1. Genetics in the Management of Leukemia and Lymphoma
Terry Hayes, M.D., Ph.D. 1

2. Overview
Many subtypes of acute and chronic leukemias are associated with specific changes in the DNA.
These genetic lesions can be used for diagnosis and prognosis.
Understanding the molecular defects may lead to improvement in therapeutic approach. 2

3. Types of Genetic Lesions in Hematologic Malignancies
Translocations that result in abnormal gene expression due to placement of genes near enhancer or promoter elements of other genes.
Translocations that produce chimeric fusion proteins with biologic activity.
Mutations in known oncogenes/tumor suppressor genes. 3

4. Consequences of Recurring Chromosomal Translocations
Gene A
Gene B
Altered expression of
normal gene B protein
Leukemia, Lymphoma A
Expression of
fusion proteins B
Leukemia, Lymphoma, Sarcoma 4

5. 5

6. 6

7. t(8;14) 7

8. Consequence of Translocation
The immunoglobulin gene causes an abnormal or altered expression of the normal protein produced by the other gene.
It is the abnormal expression, timing and level that are associated with the malignant phenotype. 8

9. Myelodysplastic Syndromes9

10. Myelodysplastic Syndromes
Refractory anemia
Refractory anemia with ringed sideroblasts
Refractory anemia with excess blasts
Refractory anemia with excess blasts in transformation
Chronic myelomonocytic leukemia 10

11. Genetic Abnormalities in MDS
Found in almost 50% of cases.
Non-random.
Different mutations associated with particular types of MDS. 11

12. Common Cytogenetic Abnormalities in MDS
Abnormality Incidence
Loss of all or part of chr %
Trisomy %
Loss of all or part of chr %
Deletion of 11q, 12p, 20q 1-7%
Deletion of X or Y 2-5% 12

13. Genes Found on 5q- IL-4 IL-5 GM-CSF M-CSF PDGF receptor CD-14
M-CSF receptor
(fms oncogene) 13

14. 5q- Ringed Sideroblast 5q-, most common abnormality in MDS
In 27% of MDS
Associated with refractory anemia and refractory anemia with ringed sideroblasts 14

15. Abnormalities Associated with 5q-
Megaloblastic change
Abnormal erythroid precursors 15

16. Abnormalities Associated with 5q-
Micromegakaryocyte
Pseudo Pelger-Huët Neutrophil 16

17. Myelodysplastic Syndromes
Mutations in ras genes: N-ras, K-ras, H-ras
Found in 10 to 25% of MDS patients
Ras gene mutation most common in CMML 17

18. Secondary MDS After exposure to epipodophyllotoxins and anthracyclines
Balanced translocations involving:
3q26
11q23
21q22 18

19. FISH in MDS 5q- Trisomy 8 Monosomy 7 Fluoroscein labeled 5p
Rhodamine labeled 5q
Red centromere probe 19

20. Prognostic Subgroups in MDS
Poor
translocations involving chromosomes 1, 3, 6
Variable
deletions of chromosomes 11q, 13q, 12q, 9q
trisomy 8
Good
deletions of 5q, 20q; trisomy 21
Papenhausen et al., Cancer Control 1997; 4: 389. 20

21. Acute and Chronic Leukemias21

22. FAB Classification Lymphoid L1 L2 L3 Myeloid M0 l M4 M1 l M5 M2 l M622

23. FAB Classification and Associated Genetic Abnormalities
FAB Common Associated
Subtype Name Gene Change
M0 Acute myeloblastic leukemia inv(3q26)
with minimal differentiation (3%) and t(3;3) 1%
M1 Acute myeloblastic leukemia
without maturation (15-20%)
M2 Acute myeloblastic leukemia t (8;21) 40%
with maturation (25-30%) t(6;9) %
M3 Acute promyelocytic leukemia t(15;17) 98%
(5-10%) t(11;17) 1%
t(5;17) %
NEJM 1999:341:1052 23

