This document summarizes a seminar presentation on chronic myeloid leukemia (CML). It provides a brief history of CML discoveries including identification of the Philadelphia chromosome in 1960 and the BCR-ABL1 fusion gene in 1973. It describes the epidemiology, molecular pathogenesis involving the BCR and ABL1 genes, and clinical phases of CML. The presentation covers diagnostic testing including blood counts, bone marrow aspiration, cytogenetics, and molecular testing. It discusses current treatment with tyrosine kinase inhibitors and monitoring treatment response using blood counts, cytogenetics and RT-PCR. The document concludes with information on resistance to treatment due to BCR-ABL1 kinase domain mutations.

1. INTEGRATED SEMINAR
TOPIC: Chronic myeloid leukemia
Moderator: Dr Amrit Sarmah
Assistant Professor, Dept of Pathology
Tezpur Medical College Hospital
Presented by: Dr Lekhraaj Gautam
3rd year PGT, Dept of Pathology
Tezpur Medical College Hospital

2. A Brief History….
In 1845, John Hughes Bennett, an Edinburgh pathologist,
described a “Case of Hypertrophy of the Spleen and Liver
in which Death Took Place from Suppuration of the Blood.”
Rudolf Virchow coined the term “leukemia,” in 1872.
Ernst Neumann established the bone marrow as the origin
of leukemia and of blood cells in general.

3. In 1951, William Dameshek posited that CML belongs
to a larger group of related disorders which he
accordingly named myeloproliferative disorders.
A fundamental experimental breakthrough by
Philadelphia cytogeneticists Peter Nowell and David
Hungerford followed in 1960.
In 1973, Janet Rowley recognized that Ph was not just a
shortened chromosome 22, but was in fact the product
of a reciprocal translocation between chromosomes 9
and 22.

5.  Chronic myeloid leukemia (CML) (chronic granulocytic leukemia, CGL) is a clonal
myeloproliferative neoplasm arising from neoplastic transformation of
pluripotent stem cell
 Consistently associated with BCR-ABL 1 fusion gene located in the Philadelphia
chromosome

6. Epidemiology:
Annual incidence of 1―2 cases per 100 000 population
M>F
Median age at diagnosis 4th to 5th decade.
Prevalence is increasing.
Predisposing factors:
Largerly unknown
Radiation exposure
Genetic predisposition

8. BCR gene
BCR- long arm of chromosome 22 (22q11)
The BCR gene encodes a 160-kDa serine- threonine kinase.
Has several functional domains- autophosphorylates and transphosphorylates several
protein substrates.
ABL1 Gene
Human homologue of the viral ABL oncogene carried by thr Ableson murine leukemia
virus.
Human Abl protein: 145- kDa protein.
Tyrosine kinase enzyme activity
Shuttles between nucleus and cytoplasm where it performs regulation of cell cycle
and cytoskeletal molding respectively
Loss of this region(as occurs in BCR-ABL) results in high constitutive kinase enzymatic
activity.

9. p190
p210
p230

11. Schematic diagram of molecular signaling pathways activated in CML

12. Phases of Disease:
CML-Chronic Phase
CML-Acclerated Phase
CML-Blast phase

13. Chronic Phase:
Peripheral Blood Findings:
Leukocytosis
Neutrophils in various stage of maturation
Hypersegmented polymorphs
Basophilia
No significant dysplasia
Blast < 10%
Normocytic normochromic anaemia
Thrombocytosis in majority of cases.

14. leucocytosis with all stages of
myeloid ceils from blast cells
to neutrophils.
Blood film shows myeloblasts,
promyelocytes and large number
of myelocytes. ‘myelocyte bulge’ is
a characteristic feature in Ph +ve
untreated CML.

15. LAP Score:
Neutrophil Alkaline Phosphatase score is markedly diminished to
0-20 (Normal: 40-100)
The mRNA for alkaline phosphatase is undetectable in
neutrophils of patients with CML.

