1
Supplementary Material
2
Contents
3
Supplementary Materials and Methods
4
Supplementary Figures 1 and 2
5
Supplementary Tables 1, 2, 3 and 4
6
Supplementary References
7
8
Supplementary Material and Methods
9
Patients and samples
10
Between 04/1995 and 07/2012, a total of 438 pediatric T-LBL patients were registered in the
11
NHL-BFM study center for the trials NHL-BFM95 and EURO-LB02 after informed consent.
12
The accompanying molecular research was approved by the ethical committees of Hannover
13
Medical School and Justus Liebig University Giessen, Germany. Diagnosis of T-LBL was
14
made according to WHO Classification of Hematological Malignancies and European
15
childhood lymphoma pathology panel.1 Patients were treated according to an ALL-BFM-type
16
treatment strategy for LBL as described previously.2 Data on patients characteristics,
17
diagnostics, treatment, and outcome were obtained from the clinical trial databases and
18
correlated with experimental results.
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PCR amplification of genomic sequences and Sanger sequencing
20
A total of 114 DNA samples of pediatric T-LBL patients were used for validation of PIK3R1,
21
PIK3CA, and PTEN using Sanger sequencing, and 99 DNA samples were available for NRAS,
22
and KRAS validation. The isolation of DNA from lymphoma material has been described
23
earlier.3
24
Mutational analysis was carried out by Sanger sequencing of an appropriate genomic region
25
of the genes PIK3R1, PIK3CA, PTEN, NRAS, and KRAS after PCR amplification. For
26
amplification of PIK3R1, PIK3CA, PTEN sequences, Q5 High Fidelity DNA Polymerase was
27
used, for amplification of NRAS and KRAS, we used OneTaq DNA polymerase (New
28
England Biolabs, Frankfurt/Main, Germany) using standard buffers and conditions.
29
Amplification of KRAS exon 2 required using GC buffer. For GenBank accession numbers
30
and primer sequences refer to Supplemental Table 1. PCR products were sequenced by LGC
31
Genomics (Berlin, Germany) or on an ABI 3730xl Genetic Analyzer (Life Technologies).
32
Base-pair substitutions were verified twice, frameshift mutations once. Multiple frameshifts in
33
a single PCR product were analyzed in detail by subcloning into Topo TA cloning vectors
34
(Life Technologies) and subsequent sequencing.
35
Statistical Analysis
36
Probability of event-free survival (pEFS) was calculated according to Kaplan and Meier with
37
differences compared by log-rank test. pEFS was calculated from date of diagnosis to first
38
event (death from any cause, relapse, resistant disease or second malignancy) or to the date of
39
last follow-up. Patients lost to follow-up were censored at date of last follow-up-examination.
40
Cumulative incidence functions for relapse were constructed by the method of Kalbfleisch
41
and Prentice, and compared with Gray’s test. Differences in the distribution of individual
42
parameters among patient subsets were analyzed using χ² or Fisher’s exact test.
Supplementary Figures
Supplementary Figure 1: Probability of EFS according to PI3K-AKT pathway mutational status.
Supplementary Figure 2: Probability of EFS according to NOTCH1 mutation (a) and loss of
heterozygosity at chromosome 6q (LOH6q) status (b).
Supplementary Tables
Supplementary Table 1: Primer sequences used for amplification of PIK3R1, PIK3CA, PTEN,
NRAS and KRAS. Some of the primers have been published.4-8
Region
PIK3R1
exons
16+17
PIK3CA
exon 10
exon 21
PTEN
exon 7
KRAS
exon 2
NRAS
exon 2
exon 3
Forward primer
Reverse primer
Product length
(bp)
GCTGGGAAACCATAGTGAAA
CTACAAGATGTTCCAAACTCAG
548
TTGCTTTTTCTGTAAATCATCTGTG
TGGGGTAAAGGGAATCAAAAG
CTGCTTTATTTATTCCAATAGGTATG
CCTATGCAATCGGTCTTTGC
321
525
TGACAGTTTGACAGTTAAAGG
CTTCTCAGTTAACCATCCTTG
466
AAGCGTCGATGGAGGAGTTT
TGGACCCTGACATACTCCCA
545
GGCCGATATTAATCCGGTGT
AACAGCACAAATAAAACAGTCCAG
TCCGACAAGTGAGAGACAGG
TGGTAACCTCATTTCCCCATA
332
627
Supplementary Table 2: Detailed description of identified mutations. Numbering of base pairs and amino acids according to GenBank accession
indicated.
