untitled h1
untitled h2

UNIVERSIDADNACIONALAUTÓNOMADEMEXICO
HOSPITALGENERALDEMÉXICO"DR.EDUARDOLICEAGA"
EFFECT OF METFORMIN ON ABCB1 AND AMPK **GENE**  
  
EXPRESSION INADOLESCENTS WITH **ACUTE**
LYMPHOBLASTIC **LEUKEMIA.**
DANIELORTIZMORALES,MD,MSc HeadofPediatricHemOnc
effect of metformin on abcb1
background acute lymphoblastic leukemia

Acutelymphoblasticleukemia(ALL)isamalignantneoplasmthatoriginatesfromlymphoidprogenitorsin the bonemarrow.Inmostcountriesitis themostcommonmalignancyinchildren,andis themost frequent causeofdeathsecondarytocancerinchildrenunder20yearsofage.1,2,3 IntheUnitedStates, the incidence of ALL varies according to ethnicity, with Hispanics being the most affected, with figures up to 40.9casesper million.4 InMexico,accordingtotherecordsofthepopularinsuranceprogram,leukemiahas a prevalence of 49.8%,in the period2007-2012, 83% of these correspond toALL.5 ClinicalcharacteristicatdiagnosisofALLincludeanemic,hemorrhagicandinfiltrativesyndromes. 3 ALLin childrenhashadprogressiveimprovementdue tomultimodal therapyand risk stratification,offering the patient an intensified treatment depending on the risk of relapse. This risk stratification is based on the patient's clinicaldata,biologicalcharacteristicsoftheleukemiccellsandearlyresponsetotreatment,which predict thelikelihoodofrelapse.3 Thesehelptoselecttheintensityoftreatment,toachievethebestchance of cure.Patientageandleukocytecountatdiagnosisareamongthemostimportant,basedonthis,ageless than1yearandgreaterthan10yearsconfersanadverseprognosis.6,7 Geneticalterationshavealsobeen related to theprognosisof children with ALL. 8-12 Early response to treatment refers to the time required to eliminate the bulk of the leukemiccell population toundetectablelevels;this is thesinglemostpowerfulprognostic factorinpediatric ALL. Measurementof submicroscopiclevels of minimalresidualdisease(1leukemiccellin104-5 normalcells) canbeobtainedby polymerasechainreactionamplificationorflowcytometry. 13,14 Theriskoftreatment failure and death is 3 to 5 times higher in patients with minimal residual disease levels of 0.01% or greaterat the end of induction than thosewith levelsless than 0.01%.15
effect of metformin on abcb1
treatment of all

Thecombination ofchemotherapy agents achieves remissionin 80-90%of childrenwith ALL,especially with intensiveprotocols of6-8drugsin4-8weeksintheremissioninductionandconsolidationphases, achieving in our days rates of up to 85% event-free survival and 90% overall survival, in developed countries.3 Current regimens include induction therapy consisting of 4-6 weeks and include steroid, vincristine, asparaginase, anthracyclines and intrathecal therapy. Consolidation includes 6 months of a combination of intensechemotherapythatisdesignedtoconsolidateremissionandpreventthedevelopmentofleukemia  
  
 in the central nervous system. This phase includes high-dose methotrexate. Andfinally, patients receive maintenancetreatmentwithlow-intensitychemotherapyfor18to30months.3 Anotherimportantpartof the treatment istherapy directedto the central nervous system. 16, 17 Treatmentfailureoccursin15-20%ofchildrenwithALLindevelopedcountries,leadingtolowercurerates afterrelapse.Whenrelapseoccursduringtreatment,theprobabilityofachievingasecondremissionis5070% and only 20-30% of patientsare cured. 18,19
effect of metformin on abcb1
all in adolescents

Thetreatmentofadolescents withALL hasalwaysbeenchallenging,asthemerefactofbeingolderthan10 years carries an adverse prognosis. These patients diagnosedwith ALL have shown very little prognostic benefit compared to younger patients with ALL younger than 10 years. It has even been reported that adolescents andyoungadultshave 7.7 to10.5 timeshigherriskofrelapsecomparedtochildrenbetween1 and10 yearsofage.20 Thefactorsthatcausethisagegrouptohaveworsesurvivalratesareunknown.Some proposalsareheterogeneityinthebiologyofleukemia,hostfactors,bothphysiologicalandpsychosocial, and the therapeuticapproachproposed bythehealth care team.21-23
effect of metformin on abcb1
multidrug resistance (mdr)

Despite advances in treatment strategies, approximately 20% of patients will experience relapse. One of themain theoriesof relapseis thepresenceofmultidrug resistance.Severalmechanisms responsiblefor drugresistancehavebeendescribed,bothinvivoandinvitro.Oneofthemostimportantmechanismsis the increased expression of ATP-binding cassette transporters or ABC transporters (ATP-binding cassette transporters).Thisisasuperfamilyof49human ABCgenes,whichareclassifiedinto8subfamiliesfrom ABCAtoABC-GandANSA,basedonthedegreeofhomologyoftheirsequences.24 Thesegenesare specialized inenergy-dependentcellulartransportandareinvolvedinawiderangeofevents,suchas removal of harmful substances,secretion of toxins,mobilizationof ions andpeptides, as wellas cell signaling.25 Within this family, ABCB1/MDR1, ABCC1/MRP1, ABCC3/MRP3 and ABCG2/BCRP genes havebeen evaluated inseveralstudies,withcontroversialresultsindrugresistanceinpediatricALL.26-30 Inclusively, Rahgozar,et al.demonstratedanincreased riskofobtainingpositiveminimal residualdiseaseinexpression ofABCA2, ABCA3, MDR1and MRP1 genes.31,32,33
effect of metformin on abcb1
metformin and cancer

