"Emergence de new ALK mutations at relapse of neuroblastoma". Dr Gudrun Schleiermacher, MD PhD, Institut Curie, Paris France 2014 ASCO annual meeting

1. Emergence of new ALK mutations
at relapse of neuroblastoma
Gudrun Schleiermacher MD PhD
Institut Curie, Paris
JCO, in press

2. Background
• Cancer : frequent secondary progression, resistance to conventional
chemotherapy : therapeutic challenge
• Selection for genetic alterations during treatment
• Subclonal driver mutations might play a role in tumor progression
• Presence of driver mutation-harboring subclones at diagnosis, which
might expand at relapse, has been linked to adverse outcome in
hematological malignancies (Landau et al, 2013)
• Different models of clonal evolution (Ding et al, 2011) :
– Dominant clone evolves into relapse clone
– A minor clone carrying the vast majority of primary tumor mutations
escapes and expands at relapse
Presented by: Gudrun Schleiermacher

3. Hallmark of neuroblastoma :
clinical heterogeneity
Improvement of EFS in NB (all stages) over the last decades
But : despite significant progress for lower stages, survival in stage 4 disease does still not
exceed 40% (mortality linked to metastatic tumor progression)
Observation only Surgery Chemotherapy
Chemotherapy
High dose chemotherapy with
autologous stem cell rescue
Surgery
Radiotherapy
Immunotherapy
Maintenance treatment
Moroz et al, 2011
Treatment of neuroblastoma :

4. ALK TRK plays a role in NB oncogenesis
Germline Mutations of ALK
in neuroblastoma families
• Activating ALK mutations in 8 – 10% of all NB at diagnosis (Mosse et al, Janoueix-Lerosey et al, Chen et al,
George et al 2008; De Brouwer et al, 2010)
• Report of a relapse-specific ALK mutation (Martinsson et al, 2011)
• Objectives of this study : Determine the frequency of ALK mutations at
relapse and their role in clonal evolution of NB
Somatic Mutations of ALK
SigP
LBD
TM
Kinase
domain
1
26
391
401
1031
1057
1116
1392
1620
Y1278S TAC>TCC
N
T
IRS-1, SHC,
RAS-MAPK
PI3K, AKT,
mTOR
ALK inhibitors
Crizotinib®
MYCN
ALK
MYCN ALK
Genomic amplification of ALK (<2%)

5. Methods
• 54 paired diagnosis – relapse NB tumor samples (France, Sweden, Belgium)
• 2 cell lines with corresponding tumor sample from which cell lines were
established
• Sanger sequencing
• Deep sequencing (IonTorrent PGM®, LifeTechnologies) when an ALK mutation
was seen in only one of the paired samples (7 cases)
• Resequencing of hotspots exon 23 and exon 25
• Depth of coverage : 100,000x
• 4 control cell lines (CLB-Car, SKNDZ, SJNB12, SKNAS)
Presented by: Gudrun Schleiermacher

6. Alk mutation
R1275Q
(CGA>CAA)
No Alk mutation
Michel Peuchmaur
Heterogeneity of ALK mutations in NB
Ganglioneuroblastoma
Local progression,
Treatment by surgery
Presented by: Gudrun Schleiermacher

7. 5/54 new ALK mutations
at relapse
All pts with ALK mutations
at diagnosis also had ALK
mutations at relapse
Results (Sanger sequencing): 14/54 Alk mutations
Clinical information ALK mutation
ALK
Detection by Sanger
Patient N°
Age at diag
(months)
stage (INSS)
Interval diagnosis-
relapse (months)
relapse type
FU
(months from
diagnosis)
Outcome Genomic profile AA change Diagnosis Relapse
NBG03 50 4 23 loc 29 DOD MNA F1174L pos pos
NBG14 90 4 10 loc 55+ NED S F1174L pos pos
NBG21 41 2b 11 met 17 DOD MNA F1174L pos pos
NB0175 101 2b 93 loc +meta 150 DOD S Y1278S pos pos
NB0399 0.2 4s 6 meta 136 DOD N R1275Q pos pos
NB0824 3 4 3 meta 16 DOD N F1174L pos pos
NB1269 14 4 10 loc 11 DOD S L1196M pos pos
NB1224 24 2b 4 loc 14 NED S R1275Q pos pos
NB0073 3 4s 7 meta 272 NED N T1151R pos pos
NBG05 37 4 45 loc +meta 50 DOD MNA R1275Q neg pos
NBG12 12 4 9 meta 9 DOD S F1174S neg pos
NBG17 29 4 13 meta 24 DOD MNA F1174L neg pos
NB1382 4 4 51 loc +meta 63 DOD S Y1278S neg pos
NB0308 3 2b 21 loc 93 NED N F1174L neg pos
Presented by: Gudrun Schleiermacher

