1. Case 11: Near-haploid B lymphoblastic leukemia with an apparent hyperdiploid karyotype
Rebecca King MD
Sarah Choi MD PhD, Peter Papenhausen PhD, and Gerald Wertheim MD PhD
The Children’s Hospital of Philadelphia
Perelman School of Medicine at the University of Pennsylvania
Philadelphia, PA

2. Clinical History 6.7 60% blasts 17.5 65
10 yo boy presented with bone pain and fatigue
17.5 65
6.7
60% blasts
Choi SM et al. J Hematopathology, 2013.

3. Flow cytometry: B lymphoblastic leukemia
Positive
Negative
CD19
CD3
CD10
CD20
CD22
s/cIgM
CD34
Kappa
CD38
Lambda
HLA-DR
MPO
TdT

4. 52,XY,+X,+Y,+14,+14,+21,+21[cp20].
Choi SM et al. J Hematopathology, 2013.

5. ETV6/RUNX1 (TEL/AML1) FISH
Chr 12
X 2
ETV6/RUNX1 (TEL/AML1) FISH. Four RUNX1 signals (chromosome 21, red) and two ETV6 signals (chromosome 12, green) are identified.
Chr 21
X 4
Choi SM et al. J Hematopathology, 2013.

6. Diagnosis: Hyperdiploid karyotype? B lymphoblastic leukemia
52,XY,+X,+Y,+14,+14,+21,+21[cp20].

7. All suggest that the original clone is near-haploid:
Several features of this patient’s karyotype are atypical for true hyperdiploidy.
52,XY,+X,+Y,+14,+14,+21,+21
Gain of chromosome Y uncommon
Tetrasomy for chromosomes other than 21
Lack of trisomy 4, 10, 17
All suggest that the original clone is near-haploid:
26<1n>,XY,+14+21

8. Hypodiploid B-ALL shows non-random chromosome retention
Non random retention of chromosomes XY, 21, 14, and 18 for unknown reasons
Safavri et al. Leukemia, 2012.

9. What if you have no hypodiploid clone and you suspect masked hypodiploidy?
However,
52,XY,+X,+Y,+14,+14,+21,+21[cp20].

10. Basic principle of SNP Array
Uses genome-wide single nucleotide polymorphism probes
Identifies both copy number alterations and loss of heterozygosity.
Detect copy number alterations ≥15 SNPs in number and regions of homozygosity>5 Mb.
Genome wide single nucleotide polymorphism (SNP) microarray analysis was carried out using the Illumina CRC BeadChip. This chip contains disease-associated gene centric content, with >800,000 SNP loci from chromosomes 1-22, >29,000 loci from the X chromosome and >2,000 loci from the Y chromosome. Nucleotide positions are determined according to the hg19 (February 2009) assembly of the human reference sequence. Analysis of this data is targeted to detect copy number alterations greater than or equal to 15 SNPs in number and regions of homozygosity greater than 5 Mb in size. Balanced rearrangements, such as translocations or inversions, will not be detected by this array unless they are accompanied by an alteration in copy number. Abnormalities present in less than 20% of cells may also be undetectable. Any additional analyses carried out on this specimen are reported separately.

11. 850KCyto-SNP array (Illumina)
AA=0
BB=1
AB= 0.5
B-allele frequency
0=2 copies
Log R ratio
Limitation: Balanced rearrangements will not be detected on this platform

12. Utility of SNP: Interstitial deletion
Region of LOH
Decreased copy number

13. Utility of SNP: Copy-neutral LOH
Regions of LOH
No change in copy number
Report if >5Mb in length

14. SNP analysis: Chromosome 14
Homozygous for allele A (AA)
Heterozygous (AABB)
Fig 4 SNP analysis of representative disomic and tetrasomic chromosomes. Bottom y-axis histogram represents copy number state and top y-axis histogram segregates allele calls based on dosage (A=+0.5, B=-0.5). (a) SNP analysis of chromosome 14.
Bottom histogram shows signal levels consistent with four copies of all loci analyzed. Top histogram demonstrates the mid-line (0 line) heterozygosity line throughout the analyzed region.
Homozygous for allele B (BB)
Log dose = 4
Copy Number 4n

