20%. The differences at 1year were significant. In this study,prior paclitaxel treatment did not influencethe responses attained withdocetaxel (Table 2).The Camps et al[27] phase III studycompared two different docetaxel doseschedules (75 mg/m² every 3 weeksvs 36 mg/m² weekly for 6 weeks, every8 weeks). Preliminary observationsin 179 patients show responserates of 11% and 7%, time to progressionof 3.4 and 3.5 months, and mediansurvival of 6.3 and 6.1 months, respectively.Eighteen percent of patients hadreceived prior paclitaxel-based treatment.No substantial differences in toxicitywere observed (Table 2).Hanna et al conducted the largestphase III randomized trial in secondlinetreatment,[28] comparing docetaxelat 75 mg/m² every 3 weeks withpemetrexed at 500 mg/m² every 3weeks. In the pemetrexed arm, folicacid (350-1,000 μg daily) and vitaminB₁₂ (1,000 μg every 9 weeks)were administered. Patients were stratifiedaccording to performance status,best response to prior chemotherapy,number of prior chemotherapyregimens, time since last chemotherapy,prior platinum- and taxane-basedchemotherapy, and baseline homocysteineserum levels. Ninety percent ofpatients had received prior cisplatintreatment and 26% had received priortaxanes. The median number of cyclesadministered was 4 in both arms.Response rates were 8.8% in thedocetaxel arm and 9.1% in the pemetrexedarm, respectively. Time to progressionwas 2.9 months in both arms.Median survival was 7.9 and 8.3months, respectively. One-year survivalwas 29.7% in both arms (Table2). While response, time to progression,and survival were similar in thetwo arms, in the pemetrexed arm lesssevere neutropenia, fewer hospitalizations,and less need for ancillary measureswere observed. This study opensthe gates for new second-line chemotherapycombinations (Figure 2).

