Molecular genetics of ALLSabina Chiaretti, MD, PhD

IV International Eurasian Hematology CongressAntalya, 9-13 October 2013

Acute lymphoblastic leukemia (ALL)

ALL is a malignant disorder characterized by the uncontrolledproliferation and accumulation of immature cells

In children, the CR rates and long-term survival reach 95% and 80%

In adults the 5-year OS rate is 30-40%

Moorman AV Blood (2010)

ALL affects both children -representing the mostcommon neoplasm in childhood- and adults withthe highest incidence in children aged 2-5

Adolescents and young adults (AYAs) show an improved survival when treated with pediatric-like protocols

Pui CH NEJM (2006)

Molecular genetics of B-lineage ALL

Courtesy of Chiaretti S.

Recurrent chromosomal rearrangments

IKZF1PAX5EBF1

CDKN2ACDKN2B

PTENRB1ETV6ERG

CD200TOX

Copy number aberrations

A considerable portion of patients lacksrecurrent gross chromosomal alterations andare considered “genetically unclassified”

Chiaretti et al, Haematologica 2013

How to analyze these cases?

• Gene expression profling

• CNA

• NGS

• ETP leukemias

• BCR/ABL1-like cases

• Hypodiploid cases

• TP53 disruption

ETP in Children

Represent 12% of T-ALLCoustan-Smith et al, Lancet Onc, 2009

ETP-ALL and Myeloid like T-ALL

MPOLYZCTSB,G,SELA2MMP8PRTN3ECP

Myeloid antigens:

Myeloid transcription factors:

Granule proteins:

MNDACEBPBCEBPACEBPDKLF4MAFBMXD1

CD11BCD66CCD24CD83CD114 (CSF3R)

0

0.05

0.1

0.15

0.2

0.25

T-ALL

Myeloid-like clusterAML

HDCD2+

miR-223

Myeloid-like T-ALL

Other T-ALL

At variance from pediatric cases, this group (10%) was recognized by GEP.Chiarettti et al, Haematologica 2010

By NGS, ETP-leukemias are characterized by mutations in signaling pathways and genes of myleoid lineage

Zhang J et al, Nature 2012

Identification of typical AML mutations in immature T-ALL of adult patients:IDH1, IDH2, DNMT3A, FLT3 and NRAS.Prominent role of ETV6 mutations (Van Vlierberghe et al, JEM 2011)

Zhang et al, Nature 2012

AML T-ALLETP

Other mutations in T-ALL

• NT5C2 5'-nucleotidase enzyme thatinactivates purine analogs: mutations detectedin 19% relapse T cell ALLs (Tzoneva et al, NatMed 2013).

• CNOT3 mutations identified in 8% adult T-ALLs, and able to cause tumors in Drosophila(De keersmaecker et al, Nat Gen 2013).

GEP previously identified the presence of aBCR/ABL-like subgroup

Haferlach et al, Blood 2005Chiaretti et al, CCR 2005

Anova analysis (p ≤ 0.00001) between BCR/ABL1-like (n=17), BCR/ABL1+ (n=73) and BCR/ABL1- (n=61) cases

366 known differentially expressed genes.

Tight clustering between BCR/ABL1-like and BCR/ABL1+.

Roughly 80 genes more highly expressed in BCR/ABL1–like.

BCR/ABL1-like

BCR/ABL1-

BCR/ABL1+

B NEG Ph +Ph-like

Affymetrix

normalized

values

CRLF2 expression

Significantly higherexpression levels ofCRLF2

Limited to a subset ofBCR/ABL1-like cases(n=7/17, 41%).

BCR-ABL1-like subset in childrenGEP: identification of a subset ofchildren (COALL and DCOG),accounting for 17% of cases, witha BCR/ABL1-like profile andfrequent deregulation of CRLF2.

Clinical findings: worse outcome,increased WBC count, poorresponse to L-ASP and DNM.

