“Evoluzione clonale delle

mutazioni di FLT3 nella LMA”

Tiziana Ottone

Dipartimento di Biomedicina e Prevenzione

Università di Roma Tor Vergata

Firenze, 22 novembre 2012

The FLT3 receptor

FLT3 receptor

Proliferation  

Differentiation  

The FLT3 gene Tyrosine kinase receptor

Mutations

Survival

Constitutively activated tyrosine

kinase receptor

Leukemogenesis

Juxtamembrane region (JM)

TK1

TK2

FLT3 TKD mutations exon 20

-  Missense point mutations/deletions

codon 835, 836

- Codons 832, 837, 839, 840, 842

FLT3 ITD mutations exon 14/15

- Insertions (deletions): -6 - > 400

bp

- Rare point mutations

FLT3 mutations in AML

FLT3 w/t FLT3 ITD

FLT3 w/t

FLT3 TKD

FLT3 mutations in AML

 Are almost exclusively found in AML (7-10% TKD and 20-25% ITD)

 FLT3 ITD hyperleukocytosis and a high percentage of bone marrow blast cells

  FLT3 mutations are frequently associated with NC-AML and NPM1 mutations

 FLT3 ITD is a poor prognostic factor with negative impact on disease-free and overall

survival in patients with intermediate-risk cytogenetics AML (NC-AML)

 Allogenic HSCT in 1° CR should be considered in patients with FLT3 ITD

FLT3 is a potential therapeutic target in AML

PKC412 CEP701

The success of FLT3 inhibitors when used as single agents has thus far

been limited by transient responses and the emergence of resistance

Inhibit multiple kinases, among them FLT3

Sorafenib AC220 Are more specific for FLT3 and much more potent FLT3 inhibitors

Several laboratory and clinical studies have shown that FLT3 is a promising therapeutic

target and various FLT3 inhibitors are currently under investigation in phase II-III

clinical trials

FLT3 ITD evaluation in AML patients

FLT3 mutations are unstable, and mutational status may change during the disease

course following chemotherapy.

However, it was unclear whether in such cases the FLT3 ITD aberration

was acquired at relapse or, alternatively, if the mutation was present at

low levels already at the time of first diagnosis.

FLT3 ITD-ve / FLT3 ITD+ve

FLT3 ITD+ve / FLT3 ITD-ve

FLT3 ITD

FLT3 wt

FLT3 wt

FLT3 wt

FLT3 wt

FLT3 wt

FLT3 wt

Below the detection limit of

conventional assays

Survival advantage because they

are resistant to chemotherapy

FLT3 ITD

FLT3 ITD

FLT3 ITD

FLT3 ITD

FLT3 ITD FLT3 wt FLT3 wt

FLT3 wt

FLT3 ITD

FLT3 ITD

FLT3 ITD

Diagnosis Relapse

FLT3 wt

FLT3 wt

FLT3 wt

FLT3 wt

FLT3 wt

FLT3 wt

FLT3 wt

FLT3 ITD FLT3 ITD

FLT3 ITD

FLT3 ITD

FLT3 wt

FLT3 wt

FLT3 wt

FLT3 wt FLT3 ITD

FLT3 ITD

FLT3 ITD

FLT3 ITD-ve / FLT3 ITD+ve

FLT3 ITD evaluation in AML patients

To gain further insights into the significance of small FLT3 ITD clones and the kinetics

of clonal evolution of cells carrying FLT3 ITD in NC-AML

6 AML patients

FLT3 wt at diagnosis

6 AML patients

FLT3 ITD at relapse

By conventional PCR FLT3 w/t

FLT3 w/t

FLT3 ITD

FLT3 ITD

FLT3 ITD

FLT3 ITD

FLT3 ITD

FLT3 wt

FLT3 wt

FLT3 wt

FLT3 wt

FLT3 ITD ?

