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.
ABSTRACT: Several decades of chemotherapy trials in nonâsmall-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 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 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). 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).In the Southwest Oncology Group study, 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).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. 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).No single chemotherapy combinationhas demonstrated an overall superiorityto any other in survivalbenefit[9,10] or in quality of lifein 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. However, noconsistent second-line treatment hadbeen undertaken until recently. TheBritish randomized study highlightedthe point that time to progressionoccurred early on, after three to fivecycles of chemotherapy.Customized chemotherapy canhelp to improve outcome by selectingpatients 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.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 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. The largestgemcitabine study, including 83 patients,attained a partial response of19%, including patients who had previouslyresponded to gemcitabine/cisplatinchemotherapy.Pemetrexed (Alimta) has been testedin patients with progressive diseasewithin 3 months after first-linechemotherapy or progression whilebeing treated with first-line chemotherapy. 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.
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). Furthermore, paclitaxelyielded a significantly lowergrowth inhibition than docetaxel inHER2/neu-overexpressing MCF/18cells (a MCF-7 human breast cancercell line transfected with HER2/neu). 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.Akt regulates cell survival by phosphorylatingdownstream apoptotic targets(BAD, procaspase 9, Forkheadfamily of transcription factors, NFkappaBregulator IKK) (Figure 1).The loss of phosphatase PTEN leadsto activation of Akt. 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.
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%.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.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 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  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 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, 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. 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.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. 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) and ribonucleotidereductase M1 polypeptide(RRM1) 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. 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. 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. 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).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.Surrogate markers in antiangiogenesisclinical trials are fully reviewedelsewhere. Increased VEGF expressionalso results from COX2-generatedprostaglandin E2 (PGE2).Interestingly, K-ras mutations activate the Raf/MEK/MAPK and PI3K/Aktpathways, resulting in increased transcriptionof COX2 mRNA.Experimental work suggesting thatK-ras mutations are associated withlower DNA repair capacity leads usto speculate that K-ras mutations mayconfer a chemotherapy advantage.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. 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).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. Similarly,irinotecan/cetuximab has achieved asignificant number of responses inpatients previously treated with irinotecan. 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.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.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). 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). 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. 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. 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.
The authors have nosignificant financial interest or other relationshipwith the manufacturers of any productsor providers of any service mentioned in thisarticle.
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