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MDS

MDS. Anomalie morfologiche Emopoiesi inefficace Midollo pieno e periferia vuota. Azienda Ospedale-Università S.Anna Ferrara. Overt growth. apoptosis. Interaction BM stroma. autoimmunity. Initiated cell Clonal expansion MDS Leukemia. Genetic/epigenetic aberrations.

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MDS

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  1. MDS Anomalie morfologiche Emopoiesi inefficace Midollo pieno e periferia vuota Azienda Ospedale-Università S.Anna Ferrara

  2. Overt growth apoptosis Interaction BM stroma autoimmunity Initiatedcell Clonal expansionMDS Leukemia Genetic/epigenetic aberrations Chromosome changes Alteration of GEP

  3. Lesioni citogenetiche nelle MDS • Meccanismi • Descrizione delle anomalie e di alcune entità • Relazione con la patogenesi e con la clinica

  4. Environment • Benzene • Smoking • Pesticides • Organic chemicals • Heavy metals • DNA polymorphisms • DNA repair genes • Carcinogen-metabolizing-genes Cumulative exposure / genetic susceptibility Initiatedcell Clonal expansionMDS Leukemia Cumulative exposure / genetic susceptibility • Iatrogen • Alkylating agents • Anti topoisomerase-II • Radiation therapy • Inherited predisposition • Neurofibromatosis (NF1 – Ras pathway) • Fanconi – Blooom (gene repair) • AML1 mutations

  5. SMD e leucemie secondarie • Rischio attuariale 0.25-1% per anno da 2 a 5-7 anni dalla fine della chemioterapia • Rischio dose dipendente e aumenta esponenzialmente dopo i 40 anni • Agenti alchilanti • -7/7q-, t(3;21), RAS; • -5/5q-, -7/7q-, 17p, 3p, 12p, complex • Inibitori delle topoisomerasi II • 11q23, t(8;21) e t(3;21), inv(16) • t(15;17)(bimolano) • 11p15 (NUP98) • Normal karyotype (10-15%) • FLT3 ITD, MLL • Other abnormalities

  6. MECHANISMS OF ALKYLATING AGENTS CELLULAR INJURY • Monoadduct formation • Extracyclic oxygen • Intrastrand cross-links • Interstrand cross-links DNA accessibility No strand separation No transcription Mutagenesis DNA loss • Blocking events • Base mispairing

  7. SMD e leucemie secondarie • Rischio attuariale 0.25-1% per anno da 2 a 5-7 anni dalla fine della chemioterapia • Rischio dose dipendente e aumenta esponenzialmente dopo i 40 anni • Agenti alchilanti • -7/7q-, t(3;21), RAS; • -5/5q-, -7/7q-, 17p, 3p, 12p, complex • Inibitori delle topoisomerasi II • 11q23, t(8;21) e t(3;21), inv(16) • t(15;17)(bimolano) • 11p15 (NUP98) • Normal karyotype (10-15%) • FLT3 ITD, MLL • Other abnormalities

  8. TOPOISOMERASE ENZYME FUNCTIONS DNA STRAND PASSAGE THROUGH THE BREAK REDUCTION OF DNA TWISTING AND COILING ESSENTIAL FOR DNA PACKAGE AND ACCESSIBILITY Covalent linkage to the 5’ DNA terminus Religation of the cleaved DNA DNA double-strand break

  9. POSSIBLE MECHANISMS FOR THE DEVELOPMENT OF CHROMOSOME TRANSLOCATIONS IN LEUKEMIA Chromosome translocation IMMORTALIZATION t(6;11)(q27;q23) NO DNA RELIGATION DIRECT INTERACTION AT TOPO-II CONSENSUS BINDING SITES Cell death APOPTOTIC STIMULI CHROMATIN FRAGMENTATION

  10. Fattori occupazionali e rischio leucemico • Il benzene e le radiazioni ionizzanti sono i soli agenti che in modo conclusivo sono stati dimostrati essere carcinogenici per il sistema ematopoietico • Altri tipi di esposizione ambientale sono sospettati di essere coinvolti nella eziopatogenesi di queste patologie: • Idrocarburi aromatici e solventi organici • Pesticidi • Altri agenti chimici • 1,3-butadiene, stirene, ossido di etilene, formaldeide • Campi elettromagnetici • Agenti infettivi

  11. Qual è l’esposizione della popolazione al benzene? I dati ambientali sono tratti da: “Aria a Ferrara” a cura di: G. Garasto, E. Canossa (ARPA sezione di Ferrara ), A. De Togni (A.USL di Ferrara) Dinamiche uguali anche nei centri urbani con meno di 50.000 abitanti Quali interventi preventivi? 1)composizione benzine 2) alimentazione veicoli 3) Limitazioni traffico 4) Fonti produttive

  12. Potenziali meccanismi genotossici e conseguenze • Il lavoratori esposti al benzene che sviluppano neoplasie ematologiche mostrano dei polimorfismi nelle vie metaboliche, che predispongono all’accumulo di intermedi del metabolismo del benzene altamenti genotossici.

