VALORE PROGNOSTICO DEL GENOTIPO NPM1 MUTATO/FLT3-ITD NEGATIVO ED ELEVATI LIVELLI DI APOPTOSI SPONTANEA DETERMINATI IN CITOMETRIA A FLUSSO NELLA LAM

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1 VALORE PROGNOSTICO DEL GENOTIPO NPM1 MUTATO/FLT3-ITD NEGATIVO ED ELEVATI LIVELLI DI APOPTOSI SPONTANEA DETERMINATI IN CITOMETRIA A FLUSSO NELLA LAM G. Del Poeta Dept of Hematology,, University Tor Vergata Roma, Italy

2 Struttura del gene NPM1 Gene Proteina (294 aa) NES NES NLS NoLS W W Dominio di oligomerizzazione Dominio di legame agli istoni Dominio di legame agli acidi nucleici NUCLEO CITOPLASMA Regolazione della sintesi proteica Crescita, proliferazione e apoptosi cellulare Stabilità genomica

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5 Le mutazioni del gene NPM1 nelle LMA NES 4 5 NES Roti G. et al. J Mol Diagn NLS NoLS DELOCALIZZAZIONE CITOPLASMATICA Falini B. et al. N Engl J Med 2005

6 Importanza clinica dello screening mutazionale di NPM1 nella LMA Anomalia genetica più frequente nella LMA NPM1c + nella LMA dell adulto NPM1 nella LMA NPM1 nella LMA-CN NPM1 w/t 65% NPM1c+ 35% NPM1 w/t 40% NPM1c+ 60%

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8 MECHANISM OF APOPTOSIS TRIGGERS MODULATORS DNA damage growth factor deprivation Fas/TNF Ceramide Glucocorticoids EFFECTORS p53 bcl-2 family oncogenes (myc,, E1A) FADD/TRAF/MORT Cell cycle regulators CLEAVAGE SUBSTRATES Cysteineproteases (Caspases) DEATH protein fragmentation (Annexin V) DNA fragmentation

9 Bax/bcl-2 Heterodimerization BAX BCL-2 APOPTOSI BAX BCL-2 BAX-BAX BAX-BAX BAX-BCL-2 BAX-BCL-2 BCL-2/BCL-2

10 INTRODUCTION NPM1-Mt interacts with p53 and its regulatory molecules (ARF,Hdm2/Mdm2), thus lowering cell proliferation and increasing apoptosis; Moreover,, AML patients show co-existing frequently NPM-Mt Mt and FLT3-ITD which increase potential for cell proliferation. Genes and proteins involved in apoptosis, such as bcl-2 and bax,, have been demonstrated to be relevant in response to treatment and outcome.

11 RATIONALE The principal aims of our clinical research were : I) to correlate NPM1-Mt or FLT3-ITD with bax/bcl-2 ratio levels, as a measure of spontaneous apoptosis; II) to assess the independent significance of NPM1 and FLT3-ITD. prognostic

12 PATIENT CHARACTERISTICS I Number 222 Males 120 Females 102 Median age 60 yrs Treatment range (21-76) <60 yrs (101) AML10/12 >60 yrs (121) AML13-like Median WBC 18.3 x10 9 /L range CR rate (%) 64 Median Survival 42.6 week Median CCR 45.5 week Transplant* autologou 12 allogeneic *censored at the time of BM or PBSC infusio

13 PATIENT CHARACTERISTICS II FAB classes n % M0 21 (10) M1 49 (22) M2 56 (25) M4 32 (14) M5 58 (26) M6 6 (3) Cytogenetics n = 178 n % Favorable 13 7 Intermediate Poor 73 4

14 MATERIALS AND METHODS Bcl-2 and Bax proteins were determined by multicolor flow cytometry on an Epics XL instrument: i) Anti-CD13/anti-CD33 PE MoAbs were added to mononuclear cells.. ii) After,the blast cells were fixed and permeabilized iii) Samples were incubated at 4 C for 30 min with anti-bcl-2 FITC or anti-bax. MoAb (Clone Ab-2, Calbiochem).

15 LAM M2 Blast Gate

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17 Mean = 55

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19 Mean = 235

20 MATERIALS AND METHODS Bcl-2 and bax were evaluated as Mean Fluorescence Intensity Ratio (RMFI) and then the results were expressed as an Index (bax/bcl-2). RMFI MFI of positively stained cells (bcl-2 or bax) MFI of cells stained with an isotype control antibody. Index (bax/bcl-2) RMFI bax RMFI bcl-2

21 MATERIALS AND METHODS The threshold for considering AML cases as apoptotic was set at the bax/bcl-2 median value > 0.35 (range( ) No of patien ,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 Bax/bcl-2

22 Tecniche di laboratorio per lo screening mutazionale di NPM1 e FLT3-ITD Sequenziamento genico D-HPLC LightCycler PCR PCR quantitativa Real Real-Time Elettroforesi capillare (EC) RT-ASO-PCR / semi nested ASO-PCR

23 NPM1 mutations and FLT3-ITD were detected by multiplex PCR and capillary gel electrophoresis (Noguera, Leukemia, 2005).

