The
new england journal
of
medicine
n engl j med 371;11 nejm.org September 11, 2014
1005
The authors’ full names, academic de‑
grees, and affiliations are listed in the Ap‑
pendix. Address reprint requests to Dr.
Mullighan at St. Jude Children’s Research
Hospital, 262 Danny Thomas Pl., Mail
Stop 342, Memphis, TN 38105; or at
charles . mullighan@ stjude . org.
Drs. Roberts and Li and Drs. Downing,
Hunger, Willman, Zhang, and Mullighan
contributed equally to this article.
N Engl J Med 2014;371:1005-15.
DOI: 10.1056/NEJMoa1403088
Copyright © 2014 Massachusetts Medical Society.
BACKGROUND
Philadelphia chromosome–like acute lymphoblastic leukemia (Ph-like ALL) is char-
acterized by a gene-expression profile similar to that of BCR–ABL1–positive ALL,
alterations of lymphoid transcription factor genes, and a poor outcome. The fre-
quency and spectrum of genetic alterations in Ph-like ALL and its responsiveness
to tyrosine kinase inhibition are undefined, especially in adolescents and adults.
METHODS
We performed genomic profiling of 1725 patients with precursor B-cell ALL and
detailed genomic analysis of 154 patients with Ph-like ALL. We examined the
functional effects of fusion proteins and the efficacy of tyrosine kinase inhibitors
in mouse pre-B cells and xenografts of human Ph-like ALL.
RESULTS
Ph-like ALL increased in frequency from 10% among children with standard-risk
ALL to 27% among young adults with ALL and was associated with a poor outcome.
Kinase-activating alterations were identified in 91% of patients with Ph-like ALL;
rearrangements involving ABL1, ABL2, CRLF2, CSF1R, EPOR, JAK2, NTRK3, PDGFRB,
PTK2B, TSLP, or TYK2 and sequence mutations involving FLT3, IL7R, or SH2B3 were
most common. Expression of ABL1, ABL2, CSF1R, JAK2, and PDGFRB fusions re-
sulted in cytokine-independent proliferation and activation of phosphorylated STAT5.
Cell lines and human leukemic cells expressing ABL1, ABL2, CSF1R, and PDGFRB
fusions were sensitive in vitro to dasatinib, EPOR and JAK2 rearrangements were
sensitive to ruxolitinib, and the ETV6–NTRK3 fusion was sensitive to crizotinib.
CONCLUSIONS
Ph-like ALL was found to be characterized by a range of genomic alterations that
activate a limited number of signaling pathways, all of which may be amenable to
inhibition with approved tyrosine kinase inhibitors. Trials identifying Ph-like ALL
are needed to assess whether adding tyrosine kinase inhibitors to current therapy
will improve the survival of patients with this type of leukemia. (Funded by the
American Lebanese Syrian Associated Charities and others.)
ABSTRACT
Targetable Kinase-Activating Lesions
in Ph-like Acute Lymphoblastic Leukemia
K.G. Roberts, Y. Li, D. Payne‑Turner, R.C. Harvey, Y.‑L. Yang, D. Pei, K. McCastlain,
L. Ding, C. Lu, G. Song, J. Ma, J. Becksfort, M. Rusch, S.‑C. Chen, J. Easton, J. Cheng,
K. Boggs, N. Santiago‑Morales, I. Iacobucci, R.S. Fulton, J. Wen, M. Valentine, C. Cheng,
S.W. Paugh, M. Devidas, I‑M. Chen, S. Reshmi, A. Smith, E. Hedlund, P. Gupta,
P. Nagahawatte, G. Wu, X. Chen, D. Yergeau, B. Vadodaria, H. Mulder, N.J. Winick,
E.C. Larsen, W.L. Carroll, N.A. Heerema, A.J. Carroll, G. Grayson, S.K. Tasian,
A.S. Moore, F. Keller, M. Frei‑Jones, J.A. Whitlock, E.A. Raetz, D.L. White, T.P. Hughes,
J.M. Guidry Auvil, M.A. Smith, G. Marcucci, C.D. Bloomfield, K. Mrózek,
J. Kohlschmidt, W. Stock, S.M. Kornblau, M. Konopleva, E. Paietta, C.‑H. Pui, S. Jeha,
M.V. Relling, W.E. Evans, D.S. Gerhard, J.M. Gastier‑Foster, E. Mardis, R.K. Wilson,
M.L. Loh, J.R. Downing, S.P. Hunger, C.L. Willman, J. Zhang, and C.G. Mullighan
Original Article
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n engl j med 371;11 nejm.org September 11, 2014
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The
new england journal
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A
cute lymphoblastic leukemia (ALL)
is the most common childhood cancer
and a major cause of illness and death in
adults.