  13. KARYOTYPE IN MDS -5/5q- -7/7q- 17p- 11q- 12p- 6p- 3p- }t-MDS 3% 20% +8 +11/+21 +4/+13 normal t(6;9) 3q21q26 11q23 t(3;5) t(1;3) t(3;21) t(7;11) 22% 55% Cryptic deletion by FISH rare i(17q) 13q- 20q- t(5;12) t(3;12)

  14. Chromosome deletion Uniparental disomy Sole defect in 33% of MDS It affects region commonly deleted Chr 1,6,11,13 Maciejewski, personal communication Naples, june 19-20° , 2006 deletion 5* 5 5* 5q- No repair Abnormal oncosuppressor break Repair 5♀ 5♂ translocation der(5q) 5*

  15. Incidenza di differenti gruppi citogenetici nei differenti gruppi di età nelle AML aumento riduzione Bacher et al, Haematologica 2005:90;1502

  16. Frequenza esposizione esposizione a pesticidi e solventi in pazienti con MDS e controlli sani Sottotipo di MDS Rigolin et al, BJH 1998;103:189

  17. SMD ed esposizione a pesticidi e solventi Rigolin et al, BJH 1998;103:189

  18. SMD ed esposizione a pesticidi e solventi Rigolin et al, BJH 1998;103:189

  19. Telomeres are short tandem DNA repeats conferring chromosome stability Telomere erosion with proliferation Normal genome structure Normal physiology Extended proliferative lifespan Early telomerase activation Genome rearrangements Transformed phenotypes Extended proliferative lifespan telomere length Genomic instability Late telomerase activation Replicative senescence, apoptosis Cell divisions

  20. Telomeri ed esposizione Rigolin et al, EJH 2004;73:351

  21. CLASSIFICATION OF MYELODYSPLASTIC SYNDROMES FAB CATEGORY CORRESPONDING WHO CATEGORY (Frequency) REFRACTORY ANEMIA R.C. WITH MULTILINEAGE DYSPLASIA 5Q- SYNDROME RA Dysplasia > 10% cells in ≥ 2 lineages REFRACTORY ANEMIA WITH R.S. RCMD with R.S. RARS Dysplasia > 10% cells in ≥ 2 lineages RAEB RAEB I (≤10% BM Bls) RAEB II (11-20% BM Bls) RAEB-t AML

  22. CYTOGENETIC ANOMALIES BY FAB CLASSES OF MDS Percent of abnormal karyotypes * with additional aberrations / complex karyotype

  23. Karyotype in MDS with and without multilineage dysplasia Good Interm. Poor Normal -Y; 5q-, 20q- OTHERS -7; complex % OF PTS % OF PTS % OF PTS WHO Type Malcovati et al, JCO, 2005

  24. 5q- single, normal, 20q-, -Y 5q translocations, 9q-, 12p- others -7, complex Tetrasomy 8

  25. SPECIFIC CYTOGENETIC ENTITIES IN MDS 20q- Tetrasomy 8 TRISOMY 8 COMPLEX 17p-/p53 5q- SYNDROME -7/7q- Good Intermediate Poor

  26. 5q- chromosome del(5)(13q33)

  27. Minimal regions of deletion in 5q- chromosome MDS/AML and t-MDS 5 IL9 SMAD5 D5S89 D5S399 SPOCK D5S479 AFMB35 D5S1983 D5S414 EGR1 Fairman et al 2,4 Mb q11 q14 q22 q23 q31 q33 q34 q35 Zaho et al 1-1,5 Mb Horrigan et al 700 Kb 5q- syndrome D5S413 SPARC MEGF G3PB GLRA1 Jaju et al 1,5 Mb micro-Rna NPM+/- mice develop MDS evolving to AML P.P. Pandolfi, Nature, 2005 NPM

  28. 5q- syndrome • 5q- as the sole anomaly • Absence of additional cryptic aberrations (deletions, FLT3, RAS, p53 mutations) • Additional chromosome anomalies = negative impact • Distinct GEP (down-regulation of 5q genes) • M/F 1:4 • Macrocytic anemia • Mononuclear Mk-cytes • Reduced BFU-E growth, normal CFU-GM growth • Erythroid hypoplasia • Favourable clinical course • Leukemic transformation rare • Transfusion dependance = negative impact • Response to lenalidomide

  29. Frequency of Cytogenetic Response According to Karyotype Complexity List et al. 355 (14): 1456,  October 5, 2006

  30. ruEPO + G-CSFresponse in low risk MDS is associated with the expansion of cytogenetically normal cells and of their CD34+ progenitor cells CD34+ cells in MDS with 5q- before treatment with EPO CD34+ cells in MDS with 5q- after treatment with EPO Rigolin GM et al BJH 2004

