2. PLAN
Introduction
BCR ABL and its Action
Clinical and Laboratory Features
Goals and Concepts of Management
Diagnostic modalities
Tyrosine Kinase Inhibitors
Other Modalities of treatment
Monitoring Of therapy
3. Chronic Myelogenous Leukemia
CML is a MPD characterized by increased granulocytic
cell line, associated with erythroid and platelet
hyperplasia.
The diagnosis of CML is established by identifying a
clonal expansion of a hematopoietic stem cell
possessing a reciprocal translocation between
chromosomes 9 and 22.
4. THE BCR-ABL MUTATION
This translocation results in the head-to-tail fusion of the
breakpoint cluster region (BCR) gene on chromosome
22q11 with the ABL1 (named after the abelson murine
leukemia virus) gene located on chromosome 9q34.
5. The Ph Chromosome: t(9;22)
22
bcr
abl
Ph ( or 22q-)
bcr-abl
FUSION PROTEIN
WITH TYROSINE
KINASE ACTIVITY
9 9 q+
6. Head to tail fusion of BCR to ABL occurs following
9:22 translocation
This region is transcribed into BCR-ABL mRNA
BCR-ABL protein is produced(p210)
This protein Dimerises and becomes active tyrosine kinase
Many intracellular kinases are phosphorylated(eg c-myc,shc)
Cell proliferates,evades apoptosis
10. CLINICAL FEATURES
30% patients are Asymptomatic at diagnosis.
Symptoms are usually Non specific(Fatigue,anorexia,weight loss)
Symptoms because of splenomegaly are common(abdominal pain,
dragging sensation in abdomen)
Rare presentations include Vasoocclusive disease, cerebrovascular
accidents, myocardial infarction, venous thrombosis, priapism, visual
disturbances, and pulmonary insufficiency.
11. LABORATORY FEATURES
Elevated white blood cell counts (WBCs), with increases in both immature
and mature granulocytes, are present at diagnosis. Usually <5% circulating
blasts and <10% blasts and promyelocytes are noted, with the majority of
cells being myelocytes, metamyelocytes, and band forms.
Platelet counts are almost always elevated at diagnosis,
Mild degree of normocytic normochromic anemia is present.
Leukocyte alkaline phosphatase is low in CML cells.
12. LABORATORY FEATURES
The marrow is hypercellular (granulocytic hyperplasia)
Reticulin fibrosis
Hyperuricemia and hyperuricosuria
Serum vitamin B12-binding proteins are increased.
Pseudohyperkalemia, and spurious hypoxemia and hypoglycemia.
14. ACCELERATED PHASE
(WHO DEFINITION)
Blasts 10-19% in peripheral blood and or bone marrow.
Peripheral Basophilia ≥20% in peripheral blood.
Persistent thrombocytopenia(<100 x109 )
Persistent thrombocytosis(>1000 x109 )
Increasing spleen size and white blood count despite therapy.
Cytogenetic evidence of clonal evolution
10-19%
15. BLASTIC PHASE
(WHO DEFINITION)
Blasts >20% in peripheral blood and or bone marrow.
Extramedullary blast proliferation
Large cluster of Blasts in bone marrow Biopsy.
>20%
16. Clinical progression of CML
Chronic phase
Median 5–6
years
stabilization
Accelerated
phase
Median duration
6–9 months
Blast crisis
Median survival
3–6 months
Advanced phases
ACQUISITION OF
NEW
MUTATIONS LIKE
TRISOMY 8, P53
MUTATION.
BLAST CRISIS IS
CONVERSION OF
CML INTO AML
This acquisition of new
mutations is called as
CYTOGENIC CLONAL
EVOLUTION
18. Response to treatment can be defined under three sequential
categories.
1.Hematological Response
2.Cyto-Genetic Response(CyR)
3.Molecular Response(MR)
19. Definitions of Responses to Treatments
Hematologic Response
Complete Hematologic response
1) Normal PB counts (WBC < 10 and plt < 450)
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
(i.e. 25 →0.025)
Amount of Dz
1X1012
1X1011
1X1010
1X10 8-9
CURE???
20. Head to tail fusion of BCR to ABL occurs following
9:22 translocation
This region is transcribed into BCR-ABL mRNA
BCR-ABL protein is produced(p210)
This protein Dimerises and becomes active tyrosine kinase
Intracellular kinases are phosphorylated(eg c-myc,shc)
Cell proliferates,evades apoptosis
Hematological Response is
measure by normalization of
leukocytes and platelet count
and decrease in spleen size
Various Guidelines suggest that Complete
hematological response should be obtained
within 3 months of initiating therapy
21. Complete hematologic response
Complete normalization of PB counts, leukocyte count < 10 x 109/L
Platelet count < 450 x 109/L
No myelocytes, promyelocytes, or blasts in PB
No palpable splenomegaly
No disease symptoms
DONE BY : CBC
22. Head to tail fusion of BCR to ABL occurs following
9:22 translocation
This region is transcribed into BCR-ABL mRNA
BCR-ABL protein is produced(p210)
This protein Dimerises and becomes active tyrosine kinase
I Intracellular kinases are phosphorylated(eg c-myc,shc)
Cell proliferates,evades apoptosis
Cyto-genetic response is
measured by quantifying 9:22
transolocation.