1
ALL encompasses a number of distinct
entities characterized by chromosomal rear-
rangements, structural variations, and sequence
mutations that perturb lymphoid maturation,
cell proliferation, cell-growth suppression, and
epigenetic regulation.
2
Our understanding of the
genetic basis of ALL has been transformed by
genomewide profiling studies that have identi-
fied multiple targets of recurring genetic altera-
tions and have defined new subtypes of ALL.
Childhood ALL is more commonly of B-cell
than T-cell lineage and includes cases associated
with hyperdiploidy, hypodiploidy, and chromo-
somal rearrangements resulting in chimeric fu-
sion genes, including ETV6–RUNX1, TCF3–PBX1,
and BCR–ABL1, as well as rearrangements of MLL
and CRLF2. As compared with younger children
with ALL, adolescents and adults with ALL have
inferior outcomes, partly because of the lower
frequency of favorable genetic features such as
ETV6–RUNX1 and hyperdiploidy, as well as the
higher frequency of BCR–ABL1
3
; however, the
genetic basis of ALL in adolescents and adults is
poorly defined.
One subtype of precursor B-cell ALL is BCR–
ABL1–like, or Philadelphia chromosome–like
(Ph-like), ALL. Patients with Ph-like ALL do not
have the BCR–ABL1 fusion protein expressed
from the t(9;22)(q34;q11.2) Philadelphia chro-
mosome yet have a gene-expression profile
similar to that of patients with BCR–ABL1
ALL.
4,5
Deletions or mutations of the lymphoid
transcription factor gene IKZF1 (encoding Ikaros)
are a hallmark of both BCR–ABL1–positive ALL
and Ph-like ALL,
4,6
and Ph-like ALL in children is
associated with poor outcomes.
4,5,7-10
Transcrip-
tome sequencing and whole-genome sequencing
in 15 children with Ph-like ALL identified chro-
mosomal rearrangements or sequence muta-
tions deregulating cytokine receptor and tyro-
sine kinase genes in all 15.
11
In addition, there
have been recent reports of patients with refrac-
tory Ph-like ALL and the EBF1–PDGFRB fusion
who have a remarkably good response to therapy
with tyrosine kinase inhibitors.
12,13
Because the
full spectrum of kinase-activating genetic altera-
tions in Ph-like ALL, their effect on outcomes in
adolescents and young adults, and their poten-
tial for therapeutic targeting are unknown, we
performed a detailed genomic analysis of 1725
children, adolescents, and young adults with pre-
cursor B-cell ALL.
Methods
Study Design
We studied 2013 patients with precursor B-cell
ALL, 1725 of whom had material available for
microarray gene-expression profiling; 1589 of
these 1725 patients had single-nucleotide-poly-
morphism microarray profiling performed. The
cohort included 330 children with National Can-
cer Institute–classified, standard-risk precursor
B-cell ALL (age range, 1 to 9 years; and periph-
eral-blood leukocyte count at diagnosis, <50,000
per cubic millimeter), 853 children with high-risk
precursor B-cell ALL (age range, 10 to 15 years;
leukocyte count, ≥50,000 per cubic millimeter;
or both), 374 adolescents (age range, 16 to 20
years), and 168 young adults (age range, 21 to
39 years) (Table S1 in Supplementary Appendix
1 and Fig. S1 in Supplementary Appendix 2, avail-
able with the full text of this article at NEJM.org).