A phase II second-line randomizedtrial of irinotecan/cisplatin vs cisplatinalone was carried out in patientspretreated with taxanes/gemcitabine.[29] Response rates were 24% and8.3%, respectively. Time to progressionwas 2.5 and 2 months, respectively.Median survival was identicalin both arms (9 months). One-yearsurvival was 40.5% and 31.2%, respectively(Table 2). Although the numberof patients included in the trial wassmall, the results were promising forthe use of the novel combination.EGFR inhibitors have also beenused as targeted therapies, attainingmeaningful response rates rangingfrom 11% to 18.4%, with median survivaltimes of 6.5 to 8.4 months, and1-year survival rates of 29% to40%.[30,31] These outcomes are relevant,especially considering that mostof these patients had received morethan two prior chemotherapy regimens.Skin toxicity was linked to bettersurvival in some of these trials(Table 2). The combination of cetuximab(Erbitux), the monoclonal antibodyagainst the extracellularligand-binding domain of EGFR, plusdocetaxel yielded a 25% response ratewith a 7.5-month median survival.[32]Tailored and TargetedChemotherapy in NSCLCIt is well known that randomizedtrials can be falsely negative if patientgenetic criteria are not taken into consideration,because a beneficial effectin responding patients can be dilutedby a large number of nonrespondingpatients or a beneficial effect in responderscan be reversed by a negativeeffect in nonresponders.[33] Theanalysis of molecular signatures canlead to the selection of patients mostlikely to respond to a given chemotherapyregimen (Figure 2). As a firststep, excision repair cross-complimenting1 (ERCC1)[6] and ribonucleotidereductase M1 polypeptide(RRM1)[11] mRNA expression levelscan be used to identify a largesubset of patients who should benefitfrom gemcitabine/cisplatin.Along the same lines, sensitivityto antimicrotubule drugs is modulatedby the expression of microtubuleassociatedprotein 4 (MAP4). MAP4promotes microtubule polymerization,and reduced MAP4 expression decreasesmicrotubule polymerizationand increases vinca alkaloid sensitivity.Conversely, elevated MAP4 expressionpromotes microtubulepolymerization and increases taxanesensitivity.[34] HER2/neu expressioncan also help to distinguish betweenpaclitaxel- and docetaxel-sensitivepatients.[19,20] The clinical value of MAP4 and HER2/neu merits beingvalidated in phase II randomized studies(Figure 2).The same need to include geneticassessment in selection criteria of clinicaltrials is of the highest priority instudies of targeted therapies. The effectivenessof EGFR inhibitors hasbeen slightly obscured by a lack ofanalysis of the dysfunctional EGFRand PI3K signaling pathways. Dissectingthe molecular events associatedwith PI3K and mitogen-activatedprotein kinase (MAPK) deregulationcan have important implications inthe design of clinical trials with PI3Kand MAPK inhibitors.[35] The developmentof phosphorylation-specificantibodies that allow for detectionof activated signaling molecules inparaffin-embedded biopsy tissuesmakes it possible to examine the multiplenodes in the PI3K and MAPKpathways (Figure 1).Overexpression of the insulin-likegrowth factor 1 receptor (IGF-1R)abrogates the effect of trastuzumaband EGFR inhibitors. Also, the lossof PTEN leads to PI3K and Akt hyperactivity.EGFR-HER-2 heterodimersinduce a stronger and moresustained proliferative signal thanEGFR homodimers.[36] Gain-offunctionmutations in oncogenes canactivate PLC, Ras-MEK (mitogen-activatedprotein/extracellular signalregulatedkinase)-MAPK, PI3K andits target Akt, S6, Src, and signalingtransduction activators of transcription(STATs).[23,37] Active Aktphosphorylates a number of substratesinvolved in apoptosis and cell cycleregulation, including the Bcl-2 familymember BAD, Forkhead transcriptionfactors, caspase-9, IkappaBkinase, p21, p27, mTOR and nitricoxide synthase (NOS) (Figure 1).[23]HER2/neu signaling activates PI3Kand Akt and the downstream kinasemTOR and increases hypoxia-induciblefactor 1-alpha (HIF-1-alpha),which mediates vascular endothelialgrowth factor (VEGF) expression.[38]Surrogate markers in antiangiogenesisclinical trials are fully reviewedelsewhere.[39] Increased VEGF expressionalso results from COX2-generatedprostaglandin E₂ (PGE₂).[40]Interestingly, K-ras mutations activate the Raf/MEK/MAPK and PI3K/Aktpathways, resulting in increased transcriptionof COX2 mRNA.[41]Experimental work suggesting thatK-ras mutations are associated withlower DNA repair capacity leads usto speculate that K-ras mutations mayconfer a chemotherapy advantage.[42]At both preclinical and clinical levels,it has been observed that COX2expression is associated with inducibleNOS (iNOS).[43,44] Molecularoxygen is required for the productionof nitric oxide by NOS. It has beendemonstrated that nitric oxide mediateschemosensitivity in tumor cellsand hypoxia-induced drug resistanceis in part the result of downstreamsuppression of endogenous nitric oxideproduction.[45] In paclitaxel/carboplatin-treated patients, we observedmedian survival of 8.5 months forthose with low COX2 mRNA levelsand 13.9 months for those with highlevels (P = .006).[11]The analysis of the PI3K and Ras/Raf/MAPK signaling pathways (Figure1) can contribute to the developmentof new combinations ofchemotherapy with targeted therapies(Figure 2). For instance, gefitinib ishighly active in tumor cell lines overexpressingHER2/neu.[46] Similarly,irinotecan/cetuximab has achieved asignificant number of responses inpatients previously treated with irinotecan.[47] These results raise the hypothesisthat EGFR inhibitors canattenuate chemoresistance by downregulatingAkt.It has been widely recognized thatit is necessary to combine several targetdrugs to block multiple nodes ofthese signaling pathways in order toattain a significant reduction inchemoresistance. The specific pointof targeted therapies in abrogatingchemoresistance has not been fullyunfolded in the design of clinical trials.Pharmaceutical firms should contributeto the development of new,more dynamic and flexible clinicaltrials testing the simultaneous combinationof several targeted therapiesplus chemotherapy, as a major meansto improving survival in NSCLC. Figure2 illustrates some of these newtargeted therapies, including bortezomib(PS-341), a selective inhibitor of the proteasome that causes proteolysisof IkappaB, the endogenousinhibitor of NFkappaB.[48]It has also been observed that theassociation of tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) with bortezomib can reducethe antiapoptotic protein c-FLIP, thusreducing bortezomib resistance.[49]In these signaling pathways, the roleof Akt (Figures 1 and 2) is crucial.Hence, the association of chemotherapywith direct Akt inhibitors or Aktupstream inhibitors is of the utmostimportance.PemetrexedPemetrexed is a multitarget antifolatethat inhibits thymidylate synthase(TS), glycinamide ribonucleotideformyl transferase(GARFT), and aminoimidazole carboxamideribonucleotide formyl transferase(AICARFT), which areessential in the synthesis of purinesfor DNA and RNA. It also interfereswith dihydrofolate reductase (DHFR),which regenerates tetrahydrofolate(Figure 3).[50,51] Increased expressionof DHFR has been correlatedwith methotrexate resistance. Pemetrexedenters cells primarily throughthe reduced folate carrier (RFC) andundergoes extensive intracellularpolyglutamation by folypolyglutamatesynthase (FPGS).[50,52]Pemetrexed pentaglutamate is mostactive against TS, followed by DHFR,GARFT and AICARFT (Figure2).[50] The expression of TS is a majortarget of 5-FU, and its correlationwith response in colorectal cancer patientshas been widely studied. TSmRNA can be used as a predictivemarker in colorectal cancer therapy.A close association of TS with E2F-1expression has also been observed incolorectal cancer patients. The E2Ffamily of transcription factors is involvedin the transcriptional regulationof several DNA synthesisenzymes and common chemotherapeutictargets such as DHFR, TS, andribonucleotide reductase (Table3).[53] E2F-1 is the transcription fac-tor most closely related to TS expression,whereas E2F-4 has been shownto be a regulator of DHFR expression.Significant correlations havebeen observed between E2F-1/E2F-4and RFC mRNA expression.[53] Inaddition, folate derivates participatein single-carbon transfers in severalreactions, including the synthesis ofnucleotides and methionine.Methionine is the precursor of Sadenosylmethionine,involved in multipletransmethylation reactions,including DNA methylation. Methylenetetrahydrofolate reductase(MTHFR) converts 5,10-methylenetetrahydrofolate,the methyl donor inTS, into 5-methyltetrahydrofolate, thepredominant circulatory form offolate. 5-methyltetrahydrofolate is acosubstrate for homocysteine remethylationto methionine by the vitaminB₁₂-dependent methioninesynthase.[54] Individuals with aC-to-T substitution at base 677 of theMTHFR gene (amino acid changeA222V) have reduced enzyme activityand higher homocysteine and lowerfolate levels than those without thissubstitution.[55,56]ConclusionsPemetrexed is a new cytotoxic drugwith promising activity in NSCLC andwill contribute greatly to second-linetreatment. We can envision that intailoring pemetrexed therapy, simultaneousRT-QPCR of gene transcripts(Table 3) can aid in selecting the groupof patients who will most benefit frompemetrexed-based treatment.

