Azathioprine and acquired aplastic anemia

1
Azathioprine and acquired aplastic anemia

• Morey Blinder
• April 29, 2005
• Hematology-Oncology Grand Rounds

2
Case report - T.F.

• 32 yo WM with a history of Crohns disease
  diagnosed in 1992. Complicated by abscesses/
  fistulas in past.
• Started on Imuran 200 mg PO q.d. (azathioprine)
  June 2004.
• Traveled to Mexico early July 2004 - CBC was
  normal.
• GI clinic - 7/28/04 with 3 day history of fever,
  rectal pain and was thought to have perianal
  fistula and was admitted.
• CBC Hgb 8.2 Hct 22.9 WBC count 600 (ANC 0)
  platelet count 42,000
• Azathioprine was discontinued.
• Blood cultures were positive for E. coli and S.
  viridans
• Treated with antibiotics G-CSF and blood product
  support.

3
Hospital course

• 7/31/04 - Surgery for perianal abscess drainage
  and debridement of necrotic material
• 8/5/04 CBC Hgb 6.4 Hct 17.9 WBC count 300 (ANC
  0) platelet count 5,000
• Hematology consult
• Bone marrow exam Markedly hypocellular (lt10)
  ME 13
• Consider transfer to BMT for supportive care
• Consider causes of severe marrow suppression

4
Thiopurine Drugs

• Thiopurine Drugs, 6-MP (right), AZA, and 6-TG are
  used as
• - chemotherapeutic agents to treat leukemia
• - immunosuppressive agents to treat IBD,
• autoimmune diseases, and following solid
  organ transplantation.
• The exact mechanism of the effects of AZA and
  6-MP is unknown
• - Interfere with DNA and RNA synthesis and
  chromosomal replications
• - Inhibit proliferation of T and B lymphocytes
• - Interfere with cytotoxicity of NK cells
• Delayed onset of action Mean of 17 weeks for
  response in CD pts
• Clinical Response rates vary in steroid sparing,
  induction or maintenance of remission.
• - IBD induces remission in 50 60 pts and
  allows steroid withdrawal in 70 of pts
• Side effects include
• - Fever, chills, nausea, vomiting, anorexia,
  diarrhea, bone marrow suppression
• - Rash, hepatotoxicity

5
Efficacy of AZA in Treatment of Active CD
Su and Lichtenstein. Gastroenterology Clinics.
2004
6
Azathioprine/6-MP treatment Early studies
Study pts. Disease leukopenia
pancytopenia Kolle, 1969 30 JRA/Stills
17 3 Lorenzen, 1969 40 Various
25 4 Ginzler, 1975 73 SLE
8 6 Singleton, 1979 59 Crohns disease
15 0 Pollack, 1980 160 Renal transplant
25 0 Present, 1980 68 Crohns disease
10 2 Mertens, 1981 300 Various
5 4 Hass, 1982 56 MS/Myasthenia
NR 0 Hall, 1985 34 Renal transplant
9 3 Kissel, 1986 64 Various
22 1 Kvein, 1986 32 JRA
NR 2 Hohfield,1988 105 Myasthenia
16 3 Total 1021 28 (2.7)
Antsey et.al. J. Royal Society of Med. 1992
7
Azathioprine Metabolism
McLeod and Siva. Pharmacogenomics. 2002
8
Azathioprine Toxicity
Baker. Reviews in Gastroenterological Disorders.
2003
9
Pharmacogenetics TPMT

• Polymorphisms in a single gene Thiopurine
  S-Methyltransferase (TPMT) - influence metabolism
  of thiopurine-based drugs
• Various TPMT alleles with point mutation(s) in
  the ten exons and nine introns as well as the 5
  promoter region have been associated with
  deficient, intermediate, or normal/high activity.
• Trimodal pattern of inheritance Autosomal
  Codominant
• - 0.3 of the population Low/No TPMT activity
• - 11 of the population Intermediate TPMT
  activity
• - 89 of the population Normal/High TPMT
  activity
• Nomenclature Wild type allele TPMT1
• Silent Variant alleles TPMT1s
• Mutant Alleles TPMT2 TPMT18

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TPMT

• TPMT is a cytosolic enzyme which catalyzes the
  S-methylation of the immunosuppressive/cytotoxic
  thiopurine drugs 6-Thioguanine (6-TG),
  6-Mercaptopurine (6-MP) and Azathioprine (AZA).
• No natural substrate is known for TPMT, and it
  has no known involvement in endogenous metabolic
  pathways.
• Multiple transcripts (3.2Kb, 1.7Kb, 1.0Kb) are
  expressed in lung, liver, skeletal muscle,
  kidney, RBCs, and WBCs
• In the absence of exposure to thiopurines, TPMT
  deficiency has no known effect on human health.

