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Leukemia (2018) 32, 931–940

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ORIGINAL ARTICLE
RAS pathway mutations as a predictive biomarker for
treatment adaptation in pediatric B-cell precursor acute
lymphoblastic leukemia
IS Jerchel1, AQ Hoogkamer1, IM Ariës1, EMP Steeghs1, JM Boer1, NJM Besselink2,3, A Boeree1, C van de Ven1,
HA de Groot-Kruseman4, V de Haas4,5, MA Horstmann6,7, G Escherich6,7, CM Zwaan1, E Cuppen2,3, MJ Koudijs2,3, R Pieters4,5
and ML den Boer1,4

RAS pathway mutations have been linked to relapse and chemotherapy resistance in pediatric B-cell precursor acute lymphoblastic
leukemia (BCP-ALL). However, comprehensive data on the frequency and prognostic value of subclonal mutations in well-defined
subgroups using highly sensitive and quantitative methods are lacking. Targeted deep sequencing of 13 RAS pathway genes was
performed in 461 pediatric BCP-ALL cases at initial diagnosis and in 19 diagnosis-relapse pairs. Mutations were present in 44.2% of
patients, with 24.1% carrying a clonal mutation. Mutation frequencies were highest in high hyperdiploid, infant t(4;11)-rearranged,
BCR-ABL1-like and B-other cases (50–70%), whereas mutations were less frequent in ETV6-RUNX1-rearranged, and rare in TCF3-PBX1-
and BCR-ABL1-rearranged cases (27–4%). RAS pathway-mutated cells were more resistant to prednisolone and vincristine ex vivo.
Clonal, but not subclonal, mutations were linked to unfavorable outcome in standard- and high-risk-treated patients. At relapse,
most RAS pathway mutations were clonal (9 of 10). RAS mutant cells were sensitive to the MEK inhibitor trametinib ex vivo, and
trametinib sensitized resistant cells to prednisolone. We conclude that RAS pathway mutations are frequent, and that clonal, but
not subclonal, mutations are associated with unfavorable risk parameters in newly diagnosed pediatric BCP-ALL. These mutations
may designate patients eligible for MEK inhibitor treatment.

Leukemia (2018) 32, 931–940; doi:10.1038/leu.2017.303

INTRODUCTION subclonal mutations), and did not compare mutation frequencies

The prognosis of children with B-cell precursor acute lympho- between cytogenetic subtypes.22–26 Mutations were reported in
blastic leukemia (BCP-ALL) has improved considerably over the the context of recent genomic studies but not studied for their
past decades, and nowadays about 80% of the patients are cured.1 prognostic impact in isolation. Therefore, the clinical significance
However, especially medium- and high-risk cases still show of RAS mutations is still debated, and it is unknown whether the
dissatisfying rates of relapse despite intense chemotherapy.2–4 recent inclusion of minimal residual disease (MRD) levels as risk
Relapsed ALL is treated with the same drugs as the initial criterion influences the prognostic effect of RAS pathway
leukemia, but cells of relapsed cases are more resistant.5 New, mutations in contemporary protocols. Recent studies found that
targeted approaches are therefore warranted. mutations in NRAS, KRAS, FLT3 and PTPN11 are more frequently
Activating mutations in KRAS, NRAS and HRAS are among the observed at relapse (34–38%), and in part confer a poor
most frequent mutations in cancer.6–9 The RAS GTPases are prognosis.26–29 We previously observed in a small cohort of 26
convert extracellular growth signals into a complex intracellular patients that RAS pathway mutations are more frequent in ex vivo
response. Upon activation of a growth factor receptor, RAS- prednisolone-resistant cases, which are sensitized to prednisolone
guanidine exchange factors (e.g. SHP2 encoded by PTPN11) by RAS pathway inhibition.30 Backtracking has shown that RAS-
activate RAS by enabling binding of GTP, which activates the mutant relapse-forming clones may exist as small subclones at
RAF and PI3 kinases and RALGDS. The RAF–MEK–ERK kinase axis is initial diagnosis.27,31,32 However, these retrospective analyses rely
crucial for mediating the oncogenic effects of RAS, demonstrated on selected cases, and the predictive value of (sub)clonal RAS
by the efficacy of MEK inhibitors in RAS-mutated cancers.10–12 pathway mutations at initial diagnosis of BCP-ALL treated in
Clinical trials are ongoing, and benefits were reported for RAF- contemporary protocols is unknown.
mutated melanomas, ovarian cancer and thyroid cancer.13–18 Here we report deep next-generation sequencing of 13 RAS
RAS mutations have been initially reported in about 15% of pathway genes together with a risk-stratified survival analysis in a
pediatric BCP-ALL.19–21 However, these studies were often clinically and biologically well-characterized cohort of 461 initial
restricted to N- and KRAS, certain subtypes or high-risk groups, diagnosis patients with BCP-ALL, treated according to a con-
or were technically limited in sensitivity (therefore overlooking temporary, MRD-based ALL treatment protocol (DCOG ALL10). In

