V Reunión de la Sociedad Asturiana de Hematología y Hemoterapia
Gijon, 9 de marzo de 2012
Diagnóstico y monitorización de
hemopatías malignas
JM Ribera
Servicio de Hematologia Clínica
ICO-Hospital Germans Trias i Pujol
Badalona
Universidad Autónoma de Barcelona
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
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Imaging studies: X-ray, CT, MRI, PET scan
Isotopic studies
“Omic” studies: genomic, proteomic, metabolomic
Nanotechnology
Importance of diagnostic methods
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Accuracy of diagnosis
Staging
Prognostic assessment
Analysis of response to therapy
Follow-up
Diagnostic work-up in ALL
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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
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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+
CD10CD10+, cyIgCig+, sIgsIg+
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+, CD1asCD3+, 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
Phenotypic study
Sensitivity: 10-4 (4 colors), ≥10-5 (>4 colors)
Cytogenetics
Hyperdiploidy
52,XY,+X,+6,+14,+17,+21,+mar
Hypodiploidy
41,XX,-4,-9,add(9)(p21),-15,-20,-22
Sensitivity: 10-2
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
Burkitt ALL
46, XY, t(9;22)(q34.1;q11.2)
ALL FISH
BCR-ABL
IgH/c-MYC
nuc ish(ABL1x3),(BCRx3),(ABL1con BCRx2)[90/100]
Sensitivity: 5x10-2
IgH & TCR rearrangements
IgH clonal
TCR clonal
FR1
(310-360pb)
310pb
Sensitivity: 10-4 – 10-5 (RQ-PCR)
Quantification of the amount of mRNA transcripts
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
RQ-PCR
Standard curve
Linear dynamic range (5 Logs)
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
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/MLL
BCR/ABL
MRD
BM
No
According to
diagnosis
According to
diagnosis
Diagnosis
BM
Always
No
No
Relapse
BM
Optional
No
No
Precursor BALL
T-ALL
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-PCR
t(8;14)(q24;q32)
C-MYC-IgH
1%
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
Main genetic abnormalities in T-ALL
Abnormality
Genes involved
Incidence
Molecular
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/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
Genetic Heterogeneity in Adult ALL
Pui C.-H., Jeha S. Nature Rev Drug Discovery 2007; 6:149-165
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 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
Usefulness of diagnostic work-up
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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
CIR and EFS according to CD20 expression and WBC
in adult ALL
Maury, S. et al. Haematologica 2010;95:324-328
Outcome by CD20 expression and therapy
according to age subgroups
Protocol
Young
Elderly
Thomas, D. A. et al. Blood 2009;113:6330-6337
T-ALL: prognostic value of differentiation
stage/phenotype
GMALL protocols
Baak U et al, Leukemia 2008
Prognostic impact of HOX11/TLX1 in adult T-ALL
Bergeron, J. et al. Blood 2007;110:2324-2330
Impact of BAALC expression on survival in adult T-ALL
Baldus, C. D. et al. J Clin Oncol; 25:3739-3745 2007
Effect of NOTCH1 mutation status on long-term
prognosis in childhood T-ALL
Breit, S. et al. Blood 2006;108:1151-1157
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OS in adult T-ALLALL
according to NOTCH1 and/or FBXW7 mutations and
chemotherapeutic protocol
Asnafi, V. et al. Blood 2009;113:3918-3924
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EFS impact of NOTCH1-FBXW7 mutations
within ERG/BAALC expression groups
Low ERG/BAALC
High ERG/BAALC
Baldus, C. D. et al. Haematologica 2009;94:1383-1390
Genetic Alterations of IKZF1, EBF1, and BTLA/CD200 and
the Cumulative Incidence of Relapse in the Original Cohort
Mullighan C et al. N Engl J Med 2009;360:470-480
CIR according to IKZF1 deletion in BCR-ABL+ ALL
Martinelli, G. et al. J Clin Oncol; 27:5202-5207 2009
Spanish PETHEMA protocols in adult ALL
Front line
Standard
risk
ALL-SR08
High
risk
Very high-risk
(Ph+ ALL)
ALL-AR-03
Young (<55yr)
Ph+ALL08
DASACORD
Elderly PhALL
LAL Old*
Burkitt
ALL
BURKIMAB**
Elderly (>55yr)
LAL OLDPh+
* EWALL trial
**Joined with GMALL
Usefulness of diagnostic work-up
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Diagnosis
Prognosis
MRD evaluation and follow-up
Early detection of relapses
CIR among 379 children with B-lineage ALL
whose MRD levels were less than 0.01% on day 46
Stow, P. et al. Blood 2010;115:4657-4663
Prognostic significance of MRD in adult ALL
Bassan R, et al. Blood 2009; 113: 4153-4162
JM Ribera et al, ASH 2009
Usefulness of diagnostic work-up
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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 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 48x109/L
(20% N, 30% L, 50% blasts), platelet count 15x109/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
May-Grünwald-Giemsa
Flow Cytometry
CD10+ ALL with My: CD33+; CD66C++
46, XX, t(9;22) (q34.1;q11.2) [22]
FISH. BCR-ABL
BCR
ABL1
p190BCR-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
Treatment
Induction:
- 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
Molecular follow-up (RQ-PCR)
EFS. CSTIBES02 vs. ALL Ph08
Ph+ ALL
TKI era
Imatinib+CHT  Allo-SCT  Tx post TPH
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?
Thank you!
White blood cells from a patient with acute lymphoblastic
leukaemia
Lancet Oncology 2009