32 Chronic Myelogenous Leukemia CML

Question 1

CML

Answer 1

Chronic Myelogenous Leukemia

Question 2

Define CML

Answer 2

Resulting from the translocation of chromosomes 9 and 22: creating the BCR-ABL1 fusion gene

Question 3

How does BCR-ABL1 cause CML?

Answer 3

leads to constitutive activation of the ABL1 tyrosine kinase;

resulting in growth factor independent myelopoiesis

Question 4

Three phases of CML

Answer 4

1. Chronic phase: last 4-5yrs, blast counts less than 2%
2. Accelerated phase
3. Blast phase: >20% blasts

Question 5

CML chronic phase

Answer 5

1. last 4-5 yrs
2. leukocytosis with marked proliferation of granulocytes and their precursors: myelocytes and segmented neutrophils
3. basophilia
4. eosinophilia
5. thrombocytosis
6. blasts<2%

Question 6

What is Philadelphia Chromosome?

Answer 6

t(9;22) (q34;q11.2) translocation

5’BCR-3’ABL1

BCR is on chr. 22

ABL1 is on chr. 9

The changed chromosome 22 is called the Philadelphia chromosome [der(22q)].

see the figure

Question 7

Philadelphia Chromosome is found in which leukemia?

Answer 7

Mainly in CML, and some in AML [ de novo B lymphoblasts leukemia/lymphoma (B-ALL) ]

Question 8

CML accelerated phase

Answer 8

1. leukocytosis, splenomegaly, or thrombocytosis uncontrolled by therapy
2. thrombocytopenia unrelated to therapy
3. clonal cytogenetic evolution
4. basophilia: >20% of cells in the peripheral blood
5. 10-19% myeloblasts in the blood or bone marrow

Question 9

CML blast phase

Answer 9

1. > 20% blast counts
2. is marked by an acute leukemia of either myeloid or lymphoid phenotype

Question 10

CML therapies

Answer 10

1. BCR-ABL1 targeted therapy using tyrosine kinase inhibitors:
   imatinib
   dasatinib
   nilotinib
   ponatinib
   bosutinib
2. Chemotherapy
3. interferon-alpha therapies

Question 11

BCR-ABL1 fusion products

Answer 11

1. m-bcr: p190 kDa, e1a2
2. M-bcr (major): 210 kDa, e13a2, e14a2
3. u-bcr: 230 kDa, e19a2

e1, e13, e14, e19 is exon1, 13, 14, 19 of BCR
a2: 5’exon2 of ABL1

Question 12

m-bcr found in which leukemia?

Answer 12

B-ALL/LBL: 50% in adults, 80% in kids

rare in CML<1%

Question 13

M-bcr (major) found in which leukemia?

Answer 13

98% in CML

50% in adult B-ALL

20% in kids B-ALL

Question 14

u-bcr found in which leukemia?

Answer 14

rare in CML

Question 15

Molecular tests for CML diagnosis

Answer 15

1. FISH for detection of BCR-ABL1 at interphase or metaphase
2. RT-PCR tests for BCR-ABL1

Question 16

Tests for monitoring CML

Answer 16

1. ABL1 mutational analysis for acquired resistance - Nested PCR plus sequencing of ABL1-TK domain
2. FISH as a complimentary of monitoring treatment response due to lack of standardized criteria
3. FISH is not sensitive enough for monitoring CML relapse, limited analytic sensitivity
4. q-RT-PCR

Question 17

Nested PCR plus sequencing of ABL1-TK domain

Answer 17

1. First PCR with forward BCR primers and reverse ABL1 primers
2. 2nd primer sets to amplify ABL1 kinase domain
3. Sanger sequencing to identify resistance mutation

Question 18

FISH BCR-ABL1

Answer 18

Dual-color, dual-fusion approach

Green: BCR gene chr. 22q11.2
Red: ABL1 chr. 9q34
Green/red fusion signal: t(9;22)(q34;q11.2), also called der22(q), der(9)

Not a random colocalization of probe signals

LOD: 1-2% of 200 cells, 0.5% of 500 cells scored.

Mainly used as initial diagnosis not good enough for monitoring relapse

metaphase or interphase FISH

Question 19

RT-PCR BCR-ABL1

Answer 19

Qualitative or quantitative

Detect both M-bcr and m-bcr rearrangements, which account for the large majority of BCR-ABL1

rapid TAT (turnaround time)

Question 20

Qualitative RT-PCR BCR-ABL1

Answer 20

Controls for RNA extraction and RT-PCR inhibitors

Controls: GAPDH or ABL1

simple, nested or multiplex approach

Detect both M-bcr and m-bcr

Detect rare variants: u-bcr, ABL1 exon 3 (a3), these are not easily controlled by quantitative PCR

Question 21

Quantitative RT-PCR (q-RT-PCR) BCR-ABL1

Answer 21

for posttherapeutic monitoring CML

LOD: 1 in 100,000 cells

Taqman, FRET probes

standard curve for determine the copies

normalizing internal reference control: ABL1

report for: BCR-ABL1/ABL1 ratio

PCR run performance controls: high positive, low positive

1 Ct difference = 2-fold concentration difference

Question 22

Conventional CML initial diagnosis methods

Answer 22

cytogenetics can detect 95% of cases BCR-ABL1

cytogenetic also detect other chromosomal abnormalities

baseline for later testing and disease monitoring

FISH and RT-PCR detect 5% cases, which are cytogenetically cryptic

Question 23

cytogenetically cryptic

Answer 23

a genetic alteration/translocation can not be detected by cytogenetic analysis

Question 24

blasts

Answer 24

abnormal immature white blood cells

normal blast count is 0-5%

high blasts prevent red blood cells and platelets production, leading to infection, anemia, and abnormal bleeding

Question 25

Monitoring CML with cytogenetic test

Answer 25

1. Major complete response: 0% t(9;22) positive cells
2. Major partial response: 1-34% t(9;22) positive cells
3. Minor response: 35-94% t(9;22) positive cells
4. No response: >95% t(9;22) positive cells

Question 26

Monitoring CML with q-RT-PCR

Answer 26

use International Scale (IS)
1. Major molecular response (MMR): 3 log reduction = 0.1% IS
2. Complete molecular response (CMR): 4.5 log reduction
3. Favorable prognostic significance: 1 log reduction = 10% IS at 3 months and 6 months

lab must establish a conversion factor to translate their results into IS with commercial reference material or exchange specimens with an IS-calibrated reference lab

q-RT-PCR BCR-ABL1 every three months

Question 27

what does CML with q-RT-PCR - 4.5 log reduction mean?

Answer 27

4.5-log reduction is referred to as a “complete molecular response (CMR)” or a “deep molecular response (DMR).”

Doctors may refer to this as “MR4. 5.”

A 4.5-log reduction indicates that 0.0032% of cells (1 out of every 32,000 cells) have the BCR-ABL1 transcripts

Question 28

TKI resistance

Answer 28

1-log increase of BCR-ABL1 after 3 month TKI therapy

ABL1 kinase domain mutations

Sequencing used for detecting mutations and TKI selection

Question 29

TKI resistance: ABL1 kinase domain mutations

Answer 29

Most common and notorious one: ABL-1 T315I

Question 30

ABL-1 T315I

Answer 30

resistance to multiple TKIs:
imatinib
dasatinib
nilotinib

Question 31

What can a doctor do if a CML patient develop resistance to all TKIs?

Answer 31

hematopoietic stem cell transplantation

clinical trials