G. Del PoetaG. Del Poeta

DeptDept of of HematologyHematology, University , University TorTor Vergata VergataRoma, ItalyRoma, Italy

VALORE PROGNOSTICO DEL GENOTIPO NPM1MUTATO/FLT3-ITD NEGATIVO ED ELEVATI

LIVELLI DI APOPTOSI SPONTANEADETERMINATI IN CITOMETRIA A FLUSSO

NELLA LAM

Struttura del gene Struttura del gene NPM1NPM1

1 2 3 4 5 6 7 8 9 10 11 12

Dominio di oligomerizzazione

Dominio di legame agli acidi nucleici

Proteina (294 aa)W W

NUCLEO CITOPLASMA

Regolazione della sintesi proteica

Crescita, proliferazione e apoptosi cellulare

Stabilità genomica

Gene

Dominio di legame agli istoni

NES NES NLS NoLS

DELOCALIZZAZIONEDELOCALIZZAZIONE

CITOPLASMATICACITOPLASMATICA

Le mutazioni del gene Le mutazioni del gene NPM1NPM1 nelle LMA nelle LMA

1 2 3 4 5 6 7 8 9 10 11 12NES NLS NoLSNES

Falini B. et al. N Engl J Med 2005 Roti G. et al. J Mol Diagn 2006

Importanza clinica dello Importanza clinica dello screeningscreening mutazionalemutazionale di di NPM1NPM1 nella LMA nella LMA

Anomalia genetica più frequente nella LMA

NPM1cNPM1c++ nella LMA dell nella LMA dell’’adultoadulto

NPM1NPM1 nella LMA nella LMA NPM1NPM1 nella LMA-CN nella LMA-CN

NPM1 w/t65%

NPM1c+35%

NPM1 w/t40%

NPM1c+60%

MECHANISM OF APOPTOSISMECHANISM OF APOPTOSIS

DNA DNA damagedamage p53p53growth factor deprivationgrowth factor deprivation bclbcl-2 family-2 familyFasFas/TNF /TNF oncogenesoncogenes ( (mycmyc, E1A), E1A)CeramideCeramide FADD/TRAF/MORTFADD/TRAF/MORTGlucocorticoidsGlucocorticoids Cell cycle regulatorsCell cycle regulators

CysteineproteasesCysteineproteases protein fragmentationprotein fragmentation ((CaspasesCaspases)) ((AnnexinAnnexin V V))

DEATHDEATH DNA DNA fragmentation fragmentation

TRIGGERS MODULATORS

EFFECTORSCLEAVAGE

SUBSTRATES

Bax/bcl-2 Heterodimerization

BCL-2BAX

BAX-BAX

BAX-BCL-2

APOPTOSI

BAXBCL-2

BAX-BAX

BAX-BCL-2

BCL-2/BCL-2

•• NPM1-Mt NPM1-Mt interactsinteracts withwith p53 and p53 and itsits regulatoryregulatory moleculesmolecules(ARF,Hdm2/Mdm2), (ARF,Hdm2/Mdm2), thusthus loweringlowering cellcell proliferationproliferation and andincreasingincreasing apoptosisapoptosis;;

•• Moreover Moreover, AML , AML patientspatients show show coco--existingexisting frequentlyfrequentlyNPM-NPM-MtMt and and FLT3-ITDFLT3-ITD whichwhich increaseincrease potentialpotential forfor cellcellproliferationproliferation..

•• Genes and proteins involved in apoptosis, such as Genes and proteins involved in apoptosis, such as bclbcl-2-2and and baxbax, have been demonstrated to be relevant in response, have been demonstrated to be relevant in responseto treatment and outcome.to treatment and outcome.