Disclosures:

The authors have nosignificant financial interest or other relationshipwith the manufacturers of any productsor providers of any service mentioned in thisarticle.

References:

1.

Rapp E, Pater JL, Wilan A, et al: Chemotherapycan prolong survival in patients withadvanced non-small-cell lung cancer: Reportof a Canadian multicenter randomized trial. JClin Oncol 6:633-641, 1988.

2.

Non-Small Cell Lung Cancer CollaborativeGroup: Chemotherapy in non-small celllung cancer: A meta-analysis using updateddata on individual patients from 52 randomisedclinical trials. Br Med J 311:899-909, 1995.

3.

Cardenal F, López-Cabrerizo MP, AntónA, et al: Randomized phase III study ofgemcitabine-cisplatin versus etoposidecisplatinin the treatment of locally advancedor metastatic non-small cell lung cancer. J ClinOncol 17:12-18,1999.

4.

Cullen MH, Billingham LJ, WoodrofeCM, et al: Mitomycin, ifosfamide and cisplatinin unresectable non-small-cell lung cancer:Effects on survival and quality of life. J ClinOncol 17:3188-3194, 1999.

5.

Albain KS, Crowley JJ, LeBlanc M, et al:Survival determinants in extensive-stage nonsmall-cell lung cancer: The Southwest OncologyGroup experience. J Clin Oncol 9:1618-1626, 1991.