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TPMT Mutant Alleles
Schaeffeler et al. Pharmacogenetics. 2004
12
Ethnic Variations of TPMT Alleles
McLeod and Siva. Pharmacogenomics. 2002
13
Influence of TPMT genotype on duration of
Azathioprine Therapy

• 61 pts wild type
• 5 pts heterozygous (TPMT3A)

Median 39 wks
Median 2 wks
P 0.018
Black, A. J. et. al. Ann Intern Med
1998129716-718
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Correlation Between Genotype and Toxicity

• Evaluated 23 pts referred to clinic for excessive
  toxicity while receiving thiopurine drugs
• 6 TPMT homozygous mutants and 11 heterozygotes
  65
• (10 normal population)
• Several other studies have also demonstrated that
  60 70 of patients suffering from thiopurine
  induced toxicity have one or two mutant TPMT
  alleles

Evans et al J. Clin. Onc. 2001
15
Thiopurine Dosing and Polymorphisms
Evans et al. J. Clin Onc. 2001
16
TPMT Polymorphisms and Toxicity
Evans. Pharmacogenetics.2002
17
Testing for TPMT Polymorphisms

• TPMT enzyme activity assay
• Analysis of RBC Thiopurine metabolites
• Molecular genetic analysis

18
RBC TPMT Activity
TPMT Phenotype was traditionally determined by a
nonchelated radiochemical assay.

• Gold Standard for phenotyping TPMT Activity
• Activity in RBCs correlates with activity in
  other tissues
• In patients who have had a recent (30 60 days)
  RBC transfusion, TPMT activity in RBCs can be
  spurious.

14C-MMP Counted
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TPMT Activity by HPLC
TPMT Activity (nmol 6-MTG/gHb/h)
RBC lysates /- 6-TG
None (0.8)
S-adenosylmethionine
Low (17)
6-methylthioguanine (6-MTG)
Normal (36)
Quantification of fluorescent 6-MTG by HPLC
High (76)
20
TPMT Polymorphisms RBC Metabolites
6-TGN
6-MMPN
6-TGN and 6-MMPN Metabolite concentration are
measured by HPLC
21
When to Measure Metabolites
Recommendations by Prometheus Laboratories

• Following initiation of thiopurine drug therapy
• (after 3 wks to establish steady metabolic
  state)
• Following any dose adjustment
• (Intended therapeutic range 235 450
  pmol/8x108 RBC)
• When utilizing a steroid-sparing strategy
• 4. Any time of disease flare
• After six months of treatment
• (TPMT activity may increase due to an
  induction effect)
• 6. At the time of an adverse event - bone marrow
  or hepatic toxicity
• Whenever expected response is not occurring
• (To measure effects of concomitant drug use)
• Twice yearly to measure against baseline
• (Therapeutic drug monitoring)

22
Correlation between 6-TG and Genotype

• Dubinsky et al. Gastroenterology. 2000
• Individuals were genotyped using Allele specific
  PCR
• 8 pts were heterozygous for the low TPMT activity
  allele (TMPTH/TPMTL)
• 84 were homozygous wild type (TMPTH/TMPTH)
• Median 6-TG concentrations in RBCs are
  significantly higher in TPMT heterozygotes. While
  there is no correlation between 6-MMPN
  concentrations and genotype.

