1. Hematologic Malignancies CON 616, 2009
William H. Fleming, M.D., Ph.D.
Division of Hematology & Medical Oncology
Hematologic Malignancies Section
Knight Cancer Institute
OHSU

2. Blood Cell Formation

3. Acute vs. Chronic Leukemia
Acute Leukemia (AML and ALL)
excess myeloblasts or lymphoblasts
short clinical course (weeks to months)
Chronic Leukemia (CML and CLL)
accumulation of mature granulocytes or lymphocytes
longer clinical course (several to many years)

4. Acute Leukemia
A clonal, molecular abnormality of hematopoietic blast cells resulting in a failure of differentiation & uncontrolled cell proliferation
Accumulation of leukemic blast cells results in marrow replacement, organ infiltration and metabolic effects

5. Acute Leukemia: AML versus ALL
Adults % of acute leukemia is AML
Children-85% of acute leukemia is ALL
Leukemic Blast morphology
AML: cytoplasmic granules, Auer rods, more cytoplasm, 2-5 nucleoli
ALL: no cytoplasmic granules, minimal cytoplasm, 1-2 nucleoli

6. Acute Leukemia: Clinical Manifestations
Constitutional & Metabolic effects:
Weight loss
Fever
Hyperkalemia
Hyperuricemia

7. Acute Leukemia: Hematology Laboratory Findings
Decreased, normal or elevated WBC
Anemia
Thrombocytopenia
Blasts on peripheral blood smear (often)
Hypercellular bone marrow with 20% or more blasts (normal is < 5%)

8. Acute Leukemia: Clinical Manifestations
Marrow replacement, organ infiltration & metabolic effects
Marrow replacement
Neutropenia: infection
Anemia: pallor, fatigue, dyspnea
Thrombocytopenia: abnormal bruising and bleeding

9. Acute Leukemia: Clinical Manifestations
Organ infiltration
Bone pain
Hepatosplenomegaly
Lymphadenopathy
Gingival hypertrophy
Leukemic meningitis

10. AML With Minimal Differentiation (M0/M1)

11. Acute Leukemia AML vs. ALL
Cell Surface Markers by Flow cytometry
AML
CD13, CD33, glycophorin (M6), platelet antigens (M7)
ALL
B lineage: CD19, CD22, CD10 (+/-), surface Ig,
T lineage: CD2, CD3, CD5, CD7

12. AML: FAB classification
French American British classification
M0-M7 based on morphology, and special cytochemical studies
Historically, distinguishing AML M0 from ALL was a major clinical problem

13. AML FAB classification
M0,M1, M2: Myeloblasts with no, little or some granulocytic maturation
M3: Promyelocytic leukemia
M4: Myelomonocytic or eosinophilic
M5: Monocytic
M6: Erythroleukemia
M7: Megakaryoblastic
Not all that useful except for M3 or APL

14. Acute Leukemia: Blasts with Auer Rods

15. Auer rods = AML

16. Acute Leukemia: AML vs. ALL
Cytochemistry AML ALL
Myeloperoxidase
Sudan black
Non-specific esterase + (M4,5) -
PAS (M6) +
Acid phosphatase + (M6) +

17. FAB is Supplemented by Cytogenetic and Molecular analysis

18. Flow Cytometry & FISH Analysis

19. Gingival Hyperplasia

20. Chloroma (Granulocytic Sarcoma)

21. Leukemia Cutis

22. AML Clinical Features & Prognosis
Age
< 60 years: >80% remission, 20-30% DFS
> 60 years: ~60% remission, 5-15% DFS
Prior marrow disorder: Myelodysplasia (MDS)
Secondary AML (prior chemo or radiotherapy)
Response to induction therapy

23. AML Cytogenetics & Prognosis
Favorable
t(8;21), t(15;17), inv(16)
Intermediate (Most patients)
normal, +8, +21, +22, del(7q), del(9q),
Adverse
-5, -7, del(5q), abnormal 3q,
complex karyotype (> 3 -5 abnormalities)