There were few significant differences in the clini-
cal features of patients with gene-expression
profiling data available and those without such
data available (Table S2 in Supplementary Ap-
pendix 2). Samples were obtained from patients
enrolled under clinical-trial protocols of St. Jude
Children’s Research Hospital, the Children’s On-
cology Group, the Eastern Cooperative Oncology
Group, the Alliance for Clinical Trials in Oncol-
ogy (Cancer and Leukemia Group B), and M.D.
Anderson Cancer Center. The details of the treat-
ment protocols are provided in Supplementary
Appendix 2. Patients, parents, or guardians gave
written informed consent for sample collection
and research, with assent provided by older chil-
dren and adolescents. The study was approved by
the St. Jude Institutional Review Board. Data
from the study have been deposited in the Euro-
pean Genome Phenome archive under accession
number EGAS00001000654.
Next-Generation Sequencing
A total of 154 patients with Ph-like ALL under-
went detailed genomic analysis, 147 of whom
underwent one or more of the following types of
next-generation sequencing: transcriptome se-
quencing (136 patients), whole-genome sequenc-
ing (42), and whole-exome sequencing (12) of
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1007
Ph-like Acute Lymphoblastic Leukemia
tumor and matched remission DNA (Table S1 in
Supplementary Appendix 1).
14
Next-generation
sequencing was not performed for 7 patients, who
instead underwent reverse-transcriptase poly-
merase-chain-reaction analysis. Transcriptome
sequencing was also performed for 160 patients
with non–Ph-like ALL (Table S3 in Supplemen-
tary Appendix 2). Details of Ph-like ALL classifi-
cation, sequencing, and analysis are provided in
Supplementary Appendix 2.
Microarray Profiling and Functional
and Cytogenetic Assays
The details of gene expression and single-nucle-
otide-polymorphism microarray profiling, fluo-
rescence in situ hybridization, cell-line prolifera-
tion and tyrosine kinase inhibitor assays, protein
expression, and xenograft experiments are pro-
vided in Supplementary Appendix 2.
Statistical Analysis
Associations between categorical variables were
examined with the use of Fisher’s exact test. As-
sociations between Ph-like ALL status and treat-
ment outcome (event-free survival and overall
survival) were examined with the use of the
Kaplan–Meier estimator, with Peto’s estimator
of standard deviation and the log-rank test, in
each patient cohort (children, adolescents, and
young adults).
4,15,16
An event was defined as a fail-
ure to achieve remission, a relapse after remis-
sion, or the development of a second malignant
neoplasm. A multivariable analysis of event-free
and overall survival was performed with the Cox
proportional-hazards regression model.
17
Analy-
ses were performed with the use of Prism soft-
ware, version 6.0 (GraphPad Software), R software
(www.r-project.org),
18
and SAS software, version
9.1.2 (SAS Institute).
Results
Clinical Characteristics and Outcomes
Overall, 264 of 1725 precursor B-cell ALL cases
(15.3%) were identified as Ph-like ALL (Table S4
in Supplementary Appendix 2). The prevalence of
Ph-like ALL significantly increased with age, from
10% among children with standard-risk ALL and
13% among those with high-risk ALL to 21%
among adolescents with ALL and 27% among
young adults with ALL (P<0.001 for the compari-
sons of children with adolescents and children
with young adults). Furthermore, patients with
Ph-like ALL had higher leukocyte counts at pre-
sentation than did patients with non–Ph-like ALL,
both overall (106,000 vs. 59,000 per cubic milli-
meter, P<0.001) and in the different age cohorts
(Table S5 in Supplementary Appendix 2). Ph-like
ALL was more common among males than among
females (Table S5 in Supplementary Appendix 2)
and was associated with elevated levels of minimal
residual disease at the end of induction therapy in
the Children’s Oncology Group trials (Table S6
in Supplementary Appendix 2). Among patients
with Ph-like ALL, the median (±SD) 5-year event-
free survival rates for children with high-risk ALL,
adolescents, and young adults was 58.2±5.3%,
41.0±7.4%, and 24.1±10.5%, respectively, and the
5-year overall survival rates were 72.8±4.8%,
65.8±7.1%, and 25.8±9.9% (Fig. 1). Across all age
groups, these survival rates were inferior to those
among patients with non–Ph-like ALL (P<0.001
for both comparisons) (Fig. S2 in Supplementary
Appendix 2). The presence of Ph-like ALL was an
independent prognostic factor in all age groups
(Table S7 in Supplementary Appendix 2).
