Supplemental Material
Patients and Method
We included 166 consecutive patients undergoing first allogeneic transplantation of
filgrastim-mobilized blood stem cells for a hematologic malignancy between December
2008 and December 2012, whose day 0 serum specimen was available in our serum bank.
The study was approved by our Research Ethics Board. Conditioning was with
fludarabine (50 mg/m2 daily from days -6 to -2), busulfan (3.2mg/kg IV daily from day -5
to -2; pharmacokinetically targeted typically to AUC of 3750 μM/min in those receiving
TBI, and avoiding an AUC of >6000 μM/min in those not receiving TBI) and
antithymocyte globulin (Thymoglobulin 0.5 mg/kg on day -2 and 2 mg/kg on day -1 and
day 0) (Russell J.A. et al: Biology of Blood and Marrow Transplantation 13:814, 2007).
In some patients, total body irradiation was added (Russell J.A. et al: Biology of Blood
and Marrow Transplantation 16:509, 2010). Additional GVHD prophylaxis included
methotrexate (15 mg/m2 IV on day +1, and 10 mg/m2 IV on day +3, +6 and +11) and
cyclosporine (5 mg/kg po bid or 2.5 mg/kg IV bid starting on day -1). For patient
characteristics, see Supplementary Table 1. One patient whose leukemia progressed
immediately after transplantation was excluded. Three patients who developed graft
failure were also excluded. Median day 0 F-ara-A level of these 3 patients (10 ng/mL)
was similar to that of the included patients (14 ng/mL).
Blood was drawn within 15 minutes before graft infusion. F-ara-A serum levels were
measured using liquid chromatography tandem mass spectrometry as described (Ng E. et
al: Journal of Chromatography B 931:103, 2013). The limits of detection and quantitation
of F-ara-A were 0.1 ng/mL and 0.2 ng/mL, respectively.
Characteristic
n
Age (years)
Male
Female
Disease
AML
ALL
Myelodysplasia
CLL
Non-Hodgkin’s Lymphoma
Myelofibrosis
CML
Other
Disease Risk Index*
Low
Intermediate
High
Very High
Comorbidity Index (HCT-CI)**
0
1
2
3 or more (max 6)
Conditioning Regimen
FluBuATG
FluBuATG+TBI
Donor
HLA matched sibling
Other***
CMV IgG (Patient/Donor)
-/-/+
+/+/+
Unknown or indeterminate
EBV IgG (Patient/Donor)
-/-/+
+/+/+
Unknown or indeterminate
Median creatinine clearance (mL/min) *****
Patients with creatinine clearance < 90mL/min
Median follow up of all patients (days)
Median follow up of surviving patients (days)
* Armand, P et al. (Blood 120:905, 2012)
** Sorror, M et al. (Blood 106:2912, 2005)
Median (range) or
Number of patients
(%)
166
50 (18-66)
96 (58%)
70 (42%)
64 (39%)
35 (21%)
16 (10%)
15 (9%)
15 (9%)
10 (6%)
9 (5%)
2 (1%)
8 (5%)
125 (75%)
31 (19%)
2 (1%)
95 (57%)
41 (25%)
14 (9%)
16 (9%)
42 (25%)
124 (75%)
60 (36%)
106 (64%)
53 (32%)
23 (14%)
38 (23%)
48 (29%)
4 (2%)
4 (2%)
13 (8%)
12 (7%)
135 (81%)
2 (1%)
59 (35-138)
10 (6%)
457 (6-1448)
548 (91-1448)
Supplementary Table
1. Patient
Characteristics
*** 8/8 HLA allele matched (for HLA-A,B,C and DRB1) unrelated donor (n= 80), 7/8 HLA allele matched unrelated
donor (n= 25), 8/8 HLA allele matched related non-sibling donor (n= 1)
*****Estimated using Cockroft-Gault equation (Cockroft D. et al: Nephron 16:31, 1976) using average of daily serum
creatinine from day -6 to day 0, and weight from day 0.
Supplementary Table 2. Univariate analysis comparing median F-ara-A
levels between patients who did vs did not achieve clinical outcomes. MannWhitney-Wilcoxon test was used.
Outcome
Median F-ara-A
with outcome
Median F-ara-A
without outcome
p
aGVHD (gr. 2-4)
13.74
13.92
0.723
aGVHD (gr. 3-4)
14.28
13.75
0.697
Any cGVHD
16.00
13.05
0.175
“Extensive” GVHD*
14.60
13.43
0.671
CMV reactivation
15.79
13.74
0.583
PTLD
14.00
13.90
0.812
Relapse
12.75
13.96
0.580
Non-relapse death
12.46
13.92
0.212
Death
15.57
13.46
0.472
* requiring treatment with systemic immune suppression
Supplementary Table 3. Correlation between F-ara-A levels and interval outcomes.
For infections, the interval outcome tested for correlation with F-ara-A level is the
infection rate, ie, the number of infections occurring between day 0 and 83* per 365 days
at risk.