INTRODUCTION INTRODUCTION

RATIONALERATIONALE

The The principalprincipal aimsaims of of ourour clinicalclinical researchresearch werewere : :

••I) I) toto correlate correlate NPM1-Mt or FLT3-ITD NPM1-Mt or FLT3-ITD withwithbaxbax/bcl-2 ratio /bcl-2 ratio levelslevels, , asas a a measuremeasure of ofspontaneousspontaneous apoptosisapoptosis;;

••II) II) toto assessassess the the independentindependent prognosticprognosticsignificancesignificance of of NPM1 NPM1 and and FLT3-ITD.FLT3-ITD.

•• NumberNumber 222222

•• MalesMales 120120•• FemalesFemales 102102

•• MedianMedian ageage 60 60 yrsyrs rangerange (21-76) (21-76)

•• TreatmentTreatment•• <60 <60 yrsyrs (101) AML10/12 (101) AML10/12•• >60 >60 yrsyrs (121) AML13-like (121) AML13-like

•• MedianMedian WBC WBC 18.18.33 x10 x1099/L/L rangerange 1.1-305 1.1-305

•• CR rateCR rate (%)(%) 6464

•• MedianMedian SurvivalSurvival 42.42.66 weeksweeks•• Median Median CCRCCR 45.45.55 weeksweeks

•• TransplantTransplant** 46 46 autologousautologous 1122 allogeneicallogeneic**censoredcensored at the time of BM or PBSC at the time of BM or PBSC infusioninfusion

PATIENTPATIENT CHARACTERISTICS I CHARACTERISTICS I

FAB FAB classesclasses nn % %

••M0M0 2121 (10)(10)••M1M1 4949 (22)(22)••M2M2 5656 (25)(25)••M4M4 3232 (14)(14)••M5M5 5858 (26)(26)••M6M6 66 (3)(3)

Cytogenetics Cytogenetics n = 178n = 178 nn % %

••FavorableFavorable 1313 7 7

••IntermediateIntermediate 9292 52 52

•• Poor Poor 7373 4 1 4 1

PATIENTPATIENT CHARACTERISTICS II CHARACTERISTICS II

•• Bcl-2 and Bcl-2 and BaxBax proteinsproteins werewere determineddetermined by by multicolormulticolorflow flow cytometrycytometry on on anan EpicsEpics XL XL instrumentinstrument::

i) i) Anti-CD13/anti-CD33 PE MoAbs were added tomononuclear cells..

iiii) ) After,tthe blast cells were fixed andpermeabilized

iii) Samples were incubated at 4°C for 30 min withanti-bcl-2 FITC or anti-bax. MoAb (Clone Ab-2,Calbiochem).

MATERIALS AND METHODS MATERIALS AND METHODS

LAM M2

Blast Gate

Mean = 55

Mean = 235

Bcl-2 and Bcl-2 and baxbax werewere evaluatedevaluated asas MeanMean FluorescenceFluorescenceIntensityIntensity Ratio (RMFI) and Ratio (RMFI) and then then the the resultsresults werewereexpressedexpressed asas anan IndexIndex ( (bbaxax/bcl-2)./bcl-2).

MFI of MFI of positivelypositively stainedstained cellscells ( (bbcl-2 or cl-2 or baxbax)) MFI of MFI of cellscells stainedstained withwith anan isotypeisotype control control

antibody.antibody.

RMFI RMFI baxbax RRMFI bcl-2MFI bcl-2

MATERIALS AND METHODSMATERIALS AND METHODS

RMFI

Index (bax/bcl-2)

The The thresholdthreshold forfor consideringconsidering AML AML casescases asas““apoptoticapoptotic”” waswas set at the set at the bbaxax/bcl-2 /bcl-2 medianmedianvaluevalue >> 0.35 ( 0.35 (rangerange 0.01 - 9.1). 0.01 - 9.1).

