1. Chronic Myeloid Leukemia
Amer Rassam, M.D.

2. Learning Objectives Myeloproliferative disorders (MPDs)
Molecular genetics of chronic myeloid leukemia
Clinical manifestations and diagnosis of chronic myeloid leukemia
Overview of the treatment of chronic myeloid leukemia
Initial treatment of chronic myeloid leukemia in chronic phase

3. Learning Objectives Explain how to define and identify a relapse
Treatment of CML in chronic phase after failure of initial therapy
Clinical use of tyrosine kinase inhibitors for chronic myeloid leukemia
Treatment of CML in accelerated phase and blast crisis
Prognosis

4. Myeloproliferative Disorders
Chronic Myeloid Leukemia (CML)
Polycythemia Vera (PCV)
Essential Thrombocythemia (ET)
Primary Myelofibrosis (PMF)

5. Myeloproliferative Disorders
Clonal disorders of hematopoiesis that arise in hematopoietic stem or early progenitor cell.
Characterized by the dysregulated production of particular lineage of mature myeloid cells with fairly normal maturation.
Exhibit a variable tendency to progress to acute leukemia
Share abnormalities of hemostasis and thrombosis
Overlap between the clinical features

6. Introduction CML is a clonal myeloproliferative neoplasm
Dysregulated production and uncontrolled proliferation of mature and maturing granulocyte with fairly normal differentiation
Fusion of 2 genes: BCR (or chromosome 22) and ABL1 (on chromosome 9), resulting in BCR-ABL1 fusion gene
Final result: Abnormal chromosome 22 called Philadelphia (Ph) chromosome
Final product: BCR-ABL1 fusion protein, a dysregulated tyrosine kinase

7. Introduction
Uncontrolled production of mature and maturing granulocytes
Predominantly neutrophils, but also basophils and eosinophils
Triphasic or biphasic clinical course
Chronic phase, accelerated phase, blast crisis

8. Phases of CML (before Imatinib)
Advanced phases
Chronic phase
Median duration 5–6 years
Accelerated phase
Median duration 6–9 months
Blast crisis
Median survival 3–6 months

9. Epidemiology Annual incidence: 1 to 2 cases per 100,000
15% – 20% of all adult leukemias
Incidence increases significantly with age
Median age: ~ 55 years
Prevalence increasing due to current therapy
Most patients present in CP, 85%
Majority of CML-related deaths due to progression to AP/BC
50% of CML patients are asymptomatic at diagnosis
Risk factors
Exposure to ionizing radiation, the only known

10. Molecular Genetics of CML
The Philadelphia chromosome was originally detected by workers in Philadelphia.
The first genetic abnormality to be associated with a human cancer.
The result of a balanced translocation between chromosomes 9 and 22.
Derivative chromosome 22 is significantly smaller
Ph chromosome is present in hematopoietic cells from patients with CML.
Therefore, the Ph chromosome is acquired and NOT inherited through the germline.

11. Molecular Genetics of CML
The development of chronic phase CML appears to be a direct result of the BCR-ABL1 activity, which promotes its development by allowing:
Uncontrolled proliferation of transformed cells
Discordant maturation
Escape from apoptosis
Altered interaction with the cellular Matrix
The progression of CML from chronic phase to accelerated face or blast crisis is a complex, multistep process (may be related to GMP).
Also, it appears to involve the constitutive expression of the BCR-ABL1 tyrosine kinase.

