Chronic Myelogenous Leukemia (CML)

Epidemiology and Etiology:

• Median age: 40s and 50s
• Any age
• Unknown etiology
• Radiation exposure in some cases
• No inherited disposition
• Usually diagnosed in chronic phase (CP)
• Uncommonly presents in blast phase (BP)

Common sites:

• Blood
• BM
• Spleen
• Liver maybe
• Blast phase:
• Lymph nodes
• Skin
• Soft tissues

Gross features:

• Splenomegaly common

Cytologic / Histologic features:

• CP:
• Peripheral blood:
• Neutrophilic leukocytosis
• Neutrophils in different stages of maturation
• No significant dysplasia
• Absolute basophilia invariably
• Eosinophilia common
• Absolute monocytosis maybe (but usually < 3%)
• p190 BCR-ABL1 isoform – monocytosis is nearly always present
• Anemia
• Blasts < 2% usually
• Platelets normal to > 1000x10⁹/L
• Marked thrombocytosis uncommonly
• Thrombocytopenia uncommon
• BM:
• Increased cellularity
• Granulocytic proliferation
• Paratrabecular cuff of immature neutrophils is often 5-10 cells thick (N 2-3 cells)
• Mature neutrophils in the intertrabecular areas often
• Eos maybe prominent
• Blasts < 5% usually
• > 10% indicates progression
• Erythroid islands reduced in number and size usually
• Megas smaller than normal and have hypolobated nuclei (“dwarf megakaryocytes”)
• Moderate to extensive megakaryocytic proliferation (40-50%)
• Normal or slightly decreased in number
• Moderate to marked reticulin fibrosis (30%) (worse prognosis)
• Pseudo-Gaucher cells commonly
• Sea-blue histiocytes commonly
• Reduced or no iron-laden macrophages (> 80%)
• Spleen:
• Red pulp infiltration by granulocytes in different maturation stages
• Liver:
• Similar infiltrate in hepatic sinusoids and portal areas maybe
• Post PTKI treatment:
• Reduction of granulocytic cellularity
• Normalization fo megakaryopoiesis
• Regression of fibrosis
• Increase in apoptosis
• Decrease in proliferative activity
• AP:
• Associated with any of the following:
• Persistent or increasing WBC (> 10x10⁹/L) and/or persistent or increasing splenomegaly unresponsive to therapy
• Persistent thrombocytosis (> 1000x10⁹/L) uncontrolled by therapy
• Persistent thrombocytopenia (< 100x10⁹/L) unrelated to therapy
• Clonal cytogenetic evolution occurring after the initial diagnostic karyotype
• 20% or more basophils in the PB
• 10-19% blasts in the blood or BM
• BM is hypercellular with myelodysplasia often
• Large clusters or sheets of small, abnormal megakaryocytes associated with marked reticulin or collagen fibrosis is common
• BP:
• >= 20% blasts in the PB or BM, OR
• Myeloid (70%) – any combination of neutrophilic, eosinophilic, basophilic, monocytic, megakaryocytic, or erythroid
• Lymphoid (20-30%)
• Extramedullary blast proliferation
• Presumptive diagnosis if accumulations of blasts occupy focal but significant areas of the BM
• An entire intertrabecular region
• Myelocytes (neutrophilic):
• 10-18um
• n:c ratio 2:1 to 1:1
• eccentric nucleus
• oval, slightly flattened or indented
• no nucleolus
• some chromatin clumping
• perinuclear hoff sometimes
• amphophilic / pale pink cytoplasm
• both coarse azurophilic (primary) and fine lilac (specific or secondary) granules in cytoplasm
• Metamyelocytes (neutrophilic):
• 10-18um
• N:C ratio 1.5:1 to 1:1
• Nucleus indented / kidney-shaped
• Indentation less than half of width of nucleus
• Clumped chromatin with distinct regions
• No nucleolus
• Plentiful pink cytoplasm with many lilac granules and rare azurophilic granules
• Band (neutrophilic)
• Same as above but with nucleus indented greater than half of its diameter

Immunophenotype:

Marker:	Sensitivity:	Specificity:
CD34 (myeloid blasts associated with AP/BP)
TdT (lymphoid blasts associated with AP/BP)
Neutrophil alkaline phosphatase (markedly decreased in neutrophils)
Coexpression of myeloid and lymphoid antigens in BP

Molecular features:

