Supplementary Material:
Clinical utility and implications of asparaginase antibodies in acute
lymphoblastic leukemia
Chengcheng Liu,1,5 Jitesh D. Kawedia, PhD1 Cheng Cheng, PhD2 Deqing Pei, 2 Christian A.
Fernandez, PhD1 Xiangjun Cai, PhD1 Kristine R. Crews, PharmD1 Sue C. Kaste, DO3,5 John C.
Panetta, PhD1 W. Paul Bowman, MD6 Sima Jeha, MD4 John T. Sandlund, MD4 William E.
Evans, PharmD1 Ching-Hon Pui, MD4 and Mary V. Relling, PharmD1
From Departments of 1Pharmaceutical Sciences, 2Biostatistics, 3Radiological Sciences, and
4
Oncology, St Jude Children’s Research Hospital, Memphis, TN; 5University of Tennessee
Health Science Center, Memphis, TN; and 6Cook Children’s Hospital, Fort Worth, TX.
Correspondence: Mary V. Relling, St. Jude Children’s Research Hospital, 262 Danny Thomas
Place, Memphis, TN 38105-2794, USA. Phone: 901-595-2348; Fax: 901-595-8869; e-mail:
mary.relling@stjude.org
1
SUPPLEMENT

Supplemental Methods

Supplemental Tables
Table S1. Clinical features of patients with and without samples evaluable for antiasparaginase antibodies.
Table S2. Number of available blood samples at each time point.
Table S3. Number of patients having allergic reaction to Elspar in different phases.
Table S4. Clinical features and allergic reactions to Elspar.
Table S5. Clinical features and anti-Elspar antibody status.

Supplemental Figures
Figure S1. Total XV asparaginase regimen and anti-asparaginase antibody
measurements.
Figure S2. Anti-Elspar antibody level relative to the time of clinical reaction to Elspar.
Figure S3. ROC curves of the antibody tests.
Figure S4. Association between Week 7 anti-Elspar antibody level and the proportion of
patients reacted to Elspar around week 7.
Figure S5. The frequency of hypersensitivity to Oncaspar and Erwinase.

Supplemental References
2
Supplemental methods
Anti-asparaginase antibodies
A total of 2010 serum samples for antibodies to three forms of asparaginase (Elspar, Erwinase,
and Oncaspar) were measured by ELISA. Of these, 471 samples were assayed at the University
of Tennessee Health Science Center (UTHSC) and 1345 samples were assayed at St. Jude
Children’s Research Hospital (SJCRH). The positive control reference pool was prepared from
patients known to have received the specific form of asparaginase and who exhibited clinical
signs of allergic reaction and had high titers of antibodies, represented by high optical density
(OD) readings. The negative control reference pool was prepared from healthy volunteers who
had never received asparaginase treatment. A sample assayed at SJCRH was considered positive
if the natural log of its 1:400 OD reading was greater than 2.58 standard deviations above the
negative control process mean. A sample assayed at UTHSC was considered positive if the
natural log of its 1:3200 OD reading was greater than 2 standard deviations above the negative
control process mean. Samples that were not positive were considered negative. Because the
magnitudes of the scale of antibody OD readings obtained at the two facilities differed, we
normalized the OD readings based on a comparison of 194 serum samples measured for antibody
levels at both SJCRH and UTHSC. The normalization of UTHSC OD readings to the same scale
was applicable to the SJCRH readings was described by an exponential model using equation 1
(Elspar), 2 (Erwinase), and 3 (Oncaspar) to maintain consistency of OD readings between
UTHSC and SJCRH. All of the samples measured at UTHSC had their OD readings normalized
to the scale of the readings obtained at SJCRH using the following equations.
log (normalized OD) = -5.066 + 3.509 ∙ exp [0.526 × log (original OD)]
[1]
3
log (normalized OD) = -4.360 + 3.518 ∙ exp [0.806 × log (original OD)]
[2]
log (normalized OD) = -5.280 + 3.684 ∙ exp [0.520 × log (original OD)]
[3]
Estimation of antibody area-under-the-curve (AUC)
The area under the antibody-concentration-versus-time curve (AUC) was estimated using the
method of trapezoids in the 360 patients who had at least four out of five scheduled antibody
results (including week 7 and week 17 tests in the continuation phase and at least two antibody
tests in the induction phase). Induction AUC (antibody AUC between day 5 and day 34) and
continuation AUC (antibody AUC between week 7 and week 17) were estimated separately
using OD readings for each patient’s serum tested against Elspar, the front-line asparaginase
preparation (Supplemental Figure 1). Analyses were performed using the sum of the induction
AUC and continuation AUC of antibodies to Elspar asparaginase.
