1. CML Chronic myelogenous leukemia
CML accounts for % of all leukemias affecting adults

2. Hematopoietic Progenitors and CML
Acquisition of the
Philadelphia
Chromosome
Blood cell development. A blood stem cell goes through several steps to become a red blood cell, platelet, or white blood cell.

3. Leukocytosis with the presence of precursor cells of the myeloid lineage. In addition, basophilia, eosinophilia, and thrombocytosis can be seen

4. whole granulocytic lineage, including an eosinophil and a basophil

5. A promyelocyte, an eosinophil, and 3 basophils

6. Diagnostic Considerations in CML
A peripheral blood smear or bone marrow aspirate can only give a presumptive diagnosis of CML – one still needs to confirm the presence of the t (9 ; 22)
Common Peripheral Blood Findings
Leukocytosis with a ‘left shift’
Normocytic anemia
Thrombocytosis in ~ 50% of patients
Absolute eosinophilia
Absolute and relative increase in basophils
LAP score is low (not frequently
employed today)
LAP = leukocyte alkaline phosphatase.

7. Most CML Patients Are Diagnosed in the Chronic Phase
Blast Phase

8. CML

9. Bone marrow film at 400X magnification demonstrates clear dominance of granulopoiesis. The number of eosinophils and megakaryocytes is increased

12. Ph Is the Result of t(9;22)(q34;q11)
BCR
ABL
BCR
ABL {
q11
BCR Ph 22 {
q34
ABL
9q+ 9
The ABL oncogene encodes a tyrosine protein kinase. The resulting BCR/ABL fusion gene encodes a chimeric protein with strong tyrosine kinase activity.

14. p210Bcr-Abl Alone Is Necessary and Sufficient for Development of CML
Morphology of acute lymphoid leukemia cells. (A) Peripheral blood smear of mouse FN, showing predominance of immature blast cells. (B) Cytospin preparation of tumor cells from large paraspinal mass of mouse GR-1. This same immature blast cell type was detected in cytospin preparations from liver and spleen as well.

15. Two forms of the BCR/ABL mutation have been identified.
These vary according to the location of their joining regions on bcr 3' domain.
Approximately 70% of patients who have the 5' DNA breakpoint have a b2a2 RNA message
And 30% of patients have a 3' DNA breakpoint and a b3a2 RNA message. The latter is associated with a shorter chronic phase, shorter survival, and thrombocytosis
Additional chromosomal abnormalities, such as an additional or double Ph1-positive chromosome or trisomy 8, 9, 19, or 21; isochromosome 17; or deletion of the Y chromosome, have been described as the patient enters a transitional form or accelerated phase of the blast crisis as the Ph chromosome persists.

16. CML: Epidemiology and Etiology
In the US, there were 4,870 cases in 2010 and an expected 5,430 cases in 2012
Most patients present in CP
Majority of CML-related deaths due to progression to AP/BC
50% of CML patients are asymptomatic at diagnosis
Prevalence increasing steadily as a result of current therapy options
Approximately 4,870 cases in the United States in 2010 (15% of all adult leukemias) and an incidence that increases significantly with age
Median age: ~ 67
Risk factors
Prior high-dose radiation exposure
Exposure to certain organic solvents (benzene)
Age
Gender (male > female ~ 1.4:1)
Majority of cases have no known inciting factor
A very small percentage (< 0.1%) of individuals can express BCR-ABL but not develop CML
Wrong cell of origin
Multiple genetic mutations leading to non-viability of the clone
Immune surveillance

17. Epidemiology and Etiology
Incidence increases significantly with age
Median age: ~ 67 yrs
Risk factors
Prior high-dose radiation exposure
Exposure to certain organic solvents
Benzene
Carbon tetrachloride
Age
Gender (male > female ~ 1.4:1)
Majority of cases have no known inciting factor
Approximate US Prevalence of the
4 Major Types of Leukemia as of 1/1/07

18. Signs and symptoms in the chronic phase are as follows:
Fatigue, weight loss, loss of energy, decreased exercise tolerance
Low-grade fever and excessive sweating from hypermetabolism
Elevated (WBC)
Splenomegaly
Hepatomegaly
Early satiety and decreased food intake from encroachment on stomach by enlarged spleen
Left upper quadrant abdominal pain from spleen infarction

19. signs and symptoms of progressive disease:
Bleeding, petechiae, and ecchymoses during the acute phase
Bone pain and fever in the blast phase
Increasing anemia, thrombocytopenia, basophilia, and a rapidly enlarging spleen in blast crisis