• See BCR/ABL Testing Guidelines for CML
• Philadelphia chromosome containing BCR-ABL1 fusion gene – t(9;22)(q34;q11)
• 95-100% of CML, 25% of adult ALL, 5% of AML, 2-5% of pediatric ALL
• 90-95% have the characteristic t(9;22)(q34;q11.2)
• Variant translocations that involve a third or even a fourth chromosome
• Increased frequency of concomitant deletions at the reciprocal ABL1/BCR fusion on the der(9), which may be adverse prognostic factor
• But another large series of 43 cases found no difference in response rate, OS, or duration of response to Imatinib
• Prevailing opinion is same clinical, prognostic, and hematologic features as standard t(9;22)
• The translocation is detected cytogenetically in more than 95% of patients while in the other 5% of patients the fusion is gene is located on a normal appearing chromosome 9 or chromosome 22.
• Cryptic translocation that cannot be identified by routine cytogenetic analysis
• FISH, RT-PCR, or southern blot will detect
• BCR (22q11.2) fused to ABL1 (9q34) produces a BCR-ABL1 fusion protein
• Fusion protein has increased tyrosine kinase activity
• Constitutive activation of several signal transduction pathways
• The counterpart ABL-BCR fusion locus on chromosome 9q + , on the other hand, has no known pathogenic consequence
• der(9) deletions involving the reciprocal ABL1/BCR fusion (17%)
• can involve one or both of ABL1 and BCR portions
• most studies show an adverse prognosis
• but prognosis with treatment with imatinib is not clear at this time
• Breakpoints:
• At diagnosis it is important that the specific fusion subtype (which depends on position of the BCR breakpoint) is identified so that the correct fusion subtype is then monitored in follow up samples
• The breakpoints in the ABL gene locate at the 5′-end of the gene and can occur anywhere from upstream of the alternative exon Ib to downstream of exon Ia, a region that spans >300 kb
• The most common breakpoint in the ABL1 gene is upstream of exon a2 (ABL a2 breakpoint).
• breakpoints in the ABL gene invariably result in a fusion of upstream BCR sequences to exons a2 or a3 of ABL following splicing of the BCR-ABL pre-mRNA
• Depending on the breakpoint location in the BCR gene, the resultant BCR-ABL fusion transcript may express a BCR-ABL hybrid protein ranging from 185 to 230 kd, which always includes an intact functional ABL kinase domain
• Major BCR-ABL1 fusion subtype (>90% CML):
• The translocation partners in the vast majority of chronic phase CML cases are the b2a2 or b3a2 fusion transcripts that express the p210 BCR-ABL protein;
• In the BCR gene, the most frequent breakpoint is downstream of either exon e13 or e14 (previously referred to as exons b2 and b3, respectively), leading to the e13a2 or the e14a2 fusion subtypes, both of which produce p210 oncoprotein
• 97% of CML patients (e13a2 and/or e14a2)
• of note, BCR exons b2 and b3 are within the major breakpoint cluster region (M-BCR) and also known as e13 and e14, respectively
• Major breakpoint cluster region M-BCR, spanning exons 12-16 (b1-b5)
• Fusion protein p210 has increased tyrosine kinase activity
• In less than 5% of patients with CML, a break in the ABL1 gene may occur downstream of exon 2 of the ABL1 gene and give rise to an e13a3 or e14a3 fusion
• minor fusion gene (<1% CML, up to 75% of ALL):
• a breakpoint downstream of BCR exon 1 results in an e1a2 fusion gene and a p190 oncoprotein (minor fusion gene)
• associated with the highest ABL tyrosine kinase activity

· Minor breakpoint region (BCR exons 1-2) (m-BCR)

• Small amounts of p190 transcript can be detected in >90% of patients with classical p210 CML, due to alternative splicing

· Rare cases of CML have only this breakpoint product

· Distinctive for having increased numbers of monocytes

· Can resemble chronic myelomonocytic leukemia

· the e1a2 transcript that expresses a p190 BCR-ABL hybrid kinase is rarely seen in chronic phase CML

· Variant fusion subtype (< 0.5% CML)

· break occurs downstream of BCR exon 6 or BCR exon 8 (e6a2, e8a2)

· mu-BCR fusion subtype (rare, indolent type of chronic neutrophilic leukemia)

· breakpoint downstream of BCR exon e19 (e19a2)

