diagnóstico y monitorización de hemopatías malignas
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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 de Barcelona. V Reunión de la Sociedad Asturiana de Hematología y Hemoterapia Gijon, 9 de marzo de 2012. - PowerPoint PPT PresentationTRANSCRIPT
Diagnóstico y monitorización de hemopatías malignas
JM Ribera Servicio de Hematologia Clínica
ICO-Hospital Germans Trias i PujolBadalona
Universidad Autónoma de Barcelona
V Reunión de la Sociedad Asturiana de Hematología y HemoterapiaGijon, 9 de marzo de 2012
Diagnosis in Malignant HematologyFrom 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
Importance of diagnostic methods
• Accuracy of diagnosis• Staging• Prognostic assessment• Analysis of response to therapy• Follow-up
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.
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
ALL Morphology
Sensitivity: 10-2
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 onlyCD2+ 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-IIIMature T-ALL (T-IV)α/β+ T-ALLγ/δ+ T-ALL
Recommendations of the EWALL Group, 2012
Phenotypic study
Sensitivity: 10-4 (4 colors), ≥10-5 (>4 colors)
Cytogenetics
Sensitivity: 10-2
Hyperdiploidy52,XY,+X,+6,+14,+17,+21,+mar
Hypodiploidy41,XX,-4,-9,add(9)(p21),-15,-20,-22
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
Pseudodiploidy
46, XY, t(9;22)(q34.1;q11.2)Burkitt ALL
ALL FISH
IgH/c-MYC
BCR-ABL
Sensitivity: 5x10-2
nuc ish(ABL1x3),(BCRx3),(ABL1con BCRx2)[90/100]
IgH & TCR rearrangements
Sensitivity: 10-4 – 10-5 (RQ-PCR)
TCR clonal
FR1
(310-360pb)
310pb
IgH clonal
BCR/ABL - t(9;22)(q34.1;q11.2)
1 2 3 4 5 6 7
1 & 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
Standard curve
Linear dynamic range (5 Logs)
RQ-PCR
Sensitivity: 10-5- 10-6
Recommendations for genetic diagnosis and follow-up of hematologic neoplasias. ALL
(AEHH, FEHH, GCECGH, Grupo BMH)(2007)
Subtype Phase Sample Conventional cytogenetics
FISH Molecular biology
Precursor B-ALL
Diagnosis BM Always BCR/ABL, MLL BCR/ABL, AML/AF4
Relapse BM Optional Optional No
Burkitt’s lymphoma/leukemia
Diagnosis BM/Tumor tissue
Always C-MYC No
Relapse BM/Tumor tissue
Optional Optional No
Pediatric ALL Diagnosis BM Always If NK or no metaphases: TEL/AML1, PBX1/E2A, MLL, BCR/ABL
TEL/AML1, PBX1/E2A,AF4/MLLBCR/ABL
MRD BM No According to diagnosis
According to diagnosis
T-ALL Diagnosis BM Always No No
Relapse BM Optional No No
Main genetic abnormalities in B-ALL
Abnormality Genes involved
Incidence Molecular detection
t(9;22)(q34;q11) BCR-ABL Adults 30%Children 3%
RT-PCR
t(12;21)(p13;q22) TEL-AML1 Adults <1%Children 20%
RT-PCR
t(4;11)(q21;q23) MLL-AF4 Adults 5%Infants 60%
RT-PCR
t(1;19)(q23;p13) E2A-PBX1 5% RT-PCRt(8;14)(q24;q32) C-MYC-IgH 1% FISHt(17;19)(q22;p13) E2A-HLF <1% RT-PCRt(11;19)(q23;p13) MLL-ENL <1% RT-PCR
JAK 1/2/3 mutations
10% Sequencing
Recommendations of the EWALL Group, 2012
Main genetic abnormalities in T-ALLAbnormality Genes involved Incidence Molecular
detectiont(10;14)(q24;q11)t(7;10)(q34;q24)
HOX11-TCRα/δHOX11-TCβ
Adults 31%Children 7%
RT-PCR
t(5;14)(q35;q32) HOX11L2-TCRα/δ Adults 13%Children 20%
RT-PCR, FISH
t(1;14)(q23;p13) TAL1-TCRα/δ 1-3% RT-PCR
Normal 1p32 SIL-TAL1 9-30% RT-PCR
Inv(7)(p15q34), t(7;7) HOXA-TCRβ 5% FISH, RT-PCR
t(10;11)(p13;q14-21) CALM-AF10 10% FISH
t(9;9)(q34;q34) NUP214-ABL1 6% FISH
t(9;14)(q34;q34) EML1-ABL1 <1% FISH
NOTCH1 mutations NOTCH1 50% Sequencing
JAK1 mutations JAK1 18% Sequencing
Recommendations of the EWALL Group, 2012
Pui C.-H., Jeha S. Nature Rev Drug Discovery 2007; 6:149-165
Genetic Heterogeneity in Adult ALL
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
Comprehensive diagnostic approachMorphology/cytochemistryAs 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
CytogeneticsAs 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
Usefulness of diagnostic work-up
• Diagnosis• Prognosis and treatment stratification• MRD evaluation and follow-up• Early detection of relapses
Prognostic impact of genetic and molecular classification of childhood
ALL
Pui C-H. Lancet 2008;371:1030
Genetics and prognosis in adult ALL. (MRC UKALLXII/ECOG 2993, n= 1522)
Moorman, AV. et al. Blood 2007; 109:3189-97
Maury, S. et al. Haematologica 2010;95:324-328
CIR and EFS according to CD20 expression and WBC in adult ALL
Thomas, D. A. et al. Blood 2009;113:6330-6337
Outcome by CD20 expression and therapy according to age subgroups
Protocol
Young
Elderly
T-ALL: prognostic value of differentiation stage/phenotype
Baak U et al, Leukemia 2008GMALL protocols
Bergeron, J. et al. Blood 2007;110:2324-2330
Prognostic impact of HOX11/TLX1 in adult T-ALL
Baldus, C. D. et al. J Clin Oncol; 25:3739-3745 2007
Impact of BAALC expression on survival in adult T-ALL
.
