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!