8. Methods
• 54 paired diagnosis – relapse NB tumor samples
• 2 cell lines and tumor sample from which cell lines were established
• Sanger sequencing
• Deep sequencing (IonTorrent PGM®) when an ALK mutation was seen in only
one of the paired samples (7 cases)
• Resequencing of hotspots exon 23 (hotspot F1174) and exon 25 (hotspot
R1275)
• Depth of coverage : 100,000x
• 4 control cell lines (CLB-Car, SKNDZ, SJNB12, SKNAS)
Exon 25 Exon 23
Presented by: Gudrun Schleiermacher

9. Analysis of noise (control cell lines):
Y1278 R1275
F1174
Exon 25
Exon 23
Presented by: Gudrun Schleiermacher

10. Chr position /
patient n°
# reads A C G T
chr2:29432655 (A) Y1278S # reads % p-value % p-value % p-value % p-value
NB1382_D 226371 99.954 1.00E+00 0.011 1.00E+00 0.031 1.00E+000 0.004 1.00E+00
NB1382_R 179938 66.326 <1E-016 33.613 <1E-016 0.032 1.00E+000 0.029 1.82E-015
Controls (n=4) SD SD SD SD
Total of all reads 701987 99.941 NA 0.012 NA 0.043 NA 0.004 NA
Mean 99.942 2.49E-003 0.012 4.67E-003 0.043 3.07E-003 0.004 2.51E-003
chr2:29432664 (A) R1275Q # reads % p-value % p-value % p-value % p-value
NB1224_D 258308 14.164 <1E-016 0.064 <1E-016 85.755 <1E-016 0.016 1.00E+000
NB1224_R2 302176 0.035 1.00E+000 0.001 1.00E+000 99.952 1.00E+000 0.012 1.00E+000
NB1224_R4 318313 0.053 1.00E+000 0.002 1.00E+000 99.935 1.00E+000 0.011 1.00E+000
NB1224_R5 302918 28.536 <1E-016 0.121 <1E-016 71.329 <1E-016 0.013 1.00E+000
Controls (n=4) SD SD SD SD
Total of all reads 703934 0.038 NA 0.001 NA 99.952 NA 0.009 NA
Mean 0.032 2.49E-003 0.012 4.67E-003 99.921 3.07E-003 0.004 2.51E-003
PGM® analysis : cases with discordant Sanger results
Cases studied by PGM®in case of discrepancies of Sanger sequencing in different
samples
5 patients : 13 samples
2 established cell lines : 4 samples
Analysis: comparison of base frequencies observed in a given sample, at a given
position, to that observed in controls (Fisher exact test)
In some cases: no evidence of ALK mutated subclones
Presented by: Gudrun Schleiermacher

11. T 0,048 0,101 0,039
A 8,15 0,019 0,049
G 0,003 19,125 0,001
C 91,799 80,754 99,912
T 0,025 0,083 0,111 0,039
A 0,798 26,113 30,921 0,049
G 0,003 0,008 0,005 0,001
C 99,175 73,796 68,963 99,912
%%
p<10-16
p<10-16
PGM (%)
NBG17_D NBG17_R1 NBG17_R2
NB0308-RNB0308-D
NB0308
0
0,1
0,2
0,3
0,4
5
25
45
65
85
100
0
0,25
0,5
0,75
1
5
25
45
65
85
100
NB0308-D NB0308-R ControlNBG17-D NBG17-R1 NBG17-R2 Control
NBG17
PGM® analysis : evidence of subclonal events
NBG17 : Presence of an ALK mutated subclone
(TTC> TTA; 0.798%) at diagnosis
NB0308 : ALK mutation switch (TTC> TTA/TTG)
leading to the same AA change at D and R

