detailed discussion on cytogenetics in CML - Pathophysiology, treatment, TKI Resistance, Mutation analysis timing, various mutations in CML, BCR-ABL1 Variants, Significance of mutations and management.

1. Department of Clinical Haematology
Dr. Sandeep Kumar
Resident – Department of Internal Medicine

3. t(9;22)(q34;q11.2) (present in 90% cases)
Complex translocations (Variant Ph)
Masked Ph
 In 5-10 % cases

4. Differential Diagnosis
Leukemoid reaction
Juvenile myelomonocytic leukemia
Chronic myelomonocytic leukemia
"Atypical CML"
Chronic eosinophilic leukemia
Chronic neutrophilic leukemia
Other myeloproliferative neoplasms
Other Philadelphia chromosome-positive malignancies

5. Ph positive Leukaemia's
Precursor B cell
lymphoblastic leukaemia (ALL).
Childhood ALL
Adult AML

6. Molecular basics of CML
• Abelson tyrosine kinase (ABL1)
fusion ABL1 gene
breakpoint cluster region (BCR) gene
BCR-ABL1 –
p210BCR-ABL1
Constitutive
Tyrosine Kinase
activity
-Excessive
Proliferation
-ReducedApoptosis

8. • Minor BCR region (m BCR Region)
• Micro – BCR (µ - BCR)
m BCR p190BCR-ABL1 PredictsWorse outcome
Juxtaposes larger
BCR fragment to
ABL1
p230BCR-ABL1 a/w More Indolent
Course

9. • p210 BCR-ABL1
• p190 BCR-ABL1
• p230 BCR-ABL1

10. Activated BCR-ABL1
Auto-phosphorylation & Activation of Downstream pathways-
modify – Gene transcription – Apoptosis, - Skeletal
organisations & - Degradation of Inhibitory proteins.
May involve – RAS, Mitogen Activating Protein (MAP) Kinases,
Signal transducers & Activators ofTranscription (STAT),
Phosphatidyl-Inositol -3-Kinase(PI3K),MYC , etc.

11. Binding of BCR-ABL1 to Adapter Protein – GRB-2, CRK, CRK-Like
Protein, SHC (Src homology containing Protein).
BCR-ABL1 TKIs bind to BCR-ABL1 Kinase Domain (KD) – Preventing
Activation ofTransformation pathways & Inhibiting Downstream
signalling.
Plethora of Signalling pathways have been implicated in BCR-ABL1
mediated cellular transformations. – COMPLEX & REDUNDANT
TRANSFORMATION NETWORK.
Differences in SignalTransduction between CML Differentiated Cells &
Early progenitors. Beta-Catenin, Wnt1, Foxo3a,TGF-β, IL-6, PP2A,
SIRT1, - Have been implicated in Stem Cell Survival.

13. Transition to blast crisis or accelerated
phase.

14. Absolute
resistanc
e to
Imatinib
Prevents
binding of TKIs
to catalytic
(ATP) binding
site
Development
of different
ABL1 Kinase
domain

16. Study Calculation Risk definition by calculation
Sokal et al – 1984 Exp. 0.0116 × (Age in Yrs – 43.4) +
(Spleen – 7.51) + 0.188 × [(Platelet
count / 700)2 - 0.563] + 0.0887 ×
(blast cells – 2.10)
Low - <0.8
Intermediate – 0.8 – 1.2
High - >1.2
Hasford et al, 1998 0.666 when Age ≥50 years + (0.042
× Spleen) + 1.0956 when PLT >1500
× 109/L + (0.0584 × Blasts cells) +
0.20399 when Basophils >3% +
(0.0413 × Eosinophils) × 100
Low - ≤ 780
Intermediate - 781 – 1480
High - >1480
EUTOS score - Probability of the patient NOT
to be in CCgR after 18 months of Imatinib therapy.
7 × Basophils + 4 × Spleen size Low risk - ≤ 87
High risk - >87
ELTS score – EUTOS Long term
survival score
0.0025 x (age in completed
years/10)3 + 0.0615 x spleen size
below costal margin + 0.1052 x
blasts in peripheral blood + 0.4104 x
(platelet count/1000)-0.5
Low - ≤ 1.5680
Intermediate - > 1.5680 but ≤
2.2185
High - > 2.2185

21. Mutation imatinib Dasatinib Nilotinib Bosutinib Ponatinib
BCR-ABL1 Confers Confers Confers Confers Confers
BCR-ABL1 c.757T>C (Y253 H) Reduced Retains Reduced ? ?
BCR-ABL1 c.763 G>A (E255K) Reduced Retains Reduced ? ?
BCR-ABL1 c.764 A>T (E255V) Reduced Retains Reduced ? ?
BCR-ABL1 c.895 G>C (V299L) Reduced Reduced Retains ? ?
BCR-ABL1 c.895 G>T (V299L) Reduced Reduced Retains ? ?
BCR-ABL1 c.943 A>G (T315A) Reduced Reduced Retains ? ?
BCR-ABL1 c.944 C>T (T315I) *** Reduced Reduced Reduced Reduced Retains
BCR-ABL1 c.950T>G (F317C) Reduced Reduced Retains ? ?
BCR-ABL1 c.949T>A(F317I) Reduced Reduced Retains ? ?
BCR-ABL1 c.951 C>A (F317L) Reduced Reduced Retains ? ?
BCR-ABL1 c.949T>C (F317L ) Reduced Reduced Retains ? ?
BCR-ABL1 c.951 C>G (F317L) Reduced Reduced Retains ? ?
BCR-ABL1 c.949T>G (F317V) Reduced Reduced Retains ? ?
BCR-ABL1 c.1076T>G (F359C) Reduced Retains Reduced ? ?
BCR-ABL1 c.1075T>A (F359I) Reduced Retains Reduced ? ?
BCR-ABL1 c.1075T>G (F359V) Reduced Retains Reduced ? ?

22. Other
BCR-ABL1
Mutations in
CML
Properties
Mutation Frequency of
Mutation in ABL1-
mutated CML
(COSMIC)
Exon Amino Acid Change Nucleotide Change(s)
4 M244V c.730A>G 7.4%
L248V c.742C>G 2.4%
G250E c.749G>A 10.4%
Q252H c.756G>T
c.756G>C
2.9%
Y253F c.758A>T 1.8%
5 D276G c.827A>G 1.4%
E279K c.835G>A 0.3%
6 F311L c.931T>C
c.933C>A
c.933C>G
0.6%
M351T c.1052T>C 9.0%
E355G c.1064A>G 3.1%
7 V379I c.1135G>A 0.6%
L384M c.1150C>A 0.6%
L387M c.1159T>A 0.8%
H396P c.1187A>C 0.1%
H396R c.1187A>G 3.1%
8 E459K c.1375G>A 1.9%
9 F486S c.1457T>C 1.1%
Implications for targeted therapies
- UNKNOWN