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Diagnóstico y monitorización de hemopatías malignas JM Ribera Servicio de Hematologia Clínica ICO-Hospital Germans Trias i Pujol Badalona Universidad Autónoma.

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Presentation on theme: "Diagnóstico y monitorización de hemopatías malignas JM Ribera Servicio de Hematologia Clínica ICO-Hospital Germans Trias i Pujol Badalona Universidad Autónoma."— Presentation transcript:

1 Diagnóstico y monitorización de hemopatías malignas JM Ribera Servicio de Hematologia Clínica ICO-Hospital Germans Trias i Pujol Badalona Universidad Autónoma de Barcelona V Reunión de la Sociedad Asturiana de Hematología y Hemoterapia Gijon, 9 de marzo de 2012

2 Diagnosis in Malignant Hematology From clinical symptoms to DNA Anamnesis and physical examination Diagnostic techniques –CBC, biochemistry –Studies in PB, BM, other fluids, lymph node, tumor mass Morphology, cytochemistry, cytofluorometry, cytogenetics, molecular genetics –Imaging studies: X-ray, CT, MRI, PET scan –Isotopic studies –Omic studies: genomic, proteomic, metabolomic –Nanotechnology

3 Importance of diagnostic methods Accuracy of diagnosis Staging Prognostic assessment Analysis of response to therapy Follow-up

4 Diagnostic work-up in ALL Anamnesis, physical examination Complete blood count, coagulation status, serum biochemical study Serology for hepatitis and HIV EKG, LVEF (advanced age or history of cardiac disease) Chest X-ray Bone marrow aspirate (morphology, cytochemistry) Bone marrow biopsy (only if dry tap) Immunophenotypic study (BM, PB) Cytogenetics (BM, PB) FISH (BM, PB) Study of molecular rearrangements (PCR) CSF study (morphology, FCM?) HLA typing as soon as possible Storage: cells, DNA, RNA.

5 ALL. WHO Classification, 2008 B precursor ALL –B precursor ALL/B-LL, non specified –B precursor ALL/B-LL, with recurrent genetic abnormalities t(9;22); BCR/ABL 11q23; MLL t(1;19); E2A/PBX1 (TCF3/PBX1) t(12;21); ETV6/RUNX1 (TEL-AML1) t(5;14); (IL3/IgH) Hyperdiploid ALL Hypodiploid ALL Precursor T-ALL/T-LL Burkitt-like ALL (mature B-ALL) –t(8;14), t(2;8), t(8;22); C-MYC

6 ALL Morphology Sensitivity: 10 -2

7 Immunologic classification of ALL B-lineage ALL: CD19+ and CD79a+ and/or CyCD22+ CD10- CD10+, cyIg- Cig+, sIg- sIg+ Pro-B ALL (B-I) Common ALL (B-II) Pre-B ALL (B-III) Mature B ALL (B-IV) T-lineage ALL: cyCD3+ and CD7+ cyCD3+ Cd7 only CD2+ and/or CD5+ CD1a+ sCD3+, CD1a- sCD3+, anti TCR α/β+ sCD3+, anti TCR γ/δ+ Pro-T ALL (T-I) Pre-T ALL (T-II) Cortical T-ALL (T-III Mature T-ALL (T-IV) α/β+ T-ALL γ/δ+ T-ALL Recommendations of the EWALL Group, 2012

8 Phenotypic study Sensitivity: (4 colors), (>4 colors)

9

10 Cytogenetics Sensitivity: Hyperdiploidy 52,XY,+X,+6,+14,+17,+21,+mar Hypodiploidy 41,XX,-4,-9,add(9)(p21),-15,-20,-22

11 Pseudodiploidy 46,XX,t(4;11)(q21;q23) 46,XX,+8,-12,der(19)t(1;19)(q23;p13.3), +der(19)t(1;19)(q23;p13.3),-20 Sensitivity: 10 -2

12 Pseudodiploidy 46, XY, t(9;22)(q34.1;q11.2)Burkitt ALL

13 ALL FISH IgH/c-MYC BCR-ABL Sensitivity: 5x10 -2 nuc ish(ABL1x3),(BCRx3),(ABL1con BCRx2)[90/100]

14 IgH & TCR rearrangements Sensitivity: – (RQ-PCR) TCR clonal FR1 ( pb) 310pb IgH clonal

15 BCR/ABL - t(9;22)(q34.1;q11.2) & 2: Patient 1 (positive p190) 3 & 4: Patient 2 (negative p190) 5: Positive control p190 6: Negative control 7: Marker of molecular weight Quantification of the amount of mRNA transcripts