patient
T-LBL-5
T-LBL-7
T-LBL-11
PIK3R1
NM_181523.2, NP_852664.1
DNA
protein
PIK3CA
NM_006218.2, NP_006209.2
DNA
protein
T-LBL-18
T-LBL-21
c.1781G>A
T-LBL-25
T-LBL-28
T-LBL-41
c.3297A>G
T-LBL-44
T-LBL-46
c.3302G>C
T-LBL-48
PTEN
NM_000314.4, NP_000305.3
DNA
protein
c.1808C>A
p.H259Q
c.[1752_1781
delins
CCCGCGGA
A]+
[1727_1728in
sG]
p.R233fsAT
GRQVHVL*
p.F241_C250
delinsPAE
c.[1727_1728
insT]+
[1732_1733in
sGCCG]+
[1732_1733in
sATAGTAG
G]
p.R233fsSTG
RQVHVL*
p.E235fsPGR
QVHVL*
p.E235*
c.1728_1730
delins
GCCCCCCG
GGG
p.R233fsAPR
GGKTSSCT
LSSLSRYLC
VVISK*
homozygous
mutation
c.[1768_1769
insTCCACC]
+
[1727_1728in
s
TTGTAAT]
p.P246_L247
insPP
p.R231fsL*
KRAS
NM_004985.4, NP_004976.2
DNA
protein
c.38G>A
p.Gly13Asp
c.35G>C
p.Gly12Ala
NRAS
NM_002524.4, NP_002515.1
DNA
protein
p.E542K
p.H1047R
p.G1049R
c.35G>A
p.Gly12Asp
patient
T-LBL-49
T-LBL-52
PIK3R1
NM_181523.2, NP_852664.1
DNA
protein
PIK3CA
NM_006218.2, NP_006209.2
DNA
protein
c.2315_2320
del CAATAC
p.Q579_Y58
0del
c.1781G>A
p.E542K
c.2270A>G
p.N564D
c.3144A>G
p.N996S
c.2270A>G
p.N564D
c.1781G>A
p.E542K
T-LBL-53
T-LBL-55
T-LBL-57
T-LBL-58
T-LBL-60
T-LBL-62
c.1728_1729i
nsA
T-LBL-65
c.1768delins
GGGGCCCA
T-LBL-74
T-LBL-80
T-LBL-82
T-LBL-85
c.2326-2A>G
T-LBL-87
c.2270A>G
T-LBL-92
PTEN
NM_000314.4, NP_000305.3
DNA
protein
c.[1728delins p.R234fsDG
AGG]+
KTSSCTLSS
[1762_1763in LSRYLCVVI
sTCAGCCG
SK*
ACGG
p.L247fsTGR
GAAGACA
QVHVL*
AGTTCATG p.P246fsRRY
TACTT
LCVVISK*
TGAGTTCC p.P246_L247
C]+
insGWR
[1767_1768in
sGG]+
[1769_1770in
sGGTTGGA
GG]
c.1753_1754
p.F241*
delTT
c.1727_1728i p.R233fsTTG
nsA
RQVHVL*
c.1729delins
p.R233fsGT
GG
GRQVHVL*
c.3297A>G
KRAS
NM_004985.4, NP_004976.2
DNA
protein
NRAS
NM_002524.4, NP_002515.1
DNA
protein
c.34G>A
p.Gly12Ser
c.34G>A
p.Gly12Ser
c.38G>T
p.Gly13Val
c.35G>A
p.Gly12Asp
p.R233fsTTG
RQVHVL*
homozygous
mutation
p.P246fsRGP
VTCVW*
p.H1047R
p.M582_D60
5delinsI
p.N564D
patient
T-LBL-94
T-LBL-95
T-LBL-100
T-LBL-103
T-LBL-105
T-LBL-111
T-LBL-112
T-LBL-113
T-LBL-114
PIK3R1
NM_181523.2, NP_852664.1
DNA
protein
PIK3CA
NM_006218.2, NP_006209.2
DNA
protein
PTEN
NM_000314.4, NP_000305.3
DNA
protein
c.[1726_1728
delinsTGGG]
+
[1767_1768in
sAA]
c.1768_1769i
nsCC
p.P246fsQR
YLCVVISK*
p.T232fsMG
TGRQVHVL
*
p.L247fsRYL
CVVISK*
c.[1728delins
GA]+
[1768delinsG
GGGG]
c.[1729_1749
delinsCCG]+
[1732G>C]
p.R233fsETG
RQVHVL*
p.P246fsRG
VTCVW*
p.R233_Y24
0delinsPD
p.R234P
c.1725_1727
delinsCCAG
C
p.T232fsPAD
GKTSSCTLS
SLSRYLCV
VISK*
p.R233fsAT
GRQVHVL*
c.1727_1728i
nsG
KRAS
NM_004985.4, NP_004976.2
DNA
protein
c.35G>T
p.Gly12Val
NRAS
NM_002524.4, NP_002515.1
DNA
protein
c.181C>A
p.Gln61Lys
c.183A>T
p.Gln61His
c.34G>T
p.Gly12Cys
Supplementary Table 3: Frequency and outcome according to the mutational status of PI3K,
PTEN, RAS, NOTCH1, FBXW7 and loss of heterozygosity of chromosome 6q (LOH6q). PI3K,
combined data for PIK3R1, PIK3CA; RAS, combined data for KRAS and NRAS; Data on mutations
of NOTCH1, FBXW7 and LOH6q have been published earlier.