Metforminisan antidiabeticdrugusedinpatientswithtype2diabetesmellitus.Metforminisasynthetic biguanide,whosemainmechanismofactionis throughmitochondrialactivity,amongothers thatarenot welldescribed.34 Ithasbeenexperimentallydemonstratedthathighlevelsofinsulinandglucosepromote cell growthin vitro,proposinga possible mechanismofactionofmetformin.  
mechanismsdirectlyinvolvingcancerandDM2,viahyperinsulinemia,hyperglycemiaandinflammation.35,36  
  
  
Insomeepidemiologicalcohortstudiesofpatientswithtype2diabetes,inwhichtheuseofmetformin alone or combinedwith sulfonylurea, decreased the risk of developing cancer, compared to those exposed to insulin.37 Thesefindingshavebeen replicatedin differentreportswithconsistentresults.Currently, there aremanyarticlesontheuseofmetforminincancer,however,themostsolidevidenceisinbreast, colorectal, ovarianandendometrialcancerinadults;althoughmoreevidenceisrequiredtoevaluatethe therapeutic effect in other cancers.  
38 Themainantitumormechanismofantidiabeticdrugssuchasbiguanideandthiazolindiones,whichhave been showninvariouscelllinestoinhibitcancercells,islikelyduetotheirdisruptionoftheAKT/mTOR signaling pathway in various ways, resulting in differences in leukemic cell proliferation, apoptosis and chemosensitivity. Some researchers have identified activated adenosine 5'-monophosphate protein kinase, or AMPK,whichisanenergyregulatorforhomeostasis,asatargetforALLtreatmentbecauseofitseffect on cell proliferation, cell cycle regulation, and its crosstalk with critical metabolic and oncogenic pathways.39-40 Metforminhasapositivecharge,whichaccumulatesinthemitochondrialmatrixandinhibitscomplexIof the respiratory chain, preventing the oxidation of NADPH with the consequent decrease in ATP levels, whichcauses theaccumulationofAMP.WhenthereisanelevatedAMP/ATPratio,theactivity ofLBK1 (liver kinaseB1orserine/threoninekinase11STK11)responsibleforthephosphorylationandactivationof AMPK, a metabolic sensor that inhibits energy-intensive cellular processes as in neoplastic cells, is promoted. AMPKisactivateddue to thechangeinATPlevels,shifting thecellfromananabolic toacatabolicstate, restoring energy balance. 41-43 In addition, activated AMPK phosphorylates TSC2 (tuberoussclerosis complex 2)whichstimulatestheactivityofGTPaseRheb(Rashomologenrichedinbrain),withsubsequent inactivation ofmTOR(mammaliantardetofrapamycin),essentialforcellproliferationbecauseitregulates theexpression ofcyclinD1,HIF-1alpha/2alpha(hypoxia-induciblefactors),andtheso-calledYamanaka transcriptionfactors, OCT4,KLF4, SOX2,c-Myc.43-45 AMPKactivationinhibitsmTOR,andthetranscriptionalregulationofFOXO3Aprogram,whichisimportant in autophagiccelldeathofpersistentlystressedcells.Insummary,treatmentofpre-BALLandT-cellALLcell lines with AMPK activators, including metformin, has been shown to significantly induce cell growth inhibition andapoptosis.46,47 Inadditiontothesystemiceffectofmetforminoninsulinlevels,thisdrugcansuppressmalignantcellsbya direct mechanism involving cancer stem cells, epithelial-mesenchymal transition (EMT) and cellular senescence.48,49 Studies,suchasthatofJingxuan,demonstratedthattheuseofmetforminimprovesthechemosensitivityof the anthracyclics, L-asparaginase, vincristine and ethoposide, through the impact on the AKT/mTOR pathway, which are used in LAL.50 Othermechanisms involvingmetforminwith othermetabolic pathways involving the cancer response have alsobeendescribed. 43,47, 51-55 Another possible effect of metformin is on the multidrug resistance gene family mentioned above.25Although  
  
  
chemotherapy resistant cells is a fundamental problem for oncologists, there are few studies on ABCB1/MDR1. However, metformin has shown a decrease in the expression of MDR1, mainly through two pathways,thefirstoneinitiateswiththeactivationofAMPKwhichdecreasestheactivityofadenylate cyclase whichinturnreducestheactivityofPKA(proteinkinaseA),preventingthefinalphosphorylationof CREB,
necessary tocarryout the transcriptionalfunction. 56,57 Second,metformin reducesNF-kBlevels through inhibition of the TNF-alpha receptor signaling cascade. 57 Some drugs, such as vincristine, anthracyclines, epipodophyllotoxinsandtyrosinekinaseinhibitors,aresubstratesofthesereceptors,which are present in several types ofcancer. 25,58
effect of metformin on abcb1
metformin in pediatrics

Inpediatrics,metforminisadrugapprovedbytheFoodandDrugAdministration(FDA)forthetreatmentof type2diabetes,andisindicatedforchildrenover10yearsofagebasedonstudiesthatdemonstrateits safety andclinicalbenefitfortype2diabetesat thisage.59 Forthisindication,itisgenerallydosedtwice dailywith anexcellentsafetyprofileinthisagerange.60 Pharmacokineticdatainchildrenbetween12and 16yearsof agehavebeenshowntobewithin5%ofthosecomparedtocontrolsforageandgenderin adults,withC
max898ng/mlandABC(inf) 6311nghr/ml.61 Therecommendedphase2doseformetforminis 1000mg/m2/day, with the main side effects reported at the gastrointestinal andmetabolic level. 40 TheonlystudysofarwheremetforminhasbeenusedinpatientswithALLwaspublishedbyTrucco,et.al.,
where he used a traditional induction scheme with 4 drugs plus metformin in pediatric patients with refractoryandrelapsedALL,in14patients,withoutreachingdefinitiveconclusionsregardingtheresponse ofALLandmetformin,howeverhedemonstratedthattheuseofmetforminissafewithanacceptable toxicity profile.40
effect of metformin on abcb1
research question