6.

Lord RVN, Brabender J, Gandara D, etal: Low ERCC1 expression correlates with prolongedsurvival after cisplatin plus gemcitabinechemotherapy in non-small-cell lung cancer.Clin Cancer Res 8:2286-2291, 2002.

7.

Silvestri G, Pritchard R, Welch HG: Preferencesfor chemotherapy in patients with advancednon-small cell lung cancer: Descriptivestudy based on scripted interviews. Br Med J317:771-775, 1998.

8.

Smith IE, O’Brien MER, Talbot DE, etal: Duration of chemotherapy in advanced nonsmall-cell lung cancer: A randomized trial ofthree versus six courses of mitomycin, vinblastineand cisplatin. J Clin Oncol 19:1336-1343,2001.

9.

Schiller JH, Harrington D, Belani CP, etal: Comparison of four chemotherapy regimensfor advanced non-small-cell lung cancer. NEngl J Med 346:92-98, 2002.

10.

Scagliotti G, De Marinis F, Rinaldi M,et al: Phase III randomized trial comparingthree platinum-based doublets in advancednon-small-cell lung cancer. J Clin Oncol20:4285-4291, 2002.

11.

Rosell R, Scagliotti G, Danenberg KD,et al: Transcripts in pre-treatment biopsies froma three randomized trial in metastatic nonsmall-cell lung cancer. Oncogene 22:3548-3553, 2003.

12.

Georgoulias V, Papadakis E,Alexopoulos A, et al: Platinum-based and nonplatinum-based chemotherapy in advancednon-small-cell lung cancer: A randomizedmuticentre trial. Lancet 357:1478-1484, 2001.

13.

Huisman C, Smit EF, Giaconne G, et al:Second-line chemotherapy in relapsing or refractorynon-small-cell lung cancer: A review.J Clin Oncol 18:3722-3730, 2000.

14.

Crinò L, Mosconi AM, Scagliotti G, etal: Gemcitabine as second-line treatment foradvanced non-small-cell lung cancer: A phaseII trial. J Clin Oncol 17:2081-2085, 1999.

15.

Smit EF, Mattson K, Von Pawel J, et al:Alimta (pemetrexed disodium) a second-linetreatment of non-small-cell lung cancer: Aphase II study. Ann Oncol 14:455-460, 2003.

16.

Niyikiza C, Baker SD, Seitz DE, et al:Homocysteine and methylmalonic acid: Markersto predict and avoid toxicity frompemetrexed therapy. Mol Cancer Ther 1:545-552, 2002.

17.

Shepherd FA, Dancey J, Ramlau R, etal: Prospective randomized trial of docetaxelversus best supportive care in patients with nonsmall-cell lung cancer previously treated withplatinum-based chemotherapy. J Clin Oncol18:2095-2103, 2000.

18.

Fosella FV, DeVore L, Kerr RN, et al:Randomized phase III trial of docetaxel versusvinorelbine or ifosfamide in patients with advancednon-small-cell lung cancer previouslytreated with platinum-containing chemotherapyregimens. J Clin Oncol 18:2354-2362, 2000.

19.

Perez-Soler R, Kemp B, Wu QP, et al:Response and determinants of sensitivity topaclitaxel in human non-small-cell lung cancertumors heterotransplanted in nude mice.Clin Cancer Res 6:4932-4938, 2000.

20.

Witters LM, Santala AM, Engle L, et al:Decreased response to paclitaxel versusdocetaxel in HER-2/neu transfected humanbreast cancer cells. Am J Clin Oncol 26:50-54,2003.

21.

Brognard J, Clark AS, Ni Y, et al: Akt/protein kinase B is constitutively active in nonsmall-cell lung cancer cells and promotes cellularsurvival and resistance to chemotherapyand radiation. Cancer Res 61:3986-3997, 2001.

22.

Bacus, SS, Altomare DA, Lyass L, et al:Akt2 is frequently upregulated in HER-2/neupositivebreast cancers and may contribute totumor aggressiveness by enhancing cell survival.Oncogene 21:3532-3540, 2002.

23.