Array-CGH: aberratios involvingIKZF1, PAX5, TCF3, and VPREB1

Den Boer et al, Lancet 2009

In high risk ALL, RNA-seq has identified novel mutations that involve TKs in the majority of cases: they appear to have transforming capability and to respond to TKIs. (Roberts KG, et al Cancer Cell 2012 )

Haploidy and ALLThe transcritpional profiling is very different between near haploid, low hypodiploid and near diploid ALL

RTKs and RAS signaling mutations affect haploid casesTP53 and IKZF2 mutations low hypodiploid and near diploid cases

Holmfeldt et al, Nat. Gen 2013, holmfeldt et al

Inhibition of RAS and PI3K reduces tumor growth in vitro

24-31 32-39

Age cohorts

Years

Pro

babi

lity

NGS in different B-ALL age cohorts

Is/are there underlying lesion/s that could be related to the different outcome?

Chiaretti et al, Haematologica 2013

Experimental strategy

Whole ExomeGenes resulting

from the Discovery panel

Recurrentlymutated genesresulting fromthe Screening

by WES

WESDiscovery panel15 B-ALL T and N(5 children, 5 AYA,

5 adults)

WESScreening panel72 B-ALL only T(14 children, 28 AYA, 30 adults)

SangerScreening panel50 B-ALL only T(9 children, 21 AYA, 20 adults)

All the cases evaluated did not harbor any known molecular rearrangement

^Suspected problem with paired normal or purity of the tumor sample

Mutational load in B-NEG* ALL- discovery panel *No common chromosomal rearrangements

Type of mutations

8.8%

Children Adolescents Adults Average N°

mutated genes 11 9.8 7.4

N°

of m

utat

ed g

enes

Overall 135 mutated genes (9.3 mutated genes/case)

57 59 61 63 65 67 69 71 73 75 77 79^ 81 83 85^

FLT3PREX2

FLT3CSMD3

CSMD3

PREX2

Load of mutations

19

10

SNP ID

Fasteris ID

98

9

7

1314

5

10 10

1

7

14

3

FLT3 CSMD3 PREX2

Process/Pathway Response to cytokine stimulus/Hemopoiesis

G-protein coupled receptor signaling pathway/ Interaction with PTEN

Sample ID ALL_4 (63) ALL_8 (71) ALL_10 (75) ALL_13 (81) ALL_8 (71) ALL_14 (83)

Frequency 13.3% 13.3% 13.3%

Mutation type In frame deletion Missense Missense Missense Missense Missense

AA change Δ836-837 Y842N G28R P3046H R189W K904I

Affected functional domain Kinase Kinase(Autocatalysis site) - CUB DH -

COSMIC (N of mutations) Yes (748) Yes (87) Yes (23)

COSMIC overlapping mutations

No No No

Involvement in hematologic diseases

Yes, AML, ALL No No

CGC* Yes No No

PolyPhen-2 prediction (score)

nd damaging (1) damaging (0.96) damaging (1) damaging (0.97) benign (0.2)

CNA (N of genes in the lesion, sample ID)

No No No

GEP call Present Absent Present/Absent

N of coding exons 24 71 40

Features of the recurrently mutated genes

*Cancer Gene Census database, March 2011 (www.sanger.ac.uk/genetics/CGP/Census). Abbreviations. AA, amino acid; fs, frameshift; D, deletion; CNA, Copy Number Aberration; nd, not determined (The PolyPhen-2 algorithm predicts only the impact of amino acid substitutions).

Incidence of mutations in the “WES screening panel” (n=72)

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

CSM

D3EX

PH5

GRIN

2BKR

ASPA

X5PT

PRZ1

CACN

A1G

CNTN

AP5

EGFL

AMLP

HN2

LRBA

MAP

1BM

BTPS

1M

DN1

SSH1

VWDE

ABCC

2CO

L6A1

DCAF

5FB

N2

FLT3

ITGA

11KI

AA17

55M

YO6

NPH

S1N

RAS

OR8

B8SE

TD2

SLC2

5A46

SS18

L1ZD

HHC1

4AK

AP8

ANO

5CC

NE2

CNGB

1CO

L11A

1FG

FR3

GAST

GTF2

A1L,

STO

N1-

GTF2

A1L

IL1R

APL1

KIAA

1614

KLF5

LGR5

LON

RF2

LRRC

28LR

RC30

MYH

13PC

BP4

PCDH

A11

PLXN

A3PR

EX2

SCG2

TNRC

6BTR

PC7

WW

C1

Overall, the recurrence is low (maximum frequency: 4 cases, 5%)