What is the effect of metformin use on ABCB1/MDR1 and AMPK gene expression in the remission induction phasein adolescentpatientswith newly diagnosed ALL?
effect of metformin on abcb1
problem statement

ALListhemostcommononcologicdiseaseinthepediatricpopulation,representing35%ofchildhood cancer casesinworldstatistics,anduptoalmost50%accordingtoMexico`snationalstatistics.Incountrieswith limitedresources,long-termsurvivalisreportedtobegreaterthan90%. IncountriessuchasMexico,the5 yearoverallsurvivalofthisdiseasehasbeendescribedtobearound50%, and in those patients with poor prognostic factors even lower, including adolescents.  
TheexpressionoftheABCB1/MDR1andAMPKgeneinadolescents,aswellasinpatientswithALLandits  
  
 variation during treatment, iscurrentlyunknown.  
Inrecentyears,theuseofmetforminhasproventobeusefulinthetreatmentofdifferenthematological malignancies and solid tumors, such as breast cancer and leukemia, through different metabolic pathways. Althoughtheusefulnessofthisdrughasbeendemonstratedinadults,inpediatricsitisstillan unproven field. Clinical trials inchildrenandadolescentsare beginning to be conducted in refractory or relapsed solid tumors; however, thereare nonein acutelymphoblastic leukemia.  
Todate,thebenefitofmetforminuseinALLinpediatrics,especiallyinthehigh-riskgroupofadolescents, and theinteractionitmay havewithABCB1/MDR1andAMPKgeneexpression, has notbeen reported.  
Therefore,itisnecessary toevaluate theexpressionof thesegenesand theusefulnessofmetforminin adolescents with acute lymphoblastic leukemia.
effect of metformin on abcb1
justification

The description of ABCB1/MDR1 and AMPK gene expression in adolescents with ALL may help to understand theirinvolvement in the biological behaviorofALL in this population.  
The searchfornewalternativesincancertreatmenttoimprovepatient survivalis one ofthe main objectives ofcurrentresearch.Therepositioningofolddrugsincancertreatmentisoneofthelinesbeing analyzed, asis thecase ofmetformin.  
Inbasicandclinicalstudies,theantineoplasticactivityofmetforminhasbeendemonstrated.Ithasbeen studied in breast cancer, prostate cancer and hematological neoplasms, all in adults. Therefore, transferring thesestudiestootherpopulations,suchasadolescents,maycontributetoimprovethe survival rateinthis groupwith poor prognosis inALL. Thegenerationofknowledgeregardingtheusefulnessof metformininthisspecificgroupofpatientscan lay the groundwork for research on the effect of this drug in solid tumors and other pediatric neoplasms.
effect of metformin on abcb1
hypothesis

If metformin is added to standard chemotherapy during the remission induction phase in Mexican adolescents with newly diagnosed ALL, then it will decrease the level of ABCB1/MDR1 gene mRNA
expressionand increaseAMPK expression levels, at theend ofthis treatment period.
effect of metformin on abcb1
objectives

General:
- To evaluate the efficacy of metformin in modifying ABCB1 and AMPK gene expression in adolescents diagnosed with ALLduring the remission induction phase.  
Specific
- To describe the expression levels of ABCB1/MDR1 and AMPK genes in adolescents with ALL at diagnosisandin healthy controls.  
- TocomparetheexpressionlevelsofABCB1/MDR1andAMPKgenesatdiagnosisandattheendof inductioninadolescentswithALLtreatedwithconventionalchemotherapyandthoseadditionally treated with metformin.  
- To compare overall and disease-free survival in adolescents with ALL treated in relation to ABCB1/MDR1, and AMPK geneexpression.  
- To compare overall and disease-free survival in adolescents with ALL treated with conventional chemotherapy with those treatedwith convectionalchemotherapy plusmetformin.
effect of metformin on abcb1
methodology

Type and design of **study**
Type of study: randomized clinical trial Study design: analytical, experimental, longitudinal and prospective study.  
Population The population to be studied are patients with newly diagnosed ALL between 10 and21years. Eligible population: patients with newly diagnosed ALL between 10 and21years old, diagnosed at the HospitalGeneraldeMéxico "Dr. Eduardo Liceaga" fromFebruary2021 toDecember 2023.  
Inclusion, exclusion and elimination criteria
- Inclusion criteria:  
  
  
1. Patients aged between 10 and21years with a new diagnosis of ALL at the HospitalGeneraldeMéxico,Dr Eduardo Licega.  
2. Patients andlegallyauthorizedpersonwho sign the informed consent.  
- Exclusion criteria:
1. Patients with antineoplastic treatment prior to admission. 2. Patients with Down syndrome.
- Elimination criteria:
1. Patients who abandon treatment for more than a few weeks.4 2. Patients who withdraw informed consent
effect of metformin on abcb1
sample size

