Supplemental Methods
F-ara-A pharmacokinetic sampling and quantification
The F-ara-A pharmacokinetic sampling schedules and their respective participant
compliance rates are summarized in Table 2. Prior to study initiation, we had developed a Fara-A population pharmacokinetic model and identified various limited sampling schedules to
estimate an individual’s F-ara-A AUC over 24 h (i.e., after one dose) using maximum a
posteriori (MAP) Bayesian estimation.[1] Individual pharmacokinetic parameters were
determined via MAP Bayesian estimation, where the population prior (from the population
pharmacokinetic analysis) may potentially offset the loss of individual data due to the use of the
limited sampling schedule. The limited sampling schedules were designed to minimize the
scaled mean square error (sMSE), which represents both bias and variance, for the AUC
estimated using MAP Bayesian estimation compared to the AUC estimated using full
pharmacokinetic sampling (i.e., 0.5h, 0.583h (5 min after the end of the infusion), 1.5h, 4.5h,
6.5h, and 24h from the start of the 0.5h fludarabine infusion) and noncompartmental analysis.
The initial sample schedule chosen had the best sMSE with the least bias and variance. On
days -4, -3, and -2 relative to graft infusion, samples were collected at 0.583h, 1.5h, 6.5h and
24h from the start of the infusion. Only four of seven (57%) participants completed this F-ara-A
pharmacokinetic sampling schedule. Due to the unacceptably high withdrawal rate, the
sampling schedule was modified to collect samples at the same times (0.583h, 1.5h, 6.5h and
24h from the start of the 0.5h infusion) but on day -4 (i.e., the first fludarabine dose,
Supplemental Figure 2) plus a sample on day -1, drawn 24h after the start of the final
fludarabine dose. The participant compliance rate improved to 75% (12 of 16 completed) but
further improvements in compliance were desirable. Participants explained that the primary
barrier to compliance was remaining in the clinic for the 6.5h time point. Therefore, the shortest
sampling schedule available with an acceptable sMSE was used for all subsequent participants.
This F-ara-A pharmacokinetic sampling schedule was 0.5h, 0.583h, 1.5h, and 24h from the start
of each infusion). Participant compliance with this sampling schedule was 98% (84 of 86
completed).
The F-ara-A plasma concentrations were quantified using liquid chromatography-mass
spectrometry (LC-MS) assay with a dynamic range from 0.067 to 2.58 mg/L and an interday
coefficient of variation (CV) <10%.[2] Subsequently, the F-ara-A pharmacokinetic parameters
and the AUC from each participant were calculated as empirical Bayes estimates using the
POSTHOC option in the nonlinear mixed-effects modeling software NONMEM (version 7.2, Icon
Development Solutions, Ellicott City, MD). Specifically, each individual’s F-ara-A concentrationtime data from the limited sampling schedule was used with the MAP Bayesian estimations from
our prior F-ara-A population pharmacokinetic model derived from a separate population.[2,1]
The inter-compartmental clearance (Q) and apparent volume of the peripheral compartment
(V2) had to be fixed to the median values obtained previously, specifically 8.29 L/h/m2 and 43
L/m2. This was not unexpected, since 1.5h was a common inflection point on the F-ara-A
concentration-time curve between the distribution and elimination.
F-ara-ATP accumulation rate in T-cell subsets
The F-ara-ATP accumulation rate in enriched CD4+ and CD8+ T-cell subsets was
evaluated for each participant using our previously published method with minor
modifications.[3] Prior to fludarabine administration, a peripheral blood sample (60 mL) was
obtained, which subsequently underwent an enrichment and purification procedure for CD4+
and CD8+ cells as previously described.[3] Although this blood draw was obtained before
fludarabine administration, many participants had low blood counts, most likely due to their
underlying disease and previous treatments. Only 46 of the 102 (45%) participants had this
sample drawn; 45 of the 46 (98%) had sufficient total cell numbers to proceed with CD4+ and
CD8+ isolation. There were various logistical challenges for obtaining this sample, which largely
arose from the narrow (24-48h) time window between consenting for the HCT treatment plan,
which was needed to determine eligibility for this ancillary biomarker protocol, and the
administration of the first fludarabine dose. This time window was too narrow to notify study
staff of patient consent onto the ancillary biomarker study and to subsequently schedule the Fara-ATP blood sample concurrent with a clinic visit. In addition, CD4+ and CD8+ isolation could
only be performed on weekdays, which further contributed to logistical challenges.