Identification of Kinase Alterations
in Ph-like ALL
A total of 123 of 264 patients with Ph-like ALL had
high CRLF2 expression, with the frequency rang-
ing from 24% among children with standard-risk
ALL to 60% among adolescents with ALL. Among
patients with high CRLF2 expression, we identi-
fied P2RY8–CRLF2 in 45 patients and IGH–CRLF2 in
61 patients; for 17 patients, there was insuffi-
cient material for analysis. Sixty-eight patients
(55%) with CRLF2 rearrangement had concomi-
tant Janus kinase mutations, most commonly in
JAK2 (Fig. S3 in Supplementary Appendix 2).
To identify the spectrum of kinase-activating
alterations in the remaining patients with Ph-like
ALL, we performed next-generation sequencing in
30 patients with CRFL2 rearrangement and 124
patients without CRLF2 rearrangement (Tables S8
and S9 in Supplementary Appendix 1). The analy-
ses of gene-expression levels from transcriptome
sequencing recapitulated those from microarray
expression data, with clustering of Ph-like and
BCR–ABL1–positive cases (Table S10 in Supple-
mentary Appendix 1 and Fig. S4 and Fig. S5 in
Supplementary Appendix 2).
Genomic alterations activating kinase signal-
ing were identified in 91% of patients with Ph-
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like ALL and were divided into distinct subgroups
of kinase and cytokine receptor genes (Fig. 2,
and Fig. S6 in Supplementary Appendix 2). These
included fusions predicted to respond to ABL1
inhibitors (involving ABL1, ABL2, CSF1R, or PDGFRB)
(12.6% of cases); rearrangements of EPOR (3.9%)
or JAK2 (7.4%); rearrangements of CRLF2 (49.7%);
genetic alterations of IL7R, FLT3, SH2B3, JAK1,
JAK3, TYK2, and IL2RB (shown under “Other
JAK–STAT” in Fig. 2; 12.6%); Ras pathway muta-
tions, several of which were associated with hy-
podiploidy (4.3%); and uncommon fusions (e.g.,
involving NTRK3 or DGKH; 0.9%). A minority of
patients (4.8%) were not found to have a kinase-
activating alteration on transcriptome sequenc-
ing analysis, and suitable material for analysis
was not available for 3.9% of patients. The fre-
quencies of these subgroups varied with age.
Notably, ABL-class rearrangements were more
common among children, and JAK2 rearrange-
ments were more frequent among young adults
(Fig. S7 in Supplementary Appendix 2).
In the transcriptome sequencing analysis, we
identified 223 gene rearrangements in 116 of
136 patients (mean, 1.6 per patient; range, 0 to
12) (Table S11 in Supplementary Appendix 1);
115 of these were either chimeric in-frame fu-
sions or rearrangements resulting in deregulated
gene expression. Across the entire cohort, rear-
rangements activating kinase signaling were
identified in 96 of 154 patients (62%), including
35 different rearrangements (16 of which were
recurrent) in 13 kinase, cytokine, or cytokine-
receptor genes: JAK2 (10 fusion partners), ABL1
(6), PDGFRB (4), ABL2 (3), CRLF2 (2), EPOR (2),
PTK2B (2), CSF1R (1), DGKH (1), IL2RB (1), NTRK3
(1), TSLP (1), and TYK2 (1) (Table 1 and Fig. 2,
and Table S12 in Supplementary Appendix 1 and
Table S13 in Supplementary Appendix 2). All
kinase fusions retained an intact tyrosine kinase
domain (Fig. S8 in Supplementary Appendix 2)
and were found by means of fluorescence in situ
hybridization to be present in the predominant
clone at diagnosis (Fig. S9 in Supplementary Ap-
pendix 2).