16 Standard curve Linear dynamic range (5 Logs) RQ-PCR Sensitivity:

17 Recommendations for genetic diagnosis and follow-up of hematologic neoplasias. ALL (AEHH, FEHH, GCECGH, Grupo BMH)(2007) SubtypePhaseSampleConventional cytogenetics FISHMolecular biology Precursor B- ALL DiagnosisBMAlwaysBCR/ABL, MLLBCR/ABL, AML/AF4 RelapseBMOptional No Burkitts lymphoma/ leukemia DiagnosisBM/Tumor tissue AlwaysC-MYCNo RelapseBM/Tumor tissue Optional No Pediatric ALL DiagnosisBMAlwaysIf NK or no metaphases: TEL/AML1, PBX1/E2A, MLL, BCR/ABL TEL/AML1, PBX1/E2A, AF4/MLL BCR/ABL MRDBMNoAccording to diagnosis T-ALL DiagnosisBMAlwaysNo RelapseBMOptionalNo

18 Main genetic abnormalities in B-ALL AbnormalityGenes involved IncidenceMolecular detection t(9;22)(q34;q11)BCR-ABLAdults 30% Children 3% RT-PCR t(12;21)(p13;q22)TEL-AML1Adults <1% Children 20% RT-PCR t(4;11)(q21;q23)MLL-AF4Adults 5% Infants 60% RT-PCR t(1;19)(q23;p13)E2A-PBX15%RT-PCR t(8;14)(q24;q32)C-MYC-IgH1%FISH t(17;19)(q22;p13)E2A-HLF<1%RT-PCR t(11;19)(q23;p13)MLL-ENL<1%RT-PCR JAK 1/2/3 mutations 10%Sequencing Recommendations of the EWALL Group, 2012

19 Main genetic abnormalities in T-ALL AbnormalityGenes involvedIncidenceMolecular detection t(10;14)(q24;q11) t(7;10)(q34;q24) HOX11-TCRα/δ HOX11-TCβ Adults 31% Children 7% RT-PCR t(5;14)(q35;q32)HOX11L2-TCRα/δAdults 13% Children 20% RT-PCR, FISH t(1;14)(q23;p13)TAL1-TCRα/δ1-3%RT-PCR Normal 1p32SIL-TAL19-30%RT-PCR Inv(7)(p15q34), t(7;7)HOXA-TCRβ5%FISH, RT-PCR t(10;11)(p13;q14-21)CALM-AF1010%FISH t(9;9)(q34;q34)NUP214-ABL16%FISH t(9;14)(q34;q34)EML1-ABL1<1%FISH NOTCH1 mutationsNOTCH150%Sequencing JAK1 mutationsJAK118%Sequencing Recommendations of the EWALL Group, 2012

20 Pui C.-H., Jeha S. Nature Rev Drug Discovery 2007; 6: Genetic Heterogeneity in Adult ALL

21 Minimum diagnostic approach in ALL Morphology/Cytochemistry Blast percentage in BM above 25% MPO negative Immunophenotyping (minimum marker panel) CyMPO, CD117, TdT, cyCD3, CD7, cyCD79a, CD19 Cytogenetics/molecular genetics Identification of t(9;22)/ BCR-ABL and t(4;11)/MLL-AF4 Recommendations of the EWALL Group, 2012

22 Comprehensive diagnostic approach Morphology/cytochemistry As above Immunophenotyping (refined panel) First step : cyMPO, CD117, TdT, cyCD3, CD7, cyCD79a, CD19, CD13, CD33, CD34, HLA-DR Second step : - B-ALL: CD10, CD20, CD22, cyIgM, sIg, CD52, CD45, CD58, Ng2 or CD133, CD66c - T-ALL: CD1a, CD2, CD3, CD5, CD4, CD8, CD52, CD99 Cytogenetics As above; whenever possible, other approaches such as CGH and aCGH may be performed Molecular genetics Detection of fusion genes as required for risk stratification Detection of Ig/TCR rearrangements Gene expression profilling (research/diagnosis purposes) Recommendations of the EWALL Group, 2012

23 Usefulness of diagnostic work-up Diagnosis Prognosis and treatment stratification MRD evaluation and follow-up Early detection of relapses

24 Prognostic impact of genetic and molecular classification of childhood ALL Pui C-H. Lancet 2008;371:1030

25 Genetics and prognosis in adult ALL. (MRC UKALLXII/ECOG 2993, n= 1522) Moorman, AV. et al. Blood 2007; 109:

26 Maury, S. et al. Haematologica 2010;95: CIR and EFS according to CD20 expression and WBC in adult ALL

27 Thomas, D. A. et al. Blood 2009;113: Outcome by CD20 expression and therapy according to age subgroups Protocol Young Elderly

