1. Integrated genomic analysis of the iAMP21 subtype of pediatric acute lymphoblastic leukemia
Ingegerd Ivanov Öfverholm, MD, PhD
Dept of Molecular Medicine and Surgery
Clinical Genetics unit
Karolinska Institutet

2. Acute lymphoblastic leukemia
Acute lymphoblastic leukemia (ALL) - most common form of pediatric cancer
70-90 new cases /year in Sweden
Unified treatment protocols of NOPHO
90% survival with modern treatment, 20 % relapse

3. Acute lymphoblastic leukemia
Genetic alterations in immature lymphoid cells
B-cell precursor ALL

4. Acute lymphoblastic leukemia
Specific chromosomal aberrations associated with outcome
Standard risk: HeH, t(12;21) = 50%
Intermediate risk: iAMP21, t(1;19), dic(9;20)
High risk: hypodiploidy, t(9:22), KMT2A
25% without prognostic markers

5. iAMP21 in childhood ALL
Background
Intrachromosomal amplification of chromosome 21
Discovered in 2003
High median age
Primary event, retained at relapse
Intermediate/high risk marker

6. iAMP21 in childhood ALL First detected by RUNX1 FISH probes
Background
First detected by RUNX1 FISH probes
Minimal region 5,1 Mb
45 genes
Li, Harrison et al. Nature 2014: Double strand break  breakage-fusion-bridge cycles and chromotripsis
Ryan et al. Leukemia 2017: Mutations in RAS pathways
Underlying mechanism of pathogenicity still largely unknown

7. Integrated analysis of iAMP21
Aims
To investigate the genomic and transcriptional profile of iAMP21
To understand the pathogenic features of iAMP21

8. Integrated analysis of iAMP21
Material and methods
16 diagnostic samples; 2 relapse samples
34 iAMP21-negative BCP ALL
Genomic profiling
Single nucleotide polymorphism array
Whole genome sequencing
Methylation
Transcriptome profiling
RNA sequencing

9. Integrated analysis of iAMP21
SNP array
Genomic microarray platform
Patient
DNA
Control

10. Genomic structure of iAMP21
SNP array
KSALL2
KSALL11
KSALL52
KSALL12
KSALL16
KSALL17
KSALL23
KSALL26
KSALL30
KSALL35
KSALL48
KSALL49
KSALL50
Individual amplification patterns
Oscillating copy number states
Terminal deletions

11. Genomic structure of iAMP21
SNP array
High number of deletions
Genomic instability?
Chromothripsis?
Recurrent deletions
RB1 (chr 13)
SH2B3 (chr 12)
IKZF1 (chr 7) 25 20 15 10 5
B-other HeH t(12;21) iAMP21 t(1;19) t(9;22) KMT2A

12. Genomic rearrangements in iAMP21
Whole genome sequencing
Input bone marrow DNA
Fragmentation
Amplification
Sequencing

13. Genomic rearrangements in iAMP21
Whole genome sequencing
DNA sequence read alignment
Rearrangements, mutations, copy number alterations

14. Genomic rearrangements in iAMP21
Whole genome sequencing
Three iAMP21 samples
Composition of chromosome 21
Multiple rearrangements
Individual patterns, no recurrent breakpoint
Inverted rearrangements
Signs of chromothripsis and breakage-fusion- bridge cycles

15. Gene expression profile in iAMP21
RNA sequencing
DNA
transcription
RNA
translation
Protein

16. Gene expression profile in iAMP21
RNA sequencing
RNA cDNA
Fragmentation
Amplification
Sequencing
Exonic reads

17. Gene expression profile in iAMP21
RNA sequencing
Differential gene expression analysis of 46 BCP ALL cases
Unsupervised clustering based on most variably expressed genes

18. Gene expression profile in iAMP21
RNA sequencing
Altered expression of 763 genes in iAMP21; top differentially expressed genes on chromosome 21
13 overexpressed genes in the minimal region of amplification (MRA)

19. Candidate genes in iAMP21
CHAF1B
Chromatin assembly protein
Chromatin stability during DNA replication
Overexpression associated with malignancy and poor prognosis
SON
Epigenetic regulation through MLL complex
Short isoforms promote expression of leukemia promoting genes

20. Preliminary conclusions
Amplification of chromosome 21 results in a unique transcription profile
Unique overexpression of potential candidates for disease and relapse
Options for tailored treatment
Epigenetic therapies
Tyrosine kinase inhibitors

21. Acknowledgements Department of Molecular Medicine and
Surgery, CMM, Karolinska Institutet
Gisela Barbany
Ann Nordgren
Magnus Nordenskjöld
Anh Nhi Tran
Vasilios Zachariadis
Fulya Taylan
Clinical genetics lab
Nina Jäntti
Department of Women’s and Children’s health, Childhood Oncology group, Karolinska Institutet
Mats Heyman
Johan Malmros
Stefan Söderhäll
Department of Medical Sciences, Molecular Medicine, BMC, Uppsala University
Jessica Nordlund
Yanara Marincevic-Zuniga
Ann-Christine Syvänen
Gudmar Lönnerholm
Department of Oncology and Pathology, CCK, Karolinska Institutet
Dan Grandér
Katja Pokrovskaja
Per Johnsson
Linda Vidarsdóttir
Jason Tyler Serviss