Thirty patients with CRLF2 rearrangement were
studied with the use of transcriptome sequencing
and whole-genome sequencing (Fig. 2, and Fig.
S10 in Supplementary Appendix 2). We found ad-
ditional alterations activating JAK–STAT in 5 of
11 patients who had CRLF2 rearrangement but no
JAK mutations; these alterations included IL7R
mutations (4 patients), an FLT3 mutation (1 pa-
tient), and a deletion of SH2B3, which encodes
the JAK2 negative regulator LNK (1 patient). One
patient had an IQGAP2–TSLP fusion; this resulted
in overexpression of TSLP, which encodes thymic
stromal lymphopoietin, the ligand for CRLF2.
Figure 1. Kaplan–Meier Estimates of Event-free and Overall Survival
among Patients with Philadelphia Chromosome–like Acute Lymphoblastic
Leukemia (Ph-like ALL).
Panel A shows the median (±SD) rates of event‑free survival for young
adults, adolescents, and high‑risk children with Ph‑like ALL (24.1±10.5%,
41.0±7.4%, and 58.2±5.3%, respectively). Panel B shows rates of overall
survival in the three age groups (25.8±9.9%, 65.8±7.1%, and 72.8±4.8%,
respectively).
Survival Rate (%)
100
80
90
70
60
40
30
10
50
20
0
0 2
4
6
8
10
Years
B Overall Survival
A Event-free Survival
P<0.001
Children, high risk
Adolescents
Young adults
No. at Risk
Children, high
risk
Adolescents
Young adults
105
76
41
93
63
18
71
42
10
49
17
3
27
6
9
3
Survival Rate (%)
100
80
90
70
60
40
30
10
50
20
0
0 2
4
6
8
10
Years
P<0.001
Children, high risk
Adolescents
Young adults
No. at Risk
Children, high
risk
Adolescents
Young adults
105
76
41
101
63
20
85
53
12
61
28
4
37
11
13
4
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n engl j med 371;11 nejm.org September 11, 2014
1009
Ph-like Acute Lymphoblastic Leukemia
Figure 2. Recurring Kinase Alterations in Ph-like ALL.
Data are shown for 154 patients with Ph‑like ALL who underwent detailed genomic analysis, including transcriptome sequencing (RNA‑seq), whole‑genome sequencing (WGS),
whole‑exome sequencing (WES), and reverse‑transcriptase polymerase chain reaction (RT‑PCR). The cohort is divided into patients with ABL‑class fusions (ABL1, ABL2, CSF1R,
PDGFRB) responsive to dasatinib, EPOR or JAK2 rearrangements, CRLF2 rearrangements, other JAK–STAT–activating mutations (IL7R, FLT3, SH2B3, JAK1, JAK3, TYK2, IL2RB, and
TSLP), other kinase fusions (miscellaneous group, including NTRK3 and DGKH), alterations in the Ras pathway (KRAS, NRAS, PTPN11, NF1, and BRAF), and no kinase alteration.
For details of specific alterations, see Tables S9 and S12 in Supplementary Appendix 1 and Table S20 in Supplementary Appendix 3.
