1. Diagnóstico y monitorización de hemopatías malignas
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 1

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 2

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

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: 10-4 (4 colors), ≥10-5 (>4 colors)

10. Cytogenetics Hyperdiploidy Hypodiploidy 52,XY,+X,+6,+14,+17,+21,+mar
41,XX,-4,-9,add(9)(p21),-15,-20,-22
Sensitivity: 10-2

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

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

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

14. IgH & TCR rearrangements
IgH clonal
TCR clonal
FR1
( pb)
310pb
Sensitivity: 10-4 – 10-5 (RQ-PCR)

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

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

17. Conventional cytogenetics
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
Optional No
Burkitt’s lymphoma/
leukemia
BM/Tumor tissue
C-MYC
Pediatric ALL
If NK or no metaphases: TEL/AML1, PBX1/E2A, MLL, BCR/ABL
TEL/AML1, PBX1/E2A,
AF4/MLL
BCR/ABL
MRD
According to diagnosis
T-ALL

18. 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%
t(4;11)(q21;q23)
MLL-AF4
Adults 5%
Infants 60%
t(1;19)(q23;p13)
E2A-PBX1 5%
t(8;14)(q24;q32)
C-MYC-IgH 1%
FISH
t(17;19)(q22;p13)
E2A-HLF
<1%
t(11;19)(q23;p13)
MLL-ENL
JAK 1/2/3 mutations
10%
Sequencing
Recommendations of the EWALL Group, 2012

19. 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%
Normal 1p32
SIL-TAL1
9-30%
Inv(7)(p15q34), t(7;7)
HOXA-TCRβ 5%
FISH,
t(10;11)(p13;q14-21)
CALM-AF10
10%
FISH
t(9;9)(q34;q34)
NUP214-ABL1 6%
t(9;14)(q34;q34)
EML1-ABL1
<1%
NOTCH1 mutations
NOTCH1
50%
Sequencing
JAK1 mutations
JAK1
18%
Recommendations of the EWALL Group, 2012

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

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

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

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

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

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

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

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

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

33. 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:

34. Genetic Alterations of IKZF1, EBF1, and BTLA/CD200 and the Cumulative Incidence of Relapse in the Original Cohort
Figure 3. Genetic Alterations of IKZF1, EBF1, and BTLA/CD200 and the Cumulative Incidence of Any Relapse in the Original Cohort. Panel A shows the cumulative incidence of relapse in the entire original cohort of patients with B-cell-progenitor ALL according to IKZF1, EBF1, and BTLA/CD200 alteration status. Panel B shows the cumulative incidence of relapse in the entire validation cohort, including patients with BCR-ABL1-positive B-cell-progenitor ALL. Panel C shows the cumulative incidence of relapse in the validation cohort after exclusion of patients with BCR-ABL1-positive B-cell-progenitor ALL. Only IKZF1 alterations were associated with poor outcome in both the original and validation cohorts. Five-year estimates of relapse are shown in the original cohort, and 10-year estimates are shown in the validation cohort. P values are calculated with the use of Gray's test.
Mullighan C et al. N Engl J Med 2009;360:

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

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
LAL Old*
ALL-AR-03
BURKIMAB**
Young (<55yr)
Ph+ALL08
DASACORD
Elderly (>55yr)
LAL OLDPh+
* EWALL trial
**Joined with GMALL

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

38. 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:

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

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

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

42. Clinical case

43. Clinical picture: weakness, gum bleeding and fever in the last 15 days
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

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. FISH. BCR-ABL
BCR
ABL1

48. p190BCR-ABL 1 2 3 4 5 6 7 1 & 2: Patient 1 (positive p190)
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: 48

49. Treatment Induction: Consolidation-1 - Imatinib, HD-MTX, HD-ARA-C
- 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:
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 52

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? 53

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