Marker
PI3KWT
PI3Kmut
PTENWT
PTENmut
RASWT
RASmut
NOTCH1WT
NOTCH1mut
FBXW7WT
FBXW7mut
LOH6qneg
LOH6qpos
n
98
9
96
17
89
10
46
70
95
21
192
25
EFS (5 years)
77±4%
78±14%
82±4%
59±12%
78±4%
70±14%
66±7%
84±5%
76±5%
79±10%
86±3%
27±9%
p value (log rank)
0.99
0.014
0.61
0.021
0.6
<0.0001
Supplementary Table 4: Comparison of published classifier for adult T-ALL9 with data on adult
T-ALL (a), applied to our cohort of pediatric T-LBL (b) and our newly defined genetic classifier for
pediatric T-LBL (c). CIR, cumulative incidence of relapse; CI, confidence interval.
a)
Definition
NOTCHmut ± FBXW7mut + RASWT + PTENWT
all other patients
N evaluable
patients
91
81
CIR (5 years)
95% CI
p
15%
54%
9–24%
42–66%
<0.001
CIR (5 years)
95% CI
p
16%
23%
6–27%
11–35%
0.28
CIR (5 years)
95% CI
p
11%
19%
64%
1–22%
7–31%
37–91%
<0.001
b)
Definition
NOTCHmut ± FBXW7mut + RASWT + PTENWT
all other patients
N evaluable
patients
51
48
c)
Definition
GR: NOTCH1mut + RASWT + PI3KWT
IR: all other patients
HR: NOTCH1WT + PTENmut ± LOH6qpos
N evaluable
patients
35
42
14
Supplementary References
1.
Oschlies I, Burkhardt B, Chassagne-Clement C, d’Amore ES, Hansson U, Hebeda K, et al.
Diagnosis and immunophenotype of 188 pediatric lymphoblastic lymphomas treated within
a randomized prospective trial: experiences and preliminary recommendations from the
European childhood lymphoma pathology panel. Am J Surg Pathol 2011; 35: 836-844.
2.
Reiter A, Schrappe M, Ludwig WD, Tiemann M, Parwaresch R, Zimmermann M, et al.
Intensive ALL-type therapy without local radiotherapy provides a 90% event-free survival
for children with T-cell lymphoblastic lymphoma: a BFM group report. Blood 2000; 95:
416-421.
3.
Bonn BR, Rohde M, Zimmermann M, Krieger D, Oschlies I, Niggli F, et al. Incidence and
prognostic relevance of genetic variations in T-cell lymphoblastic lymphoma in childhood
and adolescence. Blood 2013; 121: 3153–3160.
4.
Chang YS, Yeh KT, Hsu NC, Lin SH, Chang TJ, Chang JG. Detection of N-, H-, and KRAS
codons 12, 13, and 61 mutations with universal RAS primer multiplex PCR and N-, H-, and
KRAS-specific primer extension. Clin Biochem 2010; 43: 296-301.
5.
Li VS, Wong CW, Chan TL, Chan AS, Zhao W, Chu KM, et al. Mutations of PIK3CA in
gastric adenocarcinoma. BMC cancer 2005; 5: 29.
6.
Samuels Y, Wang Z, Bardelli A, Silliman N, Ptak J, Szabo S, et al. High frequency of
mutations of the PIK3CA gene in human cancers. Science 2004; 304: 554.
7.
Van Vlierberghe P, Palomero T, Khiabanian H, Van der Meulen J, Castillo M, Van Roy N,
et al. PHF6 mutations in T-cell acute lymphoblastic leukemia. Nat Genet 2010; 42: 338-342.
8.
Silva A, Yunes JA, Cardoso BA, Martins LR, Jotta PY, Abecasis M, et al. PTEN
posttranslational inactivation and hyperactivation of the PI3K/Akt pathway sustain primary
T cell leukemia viability. J Clin Invest 2008; 118: 3762-3774.
9.
Trinquand A, Tanguy-Schmidt A, Ben Abdelali R, Lambert J, Beldjord K, Lengliné E, et al.
Toward a NOTCH1/FBXW7/RAS/PTEN-based oncogenetic risk classification of adult Tcell acute lymphoblastic leukemia: a Group for Research in Adult Acute Lymphoblastic
Leukemia study. J Clin Oncol 2013; 31: 4333–4342.