No kinase
alteration
ABL-class fusions EPOR or JAK2 rearranged CRLF2 rearranged Other JAK–STAT
Misc
Ras only
ABLJAK–STATOtherRas
RNA-seq
WGS
WES
RT-PCR
Age
Sex
Outcome
ABL1
ABL2
CSF1R
PDGFRB
EPOR
JAK2
CRLF2
IL7R
FLT3
SH2B3
JAK1
JAK3
TYK2
TSLP
IL2RB
NTRK3
DGKH
PTK2B
DYRK1A
KRAS
NRAS
PTPN11
NF1
BRAF
Kinase
IKZF1
PAX5
EBF1
B-cell pathway
Sample
SJBALL266_D
SJBALL021728_D1
SJBALL022244_D1
SJBALL021352_D1
SJBALL021494_D1
SJBALL020877_D1
SJBALL085_D
SJBALL020882_D1
SJBALL020012_D1
SJBALL020014_D1
SJBALL020825_D1
SJBALL010_D
SJBALL021521_D1
SJBALL269_D
SJBALL020994_D1
SJBALL020091_D1
SJBALL262_D
SJBALL022489_D1
SJBALL022245_D1
SJBALL020625_D1
SJBALL020980_D1
SJBALL020644_D1
SJBALL020812_D1
SJBALL204_D
SJBALL020832_D1
SJBALL153_D
SJBALL012_D
SJBALL021318_D1
SJBALL020475_D1
SJBALL020552_D1
SJBALL020529_D1
SJBALL020704_D1
SJBALL267_D
SJBALL022486_D1
SJBALL021748_D1
SJBALL020100_D1
SJBALL020649_D1
SJBALL020579_D1
SJBALL020824_D1
SJBALL020941_D1
SJBALL021409_D1
SJBALL020084_D1
SJBALL0214
1
1_D1
SJBALL020069_D1
SJBALL020138_D1
SJBALL020936_D1
SJBALL265_D
SJBALL021329_D1
SJBALL022246_D1
SJBALL020966_D1
SJBALL022488_D1
SJBALL021856_D1
SJBALL020081_D1
SJBALL020765_D1
SJBALL021780_D1
SJBALL021367_D1
SJBALL021866_D1
SJBALL020589_D1
SJBALL020681_D1
SJBALL021344
SJBALL020939_D1
SJBALL021814_D1
SJBALL021087_D1
SJBALL195_D
SJBALL020789_D1
SJBALL02
1
100_D1
SJBALL02
11
13_D1
SJBALL02
11
18_D1
SJBALL020077_D1
SJBALL270_D
SJBALL020145_D1
SJBALL020963_D1
SJBALL021510_D1
SJBALL021313_D1
SJBALL02
1
120_D1
SJBALL083_D
SJBALL191_D
SJBALL021083_D1
SJHYPER013_D
SJBALL020013_D1
SJBALL020087_D1
SJBALL02
1
143_D1
SJBALL021053_D1
SJBALL020853_D1
SJDOWN013_D
SJHYPO
1
10_D
SJBALL101_D
SJBALL020971_D1
SJBALL021076_D1
SJBALL020125_D1
SJBALL021813_D1
SJBALL020073_D1
SJBALL239_D
SJBALL020984_D1
SJBALL021373_D1
SJBALL020807_D1
SJBALL021415_D1
SJBALL231_D
SJHYPO109_D
SJBALL021486_D1
SJBALL063_D
SJBALL020836_D1
SJBALL015_D
SJBALL021507_D1
SJBALL021772_D1
SJBALL021413_D1
SJBALL263_D
SJBALL021058_D1
SJBALL021786_D1
SJBALL021305_D1
SJBALL021393_D1
SJHYPER120_D
SJBALL021784_D1
SJHYPER150_D
SJBALL021517_D1
SJBALL020015_D1
SJHYPO018_D1
SJBALL021720_D1
SJBALL021327_D1
SJBALL021738_D1
SJBALL020700_D1
SJBALL021794_D1
SJBALL020422_D1
SJBALL021018_D1
SJHYPER003_D
SJBALL247_D
SJBALL021398_D1
SJBALL021530_D1
SJERG021891_D1
SJHYPO147_D
SJBALL264_D
SJBALL268_D
SJHYPER146_D
SJBALL02
1
109_D1
SJHYPO020_D
SJBALL02
1
102_D1
SJHYPO123_D
SJHYPER025_D
SJHYPO146_D
SJBALL0208
1
1_D1
SJBALL020488_D1
SJBALL255_D
SJBALL021080_D1
SJBALL0
1
1_D
SJBALL021047_D1
SJBALL021644_D1
SJBALL020518_D1
SJHYPO151_D
SJBALL021722_D1
SJBALL020852_D1
SJBALL021549_D1
SJHYPER021_D
SJHYPER206_D
SJHYPER227_D
SJHYPER227_D
Lesion
Fusion Protein insertion/deletion
Deletion
Age Group
Children, standard risk
Children, high risk
Outcome
No event
Event
Sex
Female
Male
Unknown
Truncating
Young adult
Adolescent
Legend
Missense
Multiple mutations
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