This can be done by
1.Karyotyping/conventional cytogenetics
2.FISH
23. CYTO-GENETIC STUDIES
Requires a bone marrow aspirate for optimal
metaphases.
In bone marrow sample, dividing cells are
halted at interphase with the help of
colchicine and then trypsin and giemsa stains
are added in order for G-BANDING of
chromosomes.
Allows for evaluation of 9:22
translocation, Clonal evolution as well as
additional chromosomal abnormalities in the
non-Ph+ clones.
Atleast 20 interphases are examined before
reporting.
25. Cytogenetic response
Complete : No Ph+ metaphases
Partial : 1% to 35% Ph+ metaphases
Minor : > 35% Ph+ metaphases
Major : 0% to 35% Ph+ metaphases (complete + partial)
Done By: Cyto-genetics/Fish
26. Head to tail fusion of BCR to ABL occurs following
9:22 translocation
This region is transcribed into BCR-ABL mRNA
BCR-ABL protein is produced(p210)
This protein Dimerises and becomes active tyrosine kinase
Intracellular kinases are phosphorylated(eg c-myc,shc)
Cell proliferates,evades apoptosis
Molecular response is measured by
quantifying the Bcr-Abl mRNA
transcripts.
This can be done by
1.QPCR
28. QUANTITATIVE PCR FOR BCR-ABL INTERPRETATION
0 3 6 9 12 15 18 21 24 27 30 33 36
PCR Cycle Number
Amount of
Fluorescence
High Concentration
Moderate
Concentration
Low Concentration
29. Molecular Response
Major Molecular Response(MMR) : ≥ 3 log reduction in BCR-ABL mRNA or
BCR-ABL/ABL ≤ 0.1% by QRT-PCR (International Scale)
Complete Molecular Response (CMR) : No detectable BCR-ABL mRNA
using assay with sensitivity at least 4.5 logs below standardized baseline
Done By: QPCR
30. PROGNOSTIC CLASSIFICATION
SOKAL INDEX
Age
Blast percentage
Cytogenic clonal Evolution
Spleen size
Platelet counts
HASFORD INDEX
Age
Blast percentage
Basophil and Eosinophils %
Spleen size
Platelet counts
34. IMATINIB
Imatinib mesylate (Gleevec) functions through competitive inhibition at
the ATP-binding site of the Abl kinase in the inactive conformation,
which leads to inhibition of tyrosine phosphorylation of proteins involved
in Bcr-Abl signal transduction.
It shows specificity for Bcr-Abl, the receptor for platelet-derived growth
factor, and Kit TK.
Imatinib induces apoptosis in cells expressing Bcr-Abl.
35. FAQ ON IMATINIB
What is the dose of imatinib?
400mg / day (maximum dose upto 800 mg can be given)
What is the duration of treatment?
At present, treatment should be given for life. Trials in which the drug is
stopped after 2 years of achieving CMR has shown promising results.
What are the adverse effects?
Myelo-suppression is the most common hematologic side effect. Others
are fluid retention, nausea, muscle cramps, diarrhea, and skin rashes.
How to treat myelo-suppression following imatinib therapy?
withholding the drug until counts improves. Growth factors like GM-CSF
(filgrastim) can be used.
36. FAQ ON IMATINIB
Can we use imatinib in pregnant patients?
imatinib is shown to be teratogenic and embryotoxic in animal studies.so it
is avoided in pregnancy
Does imatinib affects fertility?
It has shown to cause oligospermia. So men desiring conception shold
consider sperm cryo-preservation before TKI therapy
When to review the patient for monitoring response?
After 3 months of therapy and every 3 months thereafter.
38. MONITORING RESPONSE
Patients on imatinib should be reviewed at 3rd month and investigations
to be done at that time are
1.CBC
2.Bone Marrow Cytogenetics/Blood FISH
3.QPCR
If patient had achieved hematological and complete cytological
response, then patient can be monitored with QPCR every 3 months
thereafter until MMR is aceived, then QPCR can be done every 6 months.
What is the ideal response at 3 months?
39. FAILURE CAN BE BECAUSE OF
1.Resistance to TKI
2.Cytogenic clonal evolution(i.e additional
mutations)
3.CML could be Ph negative type
4.Patient adherence problems(missing more
than 2 doses in a month)
40. Resistance to imatinib
Four mechanisms of resistance to imatinib have been described to date.