Asparaginase activity
This assay was similar to a previously reported assay,1 with a faster and more controlled process
as well as improved linearity range without sample pretreatments. The asparaginase activity in
serum was measured by an enzymatically coupled oxidation of reduced nicotinamide adenine
dinucleotide (NADH):
L-Asparagine
Asparaginase
L-Aspartate + NH4
Glutamic oxalacetic transaminase
L-Aspartate + α-Ketoglutarate
Oxaloacetate + L-Glutamate
4
Oxaloacetate + NADH
Malic dehydrogenase
Malate + NAD+
When monitored by spectrophotometry, the oxidation of NADH leads to a corresponding
decrease in absorbance at 340 nM. The rate of change of absorbance is directly proportional to
asparaginase activity.
The enzyme reaction mixture was prepared by combining 200 mL of 10x Tris-buffered saline,
400 mL of glycerol, 200 mg of α-ketoglutaric acid, 264 mg of asparagine, 200 mg of β-NADH,
400 µL of glutamic oxaloacetic transaminase, 200 µL of malic dehydrogenase, and 1400 mL
distilled and deionized water, and stored at -80ºC. (All the reagents above were from SigmaAldrich, St. Louis, MO.) Before plate reading, the enzyme reaction mixture was incubated in a
37ºC water bath for 30 min. Then patient sera were added to 96-well UV-transparent plates
(Corning. Inc, NY) in quadruplicate. The reaction was started by adding 190 µL of enzyme
reaction mixture to each well. The enzyme reaction mixture was added one row at a time, with a
20 second interval between each addition. The plate was read using a BioTek ELx808IU
Microplate Reader (BioTek, Winooski, VT) at 340 nm with a 20 second interval for 15 min. The
activity was estimated by the slope of the change of optical density over time. The linearity range
of this assay was 0.011-2.2 U/mL. Samples higher than 2.2 U/mL were diluted with sample
dilution buffer (5% bovine serum albumin in deionized water). Calibrators were established with
asparaginase standard solutions: 0.025-2.0 U/mL native E. coli asparaginase (BioVendor
Laboratory Medicine. Inc, Czech Republic) in sample dilution buffer.
Receiver-operator-characteristic (ROC) curves
5
The ROC curves of antibody tests were generated by plotting the true positive rate (sensitivity)
and corresponding false positive rate (1-specificity) for every possible threshold OD value (to
separate positive from negative samples; supplemental Figure 3). The area-under-the-curve
(AUC) of the ROC curve is a relative measure of the diagnostic accuracy of a test. An AUC of
1.0 would indicate a perfect test (100% TP, 0% FP, 100% TN, 0% FN), whereas 0.5 would
indicate a random classification. A value greater 0.9 represents a rather high accuracy, a value
less than 0.7 represents a low accuracy, and a value in between would be considered fair to
good.2 (TP, true positive; FP, false positive; TN, true negative; FN, false negative.)
6
Supplemental Tables.
Table S1. Clinical features of patients with (n = 410) and without (n = 88) samples
evaluable for anti-asparaginase antibodies.
Patients with samples
Patients without samples
n (%)
n (%)
Less than 10 years
302 (81.2)
70 (18.8)
Older than 10 years
108 (85.7)
18 (14.3)
Male
228 (81.7)
51 (18.3)
Female
182 (83.1)
37 (16.9)
White
282 (88.4)
37 (11.6)
Black
65 (91.5)
6 (8.5)
Other
63 (58.3)
45 (41.7)
B-lineage
345 (81.6)
78 (18.4)
T-lineage
65 (86.7)
10 (13.3)
Low-risk
197 (82.4)
42 (17.6)
Standard/high-risk
213 (82.2)
46 (17.8)
P
Age
0.28
Sex
0.72
Racea
0.0001
Immunophenotypeb
0.33
Treatment arm
1.0
a = Genetically determined race as described;3 b = acute lymphoblastic leukemia
immunophenotype.
7
Table S2. Number of patients with evaluable blood samples at each time point.
Number of patients
Time point of antibody test
(samples)
Day 5
401 (420)
Day 19
392 (396)
Day 34
384 (399)
Week 7
381 (408)
Week 17
367 (387)
8
Table S3. Number of patients having allergic reaction to Elspar at each phase of therapy.