The T-cells for estimation of F-ara-ATP accumulation rate were obtained prior to
fludarabine administration. The CD4+ and CD8+ T-cells were purified using anti-CD4+ or antiCD8+ magnetic microbeads (Miltenyi Biotec Inc., Auburn, CA) and an autoMACS™ automatic
magnetic cell sorter (Miltenyi Biotec Inc.) according to the manufacturer’s recommendations.
Within 2h of cell isolation, the cells were incubated with fludarabine (5 μM) in RPMI 1640 media
(not containing phenol red) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v)
antibiotic/antimycotic for 4h at 37°C in the presence of 5% CO2. This concentration is in the
range of the peak fludarabine plasma concentration (average=3.86 µM) after fludarabine
administration of 30 mg/m2/day. For the ex vivo incubation with fludarabine, the number of cells
varied slightly based on yield from the isolation procedure. A minimum of 1.25×105 cells were
needed per incubation to form adequate amounts of F-ara-ATP for accurate quantitation. The
F-ara-ATP accumulation rate is linear from 1.25×105 to 2×107 cells.[3] Most incubations were
conducted in at least two replicates, with the notable exception that twenty incubations (nine
CD4+ and eleven CD8+) were conducted with single replicates due to low cell numbers. After
incubation with fludarabine, cells were washed twice in ice-cold phosphate buffered saline,
centrifuged, and solubilized in 1 M perchloric acid. Samples were frozen at -70°C prior to F-araATP quantitation.
After thawing, the sample was centrifuged and the supernatant was neutralized to pH ~
7 with 1 M potassium carbonate. F-ara-ATP was quantitated using the LC-MS method
described previously, with the following modifications.[4] A Thermo Scientific (West Palm
Beach, FL) Hypercarb column (2 mm x 100 mm x 5 µm) was used with a flow rate of 0.35
mL/min and a column temperature of 30°C. The mobile phase consisted of 100mM ammonium
acetate pH 9.75 (A) and acetonitrile (B) with the following gradient: initial 85% A and 15% B for
0.2min, to 35% B at 1min, hold 35% B until 3.0min, then 15% B at 3.01min. Chloroadenosine
triphosphate was used as an internal standard. The F-ara-ATP quantitation assay is highly
sensitive and provides a limit of detection equivalent to 50 fmol F-ara-ATP. The units for all Fara-ATP accumulation rates were pmol/1×106 cells/4h. Of the 45 participants with sufficient
total cell numbers to proceed with CD4+ and CD8+ isolation, the F-ara-ATP concentrations could
be quantitated in CD4+ and CD8+ cells from 36 (80%) and 34 (76%) participants, respectively,
with the remainder being below the limit of quantitation.
Immediately after autoMACS™ isolation, a separate aliquot of the CD4+ and CD8+ cells
was taken for total RNA extraction in the first subset of participants. However, insufficient yield
of CD4+ and CD8+ cells resulted, because the highest priority was given to having enough cells
to conduct ex vivo incubation with fludarabine to evaluate the F-ara-ATP accumulation rate.
The low cell yield led to substantive variability and the mRNA levels of the various enzymes and
transporters involved in converting F-ara-A to F-ara-ATP in the CD4+ and CD8+ are not
available.
