CML

Chronic myelogenous leukemia

CML accounts for 15 - 20% of all leukemias affecting adults

Hematopoietic Progenitors and CML

Acquisition of the Philadelphia Chromosome

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

whole granulocytic lineage, including an eosinophil and a basophil

A promyelocyte, an eosinophil, and 3 basophils

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

1.

Leukocytosis with a ‘left shift’ 2.

Normocytic anemia 3.

Thrombocytosis in ~ 50% of patients 4.

Absolute eosinophilia 5.

Absolute and relative increase in basophils 6.

LAP score is low (not frequently employed today)

LAP = leukocyte alkaline phosphatase.

Most CML Patients Are Diagnosed in the Chronic Phase

Chronic Phase Blast Phase

CML

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

Ph Is the Result of t(9;22)(q34;q11)

q11 { BCR BCR ABL Ph 22 q34 { ABL 9 ABL BCR 9q+ The ABL oncogene encodes a tyrosine protein kinase. The resulting BCR/ABL fusion gene encodes a chimeric protein with strong tyrosine kinase activity.

p210

Bcr-Abl

Alone Is Necessary and Sufficient for Development of CML

p210 Bcr-Abl

I.

Two forms of the BCR/ABL mutation have been identified. II. These vary according to the location of their joining regions on bcr 3' domain.

III. Approximately 70% of patients who have the 5' DNA breakpoint have a b2a2 RNA message IV. 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.

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

Epidemiology and Etiolog y

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

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

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

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)

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

Sokal score

widely used prognostic index, is calculated for patients aged 5-84 years by the following equation: Hazard ratio = exp 0.0116 (age - 43) + 0 .0345 (spleen size [cm below costal margin] - 7.5 cm) + 0.188 [(platelet count/700) 2 - 0.563] + 0.0887 (% 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)

3 categories of the Sokal score

• • •

Low risk: score < 0.8

Intermediate risk: score 0.8-1.2

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%

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)

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

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 300,000-600,000 cells/μL. Upon funduscopy, the retina may show papilledema, venous obstruction, and hemorrhages.

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.

Leukocyte Alkaline Phosphatase (LAP)

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

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

2+ 0 3+ 1+ 4+

LAP Score

Example: 0 x 1+ x 2+ x 3+ x 4+ x 35 cells = 0 30 cells = 30 20 cells = 40 10 cells = 30 5 cells = 20

120 LAP Score

NCI, 2011.

Bcr-Abl Translocation and CML

Philadelphia Chromosome ABL-BCR Fusion Gene

The Cytogenetic Hallmark of CML Is the Philadelphia Chromosome

9q+ 9 Ph 22 22q- = Ph chromosome

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

Small Molecule TKIs

Molecular Mechanisms of BCR-ABL Induced Leukemogenesis

Marley et al, 2005.

RT-PCR for BCR-ABL

RT-PCR for BCR-ABL in CML

1) 2) 3) 4) 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

1 Denaturation: Heat briefly to separate DNA strands 2 Annealing: Cool to allow primers to form hydrogen bond with ends of target sequence 3 Extension: DNA polymerase adds nucleotides to the 3 ” end of each primer Primers New nucleo tides

Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

Target sequence RT-PCR = real time polymerase chain reaction.

100% 10% 1% 0.1%

Monitoring Response: Sensitivity of Strategies

Diagnosis: 10 12 Leukemia Cells Blood Counts

Complete Hematologic Response

Cytogenetics

Complete Cytogenetic Response

PCR

4.5 log = 0.0032% Undetectable Range Major Molecular Response Complete Molecular Response

BCR-ABL Kinase Activity Is Essential for CML Pathogenesis K5 62 32D 32 p21 0 BCR -A BL BCR-ABL-

0.1

Imatinib (

m

M)

0.5

1.0

5.0

10

BCR-ABL

NALM-1 cells (Ph+)

NCCN Recommendations for Evaluation of Possible CML

Consideration for a diagnosis of CML

H&P including documentation of spleen size CBC, platelets Chemistries HLA typing Bone marrow aspirate and biopsy including – Aspirate for blast percentage – Karyotype a – FISH b – Quantitative RT-PCR for Bcr-Abl c a Karyotyping is recommended to identify additional chromosomal changes not detectable by FISH; including CE, complex translocations, and Ph(-) ACAs.

b FISH is acceptable for confirming the diagnosis of CML when BMB is not feasible.

c qRT-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;

Treatment Milestones for CML

Amount of Dz Definitions of Responses to Treatments Hematologic Response

Complete Hematologic response 1) Normal PB counts (WBC < 10 x 10 9 /L and plts < 450 x 10 9 /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-log 10 reduction from initial diagnosis sample (ie, 25 → 0.025) 1X10 12 1X10 11 1X10 10 1X10 8-9

Responses Correlate With Decreasing Burden of Disease

RT-PCR = real-time polymerase chain reaction; Dx = diagnosis.

Options for Establishing the Diagnosis of CML 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)

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 BM = bone marrow; CE = clonal evolution; ACA = additional chromosomal abnormatlities . .

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

Role of the Bone Marrow Biopsy in the Diagnosis of CML

One Disease – Three Phases Chronic Phase Accelerated Phase Blast Crisis

• 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) • Blasts 10%–19% of WBCs in peripheral and/or nucleated bone marrow cells • Peripheral blood basophils ≥ 20% • Persistant thrombocytopenia (< 100 x 10 9 /L) unrelated to therapy, or persistent thrombocytosis (> 1,000 x 10 9 /L) unresponsive to therapy • Increasing spleen size and increasing WBC count unresponsive to therapy • Cytogenetic evidence of clonal evolution • 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

Imatinib Greatly Improved Survival in CML-CP (MDACC data)

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

Differential Diagnoses



Agnogenic Myeloid Metaplasia With Myelofibrosis



Essential Thrombocytosis



Myelodysplastic Syndrome



Myeloproliferative Disease



Polycythemia Vera

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

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

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

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

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

Accelerated / blast phase



Dasatinib 140 mg once daily or



Nilotinib 400 mg twice daily or



Imatinib 600-800 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

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

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 )