• in the mu-BCR region, spanning exons 17-20 (c1-c4)
• Larger fusion protein, p230 is encoded
• Prominent neutrophilic maturation and/or conspicuous thrombocytosis maybe
• The amount of BCR-ABL1 transcript can be quantitated with Q-RT-PCR
• sensitivity of BCR-ABL RQ-PCR is 0.001-0.0001%, or equivalent to detecting one cell expressing BCR-ABL RNA in 100,000- 1,000,000 normal cells
• Used for following treatment response to Gleevec
• A log 3 reduction is ideal molecular response
• If the BCR-ABL transcript is increasing, the kinase domain (drug-binding site) of the transcript can be sequenced for point mutations
• The kinase domain is amplified by PCR and then sequenced using capillary electrophoresis
• In CML, the Ph chromosome is present not only in leukemic cells of the myeloid lineage but also in cells of erythroid, B lymphoid and megakaryocytic lineages reflecting, at the molecular level, a stem cell origin for the presence of BCR-ABL translocation
• BCR-ABL1 is found in all myeloid lineages and some lymphoid cells and endothelial cells
• Secondary cytogenetic changes:
• 60-80% of cases develop additional chromosome aberrations when disease progression occurs
• May precede hematologic and clinical manifestations of a more malignant disease by several months
• May serve as valuable prognostic markers
• The treatment given during the CP influences the pattern of secondary genetic changes observed
• Changes occurring during imatinib or other TKI therapy have uncertain prognostic significance
• they may be transient, or they may become the dominant clone
• Occasional CML cases harbor such changes early in the disease
• i(17)(q10)
• +Ph
• +8
• +19
• Less common (< 10% of cases with additional changes):
• -Y
• +21
• -7
• +17
• -17
• Several balanced rearrangements typically seen in AML have been observed in disease progression of CML
• Should probably be considered as second primary changes rather than as “ordinary” secondary changes
• Clonal cytogenetic changes in Ph-negative cells:
• Most common:
• +8
• -7
• -5
• -Y
• Concerns have been raised about MDS or AML developing in imatinib-treated patients (2-10%)
• Current recommendations suggest that such findings should not lead to any immediate treatment interventions in the absence of morphologic evidence of MDS/AML
• Possible exception of -7
• BCR-ABL1 KD mutations:
• associated with an increased likelihood of subsequent disease progression in patients with TKI-treated CML
• ELN and NCCN guidelines nevertheless specifically recommend a switch to certain TKI agents when particular mutations are detected
• A significant majority of BCR-ABL1 KD mutations cluster to one of four hot spots: the ATP-binding P-loop (amino acids 248 to 256); the imatinib-binding region (amino acids 315 to 317); the catalytic domain (amino acids 350 to 363); and the activation (A)-loop (amino acids 381 to 402)
• the overwhelming majority of clinically significant mutations in ABL kinase domain are found in exon a2
• Differential sensitivity to imatinib, dasatinib, nilotinib, bosutinib, and ponatinib has been demonstrated by these diverse mutant BCR-ABL1 kinases in in vitro studies
• Because there is often, but not always, a good correlation between mutation-specific in vitro resistance and in vivo clinical responses for some, but not all, KD mutations and TKIs, the identification of the specific mutation can help to inform the optimal management strategy

· In particular, the presence of the common T315I mutation suggests that ponatinib, and no other TKI, may be effective. In addition, NCCN and ELN guidelines suggest a switch to nilotinib (not dasatinib) for patients with the V299L, T315A, or F317L/V/I/C mutations; and a switch to dasatinib (not nilotinib) for patients with the Y253H, E255K/V, or F359V/C/I mutations

§ Aside from point mutations, the BCR-ABL1 KD also commonly develops insertion/deletion mutations, including a 35-bp intronic insertion at the exon 8 to 9 junction, an L248V mutation with deletion of 81 bp of exon 4, an exon 7 deletion, and several others

· Although the clinical and drug resistance significance of most of these insertiondeletion mutations is still unclear, the very common 35-bp intronic insertion after exon 8 does not appear to mediate TKI resistance, in vitro or in vivo

§ The BCR-ABL1 KD also carries some common single nucleotide polymorphisms that appear to be wholly benign, including three nonsynonymous (K247R, F311V, Y320C), and three synonymous (T240T, T315T, E499E) variants, each of which has no known effect on TKI binding or drug resistance

§

Other features:

• Natural history is bi- or tri-phasic
• Indolent chronic phase (CP)
• Accelerated phase (AP)
• Blast phase (BP)
• Median survival 2-3 y. prior to effective therapy
• Prognostic variables:
• Most important is response to treatment at the haematologic, cytogenetic, and molecular level
• Age
• Spleen size
• Platelet count
• % myeloblasts in PB
• % basophils and eosinophils in the PB
• Myelofibrosis
• Prognosis with PTKI:
• CR rate to imatinib is 70-90%
• 5 y. PFS and OS between 80-95%
• may transform to AML or ALL

References:

• Swerdlow. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue. 4th ed. WHO Publications; 2008.
• Heim S, Mitelman F. Cancer Cytogenetics. 3rd ed. Wiley-Blackwell; 2009.

o Press RD, Kamel-Reid S, Ang D. BCR-ABL1 RT-qPCR for Monitoring the Molecular Response to Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia. J Molecular Diagnostics2013;15(5):565-576.

o Foroni et al. Technical aspects and clinical applications of measuring BCR-ABL1 transcripts number in chronic myeloid leukemia. Am J Hematol 2009;84:517-522.

o Gabert J, Beillard E, van der Velden VH, et al. Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia—A Europe against cancer program. Leukemia 2003;17:2318–2357

• Cross NCP et al. Laboratory recommendations for scoring deep molecular responses following treatment for CML. Leukemia 2015;29:999-1003.
• Luu MH, Press RD. BCR-ABL PCR testing in chronic myelogenous leukemia: molecular diagnosis for targeted cancer therapy and monitoring. Expert Review of Molecular Diagnostics 13.7 (Sept 2013):749.