24. AML Cytogenetics and Prognosis
Group CR 5 year survival
Favorable 91% %
Intermediate 86% %
Adverse 63% <15%

25. AML Mutations & Prognosis
Flt 3 (ITD) Adverse
NPM-1 mutation & no Flt3 - Favorable
MLL (PTD) Adverse
CEBPA Favorable

26. AML Treatment: Induction Chemotherapy
Anthracycline (Idarubicin) for 3 days and Cytosine arabinoside (Ara-C) for 7 days (3+7, Younger/fit patients only)
Three to 5 weeks of pancytopenia
Supportive care red cell and platelet transfusions, prophylactic antibacterial, antifungals and antivirals

27. AML: Response to Induction
Remission status determined by bone marrow at end of month following induction therapy (e.g. Day 14 & 28)
Complete remission:
Normal peripheral blood counts
Normocellular marrow with < 5% blasts and normal marrow cell maturation

28. AML Treatment: Consolidation
Following induction into Complete Remission
3-4 cycles of high dose cytosine arabinoside (HiDAC) administered approximately every 5-6 weeks OR
Bone marrow (peripheral blood stem cell) transplant
(Depends on degree of risk)

29. AML Treatment: Alternative Consolidation
One or more cycles of consolidation chemotherapy then either:
Autologous stem cell transplant after high dose chemotherapy or
Allogeneic bone marrow transplantation after high dose chemotherapy

30. AML Treatment Autologous Transplant
Advantage
Collection and subsequent infusion of patient’s stem cells allows administration of otherwise lethal doses of chemotherapy
Disadvantages
Despite CR, leukemic cells may persist in marrow, blood and stem cell product
High dose therapy more toxic than standard consolidation

31. AML Treatment: Allogeneic Transplant
Advantages
Stem cells from HLA-matched sibling or unrelated individual allow high dose therapy and are free of leukemia
Immunologic graft versus leukemia effect (GVL). Results in decreased rate of leukemic relapse

32. AML Treatment: Allogeneic Transplant
Disadvantages
Immunologic graft versus host disease (GVHD) and immunosuppressive therapy result in significant morbidity and mortality
GVHD incidence and severity increases with increasing age. (Best results in Pediatrics)
Tolerability of high dose transplant limited by patient age. (Reduced dose being evaluated)

33. AML Treatment: A risk adapted approach
Favorable
Conventional chemotherapy followed by transplant only if relapse occurs
Intermediate
Conventional chemotherapy alone or autologous or allogeneic transplant
Adverse
Conventional chemotherapy followed by allogeneic transplant

34. Current Risk Stratification OHSU Acute Leukemia Program (modified NCCN Guidelines v.1.2009)
Risk Status
Cytogenetics
Molecular Mutations
Better-risk
Inv(16)1
t(8 ;21) 1
t(16 ;16) 1
Normal cytogenetics with isolated NPM1 mutation
Intermediate-risk
Normal
+8 only
t(9 ;11)
MK negative
c-KIT3 in patients with t(8;21) or Inv(16)
Poor-risk
Complex (>3 abnormalities)
-5, -7, 5q-, 7q-
MK positive
Normal cytogenetics with isolated FLT3 mutations

35. AML: NCCN Guidelines http://www.nccn.org/index.html
National Comprehensive Cancer Network (NCCN) has issued guidelines for treatment of many cancers including AML (and other hematologic malignancies)

37. Acute Leukemia: AML versus ALL
Adults: 85% of acute leukemia is AML
Children: 85% of acute leukemia is ALL
Blast morphology
AML: cytoplasmic granules, Auer rods, more cytoplasm, 2-5 nucleoli
ALL: no cytoplasmic granules, minimal cytoplasm, 1-2 nucleoli

38. AML: FAB classification
French American British classification based on the degree of blast differentiation along different cell lineages and extent of maturation
M0-M7 based on morphology, lineage-specific cytochemical and immunologic findings

39. AML: FAB classification
M0,M1, M2: Myeloblasts with no, little or some granulocytic maturation
M3: Promyelocytic leukemia (APL)
M4: Myelomonocytic or eosinophilic
M5: Monocytic
M6: Erythroleukemia
M7: Megakaryoblastic