  31. CLINICOBIOLOGICAL FEATURES OF MDS WITH +8 It is present in all abnormal cells only in 50% of the cases Is it a primary change? • Blast cells are preferentially • involved • Increase in size of the • trisomic clone in MDS • in trasformation +8 involves Multiple cell lineages Cytogenetics on single CFU FICTION method

  32. CLINICOBIOLOGICAL FEATURES OF MDS WITH +8 Rapid onset of leukemia Gradual increase of blast cell count • increase of the size of the +8 clone • additional aberrations Possible evolution patterns Stable disease • Appearance of unrelated clones • Fluctuations of the size of the +8 clone Apoptosis Intermediate prognosis Sensitivity to IST Clinical features

  33. Hypercellular BM Peripheral cytopenia MYELODYSPLASTIC SYNDROME Functional abnormalities CLONALITY Morphologic abnormalities Ineffective hemopoiesis (apoptosis) HYPOPLASTIC MDS Hypocellular BM APLASTIC ANEMIA AUTOIMMUNITY

  34. 1 APOPTOSIS AND IMPAIRED MATURATION IN MDS • IMPAIRED MATURATION • increased TNFa lenalidomide • high IFNg • high Fas ligand • high IL6 • high TGFb APOPTOSIS in CD34+ cells c-myc/bcl2 Low-risk MDS High risk MDS

  35. 2. INTERACTION BETWEEN THE MDS CLONE AND THE MICROENVIROMENT Hemopoietic cells High CD95 in CD34+ cells FAS ligand susceptibility (trisomy 8 > normal > monosomy 7) TNFa IFNg TGFb MIP-1a CD8+ T-cells NK cells T-lymphs MHC restriction Monocytes BM stromal cells stroma Mono’s

  36. 3. AUTOIMMUNITY AND TREATMENT OF MDS Bystander cell killing MDS cells +8 +8 Immune reaction Towards a new Ag on + 8 cells 30% durable responses Oligoclonal/clonal expansion Young age DR15 HLA Interval transfusion-IST T-lymphs MHC restriction

  37. MDS CLONE and gene expression profiles • Methodological problems • MDS encompasses many diseases • BM heterogeneous, which cell to study? • Comparison with what? • Interpretation of results Choose a homogeneous one CD34+ / specific lineages Pooled samples if few cells available Normal controls other subsets of MDS Post-transcriptional regulation Complex protein-protein interactions

  38. MDS CLONE and gene expression profiles Diagnostic marker MDS CD34+ cells Pellagatti et al Blood 2006 Same pattern in IFN-stimulated normal CD34+ progenitors IFNg stimulated genes upregulated Normal controls Role in cytopenia Different subsets of MDS haploinsufficiency Down-regulation of 5q genes Upregulation of HIST gene cluster at 6p21 Upregulation of actin cytoskeleton genes Upregulation of MK-cytes genes 5q- Chromatiin remodelling Signal transduction Mk-cytes ! Pellagatti et al Blood 2006

  39. MDS CLONE and gene expression profiles upregulation of genes inducing leukemia and tumorigenesis down-regulation of genes controlling cell growth and differentiation -7 Disregulation of genes related to proliferation and blood cell function MDS CD34+ cells upregulation of genes involved in immune responses down-regulation of genes implicated in apoptosis inhibition +8 Chen et al Blood 2006

  40. MOLECULAR PATHOGENESIS OF MDS Initiatedcell Clonal expansionMDS Leukemia P R I M A R Y D E F E C T • PREDISPOSITION • Inherited/acquired • Polymorphic variants • involved in mutagen • detoxification • NF-1 mutations • DNA repair genes (FA) • SBDS (S.-D. syndrome) • AML1 mutations (FPD) • Complex genetic traits -7/7q- 3p- 12p- 17p- Gene Methylation HDACs RAS p53 FLT3 -5/5q- Epigenetic Silencing of TSG Exposure to genotoxic agents (Drugs, solvents, pesticides) +8 Activation of regulatory paths through farnesylation inv(3) 11q23

  41. PATHOGENESIS AND MODERN THERAPY OF MDS Clonal expansionMDS Leukemia Normal karyotype P R I M A R Y D E F E C T 3p- 12p- 17p- Gene Methylation HDACs RAS FMS/p53 -7/7q- lenalidomide -5/5q- 5q- DNA-hypomethylating 5-AZA Epigenetic Silencing of TSG Exposure to genotoxic agents (Drugs, solvents, pesticides) +8 HDACs inhibitors VA (+ ATRA) Farnesyl transferase inhibitors inv(3) 11q23 Immunosuppression ATG - CsA Normal karyotype

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