Bianco R, Shin I, Ritter CA, et al: Lossof PTEN/MMAC1/TEP in EGF receptor-expressingtumor cells counteracts the antitumoraction of EGFR tyrosine kinase inhibitors.Oncogene 22:2812-2822, 2003.

24.

Knuefermann C, Lu Y, Liu B, et al:HER2/PI-3K/Akt activation leads to amultidrug resistance in human breastdenocarcinomas cells. Oncogene 22:3205-3212, 2003.

25.

Hirsch FR, Varella-Garcia M, FranklinWA, et al: Evaluation of HER-2/neu gene amplificationand protein expression in non-smallcelllung carcinomas. Br J Cancer 86:1449-1456, 2002.

26.

Brabender J, Danenberg KD, MetzgerR, et al: Epidermal growth factor receptor andHER2-neu mRNA expression in non-small-celllung cancer is correlated with survival. ClinCancer Res 7:1850-1855, 2001.

27.

Camps C, Massuti B, Jimenez AM, etal: Two second-line docetaxel administratedevery 3 weeks versus weekly in advanced nonsmall-cell lung cancer (NSCLC): A SpanishLung Cancer Group (SLCG) phase III trial (abstract2514). Proc Am Soc Clin Oncol 22:625,2003.

28.

Hanna NH, Shepherd FA, Rosell R, etal: A phase III study of pemetrexed vs docetaxelin patients with recurrent non-small-cell lungcancer (NSCLC) who were previously treatedwith chemotherapy (abstract 2503). Proc AmSoc Clin Oncol 22:622, 2003; J Clin Oncol (inpress).

29.

Vassilis G, Agelidou A, Syrigos K, et al:Second-line treatment with irinotecan (CPT-11)and cisplatin (CDDP) versus CDDP alone inpatients with advanced NSCLC pretreated with taxanes and gemcitabine: Final results of amulticenter randomized phase II study (abstract2516). Proc Am Soc Clin Oncol 22:626, 2003.

30.

Fukuoka M, Yano S, Giaccone G, et al:Multi-institutional randomized phase II trial ofgefitinib for previously treated patients withadvanced non-small-cell lung cancer. J ClinOncol 21:2237-2246, 2003.

31.

Grünwald V, Hidalgo M: Developinginhibitors of the epidermal growth factor receptorfor cancer treatment. J Natl Cancer Inst95:851-867, 2003.

32.

Mininberg ED, Herbst RS, HendersonT, et al: Phase I/II study of the recombinanthumanized monoclonal anti-VEGF antibodybevacizumab and the EGFR-TK inhibitorerlotinib in patients with recurrent non-smallcelllung cancer (NSCLC) (abstract 2521). ProcAm Soc Clin Oncol 22:2521, 2003.

33.

Betensky RA, Louis DN, Cairncross JG:Influence of unrecognized molecular heterogeneityon randomized clinical trials. J ClinOncol 20:2495-2499, 2002.

34.

Bash-Babula J, Toppmeyer D, LabassiM, et al: A phase I/pilot study of sequentialdoxorubicin/vinorelbine: Effects on p53 andmicrotubule-associated protein 4. Clin CancerRes 8:1057-1064, 2002.

35.

Choe G, Horvath S, Cloughesy T, et al:Analysis of the phosphatidylinositol 3’-kinasesignaling pathway in glioblastoma patients invivo. Cancer Res 63:2742-2746, 2003.

36.

Johnson D, Arteaga C: Gefitinib in recurrentnon-small-cell lung cancer: An IDEALtrial? J Clin Oncol 21:2227-2229, 2003.

37.

Wu K, Wang CH, D’Amico M, et al:Flavopiridol and trastuzumab synergisticallyinhibit proliferation of breast cancer cells: Associationwith selective cooperative inhibitionof cyclin D1-dependent kinase and Akt signalingpathways. Mol Cancer Ther 1:695-706,2002.

38.

Laughner E, Taghavi P, Chiles K, et al:HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1-alpha (HIF-1-alpha)synthesis: Novel mechanism for HIF-1-mediatedvascular endothelial growth factor expression.Mol Cell Biol 21:3995-4004, 2001.

39.

Davis DW, McConkey DJ, AbbruzzeseJL, et al: Surrogate markers in antiangiogenesisclinical trials. Brit J Cancer 89:8-14, 2003.