FLT3 ITD AML relapse may evolve from small subclones undetectable at diagnosis by

routine RT-PCR

Clinical and biological characteristics

of AML patients

UPN Age Sex WBC % Blast Karyotype FLT3 ITD* NPM1* FLT3 ITD* NPM1* Outcome

(x103/µl) (BM) at diagnosis at diagnosis at relapse at relapse

1 46 M 23 93 46,XY negative positive positive positive Relapsed at 11 mos, dead

2 55 F 18 95 46,XX negative positive positive positive Relapsed at 12 mos, dead

3 58 M 13 95 46,XY,i(17)(q10) negative positive positive positive Relapsed at 6 mos, dead

4 39 M 41 94 46,XY negative positive positive positive 1st Relapse at 4 mos,

2nd relapse at 1 mo. post-alloSCT‡, dead

5 59 M 30 70 46,XY negative positive positive positive Relapsed at10 mos, alive

6 53 M 28 75 46,XY negative positive positive positive Relapsed at 6 mos, alive

Abbreviations: UPN, unique patient number.

* by conventional PCR, ‡ allogenic stem cells transplantation

Gene III

pOCI1050

Ampr

Ori M13

ColE1

XbaINotI XhoI

BamHI

Sequenza casuale di 150 nucleotidi

C-mycGene III

pOCI1050

Ampr

Ori M13

ColE1

XbaINotI XhoI

BamHI

Sequenza casuale di 150 nucleotidi

C-myc

FLT3 ITD evaluation in AML patients

We developed a patient-specific RQ-PCR to implement FLT3 ITD detection

Relapsed sample FLT3 ITD

Patient-specific FLT3 ITD purification

Recombination cloning in bacteria

Patient-specific FLT3 ITD sequence

FLT3 ITD

Development of patient-specific RQ-PCR

Localization of primers and probe used for patient-specific RQ-PCR

Exon 14 Exon 15 ITD

Patient-specific forward primer

Reverse primer

Probe cDNA of FLT3 ITD

1 ATTTCAGAGAATATGAATATGATCTCAAATGGGAGTTTCCAAGAGAAAATTTAGAGTTTGGTAATTTCAGAGAATATGAATATGATCTCAAATGGGAGTTTCCAAGAGAAAATTTAGAGTTTG

AAGAGAAAATATAGAGTTTGGTAATTGCA

UPN 1

2 CAGAGAATATGAATATGATCTCAACAGAGAATATGAATATGATCTCAA

TATGATCACAACAGAGAATAAGAA

UPN 2

3 TATGAATATGATCTCAAATGGTATGAATATGATCTCAAATGG

GAATATGATCTCAAATGGTAAGAAT

UPN 3

4 AGAGAATATGAATATGATCTCAAATGGGAGTTTCCAAGAGAAAATTTAAGAGAATATGAATATGATCTCAAATGGGAGTTTCCAAGAGAAAATTTA

GTTTCCAAGAGAAAATTCAAGA

UPN 4

5 GATTTCAGAGAATATGAATATGATGATTTCAGAGAATATGAATATGAT

TTCAGAGAATATGAATATGATGATTTC

UPN 5

6 TACGTTGATTTCAGAGAATATGAATATGATCTCAAATGGGAGTTTCCAAGAGAAAATTTAGAGTTGGGATACGTTGATTTCAGAGAATATGAATATGATCTCAAATGGGAGTTTCCAAGAGAAAATTTAGAGTTT

TTTCCAAGAGAAAATTTAGAGTTGCGAT

UPN 6

FLT3 ITD patient-specific RQ-PCR

The assay showed maximum reproducible sensitivity and specificity at

10-4 in five patients and at 10-5 in one case.

Clinical and biological characteristics

of AML patients

UPN Age Sex WBC % Blast Karyotype FLT3 ITD* FLT3 ITD/ ABLx104†

NPM1A/ ABLx104 FLT3 ITD*

FLT3 ITD/ ABLx104†

NPM1A/ ABLx104 Outcome

(x103/µl) (BM) at diagnosis at diagnosis at diagnosis at relapse at relapse at relapse

1 46 M 23 93 46,XY negative 0 NA positive 9.32 x 104 NA Relapsed at 11 mos, dead

2 55 F 18 95 46,XX negative 0 7.55 x 105 positive 3.36 x 105 5.73 x 105 Relapsed at 12 mos, dead

3 58 M 13 95 46,XY,i(17)(q10) negative 4.82 x 103 1.04 x 106 positive 5.66 x 105 1.5 x 106 Relapsed at 6 mos, dead

4 39 M 41 94 46,XY negative 4.43 x 104 3.49 x 105 positive 7.72 x 106 2.55 x 105 1st Relapse at 4 mos,

2nd relapse at 1 mo. post-alloSCT‡, dead

5 59 M 30 70 46,XY negative 6.27 x 102 ND positive 3.00 x 106 ND Relapsed at10 mos, alive

6 53 M 28 75 46,XY negative 9.27x102 4.44 x 105 positive 1.26x104 2.25 x 105 Relapsed at 6 mos, alive

Abbreviations: UPN, unique patient number; NA, not applicable as this patient had a rare NPM1 mutation Km.; ND, not done because of unavailability of RNA samples.