40. Acute Leukemia: AML vs. ALL
Cytochemistry AML ALL
Myeloperoxidase
Sudan black
Non-specific esterase + (M4,5) -
PAS (M6) + (c)
Acid phosphatase + (M6) + (T)

41. Acute Leukemia: AML vs. ALL
Immunologic markers / Flow cytometry
AML: CD13, CD33, glycophorin (M6), platelet antigens (M7)
ALL:
B lineage: CD19, CD22, CD10 (+/-), surface or cytoplasmic Ig, TdT (+/-)
T lineage: CD7, CD3, TdT

42. AML-M3 (APL)

43. AML-M3 (APL) an important FAB subtype
Acute Promyelocytic Leukemia (M3)
Blasts and promyelocytes heavily granulated, Auer rods often abundant & disseminated intravascular coagulation (DIC) is common
Treatment differs from all other AML subtypes. (Differentiating agent therapy)
Favorable prognosis (>85% survival)

44. AML: Prognosis
Age
< 60 years: 80% remission, 20-30% DFS
> 60 years: 50% remission, 5-15% DFS
Prior marrow disorder: MDS or secondary AML (prior chemo- or radio-therapy)
Cytogenetic analysis of blasts: specific chromosomal abnormalities dictate blast biology and have a major impact on outcome
Response to first round of therapy

45. AML: Cytogenetics and Prognosis
Favorable
t(8;21), t(15;17), inv(16)
Intermediate
normal, +8, +21, +22, del(7q), del(9q), abnormal 11q23, others
Adverse
Autosomal monosomy (-5, -7) abnormal 3q, complex cytogenetics

46. AML: Cytogenetics and Prognosis
Group CR 5 year survival
favorable 91% 65%
intermediate 86% 41%
adverse 63% 14%

47. AML Risk & Mutational Analysis
Frequency of mutations in 872 adults < 60 yrs with normal cytogenetics
NPM1- 53%
FLT3 ITD - 31% and FLT3 TK mutations-11%
CEBPA -13%
MLL PTD- 7% and NRAS-13%
Schlenk et al. N Eng J Med 358:2008

48. AML Risk & Mutational Analysis
Significantly associated with complete remission
NPM1 mutation without FLT3 ITD
Mutant CEBPA
Younger age
Allogeneic transplant benefit in first CR was limited to patients with FLT3 ITD or wild type NPM1 and CEBPA
Schlenk et al. N Eng J Med 358:2008

49. AML Treatment: Induction Chemotherapy
Anthracycline (e.g. Idarubicin) for 3 days and Cytosine arabinoside (Ara-C) for 7 days
Several weeks of pancytopenia
Supportive care: anti-emetics, red cell and platelet transfusions, prophylactic and therapeutic antibacterial, antifungal and antiviral antibiotics

50. AML: Response to Induction
Remission status determined by bone marrow at end of month following recovery from induction therapy ( Mean Day 28-35)
Complete remission:
Normal peripheral blood counts
Normocellular marrow with < 5% blasts and normal marrow cell maturation

51. AML Treatment: Subsequent Consolidation
Following induction into Complete Remission: (favorable & ?intermediate)
3-4 Cycles of high dose cytarabine (HiDAC) administered approximately every 5-6 weeks
No subsequent therapy
Follow for evidence of relapse

52. AML Treatment: Alternative Consolidation
One or more cycles of consolidation chemotherapy (Intermediate or high risk)
Allogeneic bone marrow transplantation after high dose chemotherapy
Autologous stem cell transplant after high dose chemotherapy

53. AML Treatment: Allogeneic Transplant
Advantages
Stem cells from HLA-matched sibling or unrelated individual allow high dose therapy and are free of leukemia
Immunologic graft versus leukemia effect (GVL) translates into decreased rate of leukemic relapse. (How do we know?)

