11Intensità del segnaleDurata del segnalePossibilità di spegnerlo più o meno rapidamente
12Meccanismi di attivazione RiarrangiamentiAmplificazioniMutazioni puntiformi……….
13RiarrangiamentiSpesso conseguenza di traslocazioni cromosomiche (BCR-ABL, NPM-ALK) o di delezioni intracromosomiche (FIP1-PDGFRa)Meccanismo più frequente nelle neoplasie ematologiche che nei tumori solidi
14Autophosphorylation by dimerization Phosphorylation of substrates ABL ABLTyr TyrBCR BCRPhosphorylationof substratesP
15AmplificazioniPiù frequenti nei tumori solidi che nei tumori ematologici (ERB2 nel Ca mammario etc…)Più frequente per recettori di membranaMeccanismo di attivazione: l’aumento delle molecole in superficie ne favorisce il contatto anche in assenza di ligando
16Mutazioni puntiformiNumerosi esempi sia nei tumori ematologici (JAK2 in DMPC-Ph-negativi) che nei tumori solidi (KIT nei GIST)Meccanismo di attivazione: meccanismo di attivazione diretto del dominio TKP*amino acid substitutions
17FLT3 activation in AML (30% of cases) ITDPPPD835YSTATJAKRAS
25Cumulative Best Response at 12 and 60 months on First-line Imatinib in IRIS study 96%96%98%98%92%92%85%85%87%69%69%Complete hematologic response (CHR) = white cell count of <10 x 109, platelet count of <450 x 109, <5% myelocytes plus metamyelocytes, absence of blasts and promyelocytes from peripheral blood, absence of extramedullary involvement, and no signs of accelerated-phase (AP) or blast-crisis (BC) phase diseaseMajor cytogenetic response (MCyR) = 1%-35% Philadelphia-chromosome–positive (Ph+) metaphasesComplete cytogenetic response (CCyR) = absence of Ph+ metaphasesCumulative rates of CHR and CCyR were estimated according to the Kaplan-Meier method, in which patients who crossed over to the other treatment arm or discontinued treatment for reasons other than progression were censored at the last follow-up visit of the initial treatment periodCumulative estimated response rates increased over time for CHR, MCyR, and CCyR. Late responses are seenAn estimated 69% patients achieved CCyR at 12 monthsOf those patients who did not achieve a CCyR at 12 months (31%), 60% went on to achieve a responseTime (months)CHR (%)MCyR (%)CCyR (%)129685691897887624908060989287
26Event-free Survival and Survival Without AP/BC on First-line Imatinib 98%84%Estimated rate at 60 months (with 95%CI)93%(90-96)83%(80-87)Actual Events6.3% 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)Events were defined by the first occurrence of any of the following: death from any cause during treatment, progression to AP or BC phase of CML, loss of CHR, loss of MCyR, or increasing white blood cells (WBC)9 CML-unrelated deaths were recorded for patients on imatinib treatment (with no signs of clinical progression). The recorded events included: cardiac arrest (2), cardio-respiratory arrest, metastases to liver, pulmonary edema & acute renal failure, road traffic accident, metastatic rectal cancer, sepsis, and myocardial infarction.86 (15.5%) events were observed during treatment with imatinib. This resulted in an estimated event-free survival of 83% at 60 months.Estimated rate of survival without progression to AP/BC at 60 months is 93%
27Annual Event Rates on First-line Imatinib Year All events* AP/BC1st % %2nd % %3rd % %4th % %5th % %Annual rates of all events as well as progression to AP/BC decline over time with imatinib therapy* All deaths or loss of response including progression to AP/BC
28Survival Without AP/BC by Molecular Response at 12 months on First-line Imatinib Estimated rate at 60 monthsn= %n= % n= %CCyR with >=3 log red.p=0.007Molecular response was evaluated every 3 months after a CCyR was obtained using real-time quantitative polymerase chain reaction (RQ-PCR)The ratio of BCR-ABL:BCR transcripts was measuredResults were expressed as “log reductions” below a standardized baseline derived from a median BCR-ABL:BCR value obtained from 30 untreated patients with chronic-phase CMLMajor molecular response (MMR) was defined as ≥3 log reduction of BCR-ABL:BCR transcriptsNo patient with CCyR and MMR by 12 months progressed to AP/BCCCyR with <3 log red.p<0.001No CCyRDruker et al., ASCO 2006
29The BCR/ABL amount measured by RQ PCR mirrors the number of cells less sensitive to ImatinibImatinibImatinibPh+BCR/ABL reductionPhRPhRThe persistent Ph-positive cells less sensitive to imatinib may potentially become prone to progressionHigher the number, higher the risk!
30Two major problems with imatinib therapy Resistance/Loss of Response and ProgressionPersistence of small amount of leukemic cells
31to suppress the BCR-ABL TK Pathophysiology of imatinib resistancePathophysiology of imatinib resistanceBCR-ABLproliferationOtherdefectsImatinibBCR-ABLproliferationImatinib not ableto suppress the BCR-ABL TKClonal evolutionPoint mutationsBCR-ABL amplificationInsufficient IMA in cells
32a.a. that control the kinase activation step The map of mutations4 Critical regionsa.a. that control the kinase activation stepATP binding loopP-loopGatekeepersCatalytic domainActivation loopM244VD276GV289AM343TE355G/DH396R/PS417YL248VT277AM351T/VL387M/FG250EE255K/VF311L/IF317LF359VF382LE459KQ252R/HF486SV379IY253F/HT315IA380T
33Most frequent mutations P-loop2221201918171615141312no. of mutations1110987654321M244VL248VG250EQ252R/HY253F/HE255K/VD276GT277AP296HF311L/IT315IF317LM343TM351T/VE355G/DF359V/IV379IA380TF382LL387F/MH396R/PS417YE459K/QF486S*Soverini et al GIMEMA-CML WP data on 297 matinib resistant patients (CML, Ph+ ALL)
41Molecular response in IRIS trial Base line1.0CCRLog reduction of BCR-ABL2.03.092% BCR-ABLpositiveThe vast majority of pts rapidly had levels of residual leukaemia that were below the level of detection by cytogenetic analysis92% of pts have detectable levels of BCR-ABL up to 30 months4.0NotdetectedPre3691218242730Months
43Most patients who stop imatinib therapy, even when PCR neg, relapse!
44Michor et al., Nature 2005DifferentiatedProgenitors
45Punish the Parent not the Progeny The Imatinib concentrationin the progenitor cells, due to the action ofinflux (OCT-1) and efflux (ABCB1) proteinsseems to represent a major determinantfor the persistence-resistanceof Ph-positive progenitorsTessa Holyoake and Deb WhiteBcr-Abl off?Bcr-Abl on?Holyoake TL, Blood 2004
46Ph+ cells that survive and return 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 damageThe 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 damageImatinibBCR-ABLinhibitionApoptosisPh+ cellsImatinibBCR-ABLinhibitionPh+ cells that survive and returnto “normality”
47The strategy to eradicate the persistence of this tricky Ph+ population must be appropriate Combination therapy?Immunotherapy?
48San Luigi Hospital-University of Turin Daniela CilloniGiovanna Rege CambrinFrancesca MessaCarmen FavaFrancesca ArrugaIlaria DefilippiEmanuela MessaAlessandro MorottiEnrico GottardiEmilia GiuglianoAnna SerraMilena FavaSan Luigi Hospital-University of Turin