MATERIALS AND METHODSMATERIALS AND METHODS

Bax/bcl-2

No of patients

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

-0,3 0,0 0,3 0,6 0,9 1,2 1,5 1,8 2,1 2,4 2,7 3,0 3,3 3,6 3,9 4,2 4,5 4,8 5,1 5,4 5,7 6,0

Tecniche di laboratorio per lo Tecniche di laboratorio per lo screeningscreening mutazionale mutazionale di diNPM1 e FLT3-ITDNPM1 e FLT3-ITD

•• SequenziamentoSequenziamento genico genico•• D-HPLCD-HPLC•• LightCyclerLightCycler PCR PCR•• PCR quantitativa PCR quantitativa ““RealReal-Time-Time””•• Elettroforesi capillare (EC)Elettroforesi capillare (EC)•• RT-ASO-PCR / semi RT-ASO-PCR / semi nestednested ASO-PCR ASO-PCR

NPM1 mutationsand FLT3-ITDwere detected bymultiplex PCR andcapillary gelelectrophoresis(Noguera,Leukemia, 2005).

BIOLOGICAL RESULTSBIOLOGICAL RESULTS

101121

168

54

170

52

17

0

50

100

150

200

250

300

p

a

t

i

e

n

t

s

bax/bcl2-bax/bcl2+

NPM1-

NPM1+

FLT3ITD-

FLT3ITD+

NPM1+FLT3ITD+

BaxBax/bcl-2, NPM1-Mt and FLT3-ITD/bcl-2, NPM1-Mt and FLT3-ITD

54.5%

7.6%

24.3%23.4%

125

21

30

1015

21

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

p

a

t

i

e

n

t

s

FLT3ITD-FLT3ITD+P<0.00001

WBC WBC countcount and FLT3-ITD and FLT3-ITD

WBC<50 WBC 50-100 WBC>100

52 47 47

7

0

10

20

30

40

50

60

70

80

90

100

110

p

a

t

i

e

n

t

s

NPM1-NPM1+P<0.00001

CD34 and NPM1-MtCD34 and NPM1-Mt

CD34 - CD34+

12

1

55

37

66

7

0

10

20

30

40

50

60

70

80

90

patients

NPM1-NPM1+P<0.00001

KaryotypeKaryotype and NPM1-Mt and NPM1-Mt

Good Normal Poor

82%

Normal Karyotype (92 pts)

28

943

12

NPM1+FLT3-NPM1+FLT3+NPM1-FLT3-NPM1-FLT3+

7

30 25

10 8 9

61

72

0

10

20

30

40

50

60

70

80patients

(%)

NPM1+FLT3- NPM1+FLT3+ NPM1-FLT3+ NPM1-FLT3-

bax/bcl-2<0.3

bax/bcl-2>0.3

P = 0.0001

BaxBax/bcl-2 and NPM1-Mt/bcl-2 and NPM1-Mt

19

2

37

12

49

7

22

10

37

21

4 2

0

10

20

30

40

50

60patients

(%)

M0 M1 M2 M4 M5 M6

NPM1-NPM1+P = 0.03

FAB FAB classesclasses and NPM1-Mt and NPM1-Mt

CLINICAL RESULTSCLINICAL RESULTS

35

90

47

67

0102030405060708090

100patients

(%)

CR(%)

NPM1+FLT3ITD+

NPM1+FLT3ITD-

NPM1-FLT3ITD+

NPM1-FLT3ITD-

P = 0.0002

NPM1-Mt / FLT3-ITD and Complete NPM1-Mt / FLT3-ITD and Complete RemissionRemission (%) (%)

NPM1+FLT3-

NPM1+FLT3+

NPM1-FLT3+

NPM1-FLT3-

Overall Survival by NPM1-Mt and FLT3ITD

Days from Diagnosis

Cu

mu

lativ

e P

rop

ortio

n S

urv

ivin

g

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

0 365 730 1095 1460 1825 2190 2555 2920 3285

P=0.00007

35%

0%6%

15%

NPM1+FLT3-

NPM1+FLT3+

NPM1-FLT3+

NPM1-FLT3-

Disease free Survival by NPM1-Mt and FLT3ITD

Days from Diagnosis

Cu

mu

lativ

e P

rop

ortio

n R

ela

psin

g

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

0 365 730 1095 1460 1825 2190 2555 2920

P = 0.13

44%

0% P=0.008

Overall Survival by NPM1 and FLT3-ITD inAML pts > 60 years

Days from diagnosis

Cu

mu

lati

ve P

rop

ort

ion

Su

rviv

ing

0 365 730 1095 1460 1825 2190 2555 2920 32850.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

