inter and intra-tumoral heterogeneity in pediatric sarcoma · pathway mutations are common in...
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Inter and Intra-tumoralheterogeneity in pediatric
sarcoma
Jack Shern MDLasker Research Scholar
Pediatric Oncology BranchCenter for Cancer Research
National Cancer Institute
"It is good to have hair-splitters & lumpers.”-Charles Darwin
Rhabdomyosarcoma – “the most common soft tissue sarcoma of childhood”
Shern et al. Cancer Discovery. 4: 216-231, 2014.
Rhabdomyosarcoma: Inter-tumor heterogeneity
Can we use genetic information to further refine risk stratification?
Hawkins et al. Curr Opin Pediatr. 2014 Feb; 26(1): 50–56.
Williamson D et al. JCO 2010;28:2151-2158
ARST14B1 - Project Overview
COG histology review and clinical annotation
NCI extraction and quantification of
nucleotides and sample genotyping
RNA RNAseq – Shern Nanostring assay
Mike Arnold Nationwide ChildrensDNA
Targeted Capture and Illumina sequencing
NCI Oncogenomicspipeline to call point mutations, indels, amplifications and
deletions
Correlation between the observed genetic
alterations (RAS pathway alterations vs no RAS pathway alteration);
expression signatures and clinical outcome
2 unstained slides from
Clinically annotated RMS cases
COG ARST14B1 Summary
AKT1 CDK4 GAB1 MYCN PTEN
ALK CDKN2A HRAS MYOD1 PTPN11
ARID1A CTNNB1 IGF1R NF1 ROBO1
ATM DICER1 IGF2 NRAS SMARCA4
BCOR ERBB2 KRAS PDGFRA SOS1
BRAF FBXW7 MDM2 PIK3CA SOS2
CCND1 FGFR1 MET PKN1 TP53
CCND2 FGFR4 MTOR PTCH1
403 COG cases
Inadequate nucleotides (n=56)
347 Sequenced COG cases
66 Fusion Positive
281 Sequenced COG cases
316 Sequenced UK cases
597 Sequenced Fusion Negative
cases+
Tier I Calls - (Somatic/germline status unknown)• Hot Spot or stop or deleterious indel• High copy number amplification• Deep deletion of any included gene
Clinical Characteristics of the COG cohort
Characteristic n=347 (%)Sex
MaleFemale
234 (67)113 (33)
Age at presentation (years)MedianRange
6.40.02-37.8
Tumor HistologyAlveolarEmbryonalEmbryonal with diffuse anaplasiaEmbryonal with focal anaplasiaMixed Alveolar and EmbryonalMixed Embryonal and Spindle cellSpindle cellBotryoidRhabdomyosarcoma NOSCytology Specimen
66 (19)219 (63)
15 (4)6 (2)
2 (<1)9 (3)
16 (5)10 (3)3 (1)
1 (<1)
Anatomic GroupBladder Prostate Group 3Bladder Prostate Group 4Extremity Group 3Extremity Group 4Female GUHead and NeckOrbitalParameningeal Group 3Parameningeal Group 4ParatesticularPilot Study No DataRetroperitineum/Peritineum/Trunk Group 3Retroperitineum/Peritineum/Trunk Group 4
18 (5)6 (2)
16 (5)35 (10)
8 (2)29 (8)25 (7)
46 (13)14 (4)
64 (19)10 (3)
44 (13)30 (9)
Risk GroupLowIntermediateHigh
93 (27)131 (38)123 (35)
Variant CallsMedianRange
10-5
Mutation Summary
Identified at least one Tier 1 driver in 221/281 (80%) fusion negative cases
Percentage of cases summarized by
anatomy• TP53 pathway mutations are
common in fusion negative extremity lesions
• Female genitourinary cases account for all of the DICER1lesions
• HRAS and KRAS do not occur in orbital tumors
• MYOD1 mutations are restricted to the head
% of cases
1 tumor ≠1 genetic lesion
Hypothesis: Increased number of mutations leads to a worse outcome
Hypothesis: Fusion Negative Rhabdomyosarcoma is polyclonal?
What genes go together?