P lt 0.0001
23
Measuring RBC Metabolites for Therapeutic drug
monitoring of IBD Patients on AZA

• 170 IBD patients treated with AZA or 6-MP. Wanted
  to correlate 6-TGN with diseae activity as
  measured by IBDQ and Leukopenia.
• Inflammatory Bowel Disease questionnaire (IBDQ)
  previously validated with higher scores better
  quality of life. Patients in Clinical remission
  have a minimal score of 170
• Median 6-TGN concentrations were similar in (139
  vs. 131 pmol/8 X 108) 56 pts with active disease
  and 114 pts in remission.
• Questionable usefulness of therapeutic drug
  monitoring with 6-TGN

Lowry et al. Gut. 2001
24
TPMT Genotyping Genetic Mutational Analyses

• Genotyping has been traditionally performed by
  allele specific PCR of most common mutations
• 1. TPMT3A (Caucasian)
• 2. TPMT3C (African and Asian)
• Other methods may be used to evaluate many more
  mutations

25
Genotype/Phenotype Correlation

• Schaeffeler et al. Pharmacogenetics. 2004.
• Investigated the genotype-phenotype correlation
  of 1214 healthy blood donors to determine
  accuracy of genotyping in predicting TPMT
  phenotype.

Phenotyping HPLC Genotyping HPLC Sequencing
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Recommendations for Genotyping

• DNA based genotyping offers a clinically
  important strategy to prospectively diagnose TPMT
  deficiency and minimize the risk of hematologic
  toxicity in patients treated with Thiopurine
  medications.
• Despite encouraging data the routine use of
  TPMT genotyping to make treatment decisions is
  still limited.

Genotype-specific 6-MP starting doses for
childhood ALL
McLeod and Siva. Pharmacogenomics. 2002
27
Available laboratory testing
Laboratory tests sent to Mayo clinic
laboratory Volume Name Measurement C
ost (1/04-12/04) PRO-predict 6-MMP,6-TGN 273
36 TPMT enzyme assay TPMT 127
9 TPMT genotyping TPMT2 385
6 TPMT3A/3C
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Laboratory testing

• Samples sent to Prometheus Laboratories (San
  Diego, CA)
• PRO-Predict EnzAct (Enzyme activity)
• Result 3.5 EU lt 6.7 low activity
• 6.7 23.6 Intermediate activity
• gt 23.6 High activity
• PRO-Predict Metabolites (6-TGN and 6-MMPN)
• Result 6 -TGN 1673 (230 400)
• 6 -MMPN undetectable (lt 5700)
• 3. PRO-Predict TPMT (Genotyping)
• Result Homozygous TPMT3A

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Hospital course

• 8/6/04 Severe exfoliating rash thought to be due
  to Imipenem
• 8/9/04 Throat culture () HSV
• 8/10/04 Perirectal abscess - debridement with
  necrotic ulcer acute and chronic inflammation
  () C. difficile
• 8/11/04 Oral () HSV
• 8/19/04 Transferred to BMT floor
• 9/3/04 Discharged (day 38) 16 U pRBCs 19 U
  platelets
• CBC Hgb 10.7 Hct 30.0 WBC count 2,700 (ANC
  1,100) platelet count 15,000
• 9/10/04 CBC Hgb 11.6 Hct 33.2 WBC count 3,800
  (ANC 1,900) platelet count 40,000
• 2/8/05 CBC Hgb 13.1 Hct 37.8 WBC count 3,000
  (ANC 2,100) platelet count 129,000

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Conclusions

• Thiopurine S-methyltransferase (TPMT) plays a
  major role in metabolizing thiopurine medications
  like AZA, 6-MP, and 6-TG
• TPMT activity is polymorphic, 10 heterozygous
  and 1/300 have low/deficient activity
• Patients with low or deficient TPMT activity are
  at a significantly higher risk of rapidly
  developing hematologic toxicity when given a
  standard does of thiopurine drug.
• TPMT genotype correlates well with in vivo enzyme
  activity in erythrocytes.
• TPMT genotyping provides clinicians with a
  reliable method (especially when RBC phenotyping
  is not available) for identifying TPMT-deficient
  patients who can benefit from low doses of
  thiopurine drugs in order to reduce risk of
  toxicity.
• The availability of CLIA-certified TPMT
  genotyping has made routine clinical genotype
  testing a feasible approach for optimizing
  thiopurine therapy.
• Routine monitoring of TMPT genotype and/or
  phenotype prior to induction of thiopurine
  therapy is a cost-effective measure and provides
  one of the best established applications for
  pharmacogenetics in clinical practice to date.