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

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/MLL

BCR/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-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


Main genetic abnormalities in T-ALLAbnormality Genes involved Incidence Molecular

detection

t(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


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


Comprehensive diagnostic approach

Morphology/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)


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+


• Diagnosis

• Prognosis



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


• Diagnosis

• Prognosis



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

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

130,12

0,0320,0079

0,000010,000002 0,0000015

0,00001

0,000001

0,0000001

0,000001

0,00001

0,0001

0,001

0,01

0,1

1

10

100

1000

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+ ALL

TKI 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!

Documents

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