Tocalculatethesamplesize,we useddatafromthepublicationby RamosPeñafiel63,whereadecreasein the expression pattern of ABCB1 gene expression from high to low expression after metformin administration was 50%. The samplesizecalculation was based on the formula for estimating a proportion, witha confidence level  
 of 95%anda precisionof5%:
A sample number of 8 patients in each group is obtained, assuming a loss of 20%, 10patientsare calculated for each group.
effect of metformin on abcb1
definition of the variables to

| Variable    | Conceptual Definition              | Unitof       | Typeof              | Coding   |
|-------------|------------------------------------|-------|--------------|----------|
| measurement | variable                           |       |              |          |
| Age         | Time elapsed in years since birth. | Years | Quantitative | Not      |
| applicable  |                                    |       |              |          |  
| Gender                                                | Male or female phenotype of the person.              | Male/female            | Qualitative    | 0: male        |                |             |           |
|-------------------------------------------------------|------------------------------------------------------|------------------------|----------------|----------------|----------------|-------------|-----------|
| Dichotomous                                           | 1: female                                            |                        |                |                |                |             |           |
| Leukocyte                                             |                                                      |                        |                |                |                |             |           |
| countat diagnosis                                                       | Number                                               | of                     | total          | leukocytes     |                |             |           |
| reported on completebloodcount                                                       | X10/ml3                                              | Quantitative           | Not applicable |                |                |             |           |
| at diagnosis. Measurement of the amount of oxygen transport proteinreported ininitialcompletebloodcount.                                                       | g/dL                                                 | Quantitative           | Not applicable |                |                |             |           |
| Hemoglobinat diagnosis Platelet count at diagnosis                                                       | Measurementofthe number of platelet fragments produced by the megakaryocytes, reported in the Initialcompletebloodcount.                                                      | X10/ml3                | Quantitative   | Not applicable |                |             |           |
| Immunophenot ypeofleukemia lymphoblastic                                                       | Cellular variant of acute lymphoblastic leukemia     | B/ T                   | Qualitative    | 0: strain B    |                |             |           |
| dichotomous                                           | 1: lineageT 0: none 1: t(9;22), 2: t(12,21) , 3: t(4,11), 4: other                                                      |                        |                |                |                |             |           |
| None/ t(9;22)/ t(12,21)/ t(4,11), other                                                       |                                                      |                        |                |                |                |             |           |
| Cytogenetic                                           | Alterations inthegeneticstructure                                                      |                        |                |                |                |             |           |
| alterations                                           | of leukemic cells.                                   | Qualitative polytomous |                |                |                |             |           |
| No expression / lowexpression/ high expression expression                                                       | 0: not expressed 1: high expression 2 low expression |                        |                |                |                |             |           |
| MDR-1and AMPKgene expression.                                                       | Amplification of gene expression by RT-PCR           | Qualitative polytomous |                |                |                |             |           |
| Decreaseintotalperipheralblood blast count less than 1000 after7 days of steroid (day 0)                                                       | Present/absent                                       | Qualitative            | 0: Yes         |                |                |             |           |
| dichotomous                                           | 1: No                                                |                        |                |                |                |             |           |
| Pre-phase response with prednisone Morpholo gical statusat day+14                                                       | Presence of blasts in bone                           | M1, M2, M3             | Qualitative    |                |                |             |           |
| marrow sample per day                                 | polytomous                                           |                        |                |                |                |             |           |
| +14 referral induction                                | 0: M1 1: M2 2: M3                                    |                        |                |                |                |             |           |
| Presence of blasts in the bone marrow aspirate at the | M1, M2, M3                                           | Qualitative            |                |                |                |             |           |
| completionofremissioninduction                                                       | polytomus                                            |                        |                |                |                |             |           |
| phase.                                                |                                                      |                        |                |                |                |             |           |
| Morphological response atth eendof induction                                                       | 0: M1 1: M2 2: M3                                    |                        |                |                |                |             |           |
| Minimal                                               |                                                      |                        |                |                |                |             |           |
| residual disease atthe endof induction.                                                       | the presenceof1x10-3 blasts.                                                      | Positive /Negative     | Qualitative    | 0: positive    |                |             |           |
| Flow cytometry measurement of                         | dichotomous                                          | 1: negative            |                |                |                |             |           |
| Presence                                              | of                                                   | blasts                 | in             | bone           | Present/absent | Qualitative | 0: absent |
| Relapse                                               | marrow after remission has been                                                      | dichotomous            | 1: present     |                |                |             |           |
| achieved.                                             |                                                      |                        |                |                |                |             |           |  
  
  
  
| Time from diagnosis of leukemia to relapse   | Days                                  | Quantitative   | Not applicable   |                |
|----------------------------------------------|---------------------------------------|----------------|------------------|----------------|
| Timeto relapse Death                                              | Cessation of life                     | Present/absent | Qualitative      | 0: absent      |
| dichotomous                                  | 1: present                            |                |                  |                |
| Timetodeath                                              | Time from leukemia diagnosis to death | Days           | Quantitative     | Not applicable |
| Table1. Variable definitions.                                              |                                       |                |                  |                |
effect of metformin on abcb1
procedures