Immediate suppression of circulating CD4+/CD+ ratio in the peripheral blood
The circulating CD4+ and CD8+ counts in the peripheral blood were assessed
immediately before the first fludarabine dose (day -4) and then one day after the final
fludarabine dose (day -1). For the before fludarabine sample on day -4, the median time
between the collection of the circulating CD4+ and CD8+ count sample and the start of the first
fludarabine dose was 4 min (range: 0 – 10.0 h). Fludarabine was then administered on days 4, -3, and -2. For the after fludarabine sample on day -1, the median time between the start of
the last fludarabine dose and the collection of the circulating CD4+ and CD8+ count sample was
23.8 h (range: 20.4 – 43.9 h). All CD4+ and CD8+ data were obtained before TBI and
allogeneic graft infusion. The CD4+ and CD8+ cell counts were quantitated in a College of
American Pathologist-certified clinical laboratory. In brief, blood (100 µL) was added to an
antibody cocktail consisting of CD45 fluorescein isothiocyanate (FITC), CD4-phycoerythrin (PE),
CD8 PE-TR and CD3 PE-Cy5 (Beckman-Coulter, Fullerton, CA) in a TruCount Tube (BectonDickson, San Jose, CA) to allow for absolute count enumeration. The mixture was incubated for
15 min at room temperature in the dark followed by automated red blood cell lysis (TQ-Prep,
Beckman-Coulter). Six thousand T-cell events (CD3+) were acquired on a FC500 flow
cytometer (Beckman-Coulter) and automated data analysis was performed using TetraOne
software (Beckman-Coulter) per manufacturer’s instructions.
The percent decline in CD4+ or CD8+ counts was calculated by subtracting the after
fludarabine count from the before fludarabine count and dividing the result by the before
fludarabine count. A 100% decline means that no cells were detectable after fludarabine
administration. A negative percent decline indicates that the participant’s cell count was higher
after fludarabine administration, which occurred in three of the 102 participants. Of these three
participants, one, diagnosed with non-Hodgkin’s lymphoma, had increased CD4+ counts and
two, diagnosed with chronic lymphocytic leukemia, had increased CD8+ counts after fludarabine
administration.
Supplemental Table 1. F-ara-A pharmacokinetic sampling schedule optimal for participant
compliance
No.
No. (%) completed
Cohort
F-ara-A sampling schedulea
consented
samplingb
1
Doses 1, 2, 3 to 6.5h
7c
4 (57%)
2
Dose 1 to 6.5h
16
12 (75%)
3
Dose 1 to 1.5h; doses 2 and 3 before next dose
86d
84 (98%)
a
All times are relative to the start of the fludarabine infusion; a 24h sample (i.e., immediately
before next dose) was obtained after each dose. Doses 1, 2, 3 administered on HCT days -4, -3, 2, respectively. bOnly compliance with F-ara-A sampling; compliance with sampling after the graft
infusion is summarized in Li et al.(PMID: 24727337) cFirst two participants were ineligible
because of their TBI dose and, per IRB requirements, their data were destroyed. They are
included here because their withdrawals influenced the decision to change sampling schedules.
d
Excludes three participants whose F-ara-A sampling was missed because of delayed notification
of study staff but who remained in the study for sampling after the graft infusion.
Supplemental Table 2. Association of F-ara-A pharmacologic biomarkers with clinical outcomes in nonmyeloablative HCT recipients.
F-ara-ATP in
F-ara-ATP in
Post-fludarabine
Post-fludarabine
Post-fludarabine
CD4+ cells
CD8+ cells