54. AML Treatment: Allogeneic Transplant
Disadvantages
Toxicity of high dose chemotherapy
Immunologic graft versus host disease (GVHD) and immunosuppressive therapy result in significant morbidity and mortality
GVHD incidence and severity increases with increasing age
Tolerability of standard transplant limited by patient age

55. AML Treatment: Autologous Transplant
Advantage
Collection and subsequent infusion of patient’s stem cells allows administration of otherwise lethal doses of chemotherapy
Disadvantages
Despite CR, leukemic cells may persist in marrow, blood and stem cell product
High dose therapy more toxic than standard consolidation

56. AML: Disease Free Survival
Chemo
Auto
Sib Allo
UD Allo
CR1
25-35%
50%
60%
40%
CR2
< 5%
38%
44%
37%

57. AML Treatment: Risk adapted approach
(Patients < 60 yrs of age)
Favorable
Conventional chemotherapy followed by transplant only if relapse occurs
Intermediate
Conventional chemotherapy alone or Autologous or Allogeneic transplant
Adverse
Conventional chemotherapy followed by Allogeneic transplant

58. AML Treatment: Risk adapted approach
(Patients > 60 yrs of age)
Considerations include:
Fitness for conventional chemotherapy
Cytogenetics and molecular studies
Co-morbid conditions (transplant vs. observation)
Novel drugs eg, hypomethylating agents.

59. AML: NCCN Treatment Guidelines
National Comprehensive Cancer Network (NCCN) has issued guidelines for treatment of many cancers including AML (and other hematologic malignancies)
Details can be found at

60. AML-M3 or APL

61. AML-M3 (APL) Acute Promyelocytic leukemia
HL-60, a human AML cell line (M3) And primary human APL differentiates in vitro following treatment retinoic acid.
Initial clinical trials of retinoic acid failed. Why?

62. AML-M3 (APL) Acute Promyelocytic leukemia
HL-60, a human AML cell line (M3) And primary human APL differentiates in vitro following treatment retinoic acid.
Initial clinical trials of retinoic acid failed. Why?
Lab studies & initial clinical trials done with cis-retinoic acid. (Chemical grade cis-retinoic acid is ~5% trans. The trans isomer is active.)
Dr. Zhu Chen, Shanghi Institute of Hematology first published the all trans-retinoic acid results

63. AML-M3 (APL) Acute Promyelocytic leukemia
t(15;17) fuses PML gene with retinoic acid receptor-a (RAR-a)
PML/RAR-a protein represses RAR-a mediated gene activation and granulocyte differentiation
ATRA (all trans retinoic acid) releases this repression and allows promyelocytes to differentiate

64. AML-M3 or APL Acute Promyelocytic Leukemia (APL M3)
Blasts and promyelocytes heavily granulated, Auer rods often abundant
Disseminated intravascular coagulation (DIC) common
Treatment differs from all other AML subtypes
once had the worst prognosis now the best prognosis

65. AML-M3 or APL
Treated with a derivative of Vitamin A (all trans retinoic acid or ATRA)
Favorable prognosis if diagnosed just prior to starting chemotherapy (>80% cured)
Has chromosomal translocation, t(15;17) involving the retinoic acid receptor-a gene that blocks normal granulocyte differentiation

66. Core Binding Factor in AML
CBF is an alpha/beta heterodimeric transcription factor
Disruption of alpha and beta subunits of CBF results in a favorable prognosis (>70% cured)
t(8;21) fusion of the AML1 gene from chromosome 21q22 with the ETO gene on chromosome 8q22
Inv(16) fusion of the CBFbeta gene from chromosome 16q22 with the MYH11 gene from chromosome 16p13

67. Ras Signaling in AML
Ras proteins function as a hub of signal transduction pathways that promote cell cycling and proliferation and prevent apoptosis
Receptor tyrosine kinases (RTK) (e.g. FLT3 PDGF, FMS, c-KIT,) bind ligand and transmit signal to activate Ras
Ras and RTKs play a role in AML

68. Ras Pathway Mutations are Common in AML
FLT3 internal tandem duplication %
FLT3 point mutation %
FMS point mutation %
Kit point mutation, deletion, insertion <10%
Ras point mutations %
At diagnosis 30-50% of AML have mutations in Ras pathway and 50% have abnormal phosphorylation of ERK, indicating aberrant pathway activation

69. FLT3-ITD mutation
Mutant FLT3 receptors spontaneously dimerize leading to autophosphorylation due to constitutive activation of the tyrosine kinase
Allow autonomous, cytokine-independent growth in culture

70. FLT3-ITD mutation and AML
Associated with leukocytosis and increased marrow blast percentage
Associated with a poor prognosis due to increased relapse rate
Can be detected by PCR assay

71. What to do about AML with FLT3 mutation ?
More intensive therapy (e.g. Transplant)
Targeted therapy (in clinical trials):
- FLT3 kinase inhibitors
Farnesyl transferase inhibitors (block transport of Flt3 to the membrane)
-Novel agents

72. Leukemia & the Hematopoietic Microenvironment
How do we know there’s a significant biologic effect?

73. Leukemia & the Hematopoietic Microenvironment
How do we know there’s a significant biologic effect?
Post transplant, donor derived leukemia

74. Leukemia & the Hematopoietic Microenvironment
How do we know there’s a significant biologic effect?
Post transplant, donor derived leukemia
Resistance to imatinib (TK inhibitor for CML) occurs in some patients but the in vitro sensitivity to imatinib is unchanged.

75. AML Summary AML is a heterogeneous group of diseases
Age is one of the most important prognostic factors
State of the art cytogenetic and mutational analysis is critical to determine prognosis and to guide therapy
Novel therapeutic agents may be effective in traditional unfavorable risk patients

76. Complex Cytogenetics Revisited
Complex cytogenetics considered unfavorable
Criteria vary from > 3 to >5 cytogenetic abnormalities
Favorable cytogenetics trump complex cytogentics?
1,975 AML patients aged evaluated. Excluding normal cytogenetics and CBF, 733 patients evaluated
Breems et al. JCO:2008

77. Complex Cytogenetics Revisited
Of 733 patients with cytogenetic abnormalities: Loss of a single chromosome was associated with poor OS (12% at 4 yr). Monosomy 7 most common but other monosomies with same OS. Structural abnormalities influenced outcome only if associated with a single autosomal monosomy. >2 monosomal abnormalities very poor prognosis OS of 3%.
Breems et al. JCO:2008

78. Complex Cytogenetics Revisited

79. Complex Cytogenetics Revisited
New definition of monosomal karyotype (MK) proposed After excluding normal cytogentics and CBF subsets MK negative (MK-) Poor Prognosis: Structural abnormality but no autosomy. ( OS is 26% at 4 yr). MK positive (MK+) Very poor prognosis: > 2 autosomal monosomies or 1 autosomal monosomy and at least one structural abnormality. ~25% of non-CN non-CBF AML ( OS 4% at 4 yr).
Breems et al. JCO:2008

80. Complex Cytogenetic Revisited
Breems et al. JCO:2008

81. Elderly AML: The importance of reaching CR
Rowe Blood 103:479, 2005
Rowe Leukemia 19:1324, 2003

82. Azacitidine and Decitabine are Inhibitors of DNA Methyltransferase
Goffin & Eisenhauer. Annals of Oncology 2002; 13:1699

83. Decitabine in Elderly Patients With AML
Prospective, open-label, phase II study in elderly patients with untreated AML (N = 55)
Treatment: decitabine 20 mg/m2 on Days 1-5, every 4 weeks
Patient population
Median age: 74 years
Range: 61-87
ECOG PS 2: 18%
Poor cytogenetics: 44%
AML transformed from MDS: 35%
CR/Cri in 55 patients
AML De novo 7/ %
Transformed MDS /19 26%
Therapy related 2/ %
Poor risk cyto / %
Inter. risk cyto. 7/29 24%
*Ongoing Phase III trial
Cashen AF, et al. ASH Abstract 560.

84. Low-Dose Decitabine in Elderly Patients With AML
Preliminary results from phase II study of decitabine in elderly untreated AML patients (15 with secondary AML): N = 33
Median age: 74 years (range: 60-83)
Risk factors
> 70 years of age, AHD, poor cytogenetics, ECOG PS 2
≥ 3 (n = 16); 2 (n = 15); 1 (n = 2)
Treatment: decitabine 20 mg/m2 IV daily x 10 days/month → consolidation for 3- 5 days/month
Median cycles: ≥ 3
Median cycles to CR: 1
CR: 11/22 (50%)
Induction mortality: 4 (infections)
Blum W, et al. ASH Abstract 2957.