Decitabine for the Treatment of AML

1
Decitabine for the Treatment of AML

• Amanda F. Cashen, M.D.

2
Case Presentation

• A 67 year-old man with no significant past
  medical history was found to have pancytopenia
  when he presented to his primary physician with a
  complaint of frequent upper respiratory tract
  infections. He was otherwise feeling well.
  Physical exam was unremarkable.
• Initial CBC WBC 1,800 (37 neutrophils),
  hemoglobin 11.4, and platelets 115,000
• Bone marrow aspirate and biopsy 10 cellularity,
  39 blasts phenotype consistent with AML-M2
• The patient was initially followed without
  treatment. His blood counts changed little over
  the next two months, and he did not require any
  transfusion support. A bone marrow evaluation
  was repeated in anticipation of starting therapy,
  and the blast percent had decreased to 28.

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Case Presentation, cont.

• He was treated in the Phase III trial of
  decitabine versus supportive care for
  myelodysplastic syndrome (MDS) decitabine, 15
  mg/m2 iv over 3 hours, every 8 hours for 3 days,
  with cycles repeated every 6 weeks
• The first two cycles were administered on the
  inpatient leukemia service, but subsequent cycles
  were given at home. He had no complications of
  treatment, and his performance status was
  excellent.
• After two cycles of decitabine, restaging bone
  marrow evaluation revealed 30 cellularity with
  normal trilineage hematopoiesis and 4 blasts.
  Cytogenetics normal.
• Treated with another 6 cycles (BM p each 2
  cycles), until bone marrow evaluation performed
  after the eighth cycle showed 9 blasts?
  off-study for relapsed disease
• Altogether, he was treated with decitabine for
  one year, maintaining an excellent performance
  status throughout.

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Case Presentation, cont.

• After discontinuation of decitabine, the patient
  had worsening cytopenias. Over the next four
  months, his absolute neutrophil count fell below
  500 and his platelet count was 50-60,000. He was
  treated with erythropoietin and occasional
  transfusion of red blood cells.
• An unrelated bone marrow donor, 10/10 allele
  match, was identified, but stem cell
  transplantation was not pursued because the
  patient continued to feel well and the toxicity
  of the treatment was deemed too high.
• Bone marrow evaluation performed thirteen months
  after discontinuation of decitabine recurrent
  AML, with 10 cellularity and 40 blasts by flow
  cytometry.
• A petition for compassionate use of decitabine
  was approved by the manufacturer. The patient
  was treated as an outpatient with decitabine, 15
  mg/m2 iv on days 1-5 and 8-12.

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Case Presentation, cont.

• After the first cycle of decitabine, his WBC was
  3.4, Hgb 10.8, platelets 229.
• Bone marrow biopsy after two cycles 20
  cellularity with mild dyspoietic changes, no
  leukemia seen
• CBC prior to the third cycle WBC 4.6, Hgb 14,
  platelets 169

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DNA Methylation

• Covalent addition of a methyl group onto the
  fifth position of cytosine within CpG
  dinucleotides
• 3-5 of all cytosines are methylated in the
  vertebrate genome

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DNA Methyltransferases

• DNMT1
• Methylates hemi-methylated DNA
• Responsible for methylation during
  replicationmaintenance methyltransferase
• DNMT3a and DNMT3b
• Responsible for de novo methylation during
  embryogenesis
• Interact with histone deacetylases, may target
  them to heterochromatin

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CpG Islands

• 200 bp-5 kb regions that are GC-rich (60-70)
• Found in promoter regions of 50-60 of human
  genes (in almost all constitutively active
  housekeeping genes)
• Usually unmethylated (exceptionstransciptionally
  silent alleles for imprinted genes, inactive X
  chromosome)
• Methylation leads to transcriptional repression
  of the associated gene

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Methylation Changes in Cancer

• Overall, the DNA of malignant cells is
  hypomethylated
• Activation of oncogenes
• Chromosomal instability
• Reactivation of transposable elements
• Loss of imprinting
• However, there are localized increases in
  methylation, often at tumor suppressor genes
• Non physiologic DNA methylation probably both
  causes and results from malignant transformation

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How Does Hypermethylation Lead to Gene Silencing?

• Promoter becomes embedded in heterochromatin,
  when it would normally be in euchromatin
• Hypermethylated promoters are surrounded by
  tightly compacted nucleosomes, which contain
  de-acetylated histones

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DNMT
MBPs
HDAC
HDAC
HDAC
TF

• Effects of methylation
• Direct interference with TF binding
• MBPs inhibit transcription
• DNMT and MBPs recruit histone deacetylase?
  histone deacetylation? tighter packing of DNA

MBPsmethyl-cytosine binding proteins
--de-acetylated, compacted nucleosomes HDAChiston
e deacetylases TFtranscription factor
complex DNMTDNA methyltransferase
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Hypermethylated Genes in Acute Leukemia

• Levels of DNA methyltransferases are increased
  4-5 fold in leukemia Leukemia, 1998 12311
• Genes reported to be hypermethylated in acute
  leukemia
• Calcitonin (78-95), p15 (50-80), ER (50-90)
• Most studies performed with restriction enzyme
  analysis (limited to a few CpG sites in
  individual genes)

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Concurrent DNA Hypermethylation of Multiple Genes
in AML Can Res 1999 593730