Outcome
r
p
Any Infection**
0.160
0.038
Definite Infection***
0.181
0.019
Viral Infection
0.095
0.219
Bacterial Infection
0.165
0.032
Fungal Infection
-0.003
0.972
Any Severe**** Infection
0.049
0.525
Documented Severe**** Infection
0.043
0.567
Engraftment*****
0.157
0.044
* The interval of day 0-83 was arbitrarily chosen as we felt the F-ara-A on day 0
would more likely affect early transplant infections.
** Both clinical (presumed) and microbiologically documented infections.
*** Microbiologically documented infections (viral, bacterial or fungal,
including catheter associated infections)
**** Requiring hospitalization. If an infection occurred while patient was
hospitalized, then judgement was used to decide whether the infection would be
typically treated inpatient.
***** The first of 3 consecutive days with absolute neutrophil counts >0.5/nl.
The median engraftment day for patients with F-ara-A level in the 4th quartile
(21-104 ng/ml) vs the 1st quartile (1-9 ng/ml) was day 15 vs 13. The engraftment
was not influenced by the includion of TBI into the conditioning – the median
engraftment day in patients with vs. without TBI was day 14 vs 14 (p=0.81,
Mann-Whitney-Wilcoxon
test).
Supplementary Table 4: Multivariate analysis* of association between high fludarabine
levels and time to outcomes, using Cox regression (for death due to any cause) or FineGray regression (for all other outcomes). High fludarabine levels were defined as levels above
median (in the above vs below median analysis) or above 75th percentile (in the 4th vs 1st quartile
analysis). Abbreviations: SHR and HR = Subhazard Ratio and Hazard Ratio of outcome among
patients with high vs low F-ara-A level, ie, above vs below median in the “Above vs Below
Median” analysis, and above 75th percentile vs under 25th percentile in the “4th vs 1st Quartile”
analysis. 95% CI = 95% Confidence Interval.
Outcome
4th vs 1st Quartile
Above vs Below Median
SHR (95% CI)
p
SHR (95% CI)
p
aGVHD (gr. 2-4)
0.91(0.47-1.76)
0.78
0.86 (0.37-2.06)
0.75
aGVHD (gr. 3-4)
1.04 (0.39-2.84)
0.93
0.72 (0.2-2.25)
0.58
Any cGVHD
1.48 (0.49-4.24)
0.47
0.76 (0.44-1.22)
0.29
“Extensive” cGVHD
1.27 (0.70-2.31)
0.42
0.92 (0.44-1.92)
0.83
CMV reactivation
0.81 (0.42-1.53)
0.51
1.42 (0.641-3.11)
0.39
PTLD
0.55 (0.60-3.32)
0.61
0.96 (0.19-4.75)
0.97
Relapse
0.58 (0.20-1.67)
0.31
0.51 (0.14-1.75)
0.29
Non-relapse death
1.60 (0.64-3.97)
0.31
1.47 (0.43-4.99)
0.54
Death due to any
HR (95% CI)
p
HR (95% CI)
p
1.28 (0.70-2.34)
0.42
1.14 (0.53-2.48)
0.72
cause
* Multivariate analysis to determine whether patients with high F-ara-A level had a higher likelihood of developing the
outcome (Subhazard Ratio (HR/SHR) >1) or lower likelihood of the outcome (HR/SHR<1). Covariates (Cov) and
Competing Events (CE) considered were (1) for acute and chronic GVHD, Cov = donor type (HLA-matched sibling vs
other), gender combination (male/male vs other) and patient age (above vs below 45 years); CE= graft failure, second
malignancy, relapse and non-relapse death; (2) for CMV reactivation above our institutional threshold for preemptive
antiviral therapy (25,000 IU/ml plasma), Cov = CMV IgG serostatus pretransplant (+/+, +/-, -/+, -/-) and presence or absence
of significant GVHD (grade 2-4 aGVHD or cGVHD needing systemic therapy); CE = graft failure, second malignancy,
relapse and non-relapse death; (3) for posttransplant lymphoproliferative disorder (PTLD), Cov = donor type, patient age,
EBV serostatus pretransplant and presence or absence of significant GVHD before PTLD onset; CE = graft failure, second
malignancy, relapse and non-relapse death; (4) for relapse, Cov = disease risk index (low-intermediate vs high-very high),
donor type, gender combination and patient age; CE = non-relapse death; (5) for non-relapse death,: Cov = disease risk
index, donor type, patient age and comorbidity score; CE = relapse; and (6) for death due to any cause, Cov = disease risk
index, donor type, patient age and comorbidity index.
Supplemental Figure 1. Overall survival and non-relapse mortality. Overall
survival is estimated with the Kaplan-Meier method comparing (A) patients with Fara-A above vs below median (B) patients with F-ara-A in the 4th quartile vs 1st
quartile. Cumulative incidence of non-relapse death in (c) patients with F-ara-A
above vs below median, and (D) patients with F-ara-A in the 4th quartile vs 1st
quartile. In the competing risk regression for non-relapse death, the competing
event is relapse.
(A)
(B)
(C)
(D)