Infants less than 1 year old and the distribution of RAS isoform mutations by age
Median age: 14.4 years (3.3-21)All cases are head/neck or
parameningeal
MYOD1 mutations are associated with a dismal
outcome
0.0 2.5 5.0 7.5 10.0 12.5 15.0
EFS (Years)
0.0
0.2
0.4
0.6
0.8
1.0
Surv
ival
Pro
babi
lity
YesNoMYOD1
Event-free survival by MYOD1
0.0 2.5 5.0 7.5 10.0 12.5 15.0
EFS (Years)
0.0
0.2
0.4
0.6
0.8
1.0
Surv
ival
Pro
babi
lity
YesNoMYOD1
Log-Rank p< 0.0001
Event-free survival by MYOD1Event-free survival by MYOD1
Surv
ival
Pro
babi
lity
EFS (years)
0.0 2.5 5.0 7.5 10.0 12.5 15.0
EFS (Years)
0.0
0.2
0.4
0.6
0.8
1.0
Surv
ival
Pro
babi
lity
YesNoTP53
Event-free survival by TP53
0.0 2.5 5.0 7.5 10.0 12.5 15.0
EFS (Years)
0.0
0.2
0.4
0.6
0.8
1.0
Surv
ival
Pro
babi
lity
YesNoTP53
Log-Rank p= 0.0022
Event-free survival by TP53
0.0 2.5 5.0 7.5 10.0 12.5
EFS (Years)
0.0
0.2
0.4
0.6
0.8
1.0
Surv
ival
Pro
babi
lity
YesNoTP53
Event-free survival by TP53 (RMS Risk Intermediate only)
0.0 2.5 5.0 7.5 10.0 12.5
EFS (Years)
0.0
0.2
0.4
0.6
0.8
1.0
Surv
ival
Pro
babi
lity
YesNoTP53
Log-Rank p= 0.0231
Event-free survival by TP53 (RMS Risk Intermediate only)
Rhabdomyosarcoma cohort
TCGA cohort
TP53 mutations
Event-free survival by TP53 Event-free survival by TP53 (Intermediate Risk group only)
Surv
ival
Pro
babi
lity
Surv
ival
Pro
babi
lityEFS (years) EFS (years)
MYCN and CDK4 amplifications are poor prognostic modifiers in PAX fusion positive tumors
0 2 4 6 8 10 12
EFS (Years)
0.0
0.2
0.4
0.6
0.8
1.0
Surv
ival
Pro
babi
lity
YesNo
Event-free survival by MYCN Mutation at Enrollment, Alveolar Patients Only
0 2 4 6 8 10 12
EFS (Years)
0.0
0.2
0.4
0.6
0.8
1.0
Surv
ival
Pro
babi
lity
YesNo
p= 0.0003
Event-free survival by MYCN Mutation at Enrollment, Alveolar Patients Only
0 2 4 6 8 10 12
EFS (Years)
0.0
0.2
0.4
0.6
0.8
1.0
Surv
ival P
roba
bility
YesNo
Event-free survival by CDK4 Mutation at Enrollment, Alveolar Patients Only
0 2 4 6 8 10 12
EFS (Years)
0.0
0.2
0.4
0.6
0.8
1.0
Surv
ival P
roba
bility
YesNo
p= 0.0321
Event-free survival by CDK4 Mutation at Enrollment, Alveolar Patients OnlyEvent-free survival by MYCN amplification
Surv
ival
Pro
babi
lity
EFS (years)
Event-free survival by CDK4 amplification
Surv
ival
Pro
babi
lity
EFS (years)
Chen et al. Cancer Cell 24, 710-724, 2013
Pediatric Sarcoma: Intra-tumor Heterogeneity
Single cell sequencing
Carly Sayers Xiyuan Zhang
Skull based metastatic tumor
CD4+, CD37+, CD53+, CD74+Producing Complement and Lysozyme
G2/M – TOP2A, FOXM1, Kinesins, Centrosomal genes
Collagens, Fibrillin, IGFBP’s
BIRC3, KLF10
Atypical Neurofibroma with concern for Malignant Peripheral Nerve Sheath Tumor
Gutmann, D. H. et al. (2017)
Neoplastic Schwann cells: NRXN1, SOX10, S100B
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
IL34
Nor
mal
ized
Expr
essio
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Single Cell Cluster
01234567
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
CSF1RN
orm
alize
d Ex
pres
sion
Single Cell Cluster
00.20.40.60.8
11.21.4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
CSF1
Nor
mal
ized
Expr
essio
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Single Cell Cluster
Gutmann, D. H. Nature Reviews. (2017) Felix, J. et al. Structure (2013)
Single cell sequencing identifies IL34 as a novel therapeutic target in NF1 tumors
HDAC inhibitors are potent repressors of PAX3-FOXO1 transcriptional activity.
Abraham, J. et al. Genes Dev (2014)
Nor
mal
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Expr
essio
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Normalized Expression
Even in a “homogenous” cell culture, single cell RNAseqshows cell to cell variability
Bulk sequencing averages across the population
thereby losing information about rare cell populations
Definition of these cell populations has major
implications for our understanding of tumor
evolution and therapeutic resistance
All cells increase target gene
transcription
Cell to cell heterogeneity with
recruitment of transcription
AcknowledgmentsChildren’s Oncology Group
Corinne Linardic
Erin Rudinski
Mike Arnold
Steve Skapek
Dave Hall
Don Barkauskus
Doug Hawkins
Institute of Cancer Research UK
Janet Shipley
Anna Kelsey
Rebecca Brown
Julia Chisholm
Joanna Selfie
NCI DCEG
Kristie Jones
Belynda Hicks
NCI CCR
Javed Khan
Berkley Gryder
Young Song
Ashley Walton
Rajesh Patidar
Jun Wei
Mike Kelly
NCI, CCR, Pediatric Oncology Branch
Carly Sayers
Xiyuan Zhang
Marielle Yohe
Christine Heske
Carol Thiele
Brigitte Widemann
The Lasker Clinical Research Scholars Program at the NIHGoal – To grow the diminishing pool of talented clinical/translational researchers.
Total program duration 8 years:Years 1-5: NIH Intramural Research Program full support (salary and research support) Years 6-8: salary and/or research support of up to $500,000/year at outside institution, OR continuation in Intramural Research Program
Candidates – Early stage clinical researchers, within 10 years of completing core residency, with the ability to conduct independent research.
http://www.nih.gov/science/laskerscholar/