Patients with a newly morphological diagnosis of ALL at the Hospital General de México "Dr. Eduardo Liceaga" willbeinvited toparticipate inthistrial, andinformed consentwillberequestedfrom parentsand participants. Thedatacollectionisobtainedfromthepatientschart suchasage,gender,leukocytecount,hemoglobin, platelet countandimmunophenotype.  
Peripheral blood samples will be collected in EDTA tube. The sample will be centrifuged obtaining mononuclear cells, and the analysis ofABCB1 andAMPK expression will beperformed.This will be done on samples obtained before the start of treatment and at the end of the induction phase.Sampleswillbe collected by peripheral vein puncture, and mononuclear cells will be separated using density gradient. Mononuclear cells are separated and suspended in PBS medium and stored at -70ºC. RNA isolation is performed using TRIzol® (Invitrogen/life Technologies), and will be stored at-80ºC until use. For cDNA synthesis, 2gofRNA, finalvolumeof 20Lwillbecombinedwith 200Uof MMLVRT enzyme (Invitrogen, Carlsbad,CA,USA). ExpressionofmRNAlevelswillbemeasuredusingtheTaqMangeneexpressionassay(AppliedBiosystems Fostercity,CA,USA).Thecut-offpointbetweenlowandhighexpressionwillbedeterminedaccordingto mean values obtained in healthy controls, selected during case recruitment ina 2:1 ratio for each case.  
Regarding treatment, patients will be randomized into 2 groups, where in both cases the conventional remissioninductiontreatmentwillbegiven,andtointerventiongroup metforminwillbe addedatadose of1000mgm2Scday,dividedintotwodoses,withamaximumdoseof850mgevery8 hour,orally. Theassignmentwillbemadebymeansof theblock randomization technique,inwhich, froma tableof randomdigits,two-digitnumbersareselected,eachofwhichcorrespondstoablockof6patients,to which the interventionisassigned inthecorrespondingsequence.  
All patients will start treatment with steroid window, with prednisone at a dose of 60mgm2Scday (maximum dose 100mg/d), for 7 days (from day-7 to-1). Patients in the study group (treatmentwith conventional chemotherapy + metformin) are added metformin hydrochloride orally, at a dose of 1000mgm2Scday, divided into two doses, with a maximum doseof 850mg every8h startingfromday-7oftreatment.Onday 0theresponsetothepre-phasewithprednisone(60mgm2scd) will be assessed bymeansofblood biometry with differential countand blast count. BothgroupswillstartremissioninductionandconsolidationtreatmentaccordingtotheInstitutionalHigh Riskprotocol,whichincludesadministrationofvincristine,prednisone,daunorubicinand L-asparaginase. Patients assigned tointerventiongroup will also continue with metformin at the established doses.  
A bone marrow exam will be performed at day +14 of remission induction to determine the marrow responsetochemotherapy.Abonemarrowaspiratewillbeperformedattheendofremissioninduction to evaluatetheresponsetothisphaseoftreatmentwithmorphologicalstudyandminimalresidual disease, as well as determination of the expression of the ABCB1/MDR1 and AMPK gene with the methodology describedabove.Attheendofremissioninduction,metforminwillbediscontinuedingroupassignedtoit.  
Patientswillbefollowedupfor12monthsfromthestartoftreatment,andanyrelapsesofthediseaseor  
  
 deaths,aswellasthetimeatwhichtheywerediagnosed,willberecordedonthedatacollectionsheet.
effect of metformin on abcb1
statisticalanalysis

Data analysis will be carried out using the Statistical Package for Social Sciences (SPSS) version 25.0.  
Descriptivedatawillbeexpressedinpercentagesandabsolutevaluesforqualitativevariables,andwillbe describedinmedians,ranges,meansandstandarddeviationforquantitativevariables.  
TheKolmogorov-Smirnovtestwillbeperformedforcontinuousvariablestodetermineiftheyhavenormal distribution;iftheyhavenormaldistribution,bothgroupswillbeanalyzedbymeansofthet-studenttest;if theydonothavenormaldistribution,thechi-squaretest,Fisher'sexacttestandMannWhitneyUtestwill beusedforthebivariateanalysis.TocomparetheexpressionofABCB1/MDR1andAMPKgenesatthetime ofdiagnosisandattheendofinduction,McNemar'stestwillbeused. Forthecalculationoftheoverallanddisease-freesurvival function,theKaplanMeiersurvivaltestwillbe usedforbothgroupsandthedifferencesbetweengroups willbeanalyzedbymeansofthelogRanktest, both for overall and disease-free survival. Similarly, the overall and disease-free survival function will be analyzedwiththeKaplanMeiertestandlogrankforABCB1/MDR1andAMPKgeneexpression.
effect of metformin on abcb1
ethical and biosafety aspects

AccordingtotheGeneralHealthLawregardingresearchonhumansubjects,thisprotocolisconsidereda higherthanminimalriskresearch,sincerandomizedmethodsofassignmenttotherapeuticschemesare used.  
Thisprotocol takesintoaccount theMexicanGeneralHealthLaw regarding theprovisionofhealthcare services and health research,as wellas nationaland internationalethical standards.  
This study will be carried out with strict observance of recognized scientific principles and respect and confidential handling of the data obtained. The security mechanisms to maintain the ethical aspects of this study are:
1. ReviewofthisprotocolbytheresearchandethicscommitteesoftheHospitalGeneraldeMéxico
"Dr. EduardoLiceaga".  
2. All information obtained in this study will be made available to the Ethics and Research Committees.  
3. Theconfidentialityofthestudyinformationisassured,aswellastheidentityofthepatients.The identification data of the participants will be codified, assigning a consecutive participation number.Theidentificationinformationandtherelationbetweentheidentificationnumbersand the data obtained will be known only to the participating investigators and will be preserved confidentiallyin electronic media.  
4. Therewillbenocharge for thestudy,norwill therebeanyconsistency forparticipationin this study. Refusal to participate in the study will not influence the patient's treatment or clinical care.  
5. Informed consent and assent will be requested for participation in this study, respecting the  
  
  
autonomy ofthe participants.  
6. The principles of fairness arerespected since the participants will be randomly assignedby blocks.
The responsible investigators have no conflicts of interest in the conduct of this study or its publication.
effect of metformin on abcb1
references

