32 Chronic Myelogenous Leukemia CML Flashcards
CML
Chronic Myelogenous Leukemia
Define CML
Resulting from the translocation of chromosomes 9 and 22: creating the BCR-ABL1 fusion gene
How does BCR-ABL1 cause CML?
leads to constitutive activation of the ABL1 tyrosine kinase;
resulting in growth factor independent myelopoiesis
Three phases of CML
- Chronic phase: last 4-5yrs, blast counts less than 2%
- Accelerated phase
- Blast phase: >20% blasts
CML chronic phase
- last 4-5 yrs
- leukocytosis with marked proliferation of granulocytes and their precursors: myelocytes and segmented neutrophils
- basophilia
- eosinophilia
- thrombocytosis
- blasts<2%
What is Philadelphia Chromosome?
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
Philadelphia Chromosome is found in which leukemia?
Mainly in CML, and some in AML [ de novo B lymphoblasts leukemia/lymphoma (B-ALL) ]
CML accelerated phase
- leukocytosis, splenomegaly, or thrombocytosis uncontrolled by therapy
- thrombocytopenia unrelated to therapy
- clonal cytogenetic evolution
- basophilia: >20% of cells in the peripheral blood
- 10-19% myeloblasts in the blood or bone marrow
CML blast phase
- > 20% blast counts
- is marked by an acute leukemia of either myeloid or lymphoid phenotype
CML therapies
- BCR-ABL1 targeted therapy using tyrosine kinase inhibitors:
imatinib
dasatinib
nilotinib
ponatinib
bosutinib - Chemotherapy
- interferon-alpha therapies
BCR-ABL1 fusion products
- m-bcr: p190 kDa, e1a2
- M-bcr (major): 210 kDa, e13a2, e14a2
- u-bcr: 230 kDa, e19a2
e1, e13, e14, e19 is exon1, 13, 14, 19 of BCR
a2: 5’exon2 of ABL1
m-bcr found in which leukemia?
B-ALL/LBL: 50% in adults, 80% in kids
rare in CML<1%
M-bcr (major) found in which leukemia?
98% in CML
50% in adult B-ALL
20% in kids B-ALL
u-bcr found in which leukemia?
rare in CML
Molecular tests for CML diagnosis
- FISH for detection of BCR-ABL1 at interphase or metaphase
- RT-PCR tests for BCR-ABL1
Tests for monitoring CML
- ABL1 mutational analysis for acquired resistance - Nested PCR plus sequencing of ABL1-TK domain
- FISH as a complimentary of monitoring treatment response due to lack of standardized criteria
- FISH is not sensitive enough for monitoring CML relapse, limited analytic sensitivity
- q-RT-PCR
Nested PCR plus sequencing of ABL1-TK domain
- First PCR with forward BCR primers and reverse ABL1 primers
- 2nd primer sets to amplify ABL1 kinase domain
- Sanger sequencing to identify resistance mutation
FISH BCR-ABL1
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
RT-PCR BCR-ABL1
Qualitative or quantitative
Detect both M-bcr and m-bcr rearrangements, which account for the large majority of BCR-ABL1
rapid TAT (turnaround time)
Qualitative RT-PCR BCR-ABL1
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
Quantitative RT-PCR (q-RT-PCR) BCR-ABL1
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
Conventional CML initial diagnosis methods
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
cytogenetically cryptic
a genetic alteration/translocation can not be detected by cytogenetic analysis
blasts
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
Monitoring CML with cytogenetic test
- Major complete response: 0% t(9;22) positive cells
- Major partial response: 1-34% t(9;22) positive cells
- Minor response: 35-94% t(9;22) positive cells
- No response: >95% t(9;22) positive cells
Monitoring CML with q-RT-PCR
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
what does CML with q-RT-PCR - 4.5 log reduction mean?
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
TKI resistance
1-log increase of BCR-ABL1 after 3 month TKI therapy
ABL1 kinase domain mutations
Sequencing used for detecting mutations and TKI selection
TKI resistance: ABL1 kinase domain mutations
Most common and notorious one: ABL-1 T315I
ABL-1 T315I
resistance to multiple TKIs:
imatinib
dasatinib
nilotinib
What can a doctor do if a CML patient develop resistance to all TKIs?
hematopoietic stem cell transplantation
clinical trials
