Supplementary Appendix

This appendix has been provided by the authors to give readers additional information about their work.

Supplement to: Pui C-H, Campana D, Pei D, et al. Treating childhood acute lymphoblastic leukemia without
cranial irradiation. N Engl J Med 2009;360:2730-41.

(PDF updated April 5, 2012.)

Supplementary Appendix

Study Monitoring and Statistical Analysis

Interim analyses for safety monitoring

The major adverse events monitored for safety included deaths, seizures, grade 4 infections, and grade 3 or
4 mucositis during remission induction, the delay in the initiation of consolidation therapy, and
disseminated fungal infection until the end of second reinduction. Threshold rates were pre-specified for
each type of event based on historical data. Stopping thresholds, statistical significance and power were
determined by the sequential conditional probability ratio test (Xiong 1995). For example, for the infections
in induction, we monitored against a baseline rate of 10% and would suggest modifying the study if there
was statistical evidence that the infection rate exceeded 15%. The overall significance level would be
0.0636 with a power of 0.9713 to detect the unacceptable higher rate.

Interim Analysis Sample Size Modify if # Patients with

(Patients) Grade 4 Infections ≥
1 30 9
2 60 14
3 120 23
4 180 31
5 240 37
6 300 45
7 360 53
8 420 58
9 480 63

For the efficacy, we monitored the rate of molecular remission (i.e., <0.01% blasts in bone marrow) after
remission induction, with a rule determined by sequential conditional probability ratio tests. No interim
analysis for any outcome was planned and no such analysis was performed.

The protocol progress was reviewed and monitored for safety, efficacy and publication of results by an
independent Data Safety Monitoring Board (DSMB) every 6 months from the time that all patients enrolled
in the first 6 months have completed remission induction in April 2001 until October 2007 when the DSMB
determined that its monitoring role was completed.

Statistical design and analysis of the primary therapeutic aim

All patients in TOTAL XIIIA and XIIIB studies who had received prophylactic cranial irradiation served as
historical controls. Since the irradiation was given when the patients remained in continuous complete
remission at week 56 of continuation therapy, the comparison was conditioned on the subset of the same
group of patients who remained in continuous complete remission at the same time point on Total XV
study. The statistical inference consists of testing the null hypothesis that the distribution of subsequent
remission duration is the same between the two cohorts against the alternative that the duration is superior
when cranial irradiation is given, based on an unstratified Mantel-Haenszel test (α=β=0.10). A secondary
endpoint of proportion of isolated CNS relapse was also analyzed primarily for safety monitoring.

Xiong X. A class of sequential conditional probability ratio tests. J Am Statist Assoc 1995; 90:1463-1473.
Table 1. Remission Induction, Consolidation, and Early Continuation/Reinduction Therapy
A Remission induction
Agent Dosage Schedule
Methotrexate 1 g/m2 IV over 4 or 24 hours Day 1
Prednisone 40 mg/m2/day Days 5-32
Vincristine 1.5 mg/m2 per week Days 5, 12, 19, 26
Daunorubicin 25 mg/m2 per week Days 5, 12
L-asparaginase (Elspar) 10,000 U/m2 per dose IM (thrice weekly) Days 6, 8, 10, 12, 14, 16, (19, 21, 23)*
Cyclophosphamide 1000 mg/m2 IV Day 26
Cytarabine 75 mg/m2 per day IV Days 27-30, 34-37
Mercaptopurine 60 mg/m2 per night Days 26-39
Intrathecal cytarabine Age-dependent Day 1
Triple intrathecal Age-dependent Day 19 (8, 26)**
Intrathecal cytarabine (40, 50 or 60 mg for ages 1 to 1.99, 2 to 2.99 and ≥ 3 years, respectively).
Triple intrathecal treatments (methotrexate 8, 10 or 12 mg; hydrocortisone 16, 20 or 24 mg; and cytarabine 24, 30 or 36
mg for ages 1 to 1.99, 2 to 2.99 and ≥ 3 years, respectively); **Extra triple intrathecal treatment on days 8 and 26 for
patients with high-risk features of CNS relapse (CNS-2, CNS-3, traumatic lumbar puncture with blasts, T-cell ALL with
leukocyte count > 50x109/L, B-cell precursor ALL with leukocyte count > 100 x 109/L, or the presence of t(9;22)[BCR-
ABL1], MLL rearrangement, or hypodiploidy < 45 chromosomes).
*Extra asparaginase on days 19, 21, and 23 for patients with ≥ 1% residual leukemia cells in the bone marrow on day 19.

B Consolidation therapy
Agent Dosage Schedule
High-dose methotrexate* Targeted to 33μM (low-risk) or 65μM Days 1, 15, 29 and 43
(standard-/or high-risk)
Mercaptopurine 50 mg/m2 per night Days 1 to 56
Triple intrathecal Age-dependent Day 1, 15, 29 and 43

Methotrexate dosage was adjusted according to prior patient-specific pharmacokinetic data to achieve a steady-state
concentration of 65 μM (corresponding to an average dose of approximately 5 g/m2) in standard-risk cases, and 33 μM
(average 2.5 g/m2) in low-risk cases.