1. Smith MA, Seibel NL, Altekruse SF, et al. Outcomes for children and adolescents with cancer:  
  
challenges for the twenty-first century.J Clin Oncol 2010;28:2625-34.  
2. Linabery AM, Ross JA. Trends in childhood cancer incidence in the U.S. (1992- 2004). Cancer 2008;112:416-32.  
3. Hunger, SP, Charles GM. Acute lymphoblastic leukemia in children. N Engl J Med. 2015; 373.16:
1541-1552.  
4. Lim JY,BhatiaS,RobisonLL,Yang JJ.Genomicsof racialandethnicdisparitiesinchildhoodacute lymphoblastic leukemia.Cancer2014;120:955-62.  
5. Rivera-Luna R.DescriptiveEpidemiology inMexican childrenwith cancer under an open national publichealthinsuranceprogram.BMC Cancer. 201414:790.  
6. SmithM,ArthurD,CamittaB.Uniformapproachtoriskclassificationandtreatmentassignmentfor children withacute lymphoblasticleukemia.J Clin Oncol 1996;14:18-24.  
7. Hunger SP, Lu X, Devidas M, et al. Improved survival for children and adolescents from 1990-2005:
a report fromthe Children's OncologyGroup.J Clin Oncol 2012;30:1663-9.  
8. Moorman AV, Ensor HM, Richards SM, et al. Prognostic effect of chromosomal abnormalities in childhood B-cell precursor acute lymphoblastic leukaemia: resultsfrom theUKMedical Research Council ALL97/99randomised trial. LancetOncol 2010;11:429-38.  
9. Aricò M, Schrappe M, Hunger SP, et al. Clinical outcome of children with newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia treated between and1995 J 2005.Clin Oncol 2010;28:4755-61.  
10. Harrison CJ, Moorman AV, Schwab C, et al. An international study of intrachromosomal amplification ofchromosome21(iAMP21):cytogeneticcharacterizationandoutcome.Leukemia 2014;28:101521.  
11. MullighanCG,SuX,ZhangJ,etal.Deletionof*IKZF1*andprognosisinacutelymphoblasticleukemia.  
NEnglJ Med 2009; 360:470-80.  
12. KuiperRP,WaandersE,vanderVeldenVH,etal.IKZF1deletionspredictrelapseinuniformly treated pediatricprecursor B-ALL. Leukemia2010;24:1258-64.  
13. CampanaD.Minimalresidualdisease monitoringinchildhoodacutelymphoblasticleukemia.Curr  
  
  
OpinHematol 2012; 19:313-8.  
14. Pui CH, Pei D, Coustan-Smith E, et al. Clinical utility of sequential minimal residual disease measurementsinthecontextofrisk-basedtherapyinchildhoodacutelymphoblasticleukaemia:a prospective study.LancetOncol 2015;16:465-74.  
15. Schrappe M, Valsecchi MG, Bartram CR, et al. Late MRD response determinesrelapse risk overall. and in subsets of childhood T-cell ALL: results of the AIEOP-BFM- ALL study2000. Blood 2011;118:2077-84 16. PuiCH,CampanaD,PeiD,etal.Treatingchildhoodacutelymphoblasticleukemiawithoutcranial irradiation.NEnglJ Med 2009;360:2730-41.  
17. VeermanAJ,KampsWA,vandenBergH,etal.Dexamethasone-basedtherapyforchildhoodacute lymphoblastic leukaemia: results of the prospective Dutch Childhood Oncology Group (DCOG)
protocol ALL-9 (1997-2004). LancetOncol 2009;10:957-66.  
18. Nguyen K, Devidas M, Cheng SC, et al. Factors influencing survival after relapse from acute lymphoblastic leukemia: a Chil- dren's OncologyGroup study. Leukemia 2008;22:2142-50.  
19. RaetzEA,BhatlaT.Wheredowestandinthetreatmentofrelapsedacutelymphoblasticleukemia?  
HematologyAm Soc Hematol Educ Program2012;2012: 129-36.  
20. Wolfson JA,Richman JS,SunCL,et.al.CausesofinferioroutcomeinAdolescentsandYounAdults with Acute Lymphoblastic leukemia: across Oncology Services and Regardless of Clinical Trial Enrollment.Cancer Epidemiol Biomarkers Prev 2018;27:1133-1141.  
21. MoormanAV,ChiltonL,WilkinsonJ,EnsorHM,BownN,ProctorSJ.Apopulation-based cytogenetic study of adults with acute lymphoblastic leukemia. Blood. 2010;115(2):206-214.  
22. Moorman AV, Harrison CJ, Buck GA, et al; Adult Leukaemia Working Party, Medical Research Council/NationalCancerResearchInstitute.Karyotypeisanindependentprognosticfactorinadult acute lymphoblastic leukemia (ALL): analysis of cytogenetic data from patients treated on the MedicalResearchCouncil(MRC)UKALLXII/EasternCooperativeOncologyGroup(ECOG)2993trial. Blood. 2007;109(8):3189-3197.  
23. Veal GJ, Hartford CM, Stewart CF. Clinical pharmacology in the adolescent oncology patient. J Clin Oncol.2010;28(32):4790-4799.  
24. Dean M, Rzhetsky A, Allikmets R. The human ATP-binding cassette (ABC) transporter superfamily.  
GenomeRes. 2001; 11: 1156-1166.  
25. Moitra K, Lou H, Dean M. Multidrug efflux pumps and cancer stem cells: insights into multidrug  
  