Most frequently inolved pathways KEGG pathway Benjamini adjusted

p-valueGenes involved

Long-termpotentiation

2.2-1GRIN2B/NRAS/KRAS

Regulation of actincytoskeleton

2.5-1NRAS/KRAS/SSH1/FGFR3/ ITGA11

Acute myeloidleukemia 2.7-1 FLT3/NRAS/KRAS

Cancer 2.7-1 CCNE2/FGFR3/FLT3/NRAS/KRAS

MAPK signaling 3.5-1 CACNA1G/FGFR3/NRAS/KRAS

ECM-receptorinteraction 2.7-1 COL11A1/COL6A1/ITGA11

Preliminary* results of the screening of selectedgenes by Sanger (WGA DNA)

- 50 B-NEG ALL 20 adults, 21 AYA, 9 children -

*Validation on Tumor gDNA and Germline DNA (if available) ongoing

% o

fmut

ated

case

s

0

5

10

15

20

25

30

35

Preliminary results of the screening of selectedgenes by Sanger (WGA DNA)

- 50 B-NEG ALL 20 adults, 21 AYA, 9 children -

If the mutations found on the WGA are validated on gDNA, 40% of B-ALL cases display at least one mutation in these genes with KRAS being the most frequently mutated (20%) followed by FLT3 (14%) and NRAS (12%)

TP53 in relapsed childhood ALL (I)

In pediatric cohorts, TP53 copynumber and sequence alterationsin 12.4% of B-ALL and 6.4% ofpatients with T-ALL at relapse.Correlation with poor outcome Increased incidence in ALL1/AF4+cases

Hof J et al. JCO 2011;29:3185-3193

TP53 in relapsed childhood ALL(II)

Krentz S et al, Leukemia 2013

Holmfeldt et al, Nat Gen 2013

TP53 in low-hypodiplod ALL

TP53 aberrations found in 91.2% of childhood LH and 90% LH adult ALL; in 43.3% of children, but not in adults , mutations are detected also in non tumor cells

TP53 mutations in newly-diagnosed adultALL (I)

8.2%

98 adult patients (median age 36.5 years )at the onset of disease

62B-ALL

25 BCR/ABL1*24 No aberrations*9 ALL1/AF44E2A/PBX1

36 T-ALL

27 no aberrations*7 SIL/TAL1+1 NUP214/RAP+1 SET/NUP214+

Charetti et al, Haematologica 2013

* A minority of cases studied at diagnosis and relapse

Charetti et al, Haematologica 2013

TP53 mutations in newly-diagnosed adultALL (II)

6.4%

11.1%

TP53 mutant = 6.4% (4/62)

TP53 wild-type = 87.6% (58/62)

BCR/ABL1+ : 4% (1/25)

No aberrations : 12.5% (3/24)

E2A/PBX1+ : 0/4

ALL1/AF4+ : 0/9 B-ALL

Combining B-ALL and T-ALL, TP53mutations are detected in 14% of cases without recurrent molecular aberrations

TP53 mutant (n=8) TP53 wild-type (n=90)

Age at diagnosis (median) 30.12 36.42

Gender: M/F 6/2 54/36

WBC X109/L (median) 52.03 57.05

PLTS x109/L (median) 52.5 45

TP53 mutant (n=7) TP53 wild type (n=36) p

CR (n=30) 3 27

0.05Refractory (n=6) 3 3

Death in induction (n=7)

1 6

TP53 mutations in newly diagnosed adult ALL correlate with refractoriness to induction therapy

Cases with no molecular aberration

TP53 mutations in adult ALL increase at relapse

Diagnosis TP53 mutant : 0/10 Relapse: TP53 mutant 2/10

Though the numbers are small,the percentage of TP53 mutated cases increases at relapse

ConclusionsNovel sugroups identified andcurrently well characterized

Some of the novel molecularlesions identified could be targetedwith specific inhibitors

NRAS/KRAS FLT3RTK in high-risk ALL

Ruxolitinib,Dasatinib

?

PI3K and mTORinhibitors

QuizartinibPonatinib

Acknowledgments

Monica MessinaFulvia BrugnolettiAlina NeguliciAntonella VitaleAnna GuariniRobin Foà

Raul RabadanJiguang WangLaura Pasqualucci