12. G 0,003 0,003 0,010
A 0,046 0,4 0,006
C 6,609 32,378 0,015
T 93,342 67,22 99,970
0
0,1
0,5
3
83
100
0,2
0,3
0,4
63
43
23
Ba(PT_BM) CLB-BA Control%
p<10-16
Ba(PT_BM) CLB-BA
Cell line
Age at
diag
(months)
stage
(INSS)
Primary
sample
Cell line
established
from
FU
(months)
Outcome
Genomic
profile
AA change
Primary
sample
Cell line
CLB_Ma 9 4
Abdominal
tumor
bone marrow 16 DOD MNA F1174L neg
F1174
L
CLB_Ba 27 4
bone
marrow
bone marrow 117 NED MNA F1174L neg
F1174
L
Results (NB cell lines)
Presence of an ALK mutated subclone (6.6%) in the
Sample from which the cell line was established

13. Discussion
• Sensitivity of the PGM® deep sequencing technique:
• Mean overall coverage (control cell lines): >175,000X
• Errors in PGM:
• vary strongly according to the genome structure : homopolymers;
• possible link to sequencing errors due to polymerase slippage, errors in the chemistry
• Mean overall background variability : 0·034%+/-0·035% for each base
• Number of reads would be considered statistically different from the
background (Bonferroni correction) : a variation supported by 296 reads, or
observed with a frequency of 0·17%, would result in a statistically significant
difference from the controls (two-sided Fisher’s exact test)
• Sensitivity 0.17% (100 fold that of Sanger sequencing , 20%)
• Limits in sensitivity : quantity of analyzed material
• PCR of exon 23/25 amplicons : 50 ng of genomic DNA
• ~ 5000 (diploid) cells
• Limit of detection : 1/5000 cells; 1/10 000 haploid genomes
Presented by: Gudrun Schleiermacher

14. ALK in tumor progression
Clinical implications
Encourage new biopsy at relapse!
Search for subclones:
Evaluation of frequency of ALK mutated
subclones at diagnosis
ALK targeted treatment for cases
with subclonal ALK mutations?
further investigations in vitro and in vivo
• In NB, ALK mutations might occur as subclones at diagnosis with
secondary expansion
• selective advantage during tumor progression
• ALK-mutated and non-mutated cells might co-exist in an equilibrium
• expansion of an ALK-mutated clone upon treatment : preferential cytotoxic
effect on ALK non-mutated clones?
NB at diagnosis
NB at relapse
When considering
biomarker based ALK
targeted therapy:
Search for genetic
alteration
of ALK
Presented by: Gudrun Schleiermacher

15. Acknowledgements
Unité INSERM 830
Translational Research in
Pediatric Oncology Team
Olivier Delattre
Julie Cappo
Angela Bellini
Isabelle Janoueix-Lerosey
Centre Léon Bérard, Lyon
Valérie Combaret
Unité de Génétique
Somatique
Gaelle Pierron
Nathalie Clement
Eve Lapouble
Département de
Pédiatrie
Jean Michon
Isabelle Aerts
Clinicians and pathologists
of the SFCE
Michel Peuchmaur
Plateforme de Séquençage
Thomas Rio Frio
Quentin Leroy
Virginie Bénard
Leo Colmet Daage
Department of Medical Genetics,
Gotheburg, Sweden
Tommy Martinsson
Niloufar Javanmardi
Collaborators in Sweden:
Anna Djos
Ingrid Øra
Fredrik Hedborg*
Catarina Träger
Britt-Marie Holmqvist
Jonas Abrahamsson
Per KognerGhent University
Frank Speleman
Bram de Wilde