24. FAB Classification and Associated Genetic Abnormalities
FAB Common Associated
Subtype Name Gene Change, %
M Acute myelomonocytic leukemia 11q %
(20%) inv (3q26), t(3;3) 3%
M4eo Acute myelomonocytic leukemia inv(16), t(16;16) 80%
with abnl eosinophils (5-10%)
M5 Acute monocytic leukemia (2-9%) q %
t(8:16) %
M6 Erythroleukemia (3-5%)
M7 Acute megakaryocytic leukemia t(1;22) %
NEJM 1999:341:1052 24

25. Acute Lymphocytic Leukemia
Over 40 known translocations.
Correlate with prognosis.
Can be used to direct therapy. 25

26. B CELL DIFFERENTIATION
B cell Precursors
Early B cell
Precursors
Plasma cell
Early B cell Ig
Pre B cell
B cell
Cell Surface Markers
CD19
CALLA (CD10)
CD20
CD38
Precursor B Cell Leukemias
CLL, B Cell Lymphomas
Waldenström’s, Myeloma 26

27. ALL L1 and L2 Can be B or T cell lineage. May be hyperdiploid.
Common translocations
t(9;22), t(4;11), t(1;9) 27

28. Philadelphia Chromosome
t(9;22)(q34;q11)
Found in % of adult CML
20-30% of adult ALL
5% of childhood ALL
Results in bcr-abl fusion protein
c-abl on chromosome 9 (tyrosine kinase)
bcr on chromosome 22 (function?) 28

29. 29

30. Philadelphia Chromosome
In childhood ALL and 50% of adult ALL, bcr exon 1 is fused with c-abl exon 2
= 190 kd.
In CML and the other 50% of adult ALL, bcr exon 2 or 3 is fused to c-abl exon 2
= 210 kd.
Both are active tyrosine kinases. 30

31. 31

32. BCR-ABL Protein
Wild-type abl gene shuttles between nucleus and cytoplasm.
Constitutively active tyrosine kinase confined to cytoplasm.
Phosphorylates multiple substrates.
Can transform hematopoietic cells.
Protects hematopoietic cells from apoptosis. 32

33. 33

34. 34

35. Phosphatidylinositol-3
BCR-ABL
Phosphatidylinositol-3
kinase
RAS
RAF
STAT
MYC
AKT
JUN KINASE 35

36. STI 571 Specific Bcr-Abl inhibitor. Daily oral therapy.
Given to patients in chronic phase.
All patients achieved complete hematologic responses, and some had cytogenetic responses.
Minimal side effects. 36

37. ALL L1 or L2 t(4;11)(q21;q23) Biphenotypic markers
Infants <1 yr. or adults
>90% of infantile ALL
MLL gene translocated to AF-4
High wbc >150K, worst prognosis 37

38. ALL L1 or L2 t(1;19)(q21;p13). One fourth of childhood pre B-cell ALL.
E2A and PBX1 are transcription factors.
E2A-PBX1 fusion protein combines the transcriptional activation domain of E2A with the DNA binding domain of PBX1. 38

39. E2A-PBX1 Fusion Protein
Normal E2A gene helps regulate expression of kappa light chain gene.
PBX1 is homologous to the DNA binding region of homeobox genes
homeobox genes regulate the expression of genes critical to embryonic development. 39

40. ALL L3/Burkitt’s B cell or Burkitt's type leukemia. 5% of ALL.
Large round cells with deeply basophilic cytoplasm and vacuoles. 40

41. ALL - L3
Associated chromosomal translocations include t(8;14), t(2;8), t(8;22).
Poor prognosis with standard ALL treatment regimens, better with intensified regimens. 41

42. ALL L3/Burkitt’s Lymphoma42

43. IgH
Igl
Igk 43

44. Genetic Changes in T-ALL
Transcription factors translocated to T cell receptors in many T-cell ALL
TCR a/d at 14q11-14
TCR b at 14q34-36 44