1
Department of Pediatric Oncology, Erasmus MC – Sophia Children’s Hospital, Rotterdam, The Netherlands; 2Center for Personalized Cancer Treatment (CPCT), University Medical
Center Utrecht, Utrecht, The Netherlands; 3Center for Molecular Medicine and Cancer Genomics Netherlands, Division Biomedical Genetics, University Medical Center Utrecht,
Utrecht, The Netherlands; 4DCOG, The Hague, The Netherlands; 5Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands; 6Clinic of Pediatric Hematology and
Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany and 7On behalf of the COALL. Correspondence: Professor ML den Boer, Department of Pediatric
Oncology, Erasmus MC–Sophia Children’s Hospital, Na-1603, PO Box 2060, Rotterdam 3000 CB, The Netherlands.
E-mail: [email protected]
Received 5 December 2016; accepted 31 August 2017; accepted 20 September 2017; accepted article preview online 3 October 2017; advance online publication, 24 October 2017
 RAS pathway mutations in pediatric BCP-ALL
IS Jerchel et al
932
addition, we report links between RAS mutation status, clonality, could be identified by both platforms, indicating a low false-positive rate.
ex vivo cellular drug resistance and ex vivo response to MEK Only Exon 3 of NRAS was sequenced using Sanger sequencing in 248
inhibition. ALL10 samples since primer design for this exon in a multiplex amplicon
setting failed. Chromatograms were visually inspected for the presence of
mutations in codons 59 to 63 as well as analyzed by the R package
MATERIALS AND METHODS sangerseqR in combination with the tools above to call and annotate
detected variants.
A detailed description of all methods can be found in the online
Supplementary Data.
Clinical characteristics and statistics
Patient material 2 and patient-derived xenografts Clinical characteristics were compared using Fisher’s exact test in R
(version 3.2.1). We analyzed cases from the Dutch Childhood Oncology
This study comprised children with newly diagnosed BCP-ALL with an age
Group (DCOG) and Cooperative Study Group for Children with ALL (COALL)
range of 0–18 years. These studies were conducted in accordance with the (n = 432). We restricted the RAS pathway-mutated group to those carrying
Declaration of Helsinki; written informed consent was obtained from a verified MAPK pathway activating mutation (codons 12, 13 and 146 of
parents or guardians and approval given by institutional review boards. NRAS or KRAS and FLT3 or PTPN11). Event-free survival and cumulative
Mononuclear cells were isolated using density gradient centrifugation with incidence of relapse and non-response after induction therapy were
Lymphoprep (Axis Shield, Oslo, Norway) as described previously.33 Animal evaluated in 244 eligible cases treated within one protocol (DCOG ALL10),
experiments were approved by the animal ethics committee (EMC 2863 stratified for risk group. The COALL97/03 cohort consisted of patients
(103-12-08)). In some cases, xenografts of primary patient material were treated in the consecutive COALL 06–97 and COALL 07-03 protocols
established in three 7–12-week-old female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (n = 131). For details see Escherich et al.45 Treatment intensity in arms LR-R
(NSG) mice per patient (Charles River, Leiden, Netherlands). Leukemic cells and LR-S is comparable to DCOG ALL10 standard-risk treatment; LR-I, HR-R
were isolated and subsequently used for sequencing, western blot and and HR-S treatment is comparable to DCOG ALL10 medium-risk treatment.
trametinib (GSK1120212) cytotoxicity assays. For all samples leukemic blast In the COALL97/03 cohort, no patient was treated with an intensity
percentage was at least 90%, and in relapse samples blast percentage was comparable to that of the DCOG ALL10 high-risk arm. Cumulative
at least 80%. Subtypes were determined by karyotype, fluorescence in situ incidence of relapse and non-response was estimated using a competing
hybridization and/or fusion-gene specific PCR. BCR-ABL1-like cases were risks model and compared using Gray's test. Event-free survival
identified using microarray gene expression profiling by means of a 110 probabilities were estimated using the actuarial Kaplan–Meier method
probe set classifier.34,35 The composition of analyzed groups is described in and compared using the log-rank test. Hazard ratios were calculated in
a flow diagram (Supplementary Figure S1). SPSS v21 using Cox’s proportional hazard model. Outcome analyses were
performed in R 3.2.1, using the packages cmprsk46 version 2.2-7, mstate
Ex vivo cytotoxicity assays version 0.2.747 and survival version 2.38-3.48
Sensitivity towards chemotherapeutics was evaluated as previously
