l Intensità del segnale l Durata del segnale l Possibilità di spegnerlo più o meno rapidamente
Meccanismi di attivazione l Riarrangiamenti l Amplificazioni l Mutazioni puntiformi l ……….
Riarrangiamenti l Spesso conseguenza di traslocazioni cromosomiche (BCR-ABL, NPM-ALK) o di delezioni intracromosomiche (FIP1-PDGFRa) l Meccanismo più frequente nelle neoplasie ematologiche che nei tumori solidi
ABL Tyr BCR Autophosphorylation by dimerization P Phosphorylation of substrates
Amplificazioni l Più frequenti nei tumori solidi che nei tumori ematologici (ERB2 nel Ca mammario etc…) l Più frequente per recettori di membrana l Meccanismo di attivazione: l’aumento delle molecole in superficie ne favorisce il contatto anche in assenza di ligando
Mutazioni puntiformi l Numerosi esempi sia nei tumori ematologici (JAK2 in DMPC-Ph-negativi) che nei tumori solidi (KIT nei GIST) l Meccanismo di attivazione: meccanismo di attivazione diretto del dominio TK amino acid substitutions P P P P P P P P P P * * *
JAK RAS P P P P FLT3 activation in AML (30% of cases) P P P P ITD STAT D835Y
Imatinib Response and Disease Phase Chronic phase IFN failure (n=532) Accelerated phase (n=235) Myeloid Blast crisis (n=229) Haematological response95%83%31% Complete95%54%8% No evidence of leukaemia-12%4% Return to CP-17%18% Major cytogenetic response60%26% 15% Complete41%18%7% Partial19%8%
Ph+ Ph1 Ph+ NNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNN N NNNNNNNNNNNNNNNNNNNNNNNNNNN N NNNNNNNNN Ph1
Cumulative Best Response at 12 and 60 months on First-line Imatinib in IRIS study 96% 85% 69% 98% 92% 87% 96% 85% 69% 98% 92%
Event-free Survival and Survival Without AP/BC on First-line Imatinib Actual Events 6.3% AP/BC (n=35) 5.1% loss of MCyR (n=28) 2.5% loss of CHR (n=14) 1.6% CML-unrelated deaths (n=9) 83% (90-96) (80-87) Estimated rate at 60 months (with 95%CI) 93% 98% 84%
Annual Event Rates on First-line Imatinib YearAll events * AP/BC 1st 3.3% 1.5% 2nd 7.5% 2.8% 3rd 4.8% 1.6% 4th 1.5% 0.9% 5th 0.9% 0.6% * All deaths or loss of response including progression to AP/BC
Survival Without AP/BC by Molecular Response at 12 months on First-line Imatinib n=136 100% n= 94 95% n=138 88% Estimated rate at 60 months p<0.001 p=0.007 Response at 12 months CCyR with >=3 log red. CCyR with <3 log red. No CCyR Druker et al., ASCO 2006
PhR Imatinib Ph+ The BCR/ABL amount measured by RQ PCR mirrors the number of cells less sensitive to Imatinib BCR/ABL reduction The persistent Ph-positive cells less sensitive to imatinib may potentially become prone to progression Higher the number, higher the risk!
Two major problems with imatinib therapy Resistance/Loss of Response and Progression Persistence of small amount of leukemic cells
Other defects Imatinib Imatinib not able to suppress the BCR-ABL TK Point mutations BCR-ABL amplification Insufficient IMA in cells Clonal evolution
4 Critical regions ATP binding loop a.a. that control the kinase activation step M244VD276G P-loop Catalytic domain Activation loop The map of mutations V289AM343TE355G/DH396R/PS417Y L248VT277AM351T/VL387M/F G250EE255K/VF311L/IF317LF359VF382LE459K Q252R/H V379I F486S A380T Y253F/H T315I Gate keepers
Most frequent mutations 0 22 no. of mutations 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 M244VL248V G250EQ252R/HY253F/HE255K/VD276GT277AP296H F311L/IT315IF317LM343T M351T/VE355G/DF359V/IV379IA380TF382LL387F/MH396R/P S417YE459K/QF486S P-loop *Soverini et al GIMEMA-CML WP data on 297 matinib resistant patients (CML, Ph+ ALL)
CCR Notdetected 691218Pre3242730 4.0 3.0 2.0 1.0 Base line Log reduction of BCR-ABL 92% BCR- ABL positive Molecular response in IRIS trial Months
Ph+SC Normal SC Normal SC Normal SC Normal SC Normal SC Normal SC CML cells Ph+ progenitors Ph+ mature cells Imatinib Less sensitive progenitor Ph+SC
Most patients who stop imatinib therapy, even when PCR neg, relapse!
Michor et al., Nature 2005 Differentiated Progenitors
Holyoake TL, Blood 2004 Punish the Parent not the Progeny Bcr-Abl off?Bcr-Abl on? The Imatinib concentration in the progenitor cells, due to the action of influx (OCT-1) and efflux (ABCB1) proteins seems to represent a major determinant for the persistence-resistance of Ph-positive progenitors Tessa Holyoake and Deb White
Ph+ cells that survive and return to “normality” BCR-ABL inhibition BCR-ABL inhibition Ph+ cells Apoptosis The persistence of Ph-positive cells may be due to: - cells more resistant to imatinib - cells in which the BCR-ABL TK activity may be suppressed without a great damage Imatinib The persistence of Ph-positive cells may be due to: - cells more resistant to imatinib - cells in which the BCR-ABL TK activity may be suppressed without a great damage
The strategy to eradicate the persistence of this tricky Ph+ population must be appropriate Combination therapy? Immunotherapy?
University of Turin Daniela Cilloni Giovanna Rege Cambrin Francesca Messa Carmen Fava Francesca Arruga Ilaria Defilippi Emanuela Messa Alessandro Morotti Enrico Gottardi Emilia Giugliano Anna Serra Milena Fava San Luigi Hospital-University of Turin San Luigi Hospital-University of Turin