45. Genotypes in ALL 45

46. Prognostic Significance in ALL
Favorable prognosis:
hyperdiploid (>50 chromosomes)
t(12;21)
Poor prognosis:
t(9;22)
t(4;11)
t(1;19) 46

47. Ph Positive ALL Worst outcome in childhood ALL.
DFS 49% at 5 years if wbc at diagnosis <50,000.
DFS 20% at 5 years if wbc at diagnosis >100,000.
Patients do better with bone marrow transplantation from a HLA-matched donor.
NEJM 2000;342:998. 47

48. Survival in Ph+ ALL Transplantation Chemotherapy Alone
NEJM 2000;342:998. 48

49. Very High Risk Ph chromosome t(4;11) wbc ³ 200,000 £ 1 y.o. with MLL
Lower Risk
B lineage ALL
Age 1 to 9
wbc < 50,000 49

50. 50

51. AML M2 t(8;21)(q22;q22) 7% of AML Splenomegaly, chloromas
Gene fusion ETO/AML1 51

52. AML M2 More mature leukemic cell Prominent Auer rods.
Good prognosis with t(8;21)
t(8;21) is 15% of AML 52

53. 53

54. AML M2 8;21 Translocation
Involves AML1 gene on chromosome 21 and ETO on chromosome 8.
AML1 is a protein critically involved in binding DNA; regulates transcription of many myeloid-specific genes. Binds with CBFb.
Translocation breaks AML1 in the middle of the gene, removes the transactivation domain and replaces it with part of ETO. 54

55. AML1-CBFb Complex 55

56. Other Translocations Involving AML1
AML1 fused to beta-myosin heavy chain gene in inv(16), seen in AML M4
AML1 and TEL genes involved in 12;21 translocation seen in 3-% of B progenitor pediatric ALL.
AML1 important in substantial proportion of both AML and ALL. 56

57. AML M3 Acute promyelocytic leukemia. t(15;17).
Fuses PML with retinoic acid receptor.
DIC.
Good prognosis. 57

58. AML M3
Fuses PML gene on chromosome 15 with retinoic acid receptor on chromosome 17.
The retinoic acid receptor binds normally.
The PML gene is associated with histone deacetylators, which change the function of genes.
The histone tails wind around the DNA, and the gene is inactivated. 58

59. AML M3
Normal physiologic levels of retinoic acid cannot activate the target genes.
Pharmacologic levels of retinoic acid dissociate the histone deacetylation complex and allow the gene to activate, leading to cell differentiation.
Variant of M3 involves 11;17 translocation, with PLZF gene instead of PML.
No response to ATRA. 59

60. AML M4
Myelomonoblastic leukemia.
7-10% of AML.
M4 Eo subtype. 60

61. AML M4 Eo Inv(16) or t(16;16). Breakpoint at 16q22. >90% of M4 Eo.
Good prognosis. 61

62. M4 Eo
Involves the CBFb gene (normally bound to AML1, the gene involved in M2 AML).
CBFb gene is also involved in pediatric ALL and many rare translocations in AML; one of the most frequent targets of chromosome rearrangements in leukemia. 62

63. AML1-CBFb Complex 63

64. M4 Eo
CBFb subunit is fused to smooth muscle myosin heavy chain gene on chromosome 16.
Sequesters AML1 into functionally inactive complexes within the cytoplasm. 64

65. AML M4 or M5 t(11q23;variable). Approximately 5% of M4 and M5 AML.
MLL gene.
Poor prognosis. 65

66. MLL Gene
“MLL”: present in myeloid, lymphoid and mixed lineage leukemias.
At least 38 different translocations, most often found in M4 and M5 AML.
19 of 38 have been cloned.
Leukemias involving MLL lesion generally have a poor prognosis. 66