20. Blood count and peripheral smear findings
WBC 20,000-60,000 cells/μL, mildly increased basophils and eosinophils
Mild to moderate anemia, usually normochromic and normocytic
Platelet counts low, normal, or increased
Leukocyte alkaline phosphatase stains very low to absent in most cells
Leukoerythroblastosis, with circulating immature cells from the bone marrow
Early myeloid cells (eg, myeloblasts, myelocytes, metamyelocytes, nucleated red blood cells)

21. Bone marrow findings
Philadelphia (Ph) chromosome (a reciprocal translocation of chromosomal material between chromosomes 9 and 22)
BCR/ABL mutation
Hypercellularity, with expansion of the myeloid cell line (eg, neutrophils, eosinophils, basophils) and its progenitor cells
Megakaryocytes are prominent and may be increased
Mild fibrosis in the reticulin stain

22. Sokal score widely used prognostic index, is calculated for patients aged 5-84 years by the following equation:
Hazard ratio = exp (age - 43) (spleen size [cm below costal margin] cm) [(platelet count/700) ] (% blasts in blood - 2.1)
Good risk (average survival of 5-6 years)
Intermediate risk (average survival of 3-4 years)
Poor risk (average survival of 2 years)

23. 3 categories of the Sokal score
Low risk: score < 0.8
Intermediate risk: score
High risk: score > 1.2
The Sokal score correlates with the likelihood of achieving complete cytogenetic response, as follows:
Low-risk patients: 91%
Intermediate-risk patients: 84%
High-risk patients: 69%

24. Poor-prognosis characteristics Clinical and laboratory factors
Older age
Symptomatic presentation
Poor performance status
African American descent
Hepatomegaly
Splenomegaly
Negative Ph chromosome or BCR/ABL
Anemia
Thrombocytopenia
Thrombocytosis
Decreased megakaryocytes
Basophilia
Myelofibrosis (increased reticulin or collagen)

25. Therapy-associated factors may indicate a poor prognosis in patients with CML:
Longer time to hematologic remission with myelosuppression therapy
Short duration of remission
High total dose of hydroxyurea or busulfan
Poor suppression of Ph-positive cells by chemotherapy or interferon alfa therapy

26. Physical Examination
Splenomegaly correlates with the peripheral blood granulocyte counts
A very large spleen is usually a harbinger of the transformation into an acute blast crisis form of the disease.
Hepatomegaly also occurs, although less commonly than splenomegaly.
Leukostasis and hyperviscosity can occur in some patients, with extraordinary elevation of their WBC counts, exceeding 300, ,000 cells/μL. Upon funduscopy, the retina may show papilledema, venous obstruction, and hemorrhages.

27. Blast crisis
Increase in the bone marrow or peripheral blood blast count or by the development of soft-tissue or skin leukemic infiltrates.
Typical symptoms are due to increasing anemia, thrombocytopenia, basophilia, a rapidly enlarging spleen
Failure of the usual medications to control leukocytosis and splenomegaly.

28. Leukocyte Alkaline Phosphatase (LAP)
Naphthol AS-MX phosphate LAP at pH8.6 >
Naphthol AS-MX + Diazonium salt
(eg, Fast blue RR)
> Insoluble pigment

29. Count 100 consecutive segs and bands
LAP Score
Count 100 consecutive segs and bands
Score:
0 = no granules
1+ = occasional diffuse granules
2+ = moderate number of granules
3+ = many strongly positive granules
4+ = confluent strongly positive granules

30. 1+ 2+ 3+ 4+

31. LAP Score Example: 0 x 35 cells = 0 1+ x 30 cells = 30

32. Bcr-Abl Translocation and CML
Philadelphia Chromosome
ABL-BCR Fusion Gene
left image: Philadelphia chromosome. A piece of chromosome 9 and a piece of chromosome 22 break off and trade places. The bcr-abl gene is formed on chromosome 22 where the piece of chromosome 9 attaches. The changed chromosome 22 is called the Philadelphia chromosome.
NCI, 2011.

34. The Cytogenetic Hallmark of CML Is the Philadelphia Chromosome
9q+ Ph 9 22
22q- = Ph chromosome

35. FISH showing the BCR (green), ABL (orange), and BCR-ABL fusion signals (arrow): A=positive (contains a residual ABL signal), B=normal

37. Small Molecule TKIs
Mechanism of Action of BCR-ABL and of Its Inhibition by Imatinib.
Panel A shows the BCR-ABL oncoprotein with a molecule of adenosine triphosphate (ATP) in the kinase pocket. The substrate is
activated by the phosphorylation of one of its tyrosine residues. It can then activate other downstream effector molecules. When
imatinib occupies the kinase pocket (Panel B), the action of BCR-ABL is inhibited, preventing phosphorylation of its substrate. ADP
denotes adenosine diphosphate. with the permission of the publisher.