described.36 In brief, freshly isolated primary ALL cells were incubated
with a concentration range of prednisolone, vincristine, daunorubicin, RESULTS
L-asparaginase, 6-mercaptopurine and 6-thioguanine. After 4 days, cell Frequency and clonality of RAS pathway mutations in newly
viability was evaluated by adding MTT and measuring formazan diagnosed pediatric BCP-ALL
conversion with optical density measurement. LC50 values were calculated Targeted amplicon deep sequencing was used to identify RAS
(concentration at which 50% conversion activity was measured relative to
pathway mutations in samples from 461 children with BCP-ALL at
no-drug control cells) and compared between groups using Mann–
Whitney U-tests. Ex vivo sensitivity towards trametinib was measured initial diagnosis. Mutational hotspots in 13 key members of the
similarly (5 μM–0.6 nM). RAS pathway were analyzed with 49 amplicons. The median read
depth per amplicon was 1085 reads per sample (IQR: 527–2647)
(Supplementary Table S1 and Supplementary Figure S2).
Sequencing and code availability Most recurrent lesions were activating mutations in NRAS
DNA and RNA were isolated using Trizol reagent (Life Technologies, and KRAS (79%), followed by mutations in PTPN11 and FLT3
Bleiswijk, Netherlands), or using DNeasy (Qiagen, Hilden, Germany) in two
(9.3% and 10.1%, respectively; Figure 1a and Supplementary
cases (used only for trametinib sensitivity assay) and the cell line 697. For
TruSeq Custom Amplicon sequencing (Illumina, San Diego, CA, USA),
Figure S3). Mutations in BRAF, NF1, CBL and other genes occurred
sequencing libraries were prepared from 100 to 250 ng genomic DNA. sporadically. Overall, variants in RAS pathway genes were
Successful library preparation was confirmed using the Labchip GX observed in 44.2% of pediatric BCP-ALL cases. Clonal mutations
genomic analyzer (Caliper Life Sciences Benelux N.V., the Netherlands). (VAF ⩾ 25%) were found in 24.1% of all patients (median: 40.5%
Samples were then pooled equimolarly and sequenced on an Illumina VAF; Supplementary Figure S4) and subclonal mutations
MiSeq in paired-end reads of 250 bp each. Forty-nine amplicons of 425 bp (VAFo 25%) were exclusively found in 20.1% of patients (median:
covered mutational hotspot regions in 13 RAS pathway genes 4.3% VAF).
(Supplementary Table S1). The analysis script will be provided upon Most cases with RAS pathway mutations were high hyperdi-
request. ploid or those with rare or negative for BCR-ABL1, ETV6-RUNX1,
Sequence reads were aligned to the 1000 genomes human reference
TCF3-PBX1, MLL-rearrangement and high hyperdiploidy (51–67
sequences (version b37, GATK resource bundle; Broad Institute, Cambridge,
MA, USA) using BWA v0.7.1037 and GATK indel realigner v.3.3-0. Single- chromosomes), including non-BCR-ABL1-like ‘B-other’ and BCR-
nucleotide variants were called with Freebayes v0.9.18–24,38 Varscan v.2.3. ABL1-like cases (Figure 1b and Supplementary Table S3). Mutation
7,39 Bcftools v1.040 and GATK v3.3-0.41 The resulting variant call format files frequencies were high in BCR-ABL1-like (49.4%), B-other (41.8%),
were annotated using snpEff and snpSift v.4.1a42 and dbNSFP v.2.7.43 For high hyperdiploid (72.6%) and t(4;11)-rearranged (MLL-AF4, 73.3%)
reliable detection of high-confidence mutations, variants were filtered cases, with clonal mutations observed in 31.6%, 25.4%, 41.9% and
based on several criteria: For each sample, variants were excluded if they 20%, respectively (Figure 1b). Comparison of each subtype to the
were reported by only one caller, coverage was o100 reads, or o20 remaining BCP-ALL cases showed significantly more mutations in
reads supported the variant allele. Overall, variants were excluded if variant cases with MLL-rearrangement and high hyperdiploid karyotype
allele frequency (VAF) never exceeded 2% or distribution was unequal (odds ratio 3.8 and 5.7, respectively), and significantly less
between runs. Furthermore, variants were only considered if they were
mutations in cases with ETV6-RUNX1-, TCF3- and BCR-ABL1-
reported in the COSMIC V73 GRCh37 database,44 non-synonymous,
unlikely to be germline variants and not known SNPs (see rearrangement (odds ratios o0.3; Supplementary Table S4).
Supplementary Methods for details). Read depth per amplicon is Interestingly, within the BCR-ABL1-like group RAS pathway
summarized in Supplementary Table S1. An estimate of cases missed mutations were mutually exclusive with ABL/JAK class tyrosine
due to insufficient coverage is given in Supplementary Table S2. In a kinase fusions, but not with high CRLF2 expression
comparison with 25 samples that were sequenced previously,30 all variants (Supplementary Figure S5). ETV6-RUNX1-rearranged cases showed