67. MLL Translocation Partners
Location of
MLL Translocation Partners
AML
ALL
other
X Y
AF1p
AF1q
AF4
AF5
AF6
AF6q21
AF9
AF10
AB11
MLL
AF15
CBP
AF17
MSF
EEN
ENL
ELL
CDCrel
p300
AFX
CHROMOSOMES 67

68. AML M5 Monoblastic t(9;11)(p22;q23) Children, young adults
Skin or gums infiltrated with leukemic cells
MLL translocated to 9p22 68

69. AML M6 Erythroid leukemia. 5% of AML.
Often associated with deletions in chromosomes 5 and 7.
Poorer prognosis, often preceded by myelodysplastic syndrome.
Seen in older patients. 69

70. AML M7 Megakaryoblastic leukemia 10% of AML
Trisomy 21, inversions or translocations of chromosome 3, t(9;22); t(1;22) in infants.
Also seen in Klinefelter’s syndrome XXY. 70

71. Poor Prognostic Features in AML
Response to induction chemotherapy:
Unfavorable karyotype
Age >60 yr
Secondary AML
Poor performance status
wbc > 20,000
Unfavorable immunophenotype
Likelihood of relapse:
Unfavorable karyotype
Age >60 yr
Delayed response to induction chemo
wbc > 20,000
Female sex
Elevated LDH 71

72. Prognostic Features in AML
t(8;21), t(15;17), and inv(16) have good prognosis.
More frequent in younger patients
Abnormalities of chromosomes 5 or 7, trisomy 8, and t(9;11) have poor prognosis.
More common in older patients and patients with secondary leukemias 72

73. Overall Survival in AML
Favorable Cytogenetic Abnormalities
t(8;21)
inv (16)
t(15;17
Normal cytogenetics
Unfavorable Cytogenetic Abnormalities
Normal cytogenetics
complex
del (5q), abnl (3q), -7 -5
Grimwade, Blood 1998;92:2322. 73

74. Non-Hodgkin’s Lymphoma74

75. Burkitt’s Lymphoma t(8;14)(q24;q32) t(8;22)(q24;q11) t(2;8)(p11;q24)
c-myc to IgH
t(8;22)(q24;q11)
c-myc to Igl
t(2;8)(p11;q24)
Igk to c-myc
C-myc product regulates gene transcription. 75

76. Burkitt’s Lymphoma
c-myc breakpoint differs in sporadic and endemic Burkitt’s.
Epstein-Barr Virus:
97% of endemic Burkitt’s.
20-30% of sporadic cases. 76

77. Follicular Lymphomas t(14;18)(q32;q31).
Present in 85-90% of follicular lymphomas.
bcl-2 on chromosome 18, regulator of apoptosis.
Ig heavy chain on chromosome 14. 77

78. Mantle Cell Lymphoma t(11;14)(q13;q32)
bcl-1 or PRAD oncogene encoding cyclin D1 protein on chromosome 11, regulates cell division.
Ig heavy chain region on chromosome 14. 78

79. T Cell Lymphomas Translocations often involve T cell receptor genes
a and d chain genes on 14q11-14
b chain gene on 14q34-36
g chain gene on 7p15
Several other oncogenes
Ttg1, LylI, HOX 11
Transcription factor families 79

80. New Directions Spectral karyotyping (SKY)
Identifies all chromosomes simultaneously
Improves precision of karyotype analysis in malignant cells
Rowley et al, Blood 1999;93:2038. 80

81. 81

82. Summary: Genetic Changes in Leukemia and Lymphoma
Most identified chromosome changes are translocations that place critical hematopoietic genes (immunoglobulin or T cell receptor genes) near other genes that regulate transcription or cell proliferation.
Some translocations produce chimeric fusion proteins with abnormal biologic activity. 82

83. Summary: Genetic Changes in Leukemia and Lymphoma
Further understanding will allow us to deliver genotype-specific therapy to treat each individual malignancy in the optimal fashion. 83