CML is a hematopoietic stem cell disease always observed with t(9;22) that results in a novel fusion of the BCR (22q11.2) and ABL1 (9q34) genes. The chimeric BCR–ABL1 protein is a constitutively activated TK, and leads to autophosphorylation of the oncoprotein (ABL1) that can activate downstream signaling pathways including RAS, RAF, JNK, MYC, JAK/STAT, and nuclear factor-κB during cell proliferation, differentiation and survival.¹

CML serves as the best model for our understanding of the mechanisms of genetic abnormalities in leukemogenesis.³ CML was the first cancer to be associated with a recurring chromosome abnormality. In 1960, Nowell and Hungerford⁴ identified a consistently small G-group chromosome in leukemia cells in CML, later called the Philadelphia (Ph) chromosome after the place where it was discovered.⁴ Using novel chromosome banding techniques, Rowley⁵ demonstrated in 1973 that the Ph chromosome was actually a translocation between chromosomes 9 and 22, i.e., t(9;22), which was the second recurring chromosome translocation identified in cancer shortly after the t(8;21) was discovered in acute myeloid leukemia, also by Rowley in the same year.⁵ CML was also the first disease in which a molecular rearrangement was recognized as resulting in a novel fusion gene and a chimeric protein that was fundamental to leukemogenesis. In 1982, de Klein⁶ elucidated that the ABL1 gene at 9q34 was relocated to the Philadelphia chromosome in the t(9;22). In 1984 and 1985, Heisterkamp and associates⁷ and Groffen and colleagues⁸ identified the “breakpoint cluster region” (BCR) on the derivative chromosome 22, and later Shtivelman and co-workers⁹ and Grosveld and colleagues¹⁰ revealed that the BCR and ABL1 genes were fused together to give rise to a novel chimeric mRNA and protein in CML with t(9;22). Furthermore, CML serves as the first cancer with a rationally designed drug treatment that directly targets the molecular consequence responsible for the pathogenesis of the disease. By 1996, Druker and colleagues¹¹ were able to identify a TK inhibitor Imatinib that specifically bound to the fusion BCR/ABL1 protein and inhibited the constitutively active ABL1 gene function. Since then, many other novel chromosome abnormalities in leukemia, lymphoma, sarcoma, and recently in epithelial cancer have been discovered, and other genes and protein-specific drugs are used in patient treatment and clinical trials. Therefore, CML is a valid research model in cancer stem cell biology, normal and abnormal hematopoiesis and lineage commitment, disease progression, and in targeted drug development and therapy.³

The standard t(9;22) can be detected by conventional cytogenetic analysis in bone marrow or peripheral blood samples in more than 90% of CML patients. Presumably occurring in a stem cell, the t(9;22) is detectable in all myeloid lineage cells, including granulocytes, monocytes, erythroid precursors, megakaryocytes, and in all B and some T lymphocytes. At the time of diagnosis, the t(9;22) is usually seen in all metaphase cells analyzed from 24- or 48-hour cultures, indicative of a high mitotic index. In the remaining 2% to 10% of patients with CML, a variant of the t(9;22), involving a third (or more) chromosome can be observed. Either the chromosome 9 or 22 may not exhibit the usual abnormal banding pattern; rather, they may show a 3-way inter-chromosome translocation involving 9q34 (ABL1) and 22q11.2 (BCR), and a third chromosome. Certain chromosome bands are commonly involved in variant t(9;22), such as 1p36.1, 3p21, 11q13, 12p13, and 17q25, although all chromosomes have been observed in variant translocations.¹² Careful analysis will enable the identification of a 3- or 4-way translocation involving both chromosomes 9 and 22.

In a few cases, the t(9;22) is cryptic such that conventional cytogenetic analysis does not reveal any apparent chromosomal changes at 9q34 or 22q11.2. Instead, insertion of the BCR gene into the ABL1 gene or vice versa has been identified. FISH analysis or polymerase chain reaction (PCR) studies would be needed to detect the BCR/ABL1 fusion. However, in all patients, the BCR-ABL1 fusion, owing to the t(9;22), is the hallmark of CML as recognized by the 2008 World Health Organization classification of hematopoietic and lymphoid neoplasms.² The t(9;22) is also one of the recurring chromosome abnormalities in acute lymphoblastic leukemia (ALL), in particular in adult patients; the consequence of the t(9;22) is the chimeric mRNA and protein with the fusion between the 3′ end of the first exon of the BCR gene and the 5′ terminus of the second exon of the ABL1 gene on the derivative chromosome 22 (Ph chromosome).¹³ The breakpoints in ABL1 are always proximal to the second exon in both CML and ALL, whereas the breakpoints in BCR are variably different between CML and ALL. In CML, the breakpoints in BCR are more telomeric in the major breakpoint cluster region (M-bcr, between exons 12 and 15) or micro-bcr region (between exons 19 and 21), giving rise to the P210 and P230 fusions, respectively. In contrast, the breakpoints in the BCR gene in ALL are far more proximal to exon 2 in a minor breakpoint cluster region (m-bcr), producing the P190 fusion. The location of the breakpoints and the amount of the BCR gene that is included in the BCR/ABL1 fusion determines whether the leukemia will be CML or ALL, both showing a similar but not identical BCR/ABL1 fusion protein.¹³

The t(9;22) or its variants are detectable as a single cytogenetic abnormality in up to 90% of CML patients in the chronic phase at the time of diagnosis. In some patients, other chromosome aberrations are observed in addition to the t(9;22). The most common ones are loss of the Y chromosome, gain of chromosome 8, and a second Philadelphia chromosome, and isochromosome 17, that is, i(17q). Loss of the Y chromosome is generally related to increasing age (>70 years), and seems to have no effect on the disease course or phenotypic features. Notably, the remaining additional chromosome aberrations, namely, +8, +Ph, and i(17q), are also frequent in CML during the accelerated and blast phases. Thus, it is reasonable that these patients with t(9;22) and additional chromosome aberrations do poorly in comparison with those with t(9;22) as the sole abnormality. In fact, it has been demonstrated that such additional abnormalities indeed are associated with poor survival, and a higher risk of relapse.¹⁴ Particularly, the i(17q) is a predictor for poor cytogenetic and clinical response. It remains to be seen if the negative association of additional chromosome aberrations with poor prognosis in CML can be overcome by imatinib treatment.

The chronic phase of CML usually lasts about 3 to 5 years, and then evolves to an advanced stage. The disease evolution is accompanied in more than 60% of patients with additional chromosome aberrations, which can be reliably identified by cytogenetic analysis in many cases.¹² The most common additions are gain of chromosome 8 (