CD4+ cell count
CD8+ cell count
CD4+/CD8+ ratio
N
103
36
34
99
99
96
OR/HR1
OR/HR2
OR/HR1
OR/HR2
OR/HR1
OR/HR2
P
P
P
P
P
P
(95% CI)
(95% CI)
(95% CI)
(95% CI)
(95% CI)
(95% CI)
ANC nadir <
1.03
0.58
0.85
0.64
0.84
0.77
0.96
0.20
0.64
0.40
0.63
0.64
165/mm3
(0.3-3.5)
(0.3-1.3)
(0.4-1.7)
(0.2-1.8)
(0.4-1.7)
(0.3-2.3)
Day +28 CD3+
2.84
0.73
1.02
0.11
0.27
0.42
0.12
0.42
0.95
0.001
0.004
0.17
chimerism ≥ 95%
(0.8-10.7)
(0.3-1.6)
(0.5-2.0)
(0.0-0.4)
(0.1-0.7)
(0.1-1.4)
Grades 2-4 acute
1.08
1.10
1.45
0.67
1.13
0.41
0.84
0.64
0.12
0.24
0.63
0.01
GVHD
(0.5-2.2)
(0.7-1.6)
(0.9-2.3)
(0.3-1.3)
(0.7-1.9)
(0.2-0.8)
Grades 3-4 acute
1.26
0.83
0.57
1.28
0.69
1.04
0.81
0.67
0.20
0.75
0.50
0.96
GVHD
(0.2-8.2)
(0.4-1.9)
(0.2-1.4)
(0.3-6.0)
(0.2-2.0)
(0.2-5.3)
Chronic GVHD
0.97
0.53
0.84
0.99
0.88
1.66
0.95
0.02
0.46
0.97
0.58
0.17
(0.4-2.3)
(0.3-0.9)
(0.5-1.3)
(0.5-2.0)
(0.5-1.4)
(0.8-3.4)
Relapse
0.93
1.10
1.14
0.96
1.39
0.55
0.91
0.83
0.81
0.94
0.39
0.29
(0.3-3.0)
(0.5-2.7)
(0.4-3.4)
(0.3-2.8)
(0.7-2.9)
(0.2-1.7)
CMV reactivation
0.49
1.08
1.40
1.83
2.74
0.43
0.14
0.79
0.28
0.15
0.003
0.06
(0.2-1.3)
(0.6-1.9)
(0.8-2.6)
(0.8-4.2)
(1.4-5.4)
(0.2-1.0)
Non-relapse
0.46
0.94
0.87
2.64
0.87
2.23
0.21
0.85
0.56
0.10
0.70
0.19
mortality
(0.1-1.5)
(0.5-1.8)
(0.5-1.4)
(0.8-8.4)
(0.4-1.8)
(0.7-7.4)
Overall mortality
0.74
1.15
0.92
1.65
0.90
1.69
0.55
0.63
0.71
0.25
0.72
0.26
(0.3-2.0)
(0.7-2.0)
(0.6-1.4)
(0.7-3.9)
(0.5-1.6)
(0.7-4.2)
1 Neutropenia and day +28 chimerism analyzed as binary endpoints (OR), all others as time-to-event endpoint (HR). OR and HR are effects per
doubling of the biomarker evaluated for F-ara-A AUC, F-ara-ATP in CD4+ and F-ara-ATP in CD8+. 2OR and HR are effects per log increase of
biomarker for post fludarabine CD4+ cell count, post fludarabine CD8+ cell count and CD4+/CD8+ ratio. All analyses adjusted for Kahl risk
category (low, standard, high), donor-recipient gender (female to male, other), donor (related, unrelated).
Abbreviations: ANC: absolute neutrophil count; CMV: cytomegalovirus; F-ara-A: 9-β-D-arabinofuranosyl-2-fluoroadenine; F-ara-ATP:
fludarabine triphosphate; GVHD: graft-versus-host disease.
F-ara-A AUC
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Figure legends
Supplemental Figure 1. The schematic displays the intracellular uptake and metabolism of
fludarabine to the active metabolite fludarabine triphosphate (F-ara-ATP). Abbreviations: ecto5’-nucleotidase (CD73), human equilibrative nucleoside transporter 1 (hENT1), human
equilibrative nucleoside transporter 2 (hENT2), human concentrative nucleoside transporter 3
(hCNT3), deoxycytidine kinase (dCK), dephosphorylation by 5’-nucleotidase (CN-II),
deoxynucleotidase-1 (dNT-1), adenylate kinase (AK), and nucleoside diphosphate kinase
(NDK). Reprinted from Woodahl et al.[3]
Supplemental Figure 2. Schema of sample collection
Abbreviations: AUC: area under the concentration-time curve, AUEC: area under the concentrationeffect curve; CNI: calcineurin inhibitor, IMPDH: inosine monophosphate dehydrogenase, MMF:
mycophenolate mofetil, MPA: mycophenolic acid. The pre-transplant IMPDH [5] and MPA AUC –
IMPDH AUEC [6] results have been previously published.
Supplemental Figure 3. Association of accumulated F-ara-ATP accumulation rate with F-ara-A
AUC in related (white) or unrelated (grey) donor graft in CD4+ cells (A, R2=0.01) and CD8+ cells
(B, R2=0.00) and association of F-ara-ATP accumulation rate in CD8+ cells to F-ara-ATP
accumulation rate in CD4+ cells (C, R2=0.19)