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Association between Busulfan Exposure and Outcome in Children Receiving Intravenous Busulfan before Hematopoietic Stem Cell

Transplantation Association Between Pharmacogenomic and

Pharmacokinetic of Busulfan

On behalf of the Pediatric Disease Working Parties of the European Blood and Marrow Transplantion group

M.Ansari, R.Uppugunduri Satyanarayana, C.Peters, Y.Théoret, MA.Rezgui , M.Labuda, M.Champagne, M.Duval, H.Bittencourt, M.Krajinovic

Geneva University Hospital, Geneva, Switzerland

•  Busulfan (Bu) is an important alkylating agent for conditioning regimens that presents a narrow therapeutic index and a wide inter- and intra-patient variability of plasma exposures, especially in children

•  Therapeutic drug monitoring (TDM) with dose adjustment improves efficacy and safety of Bu-based conditioning regimens

•  In adults, an AUC of 900 to 1500µM*min (Css 616-1026 ng/ml) after first dose has been demonstrated to be a suitable target exposure range

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•  Optimal target AUC/Css in children undergoing HSCT remains unclear

•  Aim of the study: Evaluate the association between Bu iv exposure and

different outcomes after allo-HSCT in pediatrics

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Methods •  Patients:

Prospective Single-center (CHU Sainte-Justine in

Montreal) 89 children IV Bu receiving HSCT May 2000 and August 2010 CHU Sainte-Justine Institutional Review Board

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•  Bu treatment regimen:

Intravenous Bu (16 doses if MA or 8 doses if NMA):

16mg/m2/dose≤ 3 months old

0.8 mg/kg/dose for infants ≥3 months and <1 year of age

1 mg/kg/dose for children ≥1 year and <4 years old

0.8 mg/kg/dose for children ≥4 years old

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Gender Male 46 52 Race - ethnicity Caucasian 73 82.0

Diagnosis AML / ALL 31/10 35/11

MDS 17 19

Non-malignant 31 35

Conditioning Regimen

Bu + Cy 68 76

Bu + others (MA) 9 10

Bu/Flu (NMA) 12 14

GVHD Prophylaxis CSA + steroids 49 55

CSA + MTX 29 33

ATG 69 78

Stem Cell Source

BM 36 40

Cord Blood 52 58

Unrelated Donor 57 64

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Demographic Characteristics (n=89) Patients

N %

MAC=NMA results

•  Definitions of outcomes: VOD: Seattle criteria

GVHD grading: 1994 Consensus Conference on Acute GVHD Grading

Neutrophil recovery: first of 3 days ANC ≥ 0.5x109/L

Platelet recovery: first of 7 days ≥ 50x109/L

Primary graft failure: persistent pancytopenia ≥ 60 days

Secondary graft failure: pancytopenia after neutrophil recovery

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•  Pharmacokinetics:

Blood samples collected: Before Bu administration 15, 30, 60, 120, 180 and 240 min

after drug administration

Plasma Bu high-performance liquid chromatography assay

Non-compartmental analysis (WinNonlin, version 3.1)

Bu target Css: 600 - 900 ng/ml

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Parameter Median (Range) Age (years) 8.2 (0.1-20) Weight (kg) 27 (4.3 – 84.8) Height(cm) 126.8 (54-183) BMI 17.4 (13.1-29.5) BSA (m2) 0.97 (0.25-2.02) CMax (ng/mL) 844 (537-1338) CMin (ng/mL) 215 (66.4-522) CMean (ng/mL) 500 (277-870) CSS (ng/mL) 603 (295-1227) AUC (min.ng/mL) 205585 (94859-431260) Clearance (mL/min/kg) 4.10 (1.84-7.68) Bu Cumulative prescribed dose (mg) 297 (52-1530) Bu Cumulative administered dose (mg)

338 (48-1095)

Bu dose increase (%) 15.9 (-36.8 – 98)

Busulfan pharmacokinetic parameters after first dose of iv Bu

1 < 3 months, more than 60 patients > 4 years 9

•  Statistical analysis:

Univariate Analysis Kaplan-Meier method and log-rank test or Cumulative incidence with competing risk

and Gray’s test.