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 RAS pathway mutations in pediatric BCP-ALL
IS Jerchel et al
933

Figure 1. Frequency and distribution of RAS pathway mutations in pediatric BCP-ALL. (a) Overview of all clonal or subclonal mutations found
in pediatric BCP-ALL cases at initial diagnosis. Top bar represents the cytogenetic subtype. Black boxes represent clonal mutations (variant
allele frequency (VAF) ⩾ 25%) and gray boxes represent subclonal mutations (VAF o25%). (b) Frequency of clonal and subclonal RAS pathway
mutations overall and within the cytogenetic subtypes. BA, BCR-ABL1-rearranged; BAL, BCR-ABL1-like; BO, B-other; ER, ETV6-RUNX1-rearranged;
HD, high hyperdiploid; MLL, t(4;11)-rearranged; TCF3, TCF3-PBX1-rearranged. (c) Co-occurrence of RAS pathway mutations: bar heights indicate
the frequency of mutated cases carrying the number of RAS pathway mutations indicated on the x-axis. Segmentation of each bar indicates
the distribution of mutated genes.

Table 1. Incidence of clonal RAS pathway mutations among BCP-ALL patients with clinical risk factors

Risk parameter Incidence of clonal mutations among Statistics (clonal vs wild type)

Risk parameter: Yes Risk parameter: No Fisher’s Odds ratiob 95% CI

Pa

Age ⩾ 10 22/75 (29%) 90/357 (25%) 0.88

Male 55/227 (24%) 57/205 (24%) 0.56
High WBC (450/nl) 34/99 (34%) 78/331 (24%) 0.24
Down syndrome 2/16 (13%) 105/305 (27%) 0.10 0.3 0.03–1.38
CNS+ 0/1 (0%) 24/248 (10%) 1
PPR 7/17 (41%) 51/181 (21%) 0.15 2.22 0.65–7.41
MRD high d33 ALL10 16/48 (33%) 35/147 (18%) 0.01 2.99 1.27–7.05
MRD high d79 ALL10 2/8 (25%) 47/171 (20%) 1
Abbreviations: CNS+, non-traumatic puncture and 45 WBC/μl CSF with identifiable leukemic cells; patients with a traumatic lumbar puncture were not
included; d33, at the end of induction therapy (day 33); d79, at the end of consolidation therapy (day 79); PPR, prednisone poor responder, that is, ⩾ 1000
leukemic blasts/μl in peripheral blood on day 8 of induction therapy; MRD high, minimal residual disease ⩾ 10 −3; WBC, white blood cell count. aFisher’s exact
test P-values o0.05 are printed in bold font. bOdds ratios are only given if P-values in Fisher’s exact test were o0.2.

an intermediate frequency (26.6%, 10.5% clonal), and TCF3-PBX1- mutations also more frequently carried chromosome 21
or BCR-ABL1-rearranged cases had the lowest frequencies with 8% aberrations and additionally more often expressed high CRLF2
(0% clonal) and 4% (4% clonal) of cases being affected, and harbored less mutations in IKZF1 and PAX5 (Supplementary
respectively. Table S5).
Frequent secondary aberrations in clonal RAS pathway All except three NRAS mutations and three quarters of the KRAS
mutant cases were chromosome 21 aberrations, dic(9;20) mutations affected the codons 12 and 13 (Supplementary
chromosomes, 9p-deletion, PAX5 amplifications and CDKN2A/B Figure S6).
deletions. Opposed to that, significantly less BTG1 deletions Although clonal RAS pathway mutations were mutually
and ETV6 deletions were detected in cases with clonal RAS exclusive (Figure 1a), additional subclonal mutations were present
pathway mutations. Cases with subclonal RAS pathway in 41% of cases with a clonal mutation and in 49% of cases with a

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 RAS pathway mutations in pediatric BCP-ALL
IS Jerchel et al
934
Table 2. Clinical outcome of patients with clonal or subclonal RAS pathway mutations

Mutation status n EFS CIR

5-year EFS in % (s.e.) Cox’s hazard ratioa Log-rank 5-year CIR in % (s.e.) Gray’s test
Pcl Pcl

ALL10b
Wild type 135 92 (2) 8 (2)
Mutated 109 86 (3) 11 (3)

ALL10b
Wild type 135 92 (2) 8 (2)
Subclonal 59 88 (4) 11 (4)
Clonal 50 84 (5) 10 (4)
SR
Wild type 49 96 (3) 4 (3)
Subclonal 15 93 (6) 7 (7)
Clonal 10 69 (15) 4.57 (CI: 1.02-20.5) 0.027 21 (14)
MR
Wild type 75 88 (4) 12 (4)
Subclonal 42 85 (6) 12 (5)
Clonal 31 97 (3) 0.058 0 0.01
HR
Wild type 11 100 0
Subclonal 2 N/Ac N/Ac
Clonal 9 56 (16.6) 0.015 33 (17) 0.044
Abbreviations: CIR, cumulative incidence of relapse and non-response; EFS, event-free survival; HR, high risk; MR, medium risk; n/a, not applicable; Pcl, P-value
for comparison clonal vs wild-type cases; SR, standard risk. Only P-values o0.1 are shown. aOnly shown if significantly different, reference group: wild-
type cases. bAll tests stratified for risk arms of this protocol (SR, MR and HR). c5-year follow-up only reached by one patient. Values of Po 0.05 are printed in bold.

