4 15-20% de las leucemias (adultos) 3-5% " " (niños) LMCEPIDEMIOLOGíA15-20% de las leucemias (adultos)3-5% " " (niños)25-50 años (promedio 40)10 años más jóvenes que LLC
5 CARACTERÍSTICAS GENERALES Desorden clonal de una célula troncal LMCCARACTERÍSTICAS GENERALESDesorden clonal de una célula troncalCrom. Philadelphia (Ph) se encuentra en granulocitos,megacariocitos, monocitos y cél. eritroides.Gran aumento del pool granulocíticoEnfermedad bifásicaFase crónicaFase aceleradaFase aguda (Crisis blástica)
6 ETIOLOGÍA 5% causa conocida Radiaciones Agentes químicos LMCETIOLOGÍA5% causa conocidaRadiacionesAgentes químicosBenzol y derivadosSolventesInsecticidas
7 Enf. con alteración molecular única LMCEnf. con alteración molecular únicaLMCEnf. proliferativa de stem cells hematopoyéticasCurso clínico característicoCromosoma de Philadelphia (Ph)Alteración cromosómica únicaTirosina kinasa Bcr-AblAlteración molecular únicaCausa transformación de una Stem cells hematopoyética en clon maligno
8 Crom. Philadelphia (Ph) y gen bcr-abl LMCCrom. Philadelphia (Ph) y gen bcr-ablChromosome 22Chromosome 99 q+9c-bcr12-11c-abl222-11p210Bcr-AblPh(or 22q-)bcr2-11p185Bcr-Ablbcr-ablExonsIntronsCML BreakpointsALL BreakpointsablProteína de fusiónActividad Tirosina KinasaTranslocación recíprocat(9;22) (q34;q11)bcr-abl gene structure
9 Proteínas normales Bcr y Abl y sus contrapartes post-translocación
10 Actividad tirosina kinasa de la proteína Abl Tirosinas kinasas-Fosforilan tirosina-Actividad controlada endominio N-terminal.En región C-terminal dominio une a DNA y a los filamentos de actina (regular ciclo celular)
11 Funciones de la proteína Bcr Serina-treonina kinasa.Tiene homología a proteínas G.Puede participar en reparación del DNA.
12 Cytoskeletal proteins bcr-abl: Transducción de señalesTYROSINE KINASE ACTIVITYBcr-AblCytoskeletal proteinsPMYC?NucleusRAS-GAPRAS-GTPSAPKCBLCRKPI3KBAD14-3-3BCLXLMitochondriaRAS-GDPAKTERKSTAT1+5GRB-2SOSSHCDOKCRKLMEK1/2RAF-1
16 LMC Síntomas Signos Inespecíficos y graduales: Fase CrónicaCLÍNICASíntomasInespecíficos y graduales:Astenia, fatiga, cefalea, pérdida de peso, anorexiaSignosEsplenomegalia (80%)Hepatomegalia (20%)NormalNormal
17 Laboratorio Hemograma Estudio citogenético Cariograma Fish LMCLaboratorioHemogramaEstudio citogenéticoCariogramaFishRT-PRC bcr-ablFANCitometría de flujo
18 Laboratorio: Parámetros típicos (según fase) Parameter Chronic Accelerated Blast CrisisWBC count 20 x 109/L — —Blasts 1%–15% 15% 20%Basophils 20% —Platelets or normal or Bone marrow Myeloid hyperplasiaCytogenetics Ph+Bcr-Abl +
19 Laboratorio: Fase crónica (cont) LeucocitosisFórmula leucocitaria alteradaBasofiliaEosinofiliaAnemia NNTrombocitosis
20 Mielofibrosis con metaplasia mieloide LMCDIAGNÓSTICO DIFERENCIALReacción leucemoideSepsis, inflamación, Ca, hemólisisGranulación tóxica, Cuerpos de DöhleFAN aumentadasPh (-)Mielofibrosis con metaplasia mieloidePoiquilocitosis (GR en lágrima)Fibrosis medular
21 Cariograma LMC Estudio citogenético Menos sensible que RT-PCR abl-bcr 922Menos sensible que RT-PCR abl-bcr
22 Métodos moleculares para detectar bcr-abl cromosoma Ph FISHFluorescence in situ hybridisationRT-PCRReverse Transcriptasa Polimerase Chain Reaction
23 Fluorescence in situ hybridisation (FISH) Labeled DNA probe (fluorescent)InterphaseMetaphaseNuclear DNAbcr-abl geneSeparate DNA strands and add labeled probe[slide 73]Molecular Methods for Detecting bcr-abl at the Ph ChromosomeFISH detects highly specific DNA probes that have been hybridised to either interphase or metaphase chromosomes using fluorescence microscopy. FISH has a high rate of sensitivity (1/250 cells) and specificity.To detect the Ph chromosome, the fluorescent probe is designed to hybridise to the bcr-abl fusion gene..Labeled bcr-abl gene