12. Molecular Genetics of CML
BCR
ABL
BCR
ABL {
q11
BCR Ph 22 {
q34
ABL
9q+ 9

13. Ph chromosome and bcr-abl gene
9 q+ 9
c-bcr 1
2-11
c-abl
Ph (or 22q-) 22
2-11
p210Bcr-Abl
bcr
2-11
p190Bcr-Abl
bcr-abl
Exons
Introns
CML Breakpoints
ALL Breakpoints
abl
FUSION PROTEIN WITH TYROSINE KINASE ACTIVITY
bcr-abl gene structure
t(9;22) translocation

14. Philadelphia chromosome t(9;22)(q34;q11)
22q- = Philadelphia chromosome

15. Clinical Manifestations
Asymptomatic in 20-50% of patients
Fatigue 34%, weight loss 20%, excessive sweating 15%, abdominal fullness 15%, bleeding episodes 21% (platelet dysfunction).
Abdominal pain in the LUQ (enlarged spleen)
Tenderness over the lower sternum.
Acute gouty arthritis
Findings: Splenomegaly, anemia, WBC > 100,000, platelet count > 600,000

16. Peripheral Blood Pathology
Leukocytosis (median of 100,000)
Differentiation shows virtually all cells of neutrophilic series
Blasts < 2%
Myelocytes more than metamyelocytes (a classic finding in CML)
Neutrophils cytochemistry is abnormal – low LAP score
Basophilia in 90% of cases
Thrombocytosis. If low platelets – consider an other

18. CML Peripheral Blood Smear

19. CML Peripheral Blood Smear

20. Bone Marrow Pathology
Granulocytic maturation pattern same as in the peripheral blood
Increased reticulin fibrosis and vascularity
Erythroid islands are reduced in number and size
Dwarf megakaryocytes
Pseudo-Gaucher’s cells and Sea Blue histiocytes (markers of increased cell turnover)
Iron-laden macrophages are reduced or absent

21. Pseudo-Gaucher cells

22. Pseudo-Gaucher cells

23. Sea Blue Histiocyctes

24. CML – Bone Marrow

25. Diagnosis of CML Typical findings in the blood and bone marrow
Requires the detection of the Ph chromosomal or its product, the BCR-ABL1 fusion mRNA and the BCR-ABL1 protein.
Conventional cytogenetic analysis (karyotyping) – The first method
Florence and in situ hybridization (FISH) analysis
RT-PCR (The BEST)
Southern blot techniques – rarely used
Western Blotting – low sensitivity and labor intensive

26. BCR-ABL (FISH)

27. Cycle 1 yields 2 molecules Cycle 2 yields 4 molecules
RT-PCR for BCR-ABL
Qualitative RT-PCR allow for the diagnosis of CML
Quantitative RT-PCR is used to quantify the amount of disease
Allows for the identification of cryptic BCR-ABL translocations
Does not require a bone marrow aspirate for optimal results
Target sequence 1
Denaturation: Heat briefly to separate DNA strands 2
Annealing: Cool to allow primers to form hydrogen bond with ends of target sequence
Cycle 1 yields 2 molecules
Primers 3
Extension: DNA polymerase adds nucleotides to the 3” end of each primer
New nucleo-tides
Cycle 2 yields 4 molecules
Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

28. Most CML patients are diagnosed in the chronic phase
Blastic phase

29. Differential Diagnosis
Leukemoid reaction
Juvenile myelomonocytic leukemia (JMML)
Chronic myelomonocytic leukemia (CMML)
Atypical CML
Chronic eosinophilic leukemia
Chronic neutrophilic leukemia
Other myeloproliferative neoplasms
Other Ph chromosome positive malignancies

30. Accelerated Phase CML
10-19% blasts in the peripheral blood or bone marrow
Peripheral blood basophils ≥20%
Platelets < 100,000/microL, unrelated to therapy
Platelets > 1,000,000/microL, unresponsive to therapy
Progressive splenomegaly and increasing WBC, unresponsive to therapy
Cytogenic evolution

31. Blastic Phase CML
Blast crisis is generally refractory to treatment, occurs approximately 3-5 years after the diagnosis of CML and 18 months after the onset of accelerated face
Blasts in the peripheral blood ≥20% or in the bone marrow ≥30%
Large foci or clusters of blasts on the bone marrow biopsy
Presence of extramedullary blastic infiltrate (e.g., myeloid sarcoma, also known as granulocytic sarcoma or chloroma)