subclonal mutation (46.2% of all mutated patients; Figure 1c). patients harboring clonal mutations compared with wild-type
Most of these additional RAS pathway mutations were present at a cases, which resulted in a trend for better event-free survival.
VAF o10% (Supplementary Figure S4). Subclonal mutations were not predictive for outcome (Figure 2b,
blue lines).
Clinical characteristics and outcome Univariate analysis of RAS pathway status in these three risk
arms revealed that clonal but not subclonal mutations in RAS
Clinical characteristics were compared between BCP-ALL cases
pathway genes were predictive for an unfavorable outcome in the
with clonal RAS pathway mutations (n = 110, KRAS, NRAS, PTPN11
standard-risk-treated group (hazard ratio 4.6, P = 0.047), which
or FLT3) and wild-type cases (n = 235). Age, white blood cell count,
remained prognostic in a multivariate analysis including WBC and
gender, Down syndrome, CNS status at diagnosis and prednisone
age (hazard ratio 5.4, P = 0.032; Supplementary Table S9).
window response at day 8 of therapy did not differ significantly
Univariate analysis of RAS pathway mutations in the medium-
(Table 1). In DCOG ALL10 patients clonal mutations were enriched
risk and high-risk groups did not reveal statistical significant
among cases with high MRD levels (⩾10−3) after 4 weeks of
associations with event-free survival in this DCOG ALL10 study
induction treatment (33% in of MRD-high vs 18% in MRD-low
cohort.
cases, P = 0.01). This had an impact on mutation frequencies in the
The impact of RAS pathway mutations on clinical outcome was
actual treatment arms: 13.5% of cases in the standard-risk arm
also evaluated in the COALL97/03 cohort (Supplementary
carried a clonal RAS pathway mutation, compared with 21.9% in
Figure S7). These patients had been stratified into treatment arms
medium risk and 40.9% in the high-risk treatment arm
according to white blood cell count, age, immunophenotype and
(Supplementary Table S6, P = 0.02).
ex vivo drug response. Small group sizes are limiting this analysis
In contrast, subclonal mutations were not associated with poor
and differences were not statistically significant, but the trends
risk features. These mutations were significantly more common
support our results of the DCOG ALL10 study. In standard-risk-
among younger children (P = 0.04), those with low white blood
treated cases, those with clonal but not subclonal mutations more
cell counts (o 50 cells/nl, P = 0.02), and those in the medium-risk
often suffered from a relapse than did non-mutated cases,
treatment arm (P = 0.01; Supplementary Table S7).
The prognostic value of RAS pathway mutations was analyzed whereas this prognostic impact was absent in the medium-risk-
in 244 newly diagnosed BCP-ALL patients treated according to the treated group (Supplementary Figure S7).
DCOG ALL10 protocol (for baseline characteristics and cohort
composition see Supplementary Table S8 and Supplementary Ex vivo resistance to chemotherapeutic agents
Figure S1). In the total cohort, clonal and subclonal RAS pathway Ex vivo cytotoxicity data of prednisolone, L-asparaginase, vincris-
mutations did not associate with an inferior clinical outcome tine, daunorubicin, 6-mercaptopurine and 6-thiopurine were
(Table 2 and Figure 2a). In the DCOG ALL10 study, patients are available for 211 cases. RAS pathway-mutated cells were median
risk-stratified by MRD response on day 33 and day 79 into threefold more resistant to prednisolone compared with wild-type
standard-risk, medium-risk and high-risk treatment arms. In cases (Figure 3a; P = 0.024). Leukemic cells harboring clonal or
standard- and high-risk-treated cases clonal RAS pathway muta- subclonal KRAS G13 mutations were most resistant to predniso-
tions were associated with a significantly worse event-free survival lone: these cells were median 42000-fold more resistant
compared with wild-type cases, caused in part by a higher compared with wild-type cells (P = 0.001 and P = 0.006, respec-
incidence of relapse and non-response (Table 2 and Figure 2b, red tively; Supplementary Table S10, Figure 3a and Supplementary
lines). Significantly fewer relapses occurred in medium-risk-treated Figure S8). Clonal NRAS G13 mutations showed a trend, but the

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 RAS pathway mutations in pediatric BCP-ALL
IS Jerchel et al
935
100 100 Wildtype (n=132)

Cumulative incidence of relapse and

clonal RAS pathway mutation (n=52)
subclonal RAS pathway mutation (n=60)
80 80

Event−free survival (%)

pcl = 0.57 ptrend = 0.85

non−response (%)
60 60
ALL10
40 40

ptrend = 0.87
20 20
Wildtype (n=132)
clonal RAS pathway mutation (n=52)
subclonal RAS pathway mutation (n=60) pcl = 0.59
0 0
0 2 4 6 8 0 2 4 6 8
Time from initial diagnosis (years) Time from initial diagnosis (years)

100 100 Wildtype (n=49)

Cumulative incidence of relapse and

clonal RAS pathway mutation (n=10)
subclonal RAS pathway mutation (n=15)
80 80
Event−free survival (%)

pcl = 0.027 ptrend = 0.43

non−response (%)
60 60
Standard
risk 40 40

pcl = 0.22
ptrend = 0.066
20 20
Wildtype (n=49)
clonal RAS pathway mutation (n=10)
0 subclonal RAS pathway mutation (n=15) 0
0 2 4 6 8 0 2 4 6 8
Time from initial diagnosis (years) Time from initial diagnosis (years)

100 100 Wildtype (n=72)

Cumulative incidence of relapse and

pcl = 0.058 clonal RAS pathway mutation (n=33)

subclonal RAS pathway mutation (n=43)
80
Event−free survival (%)

80 ptrend = 0.053
non−response (%)

60 60
Medium
risk 40 40

ptrend = 0.17
20 20
Wildtype (n=72)
clonal RAS pathway mutation (n=33) pcl = 0.014
0 subclonal RAS pathway mutation (n=43) 0
0 2 4 6 8 0 2 4 6 8
Time from initial diagnosis (years) Time from initial diagnosis (years)

100 100 Wildtype (n=11)

Cumulative incidence of relapse and

clonal RAS pathway mutation (n=9)

subclonal RAS pathway mutation (n=2)
80 80
Event−free survival (%)

ptrend = 0.092
non−response (%)