16. Bone Marrow Aspirate in Chronic Phase:
Markedly hypercellular with replacement of fat by hyperplastic
hemopoietic with myeloid hyperplasia and
the M : E ratio is elevated.
Scattered amongst the marrow cells are macrophages with
linear striations or granular cytoplasm (pseudo-Gaucher cells),
some with sea blue coloured, granules resembling sea-blue
histiocytes.
Megakaryocytes are smaller (dwarf forms) than the normal,
megakaryocytes .

18. pseudo-Gaucher cells
(some with sea blue coloured, granules resembling sea-blue histiocytes)

19. Bone Marrow Trephine Biopsy in Chronic Phase:
Hypercellular marrow spaces
Granulocytic hyperplasia
Paratrabecular cuff of immature granulocytes is often seen 5 to
10 cell thick.( Normally 2 to 3 cells)
Blasts < 10%
Megakaryocytes – dwarf and hypolobated.
Moderate to marked increase in reticulin fibrosis.( Seen in
around 30% cases)

21. Differential Diagnosis:
Leukemoid Reaction
Atypical CML (aCML)
Chronic myelomonocytic leukemia (CMML)
Chronic neutrophilic leukemia
Juvenile myelomonocytic leukemia
G-CSF Therapy

23. Accelerated Phase of CML:

24. The finding of lymphoblasts in the peripheral blood or bone marrow (even
if < 10%) should prompt concern that lymphoblastic transformation may
be imminent, and warrants further clinical and genetic investigation.

25. Blast Phase of CML:

26. CML in Blast Phase:
 ≥ 20% blasts in the blood or bone marrow
 Presence of an extramedullary proliferation of blasts
Characterization of Blastic Phase-
Mostly myeloid lineage(70% cases)
Lymphoid(30%); B-Cell Type

29. Current Diagnostic Work up:
Clinical Evaluation
Haematological Investigations
- Complete Blood Count
- PBS Study
- Bone marrow Aspiration
- Trephine Biopsy
Molecular and Cytogenetic Studies
- Karyotyping
- FISH
- RTPCR

30. Conventional Cytogenetics:
• It is the technique that initially led to the identification of the Ph
chromosome in CML patients.
• In 95% of CML patients, the BCR-Abl 1 fusion gene will be evident as Ph
chromosome.
Advantage:
• Entire chromosomal complement is evaluated for the presence of
additional abnormalities to the Ph chromosome.
Disadvantages:
• Limited sensitivity to detect an abnormal clone.( 5 to 10%)
• It requires viable and dividing(metaphase) cells.

35. MONITORING OF TYROSINE KINASE INHIBITOR (TKI) RESPONSE:
Monitoring of response to TKI is carried out by-
Peripheral blood counts
Cytogenetic analysis
RT-PCR

36. Molecular response (MR) is evaluated by RT-Q-PCR on a patient’s sample by detection of
number of copies of fusion transcript.
Response evaluation is required every 3 months till major molecular response (MMR)is
achieved.
After achieving MMR, it may be carried out every 6 months.

37. Resistance to TKI:
Point mutations in the BCR-ABL1 kinase domain (KD) remain the
major cause of acquired resistance of TKIs.
BCR-ABL KD mutation status is required to integrate it into the
decision algorithm like switching the dose of imatinib or
switching to second line TK inhibitors or stem cell
transplantation.
BCR-ABL KD mutations can be detected by-
Direct sequencing
Direct sequencing + denaturing high performance liquid
chromatography (D-HPLC) analysis.

38. Some of the frequent BCR-ABL KD mutations are:
• M244V
• G250E
• Y253F/H - dasatinib is more effective than nilotinib in these
cases
• E255K/V - dasatinib is more effective than nilotinib in these
cases
• T315I - resistant to imatinib, nilotinib and dasatinib in these
cases
• F317L - nilotinib is more effective than dasatinib
• E355G
• F359V - dasatinib is more effective tlian nilotinib
• H 396 R/R

39. This Photo by Unknown Author is licensed under CC BY-SA-NC