24 FISH en MO y sangre periférica Alta correlación resultados Peripheral BloodMarrow% Ph+10080604020[slide 74]Correlation Between Interphase FISH Results on Bone Marrow andPeripheral BloodFISH can be performed on peripheral blood instead of the marrow and studies have shown that there is a highly significant correlation between FISH on peripheral-blood specimens and cytogenetics on bone marrow specimens.1Currently, FISH is being used experimentally to monitor remission status in Ph+ CML.2 It is also used for early detection of relapse and evaluation for mixed chimerism following SCT.3References1. Le Gouill S, Talmant P, Milpied N, et al. Fluorescence in situ hybridization on peripheral-blood specimens is a reliable method to evaluate cytogenetic response in chronic myeloid leukemia. J Clin Oncol ;18:2. Duba HC, Hilbe W, Mehringer A, et al. Hypermetaphase and interphase fluorescence in situ hybridisation for monitoring of remission status in Philadelphia chromosome positive chronic myeloid leukaemia. Int J Oncol. 2000;17:3. Tamura S, Saheki K, Takatsuka H, et al. Early detection of relapse and evaluation of treatment for mixed chimerism fluorescence in situ hybridization following allogeneic hematopoietic cell transplant for hematological malignancies. Ann Hematol ;79:
25 AMPLIFICACIÓN DE GEN abl/bcr LMCAMPLIFICACIÓN DE GEN abl/bcrRT-PCR (Reverse Transcriptasa Polimerase Chain Reaction)Más sensible que Cariograma (detecta 1 cél. malig. en >106 cél. N)Detección cél. Ph+ ( Diagnóstico y post-trasplante MO)MÉTODOObtención Cél. Mononucleares de MO( grad. de densidad)Obtención de RNATranscripción reversa (obtención cDNA)Transcriptasa reversa, “Primers”AmplificaciónDNA polimerasa, “Primers”, Oligonucleótidos ,TermocicladorVisualización del producto amplificadoElectroforesis Gel agarosa ,Bromuro de Ethidio , UVEtapas del RT-PCRPlasmaLinfocitosFICOLL; d 1.077PMN GR,
33 LMCTrasplante de progenitores hematopoyéticos: Alta morbilidad y mortalidadSurvival by Disease Stage, June 2001, based on transplants 1987 – Feb 2001.[slide 17]Allogeneic SCT, the Only Known Cure, Is Associated With High Morbidity andMortality Rates in CMLMatched Related DonorsAllogeneic SCT, involving human leukocyte antigen (HLA)-identical sibling donors, is currently considered the only potential cure for CML; unfortunately, most patients are not eligible for SCT.Access to the procedure is limited by availability of a suitable donor and patient age (generally <55 years of age). Transplantation within 1 year of diagnosis is preferred.1,2 Approximately 15% to 20% of patients with CML meet these criteria for SCT.3Five-year survival for patients transplanted during the chronic phase ranges from 54% to 70%; survival rates decrease with advanced stages of disease.The relapse rate is within the range of 13% to 20% in chronic phase CML.Matched Unrelated Donors (MUDs)SCT with MUDs identified by bone marrow-donor registries can increase the number of patients with CML who are candidates for SCT to 30%. Molecular studies can help match HLA-A, B, and DRB1.4 The survival rate at 3 years post-transplant is 40% to 57%.3 However, outcome is highly dependent on patient characteristics such as age, stage of disease, and time to transplant.Morbidity and MortalityMorbidity can be severe (eg, graft-versus-host disease [GVHD] and systemic infections). Mortality associated with the SCT procedure is approximately 10% but is highly influenced by age and match (eg, MUD transplantation may have mortality rates of 25% to 50%).5,6References1. Mughal TI, Goldman JM. Chronic myeloid leukemia: a therapeutic challenge. Ann Oncol ;6:2. Goldman JM. Chronic myeloid leukemia. Curr Opin Hematol ;4:3. Sawyers CL. Chronic myeloid leukemia. N Engl J Med ;340:4. Hansen JA, Gooley TA, Martin PJ, et al. Bone marrow transplants from unrelated donors for patients with chronic myeloid leukemia. N Engl J Med ;338:5. Faderl S, Kantarjian HM, Talpaz M. Chronic myelogenous leukemia: update on biology and treatment. Oncology (Huntingt) ;13:6. National Marrow Donor Program (NMDP) overview slide presentation. Available at: Accessed 17 June 2002.P=.0001