60 pcl = 0.015 60
High
risk 40 40 pcl = 0.044

ptrend = 0.030
20 20
Wildtype (n=11)
clonal RAS pathway mutation (n=9)
0 subclonal RAS pathway mutation (n=2) 0
0 2 4 6 8 0 2 4 6 8
Time from initial diagnosis (years) Time from initial diagnosis (years)

Figure 2. Clinical outcome of patients carrying clonal or subclonal RAS pathway mutations. Event-free survival (left panel) and cumulative incidence
of relapse and non-response (CIR, right panel) (a) within the ALL10 cohort (n = 244) and (b) divided by the three risk arms of ALL10. Abbreviations:
HR, high-risk group; MR, medium-risk group; Ptrend represents the P-value in a log-rank (EFS) or Gray-test (CIR) across all three groups, Pcl represents
the P-value in a log-rank or Gray-test comparing wild-type patients and those with a clonal RAS pathway mutation; SR, standard-risk group.

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 RAS pathway mutations in pediatric BCP-ALL
IS Jerchel et al
936

Figure 3. RAS pathway mutations and ex vivo cytotoxicity of chemotherapeutic agents. Ex vivo sensitivity of 211 primary patient samples
towards (a) prednisolone and (b) vincristine, distinguished by RAS mutation status. Only clonally mutated cases are considered. Only KRAS-
and NRAS-mutated groups are shown due to low recurrence of other mutations (see also Supplementary Data). Combined: All cases with a
clonal mutation in NRAS, KRAS, PTPN11, FLT3. Groups were compared by Mann–Whitney U-test, *P o0.05, **Po0.01. LC50-values were
evaluated by MTT assays as reported previously.

Retained Gained Lost

NRAS G12D
60 NRAS G12A
Variant allele frequency (%)

NRAS G12S
50
KRAS G12D
40 KRAS G12A
30 KRAS G12V
KRAS G13D
20 KRAS A146T
KRAS A146V
10
FLT3 D389G
5
4 FLT3 D386del
3 FLT3 D385H
2
1
0 PTPN11 G60V
PTPN11 E76Q
1s Dx.
2nst Rx

1 s Dx

2nst Rx

1 s Dx

2nst Rx

2nst Rx

1 s Dx

2nst Rx

1 s Dx
1 s Dx

2nst Rx

1 s Dx
4td R
R

tR

tR

tR

tR

tR
3rd R

tR

tR
R

R
1 D

1 D

1 D

1 D

1 D

1 D
h

Figure 4. Evolution of clonal and subclonal RAS pathway mutations between initial diagnosis and subsequent relapse in 13 BCP-ALL cases.
Variant allele frequency of all RAS pathway mutations found at initial diagnosis and/or relapse(s) is shown for cases with RAS pathway
mutations detected at either time point (13 of 19). Colors distinguish affected genes; symbols distinguish observed variants. Dx, initial
diagnosis sample; R, relapse sample.

difference was not significant (P = 0.18). NRAS and KRAS G12 consistent association of mutation status with ex vivo drug
mutations did not significantly affect resistance towards response was observed for daunorubicin, 6-thioguanine and
prednisolone, although several cases were highly resistant. 6-mercaptopurine (Supplementary Figure S10).
Mutations in PTPN11 and FLT3 were not associated with cellular
prednisolone resistance (Supplementary Table S10 and RAS pathway mutations at relapse
Supplementary Figure S8A).
Ex vivo cytotoxicity of the tubulin-inhibitor vincristine was also RAS pathway mutations were detected in 13 out of 19 cases with
reduced in RAS pathway-mutated cells (Figure 3b): Cases with a matched initial diagnosis and relapse samples (Figure 4). Ten
clonal KRAS or PTPN11 mutations were significantly more resistant mutations were found at diagnosis, and 10 at relapse (53%). From
to vincristine compared with wild-type cases (15-fold, P = 0.005 diagnosis onwards, the evolution of RAS pathway mutations
and 5-fold, P = 0.041, respectively). A trend was observed for clonal followed three distinct patterns: In three cases, a clonal RAS
NRAS G13 mutations (3-fold, P = 0.074). No significant difference pathway mutation was detected at initial diagnosis and at relapse
was observed for KRAS G12 mutations, and an inverse trend was (‘Retained’). In seven cases, a subclonal mutation (four patients) or
found in cases carrying an NRAS G12 mutation (5-fold decrease, no mutation (three patients) was observed at diagnosis, but at
P = 0.057, see also Supplementary Figure S8B). relapse a RAS pathway mutation was detected with higher VAF
In K/NRAS G13-mutated cases the glycine (G) was replaced by (‘Gained’). In six of these relapses the mutation was clonal,and in
an aspartic acid (D) in 13 of 15 cases. In contrast, the type of amino one it was subclonal. One of these cases was remarkable in that
acid being substituted at the G12 position was more variable (see five different subclones were observed at diagnosis but only one
also Supplementary Figure S6). As visualized in Supplementary of these mutations (NRAS G12S) was found at relapse. Loss of one
Figure S9 the substituting amino acid was not predictive for and selection of a second clone was observed in two cases, where
ex vivo prednisolone or vincristine resistance. they constituted the major clone in all subsequent relapses. In
In contrast, wild-type cases tended to be more resistant towards three cases, the initially observed RAS pathway mutation was not
L-asparaginase ex vivo than RAS pathway-mutated cases; however, detected at relapse (‘Lost’). For all ‘lost’mutations VAF at initial
this trend was not significant (Supplementary Table S10). No diagnosis was lower than 10%. Remarkably, in 9 out of 10 relapse