38. Molecular Mechanisms of BCR-ABL Induced Leukemogenesis
Chronic myeloid leukaemia: stem cell derived but progenitor cell driven
Marley et al, 2005.

40. RT-PCR for BCR-ABL RT-PCR for BCR-ABL in CML
Target sequence
RT-PCR for BCR-ABL in CML 1
Denaturation: Heat briefly to separate DNA strands 2
Annealing: Cool to allow primers to form hydrogen bond with ends of target sequence
Qualitative RT-PCR allows 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
Cycle 1 yields 2 molecules
Primers 3
Extension: DNA polymerase adds nucleotides to the 3” end of each primer
New nucleo-tides
Purpose of this slide is to note the proper use, advantages, and limitations of Quantitative RT-PCR to diagnose CML
Cycle 2 yields 4 molecules
Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence
RT-PCR = real time polymerase chain reaction.

43. Monitoring Response: Sensitivity of Strategies
Complete Hematologic Response
Diagnosis: Leukemia Cells
Cytogenetics
Blood Counts
PCR
Undetectable Range
Complete Cytogenetic Response
Major Molecular Response
100%
10% 1%
0.1%
Complete Molecular Response
4.5 log = %

45. BCR-ABL Kinase Activity Is Essential for CML Pathogenesis
NALM-1 cells (Ph+)
0.1
0.5
1.0
5.0 10
Imatinib (mM)
BCR-ABL-
32p210 BCR-ABL
K562
32D

46. NCCN Recommendations for Evaluation of Possible CML
H&P including documentation of spleen size
CBC, platelets
Chemistries
HLA typing
Bone marrow aspirate and biopsy including
– Aspirate for blast percentage
– Karyotypea
– FISHb
– Quantitative RT-PCR for Bcr-Ablc
Consideration for a diagnosis of CML
aKaryotyping is recommended to identify additional chromosomal changes not detectable by FISH; including CE, complex translocations, and Ph(-) ACAs.
bFISH is acceptable for confirming the diagnosis of CML when BMB is not feasible.
cqRT-PCR is recommended to establish a baseline since the majority of labs do not use the International Scale.
qRT-PCR = quantitative real-time polymerase chain reaction; CE = clonal evolution; ACAs = additional chromosomal abnormalities;

47. Treatment Milestones for CML
Amount of Dz
Definitions of Responses to Treatments
Hematologic Response
Complete Hematologic response
1) Normal PB counts (WBC < 10 x 109/L and plts < 450 x 109/L)
2) Normal WBC differential
3) No Dz symptoms
4) Normalization of the size of the liver and spleen
Cytogenetic Responses: Ph+ Metaphases
1) Complete: 0%
2) Partial: 1%–35%
3) Minor: 36%–65%
4) Minimal: 66%–95%
5) None: 96%–100%
Molecular Responses: Ratio of Bcr-Abl/Abl
Major Molecular Response
≥ 3-log10 reduction from initial diagnosis sample
(ie, 25 → 0.025)
1X1012
1X1011
1X1010
1X10 8-9

48. Responses Correlate With Decreasing Burden of Disease
How I monitor residual disease in chronic myeloid leukemia
The reduction of CML burden and the sensitivity of assays.
Routine cytogenetics will fail to detect the Ph (a CCyR) after a 1-2 log reduction in CML burden.
The detection limit of RT-PCR is approximately a 5-6 log reduction of disease burden.
RT-PCR = real-time polymerase chain reaction; Dx = diagnosis.

50. Options for Establishing the Diagnosis of CML
FISH
Can be done with interphase cells
Allows for the identification of potential duplications of the Ph chromosome
Allows for the identification of the loss of der(9) chromosome
Allows for the identification of cryptic translocations involving BCR-ABL that can be missed on karyotypes
Fails to identify CE or ACA
qRT-PCR
Can quantify the amount of disease
Allows for the identification of cryptic translocations involving BCR-ABL
Many primers sets only detect the p190 and/or p210 translocation and may miss p230 or alternative translocations
Requires consistent use of the same laboratory give different control genes
Fails to identify CE or ACA
Karyotyping
Requires BM aspirate for optimal metaphases
Allows for evaluation of CE as well ACA in Ph- clones
Occasionally, cryptic and complex translocation events may result in the missed identification of t(9;22)
BM = bone marrow; CE = clonal evolution; ACA = additional chromosomal abnormatlities. .