  
  
resistance and therapeutic development. Clin Pharmacol Ther.2011; 89: 491-502.  
26. Szakács G, Paterson JK, Ludwig JA, Booth-Genthe C, Gottesman MM. Targeting multidrug resistance incancer. NatRev Drug Discov 2006; 5:219-34.  
27. Litman T, Brangi M, Hudson E, Fetsch P, Abati A, Ross DD, Miyake K, Resau JH, Bates SE. The multidrug-resistantphenotypeassociatedwithoverexpressionofthenewABChalf-transporter, MXR (ABCG2).J Cell Sci 2000; 113:2011-21.  
28. BenderraZ,FaussatAM,SayadaL,PerrotJY,TangR,ChaouiD,MorjaniH,MarzacC,MarieJP, Legrand O.MRP3,BCRP,andP-glycoproteinactivitiesareprognosticfactorsinadultacutemyeloid leukemia. Clin Cancer Res 2005; 11:7764-72; 29. Kourti M, Vavatsi N, Gombakis N, Sidi V, Tzimagiorgis G, Papageorgiou T, Koliouskas D, Athanassiadou F.Expressionofmultidrugresistance1(MDR1),multidrugresistance-relatedprotein1(MRP1),lung resistanceprotein(LRP),andbreastcancerresistanceprotein(BCRP)genesand clinical outcome in childhood acute lymphoblastic leukemia. Int J Hematol 2007; 86:166-73.  
30. Cortez MA, Scrideli CA, Yunes JA, Valera ET, Toledo SR, Pavoni-Ferreira PC, Lee ML, Petrilli AS,
BrandaliseSR,ToneLG.mRNAexpressionprofileofmultidrugresistancegenesinchildhoodacute lymphoblasticleukemia.Lowexpressionlevelsassociatedwithahigherriskoftoxicdeath.Pediatr BloodCancer 2009; 53:996-1004.  
31. Rahgozar S, Moafi A, Abedi M, Entezar-e-ghaem M, Moshtaghian J, et.al. mRNA expression profile ofmultidrug-resistantgenesinacutelymphoblasticleukemiaofchildren,aprognosticvaluefor ABC3 and ABCA2.CancerBiology & Therapy 2014; 15:1, 35-41.  
32. MarzacC,GarridoE,TangR,FavaF,HirschP,DeBenedictisC,CorreE,LapusanS,LallemandJY, Marie JP,etal.ATPBindingCassettetransportersassociatedwithchemoresistance:transcriptional profiling inextremecohortsandtheirprognosticimpactinacohortof281acutemyeloidleukemia patients.  
Haematologica 2011; 96:1293-301.  
33. BergerW,SteinerE,GruschM,ElblingL,MickscheM.Vaultsandthemajorvaultprotein:novel roles in signal pathwayregulation and immunity.Cell Mol Life Sci 2009; 66:43-61.  
34. Chan K,TruongD, ShangariN,O'Brien PJ.Drug-inducedmitochondrial toxicity.ExpertOpinDrug Metab Toxicol.2005; 1:655-669 35. FengYH,Velazquez-TorresG,GullyC,ChenJ,LeeMH,YeungSC.Theimpactoftype2diabetesand antidiabeticdrugson cancer cell growth.J CellMolMed 2011; 15:825-36.  
36. GiovannucciE,HarlanDM,ArcherMC,BergenstalRM,GapsturSM,HabelLA,etal.Diabetesand  
  
  
cancer: a consensus report.DiabetesCare 2010;33:1674- 85.  
37. BowkerSL,Yasui Y,VeugelersP,JohnsonJA.Glucose-loweringagentsandcancermortalityratesin type diabetes2: assessing effects of time-varying exposure. Diabetologia. 2010; 53: 1631-1637.  
38. ZhangZJ,LiS,Theprognosticvalueofmetforminforcancerpatientswithconcurrentdiabetes:a systematic reviewand meta-analysis.Diabetes ObesMetab.2014; 16: 707-710.  
39. Sengupta TK, Leclerc GM, Hsieh-Kinser TT, Leclerc GJ, SinghI, Barredo JC. Cytotoxic effect of 5aminoimidazole-4-carboxamide-1-beta-4- ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy.Mol Cancer. 2007;6:46 40. Trucco M, Barredo JC, Goldberg J, et al. A phase I window, dose escalating and safety trial of metforminincombinationwithinductionchemotherapyinrelapsedrefractoryacutelymphoblastic leukemia: Metformin with induction chemotherapy of vincristine, dexamethasone, PEGasparaginaseand doxorubicin (VXLD). Pediatr Blood Cancer.2018;e27224.  
41. LeclercGM,LeclercGJ,KuznetsovJN,DeSalvoJ,BarredoJC.Metformininducesapoptosisthrough AMPK-dependent inhibition of UPR signaling in ALL lymphoblasts. PLoS One. 2013;8:e74420.  
42. Shackelford DB, Shaw RJ. The LKB1-AMPK pathway: metabolism and growth control in tumour suppression. Nat RevCancer.2009;9: 563- 575.  
43. DowlingRJ,NiraulaS,StambolicV,GoodwinPJ.Metforminincancer:translationalchallenges.JMol Endocrinol.2012; 48: R31-43.  
44. ShoreGC,PapaFR,OakesSA.Signalingcelldeathfromtheendoplasmicreticulumstressresponse.  
Curr Opin Cell Biol. 2011;23:143-149.  
45. OyadomariS,MoriM.RolesofCHOP/GADD153inendoplasmicreticulumstress.CellDeathDiffer.  
2004;11:381-389.  
46. EmamiRiedmaier A1,FiselP,Nies AT,SchaeffelerE,Schwab M.Metforminandcancer:fromthe old medicinecabinet topharmacologicalpitfallsandprospects.TrendsPharmacolSci.2013;34: 126135.  
47. PernicovaI,KorbonitsM.Metformin-modeofactionandclinicalimplicationsfordiabetesand cancer.  
Nat RevEndocrinol. 2014;10: 143-156.  
48. LeclercGM,LeclercGJ,FuG,BarredoJC.AMPK-inducedactivationofAktbyAICARismediatedby  
  