Breit, S. et al. Blood 2006;108:1151-1157
Effect of NOTCH1 mutation status on long-term prognosis in childhood T-ALL
.
Asnafi, V. et al. Blood 2009;113:3918-3924
OS in adult T-ALLALL according to NOTCH1 and/or FBXW7 mutations and
chemotherapeutic protocol
Baldus, C. D. et al. Haematologica 2009;94:1383-1390
EFS impact of NOTCH1-FBXW7 mutations within ERG/BAALC expression groups
Low ERG/BAALC
High ERG/BAALC
Mullighan C et al. N Engl J Med 2009;360:470-480
Genetic Alterations of IKZF1, EBF1, and BTLA/CD200 and the Cumulative Incidence of Relapse in the Original Cohort
Martinelli, G. et al. J Clin Oncol; 27:5202-5207 2009
CIR according to IKZF1 deletion in BCR-ABL+ ALL
Spanish PETHEMA protocols in adult ALLSpanish PETHEMA protocols in adult ALLFront lineFront line
Standardrisk
Highrisk
Very high-risk(Ph+ ALL)
Elderly Ph-ALL
BurkittALL
ALL-SR08ALL-AR-03 BURKIMAB**
* EWALL trial**Joined with GMALL
LAL Old*
Young (<55yr)Ph+ALL08DASACORD
Elderly (>55yr)LAL OLDPh+
Usefulness of diagnostic work-up
• Diagnosis• Prognosis• MRD evaluation and follow-up• Early detection of relapses
Stow, P. et al. Blood 2010;115:4657-4663
CIR among 379 children with B-lineage ALL whose MRD levels were less than 0.01% on day 46
Prognostic significance of MRD in adult ALL
JM Ribera et al, ASH 2009 Bassan R, et al. Blood 2009; 113: 4153-4162
Usefulness of diagnostic work-up
• Diagnosis• Prognosis• MRD evaluation and follow-up• Early detection of relapses
MRD as a Predictor of Relapse in Adults with Standard-Risk, Ph-negative ALL
Raff, T. et al. Blood 2007;109:910-915
Clinical case
Female, 35 years oldClinical picture: weakness, gum bleeding and fever in the last 15 daysPhisical 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 48x109/L(20% N, 30% L, 50% blasts), platelet count 15x109/L, coagulationstatus normal
Serum biochemical parametersUric acid 8.8 mg/dL, LDH 2230 U/L.
Chest X-ray: normalEKG: normalBM aspirate: 98% blasts, lymphoid appearanceCytochemistry: peroxidase negativeCytogenetics: 46, XX, t(9;22)(q34.1;q11.2)[22]Immunophenotypic study: Precursor B-ALL CD10+, with myeloid
markersMolecular biology: BCR-ABL, p190CSF study: normal
May-Grünwald-Giemsa
Flow CytometryCD10+ ALL with My: CD33+; CD66C++
46, XX, t(9;22) (q34.1;q11.2) [22]
BCR
ABL1
FISH. BCR-ABL
1 2 3 4 5 6 7
1 & 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: 130.12
p190BCR-ABL
TreatmentInduction:
- Imatinib, VCR, DNR, PDN (clinical trial CSTIBES02)- Result: Complete remission- [(BCR-ABL)/ABL]x100: 0.032
Consolidation-1- Imatinib, HD-MTX, HD-ARA-C- [(BCR-ABL)/ABL]x100: 0.0079
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: 0.00001
Imatinib post TPH-Well tolerated- [(BCR-ABL)/ABL]x100: 0.000003-Sustained complete molecular remission
130,12
0,0320,0079
0,000010,000002 0,0000015
0,000010,000001
0,00000010,0000010,000010,00010,0010,010,11101001000
2/11/2005 15/12/2005 2/1/2006 7/11/2006 3/5/2007 17/9/2007 29/4/2008 25/11/2008
Data
Molecular follow-up (RQ-PCR)
EFS. CSTIBES02 vs. ALL Ph08
Ph+ ALLTKI era
Imatinib+CHT Imatinib+CHT Allo-SCT Allo-SCT Tx post TPH Tx post TPH
PH+ ALL in the TKI eraPH+ ALL in the TKI eraUnsolved questionsUnsolved 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?
White blood cells from a patient with acute lymphoblastic leukaemia Lancet Oncology 2009
Thank you!