These are
1. Gene amplification i.e more Bcr-Abl are produced
Treated by increasing dose to 800mg
2. Enhanced expression of multidrug exporter proteins
Treated by increasing dose to 800mg
3. Mutations at the kinase site
Replace with Nilotinib or Dasatinib
4. Alternative signaling pathways functionally compensating for the
imatinib - sensitive mechanisms (SRC family of kinases get mutated
and they start phosphorylating the kinases)
Replace with Dasatinib
41. KINASE SITE MUTATIONS
Mutations at the kinase domain occur in approximately half of
imatinib-resistant chronic-phase cases and even more frequently in
the more advanced phases of the disease.
These mutations are being targeted by novel TK inhibitors that have a
different conformation than imatinib, demonstrating activity against
most imatinib-resistant mutations.
Nilotinib, like imatinib, binds to the kinase domain in the inactive
conformation. Dasatinib binds to the kinase domain in the open
conformation and also inhibits the SRC (sarcoma) family of
kinases, addressing the last mechanism of resistance.
CML with the T315I mutation is resistant to imatinib, nilotinib, and
dasatinib
43. NILOTINIB
400 mg twice daily
Associated with sudden death
Causes QT prolongation
DASATINIB
100 mg once daily
Causes pleural effusions in 22% of
patients.
Causes pulmonary arterial
hypertension
Both these drugs are now approved also as first line TKI’s for newly diagnosed
CML patients by the FDA due to their enhanced Cytogenetic and molecular
remissions within short time period
44. NEWER Drugs
Omacetaxine (formerly known as homoharringtonine) Protein
translation inhibitor
Sorafenib : Raf kinase inhibitor that down regulates down stream Bcr-
Abl targets
FTY720 (also known as fingolimod) : Activation of protein
phosphatase 2A that is essential for ABL1-mediated leukemogenesis
45. Allogenic Hematopoietic Stem Cell Transplant(HSCT)
INDICATIONS IN CP-CML:
1.Patients with T315I Mutations
2.Bcr-Abl mutations that are resistant to all TKI’s
3.Patient intolerant to TKI’s
Allogenic HSCT is no longer recommended as first line therapy in chronic
phase CML. It can be offered to candidates of accelerated or blast crisis.
Even after HSCT TKI’s should be continued till one year.
46. CHEMOTHERAPY
Currently reserved for rapid lowering of WBCs, reduction of
symptoms, and reversal of symptomatic splenomegaly.
Hydroxyurea induces rapid disease control. The initial dose is 1–4
g/d; the dose should be halved with each 50% reduction of the
leukocyte count. Unfortunately, cytogenetic remissions with
hydroxyurea are uncommon.
Busulphan, an alkylating agent that acts on early progenitor cells,
has a more prolonged effect. However not used because of its
serious side effects, which include unexpected, and occasionally
fatal, myelosuppression in 5–10% of patients; pulmonary,
endocardial, and marrow fibrosis; and an Addison-like wasting
syndrome
47. OTHER MODALITIES
Intensive leukapheresis may control the blood counts in chronic-
phase CML; however, it is expensive and cumbersome. It may also
have a role in the treatment of pregnant women, in whom it is
important to avoid potentially teratogenic drugs.
Splenectomy was used in CML in the past because of the suggestion
that evolution to the acute phase might occur in the spleen.
However, this does not appear to be the case, and splenectomy is
now reserved for symptomatic relief of painful splenomegaly
unresponsive to imatinib or chemotherapy, or for significant anemia
or thrombocytopenia associated with hypersplenism
48. HOW CAN WE MANAGE CML
PATIENTS IN MBGH HOSPITAL SETUP
49. Patients present
to us with
symptoms and
spleenomegaly
Patient is
suspected of Cml
in Peripheral
blood film
Confirm BCR-ABL
with FISH or
KARYOTYPING
Start on IMATINIB
therapy
FISH for Bcr-Abl costs Rs.4750
at SRL Religare Diagnostics(4-
7 days)
Imatinib starts from Rs.30 per
tablet
Karyotyping costs Rs.3100 at
Amolak Labs and report is
available after one month
Myeloblast: large nucleus fine chromatin and blue cytoplasmMyelocyte: large nucleus flattened on one side with pink cytoplasmic granulesMetamyelocyte : large nucleus with indented nucleus
1993: imatinib was patented in us.Europe.at that time no patents in india..so it was produced by generic companies1998: Novartis filed a petition for im.mesylate in india(after india announced that it will allow proxduct patents)2005: indian laws were enacted for product patenting and started processing requestsIndia rejected it saying that just by adding mesylate, it could not patent it for NovartisIt moved to supreme court and final verdict given in apr 2013 that modifications could not be patented.