Phase
Low-risk
Standard/high-risk
Total
Induction
2
3
5
Continuation weeks 1-6
0
54
54
Reinduction I
96
1
97
Continuation weeks 10-16
0
7
7
Reinduction II
2
0
2
Reintensification
0
4
4
Total
100
69
169
9
Table S4. Clinical features and allergic reactions to Elspar (n=410*)
All patients
Allergy
Low-risk Arm
No allergy
n
Allergy
P
n(%)
No allergy
n
n(%)
Standard/high-risk Arm
P
n(%)
n(%)
183
94 (51.4)
89 (48.6)
14
6 (42.9)
8 (57.1)
93
53 (57.0)
40 (43.0)
Allergy
No allergy
n(%)
n(%)
119
35 (29.4)
84 (70.6)
94
34 (36.2)
60 (63.8)
135
43 (31.9)
92 (68.1)
78
26 (33.3)
52 (66.7)
136
53 (39.0)
83 (61.0)
n
P
Age
Less than 10 years
302
129 (42.7) 173 (57.3)
0.36
0.59
Older than 10 years
108
40 (37.0)
68 (63.0)
Male
228
96 (42.1)
132 (57.9)
Female
182
73 (40.1)
109 (59.9)
104
47 (45.2)
57 (54.8)
White
282
128 (45.4) 154 (54.6) 0.037 146
75 (51.4)
71 (48.6)
Black
65
20 (30.8)
45 (69.2)
24
15 (62.5)
9 (37.5)
41
5 (12.2)
36 (87.8)
Other
63
21 (33.3)
42 (66.7)
27
10 (37.0)
17 (63.0)
36
11 (30.6)
25 (69.4)
B-lineage
345
157 (45.5) 188 (54.5) .0001 197 100 (50.8)
97 (49.2)
148
57 (39.2)
91 (60.8)
T-lineage
65
12 (18.5)
65
12 (18.5)
53 (81.5)
0.31
Sex
0.69
0.12
0.88
Racea
0.19
0.004
Immunophenotypeb
53 (81.5)
0
0 (0.0)
0 (0.0)
NA
0.003
a = Genetically determined race as described;3 b = acute lymphoblastic leukemia
immunophenotype; NA = not applicable.
*Patients with serum samples evaluable for anti-asparaginase antibodies (n = 410). Analysis for
all patients (n = 498) has been previously reported.4
10
Table S5. Clinical features and anti-Elspar antibody status (n=410)
All patients
Positive
Low-risk
Negative
n
Positive
P
n(%)
Negative
n
n(%)
Standard/high-risk
P
n(%)
Positive
Negative
n(%)
n(%)
119
53 (44.5)
66 (55.5)
94
47 (50.0)
47 (50.0)
135
65 (48.2)
70 (51.8)
78
35 (44.9)
43 (55.1)
136
71 (52.2)
65 (47.8) 0.013
n
n(%)
P
Age
Less than 10 years
302
182 (60.3) 120 (39.7)
Older than 10 years
108
54 (50.0)
54 (50.0)
Male
228
135 (59.2)
93 (40.8)
Female
182
101 (55.5)
81 (44.5)
White
282
Black
Other
0.07
183 129 (70.5)
54 (29.5)
0.14
0.49
14
7 (50.0)
7 (50.0)
93
70 (75.3)
23 (24.7)
104
66 (63.5)
38 (36.5)
175 (62.1) 107 (37.9) 0.005
146 104 (71.2)
42 (28.8)
65
36 (55.4)
29 (44.6)
24
16 (66.7)
8 (33.3)
41
20 (48.8)
21 (51.2)
63
25 (39.7)
38 (60.3)
27
16 (59.3)
11 (40.7)
36
9 (25.0)
27 (75.0)
B-lineage
345
213 (61.7) 132 (38.3) .0001
197 136 (69.0)
61 (31.0)
148
77 (52.0)
71 (48.0) 0.026
T-lineage
65
23 (35.4)
65
23 (35.4)
42 (64.6)
Sex
0.48
0.09
0.67
Racea
0.40
Immunophenotypeb
42 (64.6)
0
0 (0.0)
0 (0.0)
NA
a = Genetically determined race as described;3 b = acute lymphoblastic leukemia
immunophenotype; NA = not applicable.
11
Supplemental figure legends
Figure S1. Total XV asparaginase regimen and anti-asparaginase antibody measurements.