34 IFN- LMC Inhibe proliferación celular IFN- tiene efectos biológicos múltiples:Inhibe proliferación celularRegula expresón de otras citoquinasModula el Sistema immuneRespuesta citogenética: puede ocurrir a 12 a 18 mesesMejor sobrevida en casos Fase Crónica tempranaIFN- combinada con cytarabina (Ara-C): mejor respuesta[slide 19]IFN- Offers a Survival Advantage to Some PatientsIFN- is a member of a naturally occurring family of proteins that are produced in response to external mitogenic and viral stimuli. IFN- has a variety of biological effects, including inhibition of cell growth, regulation of cytokine expression, and modulation of the immune system.1Clinical trials with IFN- in patients with CML in chronic phase have shown that:The effects are dose-related; higher doses of IFN- correlate with higher rates of haematological and cytogenetic response as well as more severe adverse events.1,2Compared with busulfan or hydroxyurea, IFN- improves survival in low-risk patients with CML in chronic phase.3 The survival advantage is correlated with achieving a major cytogenetic response.IFN- in combination with cytarabine (Ara-C) increases the rate of major cytogenetic response and prolongs survival compared with treatment with IFN- alone.4The most common side-effects of Ara-C include nausea, vomiting, diarrhoea, and mucositis. Approximately 25% of patients discontinued therapy due to major side-effects.4References1. Faderl S, Kantarjian HM, Talpaz M. Chronic myelogenous leukemia: update on biology and treatment. Oncology (Huntingt) ;13:2. Silver RT, Woolf SH, Hehlmann R, et al. An evidence-based analysis of the effect of busulfan, hydroxyurea, interferon, and allogeneic bone marrow transplantation in treating the chronic phase of chronic myeloid leukemia: developed for the American Society of Hematology. Blood ;94:3. Hasford J, Pfirrmann M, Hehlmann R, et al. Writing for the Committee for the Collaborative CML Prognostic Factors Project Group. A new prognostic score for survival of patients with chronic myeloid leukemia treated with interferon-alfa. J Natl Cancer Inst ;90:4. Guilhot F, Chastang C, Michallet M, et al. Interferon alfa-2b combined with cytarabine versus interferon alone in chronic myelogenous leukemia. N Engl J Med ;337:
35 IFN-a: Buen resultado en fase crónica temprana PhasePatientsCHRMCR(n)(%)(%)Chronic <12 mo2748038>12 to <36 mo72628>36 mo42498[slide 21]IFN-a Therapy Works Best for Patients in Early Chronic PhaseIn chronic phase, the rate of complete haematological response (CHR) decreases from 80% for patients treated within 1 year of diagnosis of CML to only 49% for patients treated more than 3 years after the diagnosis was first established. The rate of major cytogenetic response (MCR) decreases from 38% to 8%, respectively.1The rate of responses for patients in accelerated phase CML is similar to that observed in late chronic phase.In blast crisis, the rate of responses decreases dramatically with only 20% of patients achieving a CHR and no patients achieving an MCR.These results indicate that the response rates of IFN-a therapy are the highest when patients are in early chronic phase. Patients in late chronic phase, accelerated phase, or blast crisis respond poorly to IFN-a therapy compared with patients in early chronic phase who are treated within 1 year of diagnosis.Reference1. Kantarjian HM, Giles FJ, O’Brien SM, et al. Clinical course and therapy of chronic myelogenous leukemia with interferon-alpha and chemotherapy. Hematol Oncol Clin North Am. 1998;12:31-80.Accelerated61527Blast Crisis520CHR = complete haematological response; MCR = major cytogenetic response.