Leukemia (2018) 931 – 940

 RAS pathway mutations in pediatric BCP-ALL
IS Jerchel et al
937
Viability at 0.14μM trametinib
120 Wildtype (n=12) 100
***
RAS mutant (n=6)

Cell survival (%)

100

% Cell survival
80
80
60
60
40
40 ***
20 20

0 0
0.001 0.01 0.1 1 10 Wildtype RAS mutant
Trametinib (μM)

Wildtype RAS mutant

†
pMEK1/2
S217-221
MEK1/2

pERK1/2
T202/T204
ERK1/2

α-Tubulin

RAS mutant BCP-ALL

120 120
Vehicle
100 100 +0.1
+0.1μM Trametinib
Cell survival (%)
Cell survival (%)

80 80

60 60

40 40

20 20

0 0
0.001 0.01 0.1 1 10 0.01 0.1 1 10 100
Trametinib (μM) Prednisolone (μg/mL)

RAS wildtype BCP-ALL

120 120 Vehicle
+0.1μM Trametinib
100 100
Cell survival (%)

Cell survival (%)

+1μM Trametinib
80 80

60 60

40 40

20 20

0 0
0.001 0.01 0.1 1 10 0.01 0.1 1 10 100
Trametinib (μM) Prednisolone (μg/mL)

Figure 5. The MEK inhibitor trametinib effectively kills RAS mutant primary BCP-ALL cells. (a) Sensitivity towards the MEK inhibitor trametinib
in primary or xenograft-derived BCP-ALL cells. Mean and standard deviation are shown. (b) Relative cell survival at 0.14 μM trametinib split up
per case. Gray circles represent wild-type cases; red triangles represent RAS pathway mutant cases. Bars represent group mean. Groups were
compared by Mann–Whitney U-test, ***P = 0.001. (c) Western blot analysis of phospho-ERK (T202/T204) and phospho-MEK (S217/S221) in a
subset of samples tested in (a). †Sample isolated after thawing, cells previously tested positive. (d) Ex vivo response to trametinib (left panels)
and sensitization towards prednisolone (right panels) in one NRAS G12D-mutant and one RAS pathway wild-type case.

cases a single, clonal mutation was observed (VAF of 27% or and Supplementary Figure S11A). This effect is also illustrated by
higher). individual data points at a fixed concentration of 0.14 μM
(Figures 5b, P = 0.001). High levels of MEK1/2 and ERK1/2
phosphorylation indicated an activated RAS pathway in mutant
MEK inhibitors as therapeutic option but not in wild-type cells (Figure 5c). The cytotoxicity of increasing
The MEK inhibitor trametinib was cytotoxic to RAS mutant but not trametinib concentrations corresponded with effective reduction
to wild-type leukemic cells in a range of concentrations (Figure 5a of ERK1/2 phosphorylation (Supplementary Figure S11B). In