Multivariate analysis Cox or Fine & Gray proportional hazards

regression models. IBM SPSS Statistics 19 and S-plus 6.1

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Neutrophil recovery: 92% - median 19 (9-48) days Platelet recovery: 89% - median 41 (9-173) days Graft failure: 9 (10%) primary graft failure (n=5)

secondary graft failure (n=4)

No impact of Css on hematopoietic recovery in multivariate analysis

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Results – Hematopoietic Recovery

Acute GVHD grade 2-4 cumulative incidence: 10% No impact of Css on aGVHD incidence in uni-/ multivariate analysis

VOD cumulative incidence: 11% Tendency (p=0.15) to a lower VOD incidence with Css < 603ng/ml – not confirmed in multivariate analysis

HC cumulative incidence: 25% Lower incidence of HC wth Css < 603ng/ml (P=0.02) – not confirmed in multivariate analysis.

Lung toxicity cumulative incidence: 8% Lower incidence of lung toxicity with Css < 603ng/ml in multivariate analysis (P=0.03)

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Results – aGVHD and Conditioning Toxicity

TRM cumulative incidence (5 years): 14% –  Infection was the lead cause of death (n=4)

Higher TRM incidence with Css > 603ng/ml in uni- and multivariate analysis (HR= 3.13; P <0.001)

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Results – Transplant-related Mortality

0 300 600 900 1200 1500 1800

Days after HSCT

0.0

0.2

0.4

0.6

0.8

1.0

TRM

Css < 603 ng/ml

Css > 603 ng/ml

26%

0%

P<0.001

Cumulative incidence of relapse at 5 years (n=58): 37%

Tendency to a higher relapse incidence with Css > 603 ng/ml in univariate analysis (47% vs. 25% - P=0.06), not confirmed in multivariate analysis

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Results – Relapse

Css < 603 ng/ml

Css > 603 ng/ml

81%

31%

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Results – Event-free Survival (EFS)

Results – Event-free Survival (EFS) Estimated 5 years EFS: 56% Lower EFS with Css > 603ng/ml in univariate analysis and multivariate analysis (HR: 4.76 - P<0.001)

Results – Event-free Survival (EFS)

Ansari et al, Therapeutic Drug Monitoring, 2013

89%

46%

Css < 603 ng/ml

Css > 603 ng/ml

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Results – Overall Survival (OS) Estimated 5 years OS: 67% Lower OS with Css > 603ng/ml in univariate analysis and multivariate analysis (HR: 6.6 - P<0.001)

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Conclusion

Outcome of pediatric patients receiving iv Bu can be predicted according to its pharmacokinetics

Worse outcome if Css > 603 ng/ml is mainly related to TRM

Targeting lower end of the range should be investigated

Do we have to lower the Bu target, change Cy by Flu or reverse Bu Cy order (Cy /Bu)?

What is the impact of pharmacogenetics ?

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Interindividual differences in drug responses are an important cause of resistance to treatment and adverse drug reactions. Identifying pharmacogenomic determinants of a given drug may allow for prospective identification of patients with suboptimal drug responses allowing for complementation of traditional treatment protocols by genotype-based drug dose adjustment.

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Posology : mg per Kg or m2 or according to genotype

Some children with a specific genetic profile are responding without side effects compared to a different group of children with a

different genetic profile who are responding but with toxicity. Give the right medication to the right child.

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Metabolic fate of busulfan. GSTA1 (glutathione-S-transferase), tetrahydrothiophene (THT), cystathionine gamma lyase (CTH), DPEP (dipeptidase/cysteinylglycinase), GGT (gamma glutamyl transferase), NAT (N-acetyl transferase), sulfolane (2,3,4,5-tetrahydrothiophene-1,1-dioxide) Cytochrome P 450 enzymes (CYPs), flavin mono oxidase (FMO).