• Bone marrow samples from 20 pts. with AML and 9
  controls
• Analysis with bisulfite genomic sequencing
• No single gene was always methylated in AML

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Concurrent DNA Hypermethylation of Multiple Genes
in AML Can Res 1999 593730
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Methylation Profiling in AMLBlood 2001 972923

• 36 PB or BM samples analyzed by bisulfite PCR
  (PCR products digested with restriction enzymes
  that only cleave methylated alleles)
• No methylation observed in normal bone marrow

Proportion of methylated alleles
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Methylation Profiling in AMLBlood 2001 972923
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Inhibitors of DNA Methylation

• Azacitidine and decitabine first synthesized in
  1964

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Inhibitors of DNA MethylationMechanisms of Action

• Azacitidine and decitabine are phosphorylated and
  then incorporated into DNA
• Covalently link DNMT? DNMT is depleted?
  demethylation occurs after repeated
  replication?re-expression of epigenetically
  silenced genes (especially tumor suppressor genes
  and cell cycle regulators)
• Induce differentiation in a variety of cell types
• May obstruct DNA synthesis
• Induce DNA damage via structural instability
• Decitabine is 5-10 fold more potent than
  azacytidine (azacytidine is incorporated into RNA
  and DNA)

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Decitabine and Leukemia

• First used to treat leukemia in 1981
• Initially studied at high doses (500-3000 mg/m2)
• Significant hematologic and non-hematologic
  toxicity (N/V/D, neurotoxicity, hepatotoxicity,
  mucositis)
• When used alone or in combination with an
  anthracycline, activity comparable to HDAC
• Then low-dose schedules investigated
• More effect on differentiation, less cytotoxicity

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Trials of high dose decitabine in AML
1 Schwartsmann G. Leukemia 1997 11(Suppl
1)S28-31. 3 Willemze R. Leukemia. 1997
11(Suppl 1)S24-27 4 Rivard GE. Leukemia
Research 1981 5453-462. 5 Momparler RL.
Pharmac Therapy 1985 30277-286. 6 Petti MC.
Leukemia. 1993 7(Suppl 1)36-41. 7 Richel DJ.
Br. J. Cancer 1991 64144-148
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Trials of low dose decitabine in AML and MDS
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The Phase III North American Trial of Decitabine
in Advanced MDS

• Phase III trial of DAC v. supportive care
• 170 patients randomized 11
• 25 had had previous therapy for MDS
• IPSS 0.5 Intermediate (Int)-1 (31), Int-2
  (44) and high risk disease (26)
• Included secondary MDS (14) and previously
  treated (27) MDS patients
• Treatment
• Supportive care
• DAC, 3 hr infusion of 15mg/m2/hr every 8 hrs on 3
  consecutive days every 6 wks for up to 10 cycles
• Primary endpoints
• Overall response rate (CRPR)
• Time to AML transformation or death

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• RR 17 (9 CR 8 PR) v. 0
• By IPSS Int-1 14 Int-2 21 high risk 13
• Median duration of response 266 days
• QOL improved with DAC, worsened with BSC
• Major DAC toxicity was myelosupression, FN
• No treatment related deaths
• Time to AML or death not stat. different, except
  in high risk subgroup

24
Phase 1 study of low-dose prolonged exposure
schedules of the hypomethylating agent
5-aza-2'-deoxycytidine (decitabine) in
hematopoietic malignancies Issa et al. Blood,
2004 1031635
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Does decitabine affect methylation in
MDS/AML?Blood, 2002 1002957

• BM specimens from 23 MDS patients, collected
  before and after treatment with decitabine,
  analyzed for methylation of p15 and p16
• Prior to treatment, 65 of pts. had
  hypermethylation of p15 none had
  hypermethylation of p16
• 12 evaluable pts. with p15 hypermethylation,
  response to DAC
• 9 had a gt25 decrease in methylation, associated
  with response in all (3 CRs)
• 1 had lt 25 decrease and 2 had an increase in
  methylation
• IHC was used to examine p15 expression in 8
  patients with p15 hypermethylation
• 4 patients had very low or absent p15 prior to
  DAC ? high expression after DAC
• 4 pts. had p15 expression comparable to normal BM
  prior to DAC ? persistent expression after DAC

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Phase II Study of Decitabine for Front-line
Treatment of Older Patients with AML

• Patients age gt 60, no prior therapy for AML
• Primary endpoint complete remission rate
• Treatment with decitabine 20 mg/m2 iv x 5 days
  q4weeks
• All patients will be treated with 2 cycles unless
  they have progression of peripheral blast count
• Patients with a complete or partial response
  after 2 cycles can get additional cycles until
  progression

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Phase II Study of Decitabine for Front-line
Treatment of Older Patients with AML

• Bone marrow collection at baseline, day 5 of
  cycle 1 and day 28 of cycle 2 for correlative
  studies
• RNA profiling as part of the Genomics of AML
  project
• DNA methylation profiling
• Pilot proteomics study

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Future Directions for Decitabine in Hematopoietic
Malignancies

• Combine decitabine with chemotherapy to sensitize
  cells to the effects of chemo (exreactivate
  apopotic or DNA repair pathways)
• Combine decitabine and HDAC inhibitors to enhance
  gene re-activation
• Use decitabine as maintenance therapy to maintain
  remission after induction chemotherapy or stem
  cell transplantation