DIAGNOSIS MANAGEMENT AND RECENT ADVANCES IN CML

DR RAJESH S

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

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.

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.

The Ph Chromosome: t(9;22)

22

bcr

abl

Ph ( or 22q-)

bcr-abl

FUSION PROTEINWITH TYROSINEKINASE ACTIVITY

9 9 q+

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

Stem cells

Myeloid stem cells

myelocytes

platelets

Lymphoid stem cells

Lymphocytes

Erythrocytes

BCR ABL MUTATION OCCURS in MYELOID STEM CELLSALL COMPONENTS OF MYELOID SERIES i.e MYELOCYTES,RBC’S AND PLATELETS ARE AFFECTED

CLINICAL AND LAB FEATURES

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.

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.

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.

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%

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%

Clinical progression of CML

Chronic phase

Median 5–6 years

stabilization

Accelerated phase

Median duration6–9 months

Blast crisis

Median survival3–6 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

GOALS, CONCEPTS OF MANAGEMENT AND DIAGNOSTIC MODALITIES

Response to treatment can be defined under three sequential categories.

1.Hematological Response

2.Cyto-Genetic Response(CyR)

3.Molecular Response(MR)

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???

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

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

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 cytogenetics2.FISH

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.

FLOURESCENT IN-SITU HYBRIDISATION

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

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 by1.QPCR

Bcr-Abl

Bcr

Abl

cDNA

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

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

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

TREATMENT WITH TYROSINE KINASE INHIBITORS

TREATMENT VARIOUS MODALITIES

HSCTPOTENTIALLY CURATIVE

TYROSINE KINASE INHIBITORS

DISEASE CONTROL

CHEMOTHERAPYPALLIATION

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.

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.

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.

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?

FAILURE CAN BE BECAUSE OF

1.Resistance to TKI2.Cytogenic clonal evolution(i.e additional mutations)3.CML could be Ph negative type4.Patient adherence problems(missing more than 2 doses in a month)

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 800mg2. Enhanced expression of multidrug exporter proteins

Treated by increasing dose to 800mg3. Mutations at the kinase site

Replace with Nilotinib or Dasatinib4. 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

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

Bcr-Abl imatinib

Mut. Bcr-Abl imatinib

Mut. Bcr-Abl dasatinib

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

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 

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.

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

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

HOW CAN WE MANAGE CML PATIENTS IN MBGH HOSPITAL SETUP

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

NAME SALT MANUFACTURER COST/DAY PER MONTH

1.RESIMAT IMATINIB RESONANCE LABS RS.30.10 903

2.LUPITIB IMATINIB LUPIN RS.170 5100

3.MITINAB IM.MESYLATE GLENMARK RS.180 5400

4.MESYLONIB IM.MESYLATE MIRACALUS RS.300 9000

5.IMATIB IM.MESYLATE CIPLA RS.300 9000

6.CHEMOTINIB IM.MESYLATE NEON RS.300 9000

7.CELONIB IMATINIB CELON RS.320 9600

8.VEENAT IMATINIB NATCO RS.352 10560

THANK YOU