C Early continuation/reinduction therapy

Week Low-risk Patients Standard- or high-risk Patients
1 Mercaptopurine + dexamethasone + vincristine Asparaginase + mercaptopurine + dexamethasone + vincristine
+ doxorubicin
2 Mercaptopurine + methotrexate Asparaginase + mercaptopurine
3 Mercaptopurine + methotrexate Asparaginase + mercaptopurine
4 Mercaptopurine + dexamethasone + vincristine Asparaginase + mercaptopurine + dexamethasone + vincristine
+ doxorubicin
5 Mercaptopurine + methotrexate Asparaginase + mercaptopurine
6 Mercaptopurine + methotrexate Asparaginase + mercaptopurine
7 Dexamethasone + vincristine + asparaginase + Asparaginase + dexamethasone + vincristine + doxorubicin
doxorubicin
8 Vincristine + asparaginase Asparaginase + vincristine + doxorubicin
9 Dexamethasone + vincristine + asparaginase Asparaginase + dexamethasone + vincristine
10 Mercaptopurine + methotrexate Asparaginase + mercaptopurine
11 Mercaptopurine + methotrexate Asparaginase + mercaptopurine + vincristine + doxorubicin
12 Mercaptopurine + methotrexate Asparaginase + mercaptopurine
13 Mercaptopurine + methotrexate Asparaginase + mercaptopurine
14 Mercaptopurine + dexamethasone + vincristine Asparaginase + mercaptopurine + dexamethasone + vincristine
+ doxorubicin
15 Mercaptopurine + methotrexate Asparaginase + mercaptopurine
16 Mercaptopurine + methotrexate Asparaginase + mercaptopurine
17 Dexamethasone + vincristine + asparaginase + Asparaginase + dexamethasone + vincristine
doxorubicin
18 Vincristine + asparaginase Asparaginase + vincristine
19 Dexamethasone + vincristine + asparaginase Asparaginase + vincristine + dexamethasone + high-dose
cytarabine
20 Mercaptopurine + methotrexate -----------------------------------
21 Mercaptopurine + methotrexate Mercaptopurine + methotrexate
22 Mercaptopurine + methotrexate Mercaptopurine + methotrexate
23 Mercaptopurine + methotrexate Cyclophosphamide + cytarabine
24 Mercaptopurine + dexamethasone + vincristine dexamethasone + vincristine
Mercaptopurine – 75 mg/m2 PO every evening for 7 days for low-risk group; 50 mg/m2 in the first 16 weeks and 75
mg/m2 thereafter for the standard- and high-risk groups. The starting dose for patients with heterozygous deficiency of
thiopurine methyltransferase was 60 mg/m2 instead of 75 mg/m2.

Dexamethasone – 8 mg/m2 PO per day in 3 divided doses for 5 days for low-risk group and 12 mg/m2 for standard-risk
group; 8 mg/m2 on days 1 to 8 and 15 to 21 during reinduction I (weeks 7 to 9) and reinduction II (weeks 17 to 19) for
both groups.

Asparaginase – 10,000 U/m2 IM thrice weekly for 9 doses during each reinduction for low-risk group; and 25,000 units
/m2 IM weekly for 19 doses for the standard- and high-risk groups; in patients with allergic reactions to E coli
asparaginase, Erwinia asparaginase 20,000 units /m2 thrice weekly during reinduction treatment for the low-risk group,
and 25,000 units /m2 twice weekly in standard-risk group; in patients with allergic reactions to both E coli and Erwinia
asparaginase, or in those for whom Erwinia asparaginase was not available, pegaspargase (Oncaspar) 2500
units /m2 per week.

Vincristine – 2 mg/m2 IV, except for weeks 7-9 and 17-19 when given at 1.5 mg/m2; Methotrexate - 40 mg/m2 IV or IM;
Doxorubicin 30 mg/m2 IV; High-dose cytarabine - 2 g/m2 IV every 12 hours for 4 doses; Cyclophosphamide - 300
mg/m2 IV; Cytarabine - 300 mg/m2 IV.

Triple intrathecal therapy - low-risk cases with CNS-1 status: weeks 7, 12, 17, 24, 32, 40, and 48; low-risk cases with
CNS-2, traumatic lumbar punctures with blasts or leukocyte count ≥ 100 x 109/L: weeks 7, 12, 17, 24, 28, 32, 36, 40, 44,
and 48; standard-risk cases: weeks 7, 12, 17, 24, 28, 32, 36, 40, 44 and 48; other standard-risk cases with leukocyte count
≥ 100 x 109/L, T-cell ALL with WBC ≥ 50 x 109/L, presence of Philadelphia chromosome, MLL rearrangement,
hypodiploidy <45, or CNS-3 status: weeks 3, 7, 12, 17, 24, 28, 32, 36, 40, 44, 48, 56, 64, 72, 80, 88 and 96.
Table 2. Continuation therapy (from week 21 to the end of therapy)*