Leukemia (2018) 931 – 940

 RAS pathway mutations in pediatric BCP-ALL
IS Jerchel et al
938
addition, we found that intrinsic resistance to prednisolone could vincristine pulses during maintenance therapy.4 This is further
be reversed by trametinib irrespective of RAS mutation status supported by RAS-mutated patients being relatively sensitive to
(Figure 5d), which corroborates our previous findings.30 asparaginase ex vivo. The prognosis of those 20.1% of patients
with subclonal mutations was similar to wild-type cases, and they
generally presented with good risk features. Therefore, subclonal
DISCUSSION mutations in RAS pathway genes should not be used to assign
The RAS pathway is the most frequently mutated pathway in patients to a higher risk group or treatment with an MEK inhibitor.
cancer, and may serve as treatment target.7,8,49 Recent reports Evaluation of 19 cases with matched diagnosis and relapse
suggest that RAS pathway mutations are recurrent in pediatric material gave an important insight into the evolution of RAS
BCP-ALL.20,27,30,31,50 However, frequency and prognostic value of pathway mutations: (1) clonal mutations at diagnosis were
subclonal mutations at initial presentation were unknown. In this preserved at relapse, (2) subclonal mutations detected at initial
study we addressed the clinical value of mutations in 13 key diagnosis were often found at relapse and (3) a single, clonal
members of the RAS pathway using deep targeted sequen- mutation was found in 9 out of 10 relapses. These observations
cing, which allowed detection of subclones down to 1% VAF. confirm that RAS pathway mutations are frequent at relapse in
Our study cohort contained 461 newly diagnosed cases and pediatric BCP-ALL.28,29,63,64 The reduced mutational diversity at
represents all subtypes and risk groups of BCP-ALL, with a relapse suggests outgrowth of a single clone with stronger
distribution comparable to the general pediatric BCP-ALL dependence on MAPK signaling, which may increase the chance
population. of success for RAS-targeted therapy.
We identified clonal RAS pathway mutations (⩾25% VAF) in NRAS and KRAS mutations increase the risk for therapy failure at
24.1% and subclonal mutations (o 25% VAF) in 20.1% of patients relapse, demanding alternative treatment options.27,28 These
at initial diagnosis. This total of 44.2% is considerably higher than patients may be eligible for MEK inhibitors such as trametinib.
the 15% mutation frequency previously detected by Sanger Leukemic cells with RAS pathway mutations were sensitive to
sequencing, and shows that RAS pathway activation cooperates trametinib, while wild-type cells were not. We further confirm our
with several primary oncogenic lesions.20,31 The vast majority of previous finding that MEK inhibition can synergize with
mutations (98%) occurred in NRAS, KRAS, FLT3 and PTPN11, prednisolone.30 Interestingly, 9 of 11 RAS-mutated cases allocated
revealing a central role of these genes in pediatric BCP-ALL. to the high-risk arm had a poor in vivo response to prednisone
We observed high mutation frequencies among high hyperdi- after 1 week of therapy. This provides a rationale to combine MEK
ploid, MLL-AF4-rearranged, BCR-ABL1-like and B-other cases, inhibitors with glucocorticoids. Notably, within our data no
moderate frequencies among ETV6-RUNX1 cases, and rarely correlation was observed between the response to prednisolone
observed them in TCF3-PBX1 and BCR-ABL1-rearranged cases, and the response to trametinib.
confirming reports of smaller sample sets.23,27,32,51 In conclusion, clonal mutations in NRAS, KRAS, PTPN11 and FLT3
The BCR-ABL1 fusion is known to require MAPK signaling, but are associated with therapy resistance. Given that clonal muta-
the low mutation frequency suggests that independent RAS tions at initial diagnosis were retained at relapse and that
pathway activation is not required for this aggressive disease.52–56 subclonal mutations often expanded at relapse, RAS pathway
BCR-ABL1-like cases, who bare strong similarities with BCR-ABL1- mutations may serve as a biomarker to identify patients eligible
positive leukemia, frequently carry RAS pathway mutations. These for MEK/ERK targeted therapy. The synergistic effect between MEK
were mutually exclusive with tyrosine kinase fusions, suggesting inhibition and prednisolone may be of additional advantage.
divergent pathogenic mechanisms. Our data further show that
While our results and supremacy of MEK inhibitors require
RAS pathway activating mutations play a role in about half of all
confirmation in independent cohorts, our data suggest screening
B-other patients. The largest group of RAS pathway-mutated cases
(a) all cases that are eligible for treatment reduction based on
was observed within high hyperdiploid cases. Our extensive
MRD and (b) patients for whom treatment intensification is
screen extends the existing knowledge by revealing frequent
advised and who may benefit from adjuvant MEK inhibitor
subclonal events.27,32,57,58 Our data advocate MEK inhibitors as a
treatment. Methodically, accurate detection of any clonal RAS
treatment option in high hyperdiploid cases with poor therapy
pathway mutation should be ensured, but detection of subclonal
response. We further identified several secondary aberrations that
co-occurred less or more frequently with RAS pathway mutations. mutations is not required.
Interestingly, RAS pathway mutant cases infrequently carried BTG1
deletions, which have recently been linked to glucocorticoid CONFLICT OF INTEREST
resistance and may represent a distinct resistance mechanism in The authors declare no conflict of interest.
RAS pathway wild-type cases.59
In the largest data set analyzed so far (n = 211), we confirmed
the association of RAS pathway mutations with prednisolone ACKNOWLEDGEMENTS
resistance.30,60,61 Additionally, RAS pathway mutant cells were This work was supported by the NWO VICI program (grant 016.126.612), the Dutch
ex vivo resistant to vincristine, but not to L-asparaginase, Cancer Society (grants AMC 2008-4265 and EMCR 2014-6998), and the KIKA
daunorubicin and thiopurines. This suggests that patients with Foundation (grants 132 and 161) and the Pediatric Oncology Foundation Rotterdam.
RAS pathway mutations could profit from intensification of The CPCT is supported by the NutsOhra Foundation (grant 1102-062). We thank Ies
these drugs. Nijman and Annelies Smouters for their help in setting up the analysis pipeline and in
Patients with high MRD levels (⩾10− 3 after induction or sequencing analysis.
consolidation) are at higher risk for relapse and treated more
intensely, also in DCOG ALL10.4,62 Patients with clonal RAS
pathway mutations treated in the DCOG standard-risk arm had AUTHOR CONTRIBUTIONS
an unfavorable clinical outcome despite negative MRD levels. ISJ and MLdB designed/performed experiments, analyzed/interpreted data and
Since the prognosis of clonally mutated patients is highly wrote the manuscript; AQH and JMB analyzed/interpreted data; IMA, NJMB, MJK
favorable in the medium-risk group (5-year event-free survival and EC designed sequencing experiments; EMPS designed/performed experi-
97%, cumulative incidence of relapse and non-response 0%; ments and obtained xenograft material; AB and CvdV obtained xenograft
Figure 2b), MRD-low risk patients with clonal RAS pathway samples, HAdG-K, MAH and GE provided samples and clinical information,
mutations may benefit from medium-risk treatment, which MLdB and RP conceptualized the study and interpreted results. All authors read,
includes intensive PEG-asparaginase and dexamethasone/ revised and approved the manuscript.

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 RAS pathway mutations in pediatric BCP-ALL
IS Jerchel et al
939
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