28 T-ALL: prognostic value of differentiation stage/phenotype Baak U et al, Leukemia 2008 GMALL protocols

29 Bergeron, J. et al. Blood 2007;110: Prognostic impact of HOX11/TLX1 in adult T-ALL

30 Baldus, C. D. et al. J Clin Oncol; 25: Impact of BAALC expression on survival in adult T-ALL

31 . Breit, S. et al. Blood 2006;108: Effect of NOTCH1 mutation status on long-term prognosis in childhood T-ALL

32 . Asnafi, V. et al. Blood 2009;113: OS in adult T-ALLALL according to NOTCH1 and/or FBXW7 mutations and chemotherapeutic protocol

33 Baldus, C. D. et al. Haematologica 2009;94: EFS impact of NOTCH1-FBXW7 mutations within ERG/BAALC expression groups Low ERG/BAALC High ERG/BAALC

34 Mullighan C et al. N Engl J Med 2009;360: Genetic Alterations of IKZF1, EBF1, and BTLA/CD200 and the Cumulative Incidence of Relapse in the Original Cohort

35 Martinelli, G. et al. J Clin Oncol; 27: CIR according to IKZF1 deletion in BCR-ABL+ ALL

36 Spanish PETHEMA protocols in adult ALL Front line Standard risk High risk Very high-risk (Ph+ ALL) Elderly Ph- ALL Burkitt ALL ALL-SR08 ALL-AR-03 BURKIMAB ** * EWALL trial ** Joined with GMALL LAL Old* Young (<55yr) Ph+ALL08 DASACORD Elderly (>55yr) LAL OLDPh+

37 Usefulness of diagnostic work-up Diagnosis Prognosis MRD evaluation and follow-up Early detection of relapses

38 Stow, P. et al. Blood 2010;115: CIR among 379 children with B-lineage ALL whose MRD levels were less than 0.01% on day 46

39 Prognostic significance of MRD in adult ALL JM Ribera et al, ASH 2009Bassan R, et al. Blood 2009; 113:

40 Usefulness of diagnostic work-up Diagnosis Prognosis MRD evaluation and follow-up Early detection of relapses

41 MRD as a Predictor of Relapse in Adults with Standard-Risk, Ph-negative ALL Raff, T. et al. Blood 2007;109:

42 Clinical case

43 Female, 35 years old Clinical picture: weakness, gum bleeding and fever in the last 15 days Phisical exam : pale, petechiae and ecchymoses on arms and legs, gum bleeding, liver enlargement (3 cm below right costal margin) Complete blood count Hb 88 g/L, hematocrit 0,24 L/L, MCV 90fL, WBC count 48x10 9 /L (20% N, 30% L, 50% blasts), platelet count 15x10 9 /L, coagulation status normal Serum biochemical parameters Uric acid 8.8 mg/dL, LDH 2230 U/L. Chest X-ray : normal EKG : normal BM aspirate : 98% blasts, lymphoid appearance Cytochemistry : peroxidase negative Cytogenetics : 46, XX, t(9;22)(q34.1;q11.2)[22] Immunophenotypic study : Precursor B-ALL CD10+, with myeloid markers Molecular biology : BCR-ABL, p190 CSF study : normal

44 May-Grünwald-Giemsa

45 Flow Cytometry CD10+ ALL with My: CD33+; CD66C++

46 46, XX, t(9;22) (q34.1;q11.2) [22]

47 BCR ABL1 FISH. BCR-ABL

48 & 2: Patient 1 (positive p190) 3 & 4: Pacient 2 (negative p190) 5: Positive control p190 6: Negative control 7: Marker of molecular weight [(BCR-ABL)/ABL]x100: p190 BCR-ABL

49 Treatment Induction : - Imatinib, VCR, DNR, PDN (clinical trial CSTIBES02) - Result: Complete remission - [(BCR-ABL)/ABL]x100: Consolidation-1 - Imatinib, HD-MTX, HD-ARA-C - [(BCR-ABL)/ABL]x100: Allogeneic SCT from a HLA-identical sibling - Conditioning regimen: cyclophosphamide + ICT - Grade 2 cutaneous acute GVHD - Chronic GVHD with limited skin involvement - [(BCR-ABL)/ABL]x100: Imatinib post TPH -Well tolerated - [(BCR-ABL)/ABL]x100: Sustained complete molecular remission

50 Molecular follow-up (RQ-PCR)

51 EFS. CSTIBES02 vs. ALL Ph08

52 Ph+ ALL TKI era Imatinib+CHT Allo-SCT Tx post TPH

53 PH+ ALL in the TKI era Unsolved questions Induction - Intensity of CHT, number of cycles? - Type of TKI. Combination of TKI? SCT - - Always? - Modality? - MRD status at SCT Maintenance after SCT - - Always or in MRD+ status? - Type of TKI - TKI + other cytotoxic/immunomodulatory drugs? - Duration?

54 White blood cells from a patient with acute lymphoblastic leukaemia Lancet Oncology 2009 Thank you!


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