24 BIOLOGICAL RESULTS

25 Bax/bcl-2, NPM1-Mt and FLT3-ITD 300 bax/bcl2- bax/bcl2+ p a t % % % NPM1- NPM1+ FLT3ITD- FLT3ITD+ NPM1+FLT3ITD+ i e n t % s

26 WBC count and FLT3-ITD p a t i e n t s WBC<50 WBC WBC> FLT3ITD- FLT3ITD+ P<

27 CD34 and NPM1-Mt p a t i e n t s NPM1- NPM1+ P< CD34 - CD34+

28 Karyotype and NPM1-Mt p a t i e n t s % Good Normal Poor 66 NPM1- NPM1+ P<

29 Normal Karyotype (92 pts) NPM1+FLT3- NPM1+FLT3+ NPM1-FLT3- NPM1-FLT

30 Bax/bcl-2 and NPM1-Mt p a t bax/bcl-2<0.3 bax/bcl-2>0.3 P = i 50 e n t s (%) NPM1+FLT3- NPM1+FLT3+ NPM1-FLT3+ NPM1-FLT3-

31 p a t i e n t s (%) FAB classes and NPM1-Mt NPM1- NPM1+ P = M0 M1 M2 M4 M5 M6

32 CLINICAL RESULTS

33 NPM1-Mt / FLT3-ITD and Complete Remission (%) p a t i e n t NPM1+FLT3ITD+ NPM1+FLT3ITD- NPM1-FLT3ITD+ NPM1-FLT3ITD- P = s 20 (%) 10 0 CR(%)

34 Overall Survival by NPM1-Mt and FLT3ITD 1,0 0,9 0,8 0,7 0,6 P= ,5 0,4 0,3 35% Cumulative 0,2 0,1 0,0 15% 6% 0% Days from Diagnosis NPM1+FLT3- NPM1+FLT3+ NPM1-FLT3+ NPM1-FLT3-

35 Disease free Survival by NPM1-Mt and FLT3ITD 1,0 0,9 0,8 0,7 0,6 P = ,5 44% 0,4 0,3 0,2 0% P=0.008 NPM1+FLT3- Cumulative 0,1 0, Days from Diagnosis NPM1+FLT3+ NPM1-FLT3+ NPM1-FLT3-

36 Overall Survival by NPM1 and FLT3-ITD in AML pts > 60 years Cumulative Proportion Surviving n = 121 pts P = Days from diagnosis NPM1mt FLT3/ITD- NPM1mt FLT3/ITD+ NPM1wt FLT3/ITD+ NPM1wt FLT3/ITD-

37 Cumulative Proportion Surviving P = Days from Diagnosis NPM1mt FLT3/ITD- Bax/bcl-2 >0.35 All other groups Cumulative Proportion Relapsing P = Days from diagnosis NPM 1mt FLT3/ITD- Bax/bcl-2 >0.35 All other groups

38 MULTIVARIATE ANALYSIS (CR and OS) Outcome, variable Odds Ratio (CI) P CR NPM1 status 1.78 ( ) 0.42 FLT3-ITD 0.52 ( ) 0.18 Int. NPM1 x FLT3-ITD 0.16 ( ) Bax/bcl-2 ratio > ( ) < Age >60 y 0.28 ( ) WBC (50-100x10 9 /L) 0.61 ( ) 0.36 WBC (>100x10 9 /L) 0.72 ( ) 0.54 Outcome, variable Hazards Ratio (CI) P OS NPM1 status 1.35 ( ) 0.19 FLT-3 ITD 0.85 ( ) 0.57 Int. NPM1 x FLT3-ITD 2.35 ( ) Bax/bcl-2 ratio > ( ) < Age > 60 y 1.06 ( ) 0.74 WBC (50-100x109/L) 1.51 ( ) 0.09 WBC (>100x109/L) 1.80 ( ) 0.01 CI, confidence intervals; Int, multiplicative interaction between NPM1 and FLT3-ITD.

39 CONCLUSIONS NPM1-mutated/FLT3-ITD negative patients exhibit high levels of spontaneous apoptosis,, thus explaining their more favorable response to therapy. NPM1 mutations in the absence of FLT3-ITD identify a subgroup of patients with favorable prognosis and in patients with both mutations FLT3- ITD dominates the leukemic phenotype conferring a poor outcome.

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41 ACKNOWLEDGMENTS M.I. Del Principe A. Venditti S. Amadori E. Ammatuna F. Buccisano S. Zaza T. Ottone S. Lavorgna F. Lo Coco F. Luciano L. Maurillo lo Reparto Ematologia Policlinico Tor Vergata Laboratorio Ematologia Policlinico Tor Vergata Laboratorio Ematologia Ospedale S.Eugenio