Week Low-risk Patients Standard- or high-risk Patients

21 Mercaptopurine + methotrexate Mercaptopurine + methotrexate
22 Mercaptopurine + methotrexate Mercaptopurine + methotrexate
23 Mercaptopurine + methotrexate Cyclophosphamide + cytarabine
24 Mercaptopurine + dexamethasone + vincristine Dexamethasone + vincristine
25 Mercaptopurine + methotrexate Mercaptopurine + methotrexate
26 Mercaptopurine + methotrexate Mercaptopurine + methotrexate
27 Mercaptopurine + methotrexate Cyclophosphamide + cytarabine
28 Mercaptopurine + dexamethasone + vincristine Dexamethasone + vincristine

*See footnote of Table 1 in the original article for the details of intrathecal treatments

Mercaptopurine – 75 mg/m2 per night for 7 days; Methotrexate - 40 mg/m2 IV or IM per week; Cyclophosphamide - 300
mg/m2 IV on day 1; Cytarabine - 300 mg/m2 IV on day 1; Dexamethasone – 8 mg/m2 PO per day in 3 divided doses for 5
days for low-risk group and 12 mg/m2 for standard-risk group; Vincristine – 2.0 mg/m2 IV (maximum 2 mg) on day 1.

Low-risk patients received daily mercaptopurine and weekly methotrexate, interrupted by pulses of dexamethasone,
vincristine, and mercaptopurine every 4 weeks (up to week 100); after which only mercaptopurine and methotrexate will
be given. Standard-risk patients received three drug pairs given in 4-week blocks: mercaptopurine plus methotrexate in
the first and second weeks, cyclophosphamide plus cytarabine in the third week (replaced by mercaptopurine and
methotrexate after week 67), and dexamethasone plus vincristine in the fourth week with added mercaptopurine between
week 68 and week 100 (replaced by mercaptopurine and methotrexate after week 100). The total duration of
continuation treatment was 120 weeks for girls and 146 weeks for boys.

The dosages of mercaptopurine and methotrexate were tailored to the limits of tolerance (leukocyte count between 1.5
and 3.0 x 109/L and absolute neutrophil count above 0.5 x 109/L); higher counts were permissible a week following
dexamethasone treatment. Thiopurine methyltransferase phenotype and genotypes were determined prospectively in all
patients, with doses of mercaptopurine lowered in those with heterozygous enzyme deficiency (e.g., 60 mg/m2 instead of
75 mg/m2; no homozygous deficient patients were observed); subsequent doses of mercaptopurine (not methotrexate)
were further reduced in patients with poor tolerance and enzyme deficiency.
Table 3. Reintensification Therapy
Agent Dosage Schedule
Dexamethasone 20 mg/m2 per day PO or IV Days 1-5
Cytarabine 2 g/m2 (every 12 hours) for 4 doses Days 1-2
Etoposide 100 mg/m2 (every 12 hours) for 5 doses Days 3-5
Asparaginase 25,000 U/m2 IM Day 6
Table 4. Clinical and biologic features of the 11 patients with isolated CNS relapse

Minimal
Patient residual
No. disease
Initial
Age at Leukocyte Day Remission Second
diagnosis count x 19 Day 46 CNS Duration remission
(Year) 109L Sex Race Subtype (%) (%) Status (Year) duration(Year)
1 2.03 86.2 Male African B-cell precursor 0.06 0.00 CNS-1 3.2 2.5
American
2 5.87 3.50 Male White Hyperdiploidy >50 0.02 0.00 CNS-1 2.97 0..43
3 4.30 34.8 Male African t(1;19) [TCF3- 1.12 0.00 CNS-2 1.9 5.52
American PBX1]
4 5.20 7.2 Male African t(1;19) / [TCF3- 0.00 0.00 CNS-2 1.8 1.8
American PBX1]
5 11.13 53.7 Male African t(1;19) / [TCF3- 0.27 0.00 CNS-2 3.1 2.5
American PBX1]]
6 1.18 656.2 Female White T Cell 0.88 0.03 CNS-3 1.9 2.3
7 1.86 567.0 Female White T Cell 0.02 0.00 CNS-2 0.4 3.6
8 5.39 657.0 Female African T Cell 2.62 1.59 CNS-2 0.4 4.2
American
9 6.06 3.4 Male White T Cell 0.00 0.00 CNS-2 1.3 2.0
10 7.08 34.1 Male White T Cell 0.14 0.00 CNS-1 2.3 2.0
11 10.90 125.8 Male White T Cell 0.00 0.00 traumatic 0.9 4.2
with
blasts
Figure 1. Comparison of continuous complete remission duration in 71 high-risk patients in Total XV
study who received chemotherapy only versus 56 historical controls who received prophylactic cranial
irradiation in Total XIII studies. The 5-year rates are means ± standard error.