To establish the relationship between GSTM1 and GSTA1 gene variations and drug exposure (Cmax, AUC, Css, Cl) or HSCT outcomes in MAC patients only (RIC excluded). Toxicity (a GVHD and VOD, hemorrhagic cystitis and lung toxicity)

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Cmax (log) AUC (log) Css (log) CL (log)

GSTM1 null genotype and PK

P=0.031 P=0.031P=0.041 P=0.071

+ = null genotype (16) - = without (27) ≥4 years of age

!""""""""""""""""""+" !""""""""""""""""""+" !""""""""""""""""""+" !""""""""""""""""""+"

Ansari et al BMT 2011

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Cmax ng/ml

Haplotype*2A

P=0.02 M-W P=0.02 P=0.02

+ = carrier (29) - = non carrier (40)

GSTA1 - PK Css ng/ml AUC ng/ml

P=0.02 P=0.01 P=0.02

Cmax//mg/kg Css //mg/kg AUC //mg/kg CL ml/min/kg

P=0.01

Ansari et al BMT 2013

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E

FS

P=0.03 62(26)

*2A*2A + 8(0)

*2A*2A - VO

D

GG 7(3)

CC 25(2)

CG 37(4)

CC 22(2) CT 40(4)

TT 7(3)

TT 7(3)

CC&CT 62(6) P=0.008

Time (Days)

GG 7(3)

CC&CG 62(6)

C-69T - *B C-1142G - *B1

P=0.008

GSTA1- clinical outcome EFS 100% vs 54%

VOD seen in 43% homozygous *B or *B1 compared with 9.6% (HR=5.3, 95%CI=1.3-21.5, P=0.009)

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!69 !513 !631 !1142 Haplotype Haplotype1carriers

*A1 C T T C 711(35.5) 451(47.3) *A2 C T G C 421(24.9) 331(34.7) *A3 C T T G 21(1.3)1 11(1) *B1 T T G G 651(32.1) 471(49.4) *B1a T C G G 51(3.1) 51(5.2) *B2 T T G C 51(3.1) 51(5.2)

Minor1allele 751(39.4) 51(2.6) 731(38.4) 721(37.8)

expected

observed

Conclusion

BMT 2013

Conclusion

It seems there is an impact of pharmacogenetics

A prospective multicentric European Blood and Marrow (EBMT)/Swiss Oncology Group (SPOG) study is ongoing to confirm these results in 150 children receiving Busulfan as well as in 50 thalassemia patients and in 400 acute lymphoblastic leukemia children (ALL SCT ped FORUM Study, iBFM 2013)

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In our model, the demographic elements (age, weight) explain 37% of the variability of Busulfan. With the addition of the PG, 54 % of the variability is explained. Hence we must study many more genes and their interaction in order to explain 100% of this variability. Thus there exists today a possible breakthrough on the dose adjustment according to the genotype.

Aknowledgements

The EBMT Paediatric Diseases Working Party and C.Peters as well as the SPOG

Switzerland Canada D.Belli H.Bittencourt Y.Daali M.A.Champagne P.Dayer C.Desjean J.Desmeules C.Drouin M.Fathi S.Dulucq F.Gumy-Pause M.Duval P.Huezo-Diaz M.Krajinovic S.Hughes M.Labuda H.Ozsahin J.F.Lauzon-Joset J.Passweg S.Malouin C.A.Siegrist S.Mezziani S.Suter A.Rezgui A.Tyagi J.Rousseau R.C.Uppugunduri G.St-Onge N.Von-der-Weid Y.Théoret Pierre Fabre Laboratories M.F.Vachon

Thank you to our pharmacogenomic plateforme of pediatric hematology and oncology (CANSEARCH research Laboratory) at

the Geneva University Medical School

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Thank you to the patients and their parents who accepted and signed a consent form for this study

marc.ansari@hcuge.ch

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www.cansearch.ch

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Css Median if rejection, death and relapse , and no event Css low..but IC large

32%

40%

70%

91%

40%

52%

85%

91%

in Group: I Css < 511ng/ml II Css 511-603 III Css 603-714 IV Css >714

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Css Quartiles OS/EFS (5y)

HR (CI) 2.15 (1.41 – 3.28) p< 0.001

HR (CI) 1.80 ( 1.30 – 2.50 ) p< 0.001

I II

III

IV

I

II

III

IV

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