40.

Fukuda R, Kelly B, Semenza GL: Vascularendothelial growth factor gene expressionin colon cancer cells exposed to prostaglandinE2 is mediated by hypoxia-inducible factor 1.Cancer Res 63:2330-2334, 2003.

41.

Araki Y, Okamura S, Hussain SP, et al:Regulation of cyclooxygenase-2 expression bythe Wnt and ras pathways. Cancer Res 63:728-734, 2003.

42.

Feng Z, Hu W, Chen JX, et al: PreferentialDNA damage and poor repair determineras gene mutational hotspot in human cancer.J Natl Cancer Inst 94:1527-1536, 2002.

43.

Rao CV, Indraine C, Simi B, et al:Chemopreventive properties of a selective induciblenitric oxide synthase inhibitor in coloncarcinogenesis, administered alone or incombination with celecoxib, a selectivecyclooxygenase-2 inhibitor. Cancer Res62:165-170, 2002.

44.

Rahman A, Dhar DK, Yamaguchi E, etal: Coexpresion of inducible nitric oxide synthaseand COX-2 in hepatocellular carcinomaand surrounding liver: Possible involvement ofCOX-2 in the angiogenesis of hepatitis C virus-positive cases. Clin Cancer Res 7:1325-1332, 2001.

45.

Matthews NE, Adams MA, Maxwell LR,et al: Nitric oxide-mediated regulation ofchemosensitivity in cancer cells. J Natl CancerInst 93:1879-1885, 2001.

46.

Moasser MM, Basso A, Averbuch SD,et al: The tyrosine kinase inhibitor ZD1839(“Iressa”) inhibits HER2-driven signaling andsuppresses the growth of HER2-overexpressingtumor cells. Cancer Res 61:7184-7188, 2001.

47.

Cunningham D, Humblet Y, Siena S, etal: Cetuximab (C225) alone or in combinationwith irinotecan (CPT-11) in patients with epidermalgrowth factor receptor (EGFR) –positive,irinotecan-refractory metastatic colorectalcancer (MCRC) (abstract 1012). Proc Am SocClin Oncol 22:252, 2003.

48.

Richardson PG, Barlogie B, BerensonJ, et al: A phase 2 study of bortezomib in relapsed,refractory myeloma. N Engl J Med348:2609-2617, 2003.

49.

Syers TJ, Brooks AD, Koh CY, et al: Theproteasome inhibitor PS-341 sensitizes neoplasticcells to TRAIL-mediated apoptosis byreducing levels of c-FLIP. Blood 102:303-310,2003.

50.

Curtin NJ, Hughes AN: Pemetrexed disodium,a novel antifolate with multiple targets.Lancet Oncol 2:298-306, 2001.

51.

Sarries C, Haura EB, Roig B, et al:Pharmacogenomic strategies for developingcustomized chemotherapy in non-small-celllung cancer. Pharmacogenomics 3:763-780,2002.

52.

Scagliotti G, Shin D-M, Kindler HL, etal: Phase II study of pemetrexed with and withoutfolic acid and vitamin B12 as front-linetherapy in malignant pleural mesothelioma. JClin Oncol 21:1556-1561, 2003.

53.

Sowers R, Toguchida J, Qin J, et al:mRNA expression levels of E2F transcriptionfactors correlate with dihydrofolate reductase,reduced folate carrier, and thymidilate synthasemRNA expression in osteosarcoma. Mol CancerTher 2:535-541, 2003.

54.

Sekhon J, Pereira P, Sabbaghian N, etal: Antisense inhibition of methylenetetrahydrofolatereductase reduces survival ofmethionine-dependent tumour lines. Br J Cancer87:225-230, 2002.

55.

Klerk M, Verhoef P, Clarke R, et al:MTHFR 677C T polymorphism and risk ofcoronary heart disease. JAMA 288:2023-2031,2002.

56.

Cohen V, Panet-Raymond V, SabbaghianN, et al: Methylenetetrahydrofolate reductasepolymorphism in advanced colorectal cancer:A novel genomic predictor of clinical responseto fluoropyrimidine-based chemotherapy. ClinCancer Res 9:1611-1615, 2003.