All patients received Elspar (ASP) at 10000 U/m2 per dose during remission induction; patients
on the SHR arm received 25000 U/m2 per week at weeks 1-19; those on the LR arm received
10000 U/m2 thrice weekly at reinduction weeks 7-9 and 17-19. Serum samples for antiasparaginase antibodies (ANTI-ASP) were collected on days 5, 19, and 34 of remission
induction and weeks 7 and 17 of continuation therapy. The area under the antibodyconcentration-versus-time curve (ANTI-ASP AUC) was estimated for induction (between day 5
and day 34) and continuation (between week 7 and week 17) using OD readings tested against
Elspar.
ASP (LR)
ASP (SHR)
( )
( )
10000 U/m2
10000 U/m2
10000 U/m2
25000 U/m2
4
7 1
Weeks 1
Induction
4
Consolidation
7 1
4
Continuation
7
10
14
(Reinduction I)
17
20
47
(Reinduction II)
ANTI-ASP
Day 5
ANTI-ASP
AUC
19
34
Induction AUC
Week 7
Week 17
Continuation AUC
12
Figure S2. Anti-Elspar antibody level relative to the time of clinical reaction to Elspar.
Anti-Elspar OD level in patient samples (dots) relative to the number of days elapsed before
(negative) or after (positive) between sample collection and clinical reaction to Elspar. The trend
lines of median (solid) and quartiles (dashed) are shown.
13
Figure S3. ROC curves of the antibody tests. Receiver operating characteristic (ROC) curves
of the antibody tests at day 34, week 7 and week 17, showing the true positive rates and
respective false positive rates for predicting or confirming clinical allergic reactions. Each point
on the curve corresponds to a specific threshold of anti-Elspar OD that putatively divides
antibody-positive from antibody-negative samples. Right y-axis shows the color key that
visualizes the mapping from the natural log of anti-Elspar OD to colors. The positive threshold
dividing positive from negative samples for logOD is -3.2 (circles). The AUC of the ROC
curves, which represents overall test performance on predictions, is shown in each graph. LR,
low-risk; SHR, standard/high-risk; Ab, anti-Elspar antibody; Rxn, clinical reaction; AUC, area
under the curve. Time of antibody tests: D34 = Day 34 of induction therapy; W7 = Week 7 of
continuation therapy; and W17 = Week 17 of continuation therapy.
AUC=0.857
False positive rate
log(anti-Elspar OD)
False positive rate
W7 Ab vs. W1-6 Rxn (SHR)
W17 Ab vs. W1-16 Rxn (SHR)
AUC=0.870
False positive rate
True positive rate
log(anti-Elspar OD)
False positive rate
True positive rate
True positive rate
log(anti-Elspar OD)
D34 Ab vs. W1-6 Rxn (SHR)
AUC=0.908
log(anti-Elspar OD)
False positive rate
AUC=0.700
True positive rate
True positive rate
True positive rate
AUC=0.782
W17 Ab vs. W7-9 Rxn (LR)
log(anti-Elspar OD)
W7 Ab vs. W7-9 Rxn (LR)
log(anti-Elspar OD)
D34 Ab vs. W7-9 Rxn (LR)
AUC=0.867
False positive rate
14
Figure S4. Association between Week 7 anti-Elspar antibody level and the proportion of
patients reacting to Elspar around week 7. Left panels: the distribution of patients with (solid
bars) and without (open bars) clinical reactions by their anti-Elspar OD at week 7. (a) Patients on
the LR arm (n = 195) with and without reactions during weeks 7-9. (c) Patients on the SHR arm
(n = 175) with and without reactions during weeks 1-6. Right panels: the positive correlation
between week 7 anti-Elspar OD and the proportion of patients having clinical reaction at (b)
weeks 7-9 (LR) and (d) weeks 1-6 (SHR). Trend lines are shown. LR, low-risk; SHR,
standard/high-risk.
15
Figure S5. The frequency of hypersensitivity to Oncaspar and Erwinase. The proportion of
patients exhibiting secondary allergic reactions and antibody positivity among the patients who
had samples tested for anti-Oncaspar (n = 85) or anti-Erwinase (n = 63) after they received
Oncaspar or Erwinase. Ab, antibody against Oncaspar (left) or Erwinase (right).
Erwinase (n=63)
Oncaspar (n=85)
Ab-negative
No reaction
15 (18%)
Ab-positive
Reaction
20 (23%)
Ab-negative
Reaction
3 (4%)
Ab-positive
No reaction
47 (55%)
Ab-positive
Reaction
4 (6%)
Ab-positive
No reaction
8 (13%)
Ab-negative
No reaction
46 (73%)
Ab-negative
Reaction
5 (8%)
16
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17