36 Quimoterapia es solo paliativa LMCQuimoterapia es solo paliativaFármacos citotóxicos oralesHydroxyureaBusulfanRespuesta Hematológica: 90%Respuesta citogenéticas: 1%–5%No modifica progresión de enfermedad[slide 23]Chemotherapy Is Only Palliative in the Treatment of CMLHydroxyurea, an inhibitor of DNA synthesis, and busulfan, an alkylating agent, have been the oral chemotherapeutic agents of choice for CML.These agents may induce complete haematological response in up to 90% of patients with CML in chronic phase, however, cytogenetic responses are rare and disease progression is not affected. Thus, these agents are generally considered to be palliative.1-5Of the 2 chemotherapeutic agents, hydroxyurea is more effective than busulfan. Therapy with hydroxyurea is associated with a superior outcome to busulfan (5-year survival 44% vs 32%, respectively) with a significantly longer median survival (58 months vs 45 months, P=.008, respectively).3,6Hydroxyurea is generally better tolerated than busulfan; the side-effects associated with hydroxyurea are rare and mild and include nausea, vomiting, diarrhoea, and mucosal and dermal ulcerations.3,7,8References1. Faderl S, Kantarjian HM, Talpaz M. Chronic myelogenous leukemia: update on biology and treatment. Oncology (Huntingt) ;13:2. The Italian Cooperatitive Study Group on Chronic Myeloid Leukemia. Interferon alfa-2a compared with conventional chemotherapy for the treatment of chronic myeloid leukemia. N Engl J Med ;330:3. Hehlmann R, Heimpel H, Hasford J, et al, and the German CML Study Group. Randomized comparison of interferon- with busulfan and hydroxyurea in chronic myelogenous leukemia. Blood. 1994;84:4. Allan NC, Richards SM, Shepherd PCA, on behalf of the UK Medical Research Council’s Working Parties for Therapeutic Trials in Adult Leukaemia. UK Medical Research Council randomised, multicentre trial of interferon-n1 for chronic myeloid leukaemia: improved survival irrespective of cytogenetic response. Lancet ;345:5. Ohnishi K, Ohno R, Tomonaga M, et al, and the Kouseisho Leukemia Study Group. A randomized trial comparing interferon- with busulfan for newly diagnosed chronic myelogenous leukemia in chronic phase. Blood ;86:6. Silver RT, Woolf SH, Hehlmann R, et al. An evidence-based analysis of the effect of busulfan, hydroxyurea, interferon, and allogeneic bone marrow transplantation in treating the chronic phase of chronic myeloid leukemia: developed for the American Society of Hematology. Blood ;94:7. Sawyers CL. Chronic myeloid leukemia. N Engl J Med ;340:8. Hill JM, Meehan KR. Chronic myelogenous leukemia. Curable with early diagnosis and treatment. Postgrad Med ;106: ,
37 Bcr-Abl: Target terapéutico LMCBcr-Abl: Target terapéuticoBcr-Abl: 95% pacientesBcr-Abl: Asociada a causaTirosina kinasa Bcr-Abl:Requerida para función celular de LMC.[slide 26]Bcr-Abl as a Therapeutic Target for CMLThe Bcr-Abl fusion protein, the product of the Ph chromosome, fulfills the criteria for an ideal molecular target in cancer because it is present in 95% of patients with CML.Extensive research has shown that Bcr-Abl is the unique pathophysiological cause of CML.Bcr-Abl tyrosine kinase activity is constitutively increased in CML cells, affecting numerous signal transduction pathways that are essential for leukaemic transformation, including increased cellular proliferation, anti-apoptotic effects, and adhesion defects.Imatinib is a specific tyrosine kinase inhibitor of the Bcr-Abl fusion protein.1Reference1. Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med ;2:
38 Imatinib: Inhibe selectivamente kinasas Kinases Inhibited Kinases Not Inhibitedv-Abl 0.1–0.3 Flt-3 >10p210Bcr-Abl 0.25 c-Fms, v-Fms >10p185Bcr-Abl 0.25 EGF receptor >100TEL-Abl 0.35 c-erbB2 >100PDGF-R 0.1 Insulin receptor >100TEL-PDGF-R IGF-1 receptor >100c-Kit 0.1 v-Src >10JAK-2 >100[slide 29]Imatinib Is Highly Selective for Multiple KinasesImatinib, which was selected from a series of compounds that inhibit protein kinases, was found to be a potent inhibitor of the Abl protein tyrosine kinase.Imatinib was assayed in vitro to determine the concentration that resulted in a 50% reduction (IC50) in tyrosine phosphorylation of specific tyrosine kinases.Imatinib is a potent inhibitor of Abl and Bcr-Abl intracellular tyrosine kinases as well as the transmembrane tyrosine kinase receptor platelet-derived growth factor receptor (PDGF-R) and c-Kit, the receptor for stem cell factor. The transcription factor (TEL) fusion proteins with Abl and PDGF-R are also inhibited.1-5Imatinib did not inhibit other protein kinases that were tested, including epidermal growth factor (EGF) receptor, the receptors for insulin, and insulin-like growth factor I (IGF-I).References1. Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med ;2:2. Carroll M, Ohno-Jones S, Tamura S, et al. CGP 57148, a tyrosine kinase inhibitor, inhibits the growth of cells expressing BCR-ABL, TEL-ABL, and TEL-PDGFR fusion proteins. Blood ;90:3. Sawyers CL, Druker B. Tyrosine kinase inhibitors in chronic myeloid leukemia. Cancer J Sci Am ;5:63-69.4. Deininger WN, Goldman JM, Lydon N, et al. The tyrosine kinase inhibitor CGP57148B selectively inhibits the growth of BCR-ABL-positive cells. Blood ;90:5. Buchdunger E, Cioffi CL, Law N, et al. Abl protein-tyrosine kinase inhibitor STI1571 inhibits in vitro signal transduction mediated by c-Kit and platelet-derived growth factor receptors. J Pharmacol Exp Ther. 2000;295:
39 Imatinib inhibe crecimiento de células Bcr-Abl positivas Imatinib Concentration (M)U937*KG1*SU DHL1*KCL22†K562†KU812†[slide 30]Imatinib Inhibits the Growth of Bcr-Abl–Positive CellsIn vitro experiments with human neoplastic cell lines demonstrated that imatinib inhibited the growth of Ph+ cells that express Bcr-Abl, but did not have a significant effect on Bcr-Abl–negative lines.1When cultured in the presence of increasing concentrations of imatinib, the 3 Bcr-Abl–positive leukaemic cell lines—K562, KCL22, and KU812—showed a dose-dependent inhibition of proliferation, whereas the 3 Bcr-Abl–negative neoplastic cell lines—U937, KG1, and SU DHL1—were relatively unaffected.1In addition, in vivo experiments showed that imatinib inhibited tumour growth in a dose-dependent manner in syngeneic mice inoculated with a growth factor-independent Bcr-Abl murine myeloid cell line.2These data suggest that inhibition of Bcr-Abl tyrosine kinase activity by imatinib is specific and results in inhibition of leukaemic cell growth and may have activity in the treatment of Bcr-Abl–positive leukaemia.1-3References1. Gambacorti-Passerini C, le Coutre P, Mologni L, et al. Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL+ leukemic cells and induces apoptosis. Blood Cells Mol Dis ;23:2. le Coutre P, Mologni L, Cleris L, et al. In vivo eradication of human BCR/ABL-positive leukemia cells with an ABL kinase inhibitor. J Natl Cancer Inst ;91:3. Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med ;2:*Bcr-Abl–negative cell lines.†Bcr-Abl–positive cell lines.