15. SNP analysis: Chromosome 2
Homozygous for allele A (AA)
LOH
Copy 2 (normal or uniparental)
Fig 4 SNP analysis of representative disomic and tetrasomic chromosomes. Top y-axis histogram represents copy number state and bottom y-axis histogram segregates allele calls based on dosage (A=+0.5, B=-0.5). (a) SNP analysis of chromosome 14. Top histogram shows signal levels consistent with four copies of all loci analyzed. Bottom histogram demonstrates the mid-line (0 line) heterozygosity line throughout the analyzed region. (b) SNP analysis of chromosome 2. Top histogram shows signal levels consistent with two copies of all loci analyzed. Bottom histogram demonstrates homozygosity at the vast majority of loci analyzed, illustrated by the absence of AB alleles on the center (0) line. This is consistent with loss of heterozygosity.
Homozygous for allele B (BB)
Log dose = 2
Copy Number 2n

16. Diagnosis:
B lymphoblastic leukemia with masked hypodiploid karyotype secondary to duplication of the hypodiploid clone

17. Recurring Chromosomal Rearrangements in B-ALL
Loh and Mullighan. CCR. 2012

18. Key Genetic Events in B-ALL
Genetics
Prognosis
Frequency (%)
Method of Detection
Hyperdiploidy (>50)
Favorable
20-30
Karyotype/FISH
t(12;21) ETV6-RUNX1
25 (peds)
FISH
t(1;19) TCF3-PBX1
Neutral 5
RT-PCR/ FISH
t(5;14) IL3-IGH
<1
Pax5 rearrangements 30
FISH and targeted molecular*
Hypodiploidy (<44)
Unfavorable
1-2
Karyotype/ FISH
t(9;22) BCR-ABL1
25 (adult), 4(peds)
RT-PCR
t(v; 11q23) MLL
CRLF2 rearrangement
5-10
Targeted molecular*
IKZF1 del or mut
15 (peds)
*Targeted molecular: Refers to various next generation and Sanger sequencing methods as well as other PCR based methods.
Mullighan. JCI. 2012
Loh and Mullighan. CCR. 2012

19. Genetic subgroups predict survival in lymphoblastic leukemia.
Hyperdiploid, E2A-PBX1, TEL-AML1
BCR-ABL1
MLL-AF4
Hypodiploidy not included due to low numbers of cases
Pui et al. Lancet, 2008.

20. Prognosis in B-ALL is affected by ploidy.
High hyperdiploid
Hyperdiploid
Hypodiploid
Secker-Walker et al. BJH

21. Near haploid and low hypodiploid groups show poor outcome relative to those with 42-45 chromosomes.
Harrison CJ et al. BJH

22. Hyperdiploid B-ALL Hypodiploid B-ALL
Common; 20-30%
>50 chromosomes
Trisomy 4, 10, 17
80% 5 yr EFS
Likely achieve cure with chemotherapy alone.
Rare; <5% of B-ALL
<46 chromosomes
High Hypodiploid(40-45)
Low Hypodiploid (33-39)
Near haploid (23-29)
30% 5 yr EFS
HSCT is standard of care.
Add picture of karyotype

23. Conclusions
Hypodiploidy portends an extremely poor prognosis in B-ALL.
Due to their rarity and propensity to duplicate their genome, these cases may be misdiagnosed as hyperdiploid B-ALL which will have prognostic and potentially therapeutic implications.
SNP arrays are a valuable tool in identifying copy-neutral LOH and can help confirm a suspected hypodiploid case.

24. Acknowledgements Sarah Choi Gerald Wertheim Peter Papenhausen
Jackie Biegel
Dale Frank
Michele Paessler