P = 0.0002

n = 121 pts NPM1mt FLT3/ITD-

NPM1mt FLT3/ITD+

NPM1wt FLT3/ITD+

NPM1wt FLT3/ITD-

Days from Diagnosis

Cu

mu

lati

ve P

rop

ort

ion

Su

rviv

ing

0 365 730 1095 1460 1825 2190 2555 2920 32850.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

P = 0.00005

NPM1mt FLT3/ITD- Bax/bcl-2 >0.35

All other groups

Days from diagnosis

Cu

mu

lati

ve P

rop

ort

ion

Rel

apsi

ng

0 365 730 1095 1460 1825 2190 2555 29200.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

P = 0.004

NPM1mt FLT3/ITD-

Bax/bcl-2 >0.35 All other groups

MULTIVARIATE ANALYSIS (CR and OS)MULTIVARIATE ANALYSIS (CR and OS)Outcome, variable Odds Ratio (CI) PCR NPM1 status 1.78 (0.44-7.27) 0.42

FLT3-ITD 0.52 (0.20-1.34) 0.18

Int. NPM1 x FLT3-ITD 0.16 (0.02-1.29) 0.085 Bax/bcl-2 ratio >0.35 16.42 (6.42-41.99) <0.0001

Age >60 y 0.28 (0.12-0.62) 0.002

WBC (50-100x109/L) 0.61 (0.21-1.77) 0.36

WBC (>100x109/L) 0.72 (0.25-2.10) 0.54

Outcome, variable Hazards Ratio (CI) POS NPM1 status 1.35 (0.86-2.11) 0.19

FLT-3 ITD 0.85 (0.49-1.48) 0.57

Int. NPM1 x FLT3-ITD 2.35 (1.03-5.40) 0.043

Bax/bcl-2 ratio >0.35 0.34 (0.24-0.49) <0.0001

Age > 60 y 1.06 (0.75-1.49) 0.74

WBC (50-100x109/L) 1.51 (0.94-2.42) 0.09

WBC (>100x109/L) 1.80 (1.12-2.87) 0.01

CI, confidence intervals; Int, multiplicative interaction between NPM1 and FLT3-ITD.

CONCLUSIONSCONCLUSIONS

•• NPM1-mutated/FLT3-ITD negative patients exhibit NPM1-mutated/FLT3-ITD negative patients exhibithigh levels of spontaneous apoptosishigh levels of spontaneous apoptosis, thus explaining, thus explainingtheir their more favorable response to therapymore favorable response to therapy..

•• NPM1 mutations in the absence of FLT3-ITD NPM1 mutations in the absence of FLT3-ITDidentify a subgroup of patients with favorableidentify a subgroup of patients with favorableprognosis and in patients with both mutations prognosis and in patients with both mutations FLT3-FLT3-ITD dominates the ITD dominates the leukemicleukemic phenotype conferring a phenotype conferring apoor outcome.poor outcome.

ACKNOWLEDGMENTSACKNOWLEDGMENTS

M.I. Del PrincipeM.I. Del PrincipeA. A. VenVendittidittiS. S. Amadori Amadori Reparto Ematologia PoliclinicoReparto Ematologia Policlinico Tor Tor Vergata Vergata

E. E. AmmatunaAmmatunaF. F. BuccisanoBuccisanoS. S. ZazaZazaT. T. OttOttooneneS. S. LavorgnaLavorgnaF. Lo F. Lo Coco Coco Laboratorio Ematologia Policlinico Laboratorio Ematologia Policlinico Tor Tor VergataVergata

F. LucianoF. LucianoL. L. MaurilMaurillo lo Laboratorio Ematologia Ospedale Laboratorio Ematologia Ospedale S.S.EugenioEugenio