51. Role of the Bone Marrow Biopsy in the Diagnosis of CML
One Disease – Three Phases
Chronic Phase
Myeloproliferative neoplasm associated with the balanced chromosomal translocation between the long arms of Chromosome 9 and 22 [t(9;22)(q34;q11.2)]
< 10% of the cells in the blood and bone marrow are blast cells (immature blood cells)
Accelerated Phase
Blasts 10%–19% of WBCs in peripheral and/or nucleated bone marrow cells
Peripheral blood basophils ≥ 20%
Persistant thrombocytopenia (< 100 x 109/L) unrelated to therapy, or persistent thrombocytosis (> 1,000 x 109/L) unresponsive to therapy
Increasing spleen size and increasing WBC count unresponsive to therapy
Cytogenetic evidence of clonal evolution
Blast Crisis
Blasts ≥ 20% of peripheral blood white cells or of nucleated bone marrow cells
Extramedullary blast proliferation
Large foci or clusters of blasts in the bone marrow biopsy

53. Imatinib Greatly Improved Survival in CML-CP (MDACC data)
FIGURE 2. Survival of patients with early chronic phase chronic myeloid leukemia treated at M. D.
Anderson Cancer Center in different eras compared with those treated with imatinib.

54. Diagnostic Considerations
Problems to be considered include the following:
Acute myeloid leukemia
Chronic myelomonocytic leukemia
Chronic neutrophilic leukemia
Thrombocythemia
Leukemoid reactions from infections (chronic granulomatous [eg, tuberculosis])
Tumor necrosis

55. Differential Diagnoses
Agnogenic Myeloid Metaplasia With Myelofibrosis
Essential Thrombocytosis
Myelodysplastic Syndrome
Myeloproliferative Disease
Polycythemia Vera

56. Goals of treatment of CML include the following:
Management
Goals of treatment of CML include the following:
Hematologic remission (normal CBC and physical examination [ie, no organomegaly])
Cytogenetic remission (normal chromosome returns with 0% Ph-positive cells)
Molecular remission (negative polymerase chain reaction [PCR] result for BCR/ABL mRNA

57. Goals of treatment
Hematologic remission normal CBC and physical examination ie, no organomegaly
Cytogenetic remission (normal chromosome returns with 0% Ph-positive cells)
Molecular remission (negative polymerase chain reaction [PCR] result for the mutational BCR/ABL mRNA), which represents an attempt for cure and prolongation of patient survival

58. Tyrosine kinase inhibitors for CML
Imatinib mesylate (Gleevec): For chronic, accelerated, and blastic phases; standard treatment of choice
Dasatinib (Sprycel): For chronic phase
Nilotinib (Tasigna): For chronic phase
Bosutinib (Bosulif): For chronic, accelerated, and blast phases
Ponatinib (Iclusig): For chronic, accelerated, and blast phases

59. Other medications for CML
Interferon-alfa: Former first-line agent; now combined with newer drugs for refractory cases
Hydroxyurea (Hydrea): Myelosuppressive agent for inducing hematologic remission
Busulfan: Myelosuppressive agent for inducing hematologic remission
Omacetaxine (Synribo): Protein translation inhibitor indicated for chronic- or accelerated-phase CML with resistance and/or intolerance to 2 or more tyrosine kinase inhibitors

60. Treatment recommendations for Chronic phase
Dasatinib 100 mg PO once daily or
Nilotinib 300 mg PO twice daily or
Imatinib 400 mg PO once daily or
Bosutinib 500 mg PO once daily or
Ponatinib 45 mg PO once daily

61. Accelerated / blast phase
Dasatinib 140 mg once daily or
Nilotinib 400 mg twice daily or
Imatinib mg PO once daily or
Bosutinib 500 mg PO once daily or
Ponatinib 45 mg PO once daily
There is a high relapse rate in patients in accelerated phase even after successful treatment; transplantation should be considered

62. Blast phase:
Patients in lymphoid blast phase can be treated with acute lymphoblastic leukemia (ALL) induction chemotherapy regimens in combination with a tyrosine kinase inhibitor
Patients in myeloid blast crisis can be treated with acute myeloid leukemia (AML) induction chemotherapy regimens in combination with a tyrosine kinase inhibitor; some patients can be treated with a tyrosine kinase inhibitor alone

63. Allogeneic bone marrow transplantation (BMT) or stem cell transplantation
Only proven cure for CML
Ideally performed in the chronic phase
Candidate patients should be offered the procedure if they have a matched or single–antigen-mismatched related donor available
Overall survival for allogeneic BMT with matched unrelated donors ranges from 31% to 43% for patients younger than 30 years and from 14% to 27% for older patients
Currently relegated to patients who do not achieve molecular remissions or show resistance to imatinib and failure of second-generation tyrosine kinase inhibitors (eg, dasatinib)