40 Imatinib: Resistencia ocurre principalmente en fase avanzada LMCImatinib: Resistencia ocurre principalmente en fase avanzadaF. crónicaF. blásticaRecaidaEstados avanzados de Cas: multiples cambios genéticosEn fase acelerada: A menudo recaen con resistencia a quimioterapia.En crisis blástica: Algunos rsponden pero recaenHaematopoieticdifferentiationBone marrow to peripheral blood[slide 48]Resistance to Imatinib Occurs Predominantly During Advanced Phase CMLCancers are characterised by multiple oncogenic events that collectively contribute to the phenotype of advanced stage disease.1In the late stages of tumour development, relapse with the development of chemotherapy resistance is common.1Not surprisingly, resistance to imatinib has been observed, mainly in patients with blast crisis CML.At that stage of disease, additional genetic abnormalities are generally observed in addition to the presence of the Ph chromosome.2These patients initially respond to imatinib but then relapse. Several investigations have studied these patients at the molecular level.2During haematopoietic differentiation in chronic phase CML, Ph+ stem cells in the bone marrow (green) self-renew or differentiate, and their mature progeny appear in the peripheral blood.During blast crisis, secondary genetic changes cause a clone of Ph+ blasts (yellow) to expand. As long as blasts are still sensitive to imatinib, the response is maintained and peripheral blasts are low.During relapse, further genetic changes occur and some Ph+ blasts acquire Bcr-Abl mutations. These cells become resistant (pink). Relapsing patients have both sensitive and resistant Ph+ cells.References1. Chu E, DeVita VT Jr. Principles of cancer management: chemotherapy. In: DeVita VT Jr, Hellman S, Rosenberg SA, eds. Cancer: Principles and Practice of Oncology. 6th ed. Philadelphia, Pa: Lippincott Williams & Wilkins; 2001:2. Gorre ME, Mohammed M, Ellwood K, et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science ;293:Ph-negativePh+ blastsPh+Ph+ imatinib-resistant blasts
41 Imatinib: Induce mayor respuesta citogenética [slide 60]Imatinib Effectively Induces a Greater Major Cytogenetic Response Rate1In the imatinib group of patients, 83% of patients achieved an MCR and only 20% of patients achieved an MCR in the IFN-a + Ara-C group.An outstanding 68% of patients achieved a complete cytogenetic response while on imatinib therapy and only 7% achieved the same result on IFN-a + Ara-C therapy.These differences were all highly statistically significant.Partial cytogenetic responses were seen in 15% and 13% of patients in the imatinib and the IFN-a + Ara-C groups, respectively.Reference1. Data on file. Novartis Pharma AG, Basel, Switzerland.*P<.001.
42 Con Imatinib: Respuesta hematológica completa más rápida 10094%Imatinib9080706055%IFN- + Ara-C% Responding5040[slide 63]Complete Haematological Responses Were Rapid With Imatinib1Using the same Kaplan-Meier approach, a statistically significantly higher proportion of patients achieved a complete haematological response with imatinib (94%) compared with IFN-a + Ara-C (54%).Complete haematological response was achieved sooner in the imatinib arm (median of 1 month) compared with IFN-a + Ara-C arm (median time of 2.5 months).Reference1. Data on file. Novartis Pharma AG, Basel, Switzerland.30201036912151821Months Since Randomisation
43 Con Imatinib: Cambios citogenéticos más rápidos 1009083%80Imatinib7060% Responding504030[slide 64]Major Cytogenetic Responses With Imatinib Were Rapid1The data were further analysed using a Kaplan-Meier approach.MCR on imatinib (median time 3 months, range 2.2 to 17 months) was achieved rapidly compared with IFN-a (median time 5.8 months, range 2.8 to 16.7 months).Reference1. Data on file. Novartis Pharma AG, Basel, Switzerland.20%20IFN- + Ara-C1036912151821Months Since Randomisation