* by conventional PCR, † by patient-specific RQ-PCR, ‡ allogenic stem cells transplantation

Copy number analysis of FLT3 ITD

by patient-specific RQ-PCR

D

R

R

D

R

D

D

R

Amplification plot following patient-specific RQ-PCR

for FLT3 ITD at diagnosis, post-induction and relapse

Relapse Diagnosis

Post

induction

FLT3 w/t

(367 bp)

Diagnosis

Fluo

resc

ence

inte

nsity

(arb

itrar

y un

its)

Fragment length

Electropherogram analysis of FLT3 ITD of UPN 3 by

gel electrophoresis and fluorescence detection

Post-induction

Fluo

resc

ence

inte

nsity

(arb

itrar

y un

its)

Fragment length

FLT3 w/t

(367 bp)

FLT3 ITD

(412 bp)

Relapse

Fluo

resc

ence

inte

nsity

(arb

itrar

y un

its)

Fragment length

FLT3 ITD

(412 bp)

Parallel analysis of FLT3 ITD and NPM1 mutation A

by RQ-PCR in UPN 3

Diagnosis Relapse

FLT3 ITD evaluation in AML patients

5 pt: normal cytogenetics

1 pt: 46,XY,i(17)(q10)

Identical at relapse in 5 evaluable cases

6 pt: NPM1 mut

By conventional RT-PCR

6 pt: NPM1 mut

UPN 1

UPN 2

UPN 4

UPN3

UPN5 UPN 6

UPN 1

UPN 2

UPN 4

UPN3

UPN5 UPN 6

Identify FLT3 ITD in 4 diagnostic samples which had been labeled as FLT3

w/t using the conventional RT-PCR

UPN 4

UPN3

UPN5 UPN 6

Copy number of patient-specific FLT3 ITDs

6.27x102 and 4.43x104 1.26x104 and 7.72x106

Diagnosis FLT3 w/t Relapse FLT3 ITD

By patient-specific RQ-PCR

FLT3 ITD by conventional RT-PCR

UPN Outcome

1 Relapsed at 11 mos, dead 2 Relapsed at 12 mos, dead 3 Relapsed at 6 mos, dead 4 1st Relapse at 4 mos, 2nd relapse at 1 mo. post-alloSCT, dead 5 Relapsed at10 mos, alive 6 Relapsed at 6 mos, alive

Post-induction

Fluo

resc

ence

inte

nsity

(arb

itrar

y un

its)

Fragment length

FLT3 w/t

(367 bp)

FLT3 ITD

(412 bp)

Fluo

resc

ence

inte

nsity

(arb

itrar

y un

its)

Fragment length

FLT3 w/t

FLT3 ITD

FLT3 w/t

FLT3 ITD

Fluo

resc

ence

inte

nsity

(arb

itrar

y un

its)

Fragment length

FLT3 ITD evaluation in AML patients

Discussion and conclusion

The instability of FLT3 ITD during AML disease course has been reported in several studies

A patient-specific RQ-PCR based strategy may unravel FLT3 ITD low levels in CN-AML

which are undetectable at presentation by conventional RT-PCR

FLT3 ITD was documented by routine PCR in 3/6 cases during remission, strongly

suggesting the need of repeated testing of FLT3 status after diagnosis and during

treatment in CN-AML

In particular, our data suggest that CN-AML patients positive for NPM1 mutations,

should be tested for FLT3 ITD on routine basis during the disease course

Although obtained in a small series, our data also suggest that presence of NPM1 mutations

carries unfavourable prognosis when accompanied by FLT3 ITD even if the latter is only

present at subclonal level.

Thanks for your attention!