Metformin Reduce the Relapse Rate on Patients With B-cell Precursor (Ph+ Negative) Acute Lymphoblastic Leukemia
Condition: Acute Lymphoblastic Leukemia
MeSH terms: Leukemia; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Leukemia, Lymphoid
Study type: Interventional

Summary: 
      Metformin's Antitumor activity were identified from differens diabetic patients trials,
      mainly associated to its mechanism of action and protein - kinase AMPK (AMP-activated protein
      kinase) activation. According to Cancer and Diabetes International Consensus from 2012,
      diabetes increases the risk for developping cancer and metformin has an protector effect
      against cancer cells and has an impact on overall survival.

      Chemotherapy drug resistance induces treatment fail in oncology. Metformin increases AMPK
      levels, blocks PI3K (phosphatidylinositol 3- kinase)/ AKT /mTOR(mammailian Target of
      Rapamycin) pathway but few evidence associated with drug resistance gene expression.

      This is an, experimental one-center study that pretends to stablish the effect of adding
      metformin 850 mg PO three times a day over the multi-drug resistance gene expression (ABCB1)
      in de novo Acute Lymphoblastic Leukemia in one 7-days cycle with prednisone as pre-treatment-
      and on the induction remission treatment.
    

Detailed description: 
      The MDR (Multidrug-Resistance) Genes are implicated on the resistance of several types of
      cancer. The most important on leukemia are the ABCB1 and ABCG2- ABCB1 are implicated on
      resistance and severity on Acute Myeloid Leukemia and pediatric Acute Lymphoblastic Leukemia.
      Those transporters use ATP for use. Metformin decrease the intracellular ATP (Adenosine
      triphosphate) reserve the by the activation of AMPK. Recently on MCF7-Adr (Michigan Cancer
      Foundation 7, breast cancer cell line) cancer cell line, Metformin block the function of the
      P-glycoprotein by the inhibition of the Nuclear Factor -kappa-B.

      The clinical evidence at this moment is limited, based on small clinical trials or
      observational studies. This study tries to evaluate the effect of the addition of Metformin
      to a standard chemotherapy regimen on patients who express high levels of expression of mRNA
      (messenger ribonucleic acid) ABCB1

      Experimental protocol

      The patients will be stratified on three groups: The high-expression, low expression and
      absent gene expression according the level mRNA of ABCB1 at diagnostic. The samples are
      obtained from mononuclear peripheral blood cells

      Extraction of total RNA Total RNA was isolated by TRIzol® (Invtirogen Life Technologies)
      according to the manufacturer´s recomendations described by Chomczynski and Sacchi.

      The concentration and purity of total RNA was determined in a UV -vis spectrophotometer
      (Thermo Scientific , Genesis 10S UV-vis). The integrity of the genetic material was confirmed
      by 1.5% agarose gel electrophoresis at 70 V for 40 min. The RNA was stored at -80°C until
      needed.

      Synthesis of cDNA Fort he synthesis of c DNA (complementary deoxyribonucleic acid) the amount
      of RNA used was 2µg for a final volume of 20 µg. The RNA was mixed with 1 µl oligonucleotide
      12-18 (INVITROGEN, Carlsbad, CA) and 1 µl de dNTPs (deoxynucleoside triphosphate) 10mM
      (Applied Biosystems, Roche). After the addition of 4 µl of Buffer 5 X (Tris- HCl
      (hydrochloric acid) 250mM, KCl (potassium chloride) 375 mM MgCl2 15mM), 2 µl de DTT
      (dithiothreitol) (0.1M) and wáter, all the mix was incubated at at 37° for two minutes and 1
      µl of MMLV(Moloney Murine Leukemia Virus Reverse Transcriptase) Reverse transcriptase- enzyme
      (200u) (INVITROGEN, Carlsbad, CA) and incubated at 37°C for 50 minutes.

      Real-time polymerase chain reaction (qRT-PCR) analysis The mRNA expression levels of the
      ABCB1 (Hs01069047), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Hs00985689) genes
      were measured using the TaqMan® gene expression assay (Applied Biosystems, Foster City, CA,
      USA). The GAPDH gene was used as an endogenous control, and each sample was analyzed in
      triplicate. The relative expression levels were calculated using the 2-ΔΔCtmethod with bone
      marrow as a calibrator. The high and low expression cut-off points were determined by the
      mean values observed in healthy donors.

      Treatment protocol All the patients recieve an induction remission treatment with an initial
      pre-induction phase with steroids (prednisone) from day -7 to day -1 (-7 ,-6 = 25mg , -5 , -4
      : 50mg , -3, -2: 75mg and on day -1 : 100mg). The induction remission treatment is based on a
      28 day treatment with prednisone (60mg/m2),Vincristine (1.5mg/m2 maxium 2mg) and Daunorubicin
      60mg/m2 on days +1, +8 and +15. If the patient archive a Complete Remission, continues with a
      consolidation therapy with sequential blocks of treatment that includes Cytarabine,
      Etoposide, Methotrexate. If the patient still on remission at the end of consolidation
      therapy continues on maintenance phase that includes 6-mercaptopurine and a weekly dose of
      methotrexate for around two years

      Response At day 28 of the induction remission treatment, according the result of bone marrow
      sample the patientes will be declared on remission (less than 5% blast) or refractory (>5%
      blast cell). If the patient present at any point of treatment an increase on bone marrow
      blast count the patient is considered on relapse.
    

Intervention name(s): Metformin
Intervention type(s): Drug
Intervention description(s): Metformin 850 mg PO three times a day plus prednisone in one 7-days cycle as pre-treatment and during the 28 days induction remission treatment, consolidation therapy and maintenance
Intervention MeSH terms: Metformin
Allocation: Non-Randomized
Intervention model: Parallel Assignment
Masking: None (Open Label)
Primary purpose: Basic Science
Keywords: Acute Lymphoblastic Leukemia (ALL), ABCB1, Metformin