  
IGF-1R-dependent and independent mechanisms in acute lymphoblastic leukemia. J Mol Signal. 2010;5:15.  
49. Kuznetsov JN, LeclercGJ, LeclercGM, Barredo JC. AMPK and Akt determine apoptotic cell death following perturbations of one-carbonmetabolism by regulatingER stress inacute lymphoblastic leukemia.Mol Cancer Ther.2011;10:437-447.  
50. PanJ,ChenC,Jiny,Fuentes-MattelE,Velasquez-TorresG,et.al.Differentialimpactof structurally differentanti-diabeticdrugsonproliferationandchemosensitivityofacutelymphoblasticleukemia cells.Cell Cycle 2012; 11:12,2314-2326.
51. Serrano M. Metformin and reprogramming into iPSCs. Cell Cycle. 2012; 11: 1058-1059.  
52. Feng YH, Wu CL, Shiau AL, Lee JC, Chang JG, Lu PJ, et al. MicroRNA- 21-mediated regulation of Sprouty2proteinexpressionenhancesthecytotoxiceffectof5-fluorouracilandmetforminincolon cancer cells. IntJ MolMed2012; 29:920-926.  
53. Dirat B, Ader I, GolzioM, Massa F,Mettouchi A, Laurent K, et al. Inhibition of the GTPase Rac1 mediatestheantimigratoryeffectsofmetformininprostatecancercells.MolCancerTher.2015; 14: 586- 596.  
54. FanC,WangY,LiuZ,SunY,WangX,WeiG,etal.Metforminexertsanticancereffectsthroughthe inhibitionoftheSonichedgehogsignalingpathwayinbreastcancer.IntJMolMed.2015;36:204214.  
55. LinCC,YehHH,HuangWL,YanJJ,LaiWW,SuWP,etal.Metforminenhancescisplatincytotoxicity bysuppressingsignaltransducerandactivatoroftranscription-3activityindependentlyoftheliver kinase B1-AMP-activated protein kinase pathway. Am J Respir Cell Mol Biol 2013; 49: 241-250.  
56. Bullock BP,Habener JF. Phosphorylation of the cAMP responseelement binding proteinCREBby cAMP-dependent protein kinase A and glycogen synthase kinase-3 alters DNA-binding affinity, conformation, andincreases net charge.Biochemistry 1998; 37: 3795-3809.  
57. KimHG,HienTT,HanEH,HwangYP, Choi JH, Kang KW,etal.MetformininhibitsP-glycoprotein expressionviatheNF-kappaBpathwayandCREtranscriptionalactivitythroughAMPKactivation.BrJ Pharmacol. 2011; 162:1096-1108.  
58. Gerrard G, Payne E, Baker RJ, Jones DT, Potter M, Prentice HG, et al. Clinical effects and Pglycoprotein inhibition in patients with acute myeloid leukemia treated with zosuquidar trihydrochloride, daunorubicinand cytarabine. Haematologica. 2004; 89: 782-790.  
59. JonesKL,ArslanianS,PeterokovaVA,ParkJS,TomlinsonMJ.Effectofmetformininpediatric patients withtype diabetes2: a randomized controlled trial. Diabetes Care. 2002;25:89-94.  
60. Silverstein JH, Rosenbloom AL. Treatmentoftype2diabetesmellitus in children and adolescents. J
Pediatr Endocrinol Metab. 2000;13(Suppl 6):1403-1409.
61. Gao X, Christensen M, Burghen GA, et al. Pharmacokinetics of met- formin in pediatric type 2  
  
  
diabetic and healthy adult subjects. Clin Pharmacol Ther. 2003;73:P46-P46.  
  
62. Ramos-Peñafiel C, Olarte-Carrillo I, Cerón Maldonado R, Miranda-Peralta E, et.al. Effect of metformin in the induction stage in patients with acute lymphoblastic leukemia and its clinical impact on survival. Rev Med Chile 2018; 146: 846-853.  
63. Ramos-Peñafiel C, Olarte-Carrillo I, Cerón-Maldonado R, Santoyo-Sánchez A, et al. Effect of metformin on the expression of Multidrug-Resistance Genes(ABCB1, ABCG2) in Acute Lymphoblastic Leukemia,invitro andin Vivo study. Blood2016;128(22):5142  
  
  
  
DR. EDUARDO LICEAGA
Chemotherapy protocol for remission induction in high risk LAL  
| 71.61.5141.31.21.10.11.21.3141516171819110   |
|----------------------------------------------|
| Medicamento
Prednisona                                              |
| 60mg/m2
Vincristina                                              |
| 1.5mg/m2
Doxorrubicina                                              |
| Doolean and social a                         |
| Intratecal
Etopósido                                              |
| Etiposso
300mg/m2
300mg/m2
Metformina                                              |
| 1000mg/m2 - Solo                             |
| grupo A
Valoración de la
prefase con                                              |
| prednisona con BH
Aspirado de médula                                              |
| ósea
Medición de                                              |
| expresión de genes
ABCB1 y AMPK                                              |  
  
e