32. Blast Phase CML – Bone Marrow

33. Blast Phase CML – Bone Marrow

34. What is the optimal frontline therapy for CML?
Clinical Debate
What is the optimal frontline therapy for CML?

35. Principles of CML treatment
Relieve symptoms of hyperleukocytosis, splenomegaly and thrombocytosis.
Hydration
Chemotherapy (Busulfan, hydroxyurea)
Control and prolonging the chronic phase (non-curative)
Tyrosine kinase inhibitors
Alpha-interferon + chemotherapy
Chemotherapy (hydroxyurea)

36. Treatment Options
Treatment decisions for patients with CML are complex, due to the variety of available options, many of which are conflicting.
Potential cure with allogeneic hematopoietic stem cell transplantation
Disease control without cure using tyrosine kinase inhibitors (TKIs)
Palliative therapy with cytotoxic agents

37. Factors influencing choice of therapy
Phase of CML
Availability of a donor for allogeneic stem cell transplant
Patient age
Presence of medical co-morbidities
Response to treatment with TKIs

38. IRIS Study Design: Imatinib Mesylate Versus IFN- + ara-C
1106 patients enrolled from June 2000 to January 2001
Imatinib Mesylate
IF:
Loss of MCR or CHR
Increasing WBC count S R
Crossover
Intolerance of treatment
Failure to achieve MCR
at 12 months*
Failure to achieve CHR at 12 months*
Request to discontinue IFN-*
IFN- a
+ ara-C
Progression
Increasing WBC count
S = screening. R = randomization.
Loss of MCR or CHR
Accelerated phase or blast crisis
Death

39. Hematologic Responses
100
96% 90
Imatinib mesylate 80 70 60
67%
% Responding 50
IFN- + ara-C 40 30 20 10 3 6 9 12 15 18 21
Months Since Randomization

40. Cytogenic Responses 100 90 83% 80 Imatinib mesylate 70 60 % Responding50 40 30
20% 20
IFN- + ara-C 10 3 6 9 12 15 18 21
Months Since Randomization

41. Overall Survival on First-Line Imatinib (IRIS Study)

42. Resistance to Imatinib occurs predominantly during advanced phase CML
Chronic Phase
Blast Crisis
Advanced stage cancers are characterized by multiple genetic changes
Patients in advanced phase often relapse with the development of chemotherapy resistance
Some patients in blast crisis CML respond to Imatinib but then tends to relapse
Relapse
Hematopoietic
differentiation
Bone marrow to peripheral blood
Ph-negative
Ph+ blasts
Ph+
Ph+ Imatinib mesylate-
resistant blasts

43. Initial Treatment Imatinib (Gleevec) Dasatinib (Sprycel)
Tyrosine kinase inhibitors are for first-line therapy in chronic phase CML
Imatinib (Gleevec)
Dasatinib (Sprycel)
Nilotinib (Tasigna)
All 3 agents are considered to be (category 1) based on the NCCN guidelines and recommendations.
Second-generation TKIs (dasatinib or nilotinib) produce faster and deeper response than imatinib

44. Treatment of CML after failure of initial therapy
No randomized trials have directly compared the efficacy of second-generation TKIs in patients with chronic phase CML who experience failure of an initial TKIs
A trial of another TKI.
Dasatinib preferred in patients with pancreatitis, elevated bilirubin or hyperglycemia
Dasatinib crosses the blood brain barrier and would therefore be preferred in patients with CNS involvement
Nilotinib might be chosen for patients with a history of pleural or pericardial effusion or disease
Dasatinib and Nilotinib can result in QT prolongation

45. Other Options Bosutinib – toxicity is a limiting factor
Ponatinib – toxicity is a limiting factor
Increase the dose of Imatinib
Omacetaxine mepesuccinate – SQ Injection
Approved by the FDA for patients resistant or intolerant to 2 or more TKIs
Hematopoietic cell transplant – the only cure
Clinical trials

46. Other Options Interferon alfa plus cytarabine Hydroxyurea Busulfan
Patients who are ineligible for HCT but have either a contraindication to a second-generation TKI or have failed to respond to treatment with available TKI
Interferon alfa plus cytarabine
Hydroxyurea
Busulfan

47. Response Criteria Hematologic response Cytogenic response
Molecular response

48. Resistance to treatment
Primary resistance – patient fails to achieve a desired response to initial treatment
Secondary resistance – patient with an initial response to a TKI ultimately relapses

49. Loss of Response T315I mutation Y253H, E255k/V and F359V/C/I mutations
Patients should be re-evaluated with a bone marrow biopsy with cytogenetics, and BCR-ABL kinase mutation analysis
T315I mutation
Resistant to all TKIs, except Ponatinib
Patient should be evaluated for SCT
Y253H, E255k/V and F359V/C/I mutations
Resistant to Imatinib and Nilotinib but sensitive to Dasatinib
F317L/V/I/C, V299L and T315A mutations
Sensitive to Nilotinib but with intermediate sensitivity to Imatinib and Dasatinib

50. Mechanisms of action TKIs
They block the initiation of bcr-abl pathway
Many TKIs also affect other signaling pathways
Dasatinib and Bosutinib inhibit both Bcr-Abl and Src kinases.
Nilotinib inhibits Bcr-Abl, c-kit and platelet derived growth factor receptor (PDGFR)
These differences in targeted pathways may be responsible for their varied clinical effects in tumors

51. Mechanisms of Action, Imatinib
Competitively inhibits the inactive configuration of the Bcr-Abl protein tyrosine kinase
Blocking the ATP binding site and thereby preventing a conformational switch to the active form
Inhibits cellular proliferation and tumor formation
Produces 95% decrease in CML colony growth
Inhibits platelet-derived growth factor and c-kit

52. GLEEVEC (Imatinib)

53. GLEEVEC (Imatinib)
Molecular consequence of the t(9;22) is the fusion protein BCR–ABL, which has increased in tyrosine kinase activity
BCR-ABL protein transform hematopoietic cells so that their growth and survival become independent of cytokines
It protects hematopoietic cells from programmed cell death (apoptosis)

54. TASIGNA (Nilotinib)

55. Drug Interaction with TKIs
They are metabolized by the CYP3A4 system – can inhibit other cytochrome P450 pathways
Therefore, they compete with Coumadin
Low TKIs levels – St. John’s wort, rifampin, carbamazepine, phenobarbital and phenytoin
High TKIs levels – diltiazem, verapamil, itraconazole, ketoconazole, clarithromycin, erythromycin and grapefruit juice

56. Side Effects of TKIs
Imatinib - Bone marrow suppression; fluid retention/edema; gastrointestinal effects; heart failure; hepatotoxicity
Dasatinib - Bone marrow suppression; pleural/pericardial effusions; pulmonary arterial hypertension; QT prolongation; aspirin like effect
Bosutinib - Bone marrow suppression; fluid retention/edema; gastrointestinal effects

57. Side Effects of TKIs
Nilotinib - Bone marrow suppression; atherosclerosis-related events; electrolyte imbalance; hepatotoxicity
Black box: QT prolongation (screening required)
Ponatinib - Bone marrow suppression; fluid retention/edema; gastrointestinal effects; heart failure; hypertension; pancreatitis; aspirin-like effect
Black box: Arterial thrombosis; hepatic toxicity

58. Pregnancy and TKIs All TKIs could be teratogenic during pregnancy
Women are advised not to become pregnant while on TKIs (any TKI)
Best effective contraception is the barrier
Woman taking TKIs are advised to avoid to breast-feeding

59. Prognosis
Improved dramatically since the incorporation of tyrosine kinase inhibitors into the initial treatment
SEER database patient’s, year 2000 and 2005
15-44 years – OS 72 versus 86%
45-64 years – OS 68 versus 76%
65-74 years – OS 38 versus 51%
75-84 years – OS 19 versus 36%
Stage of disease at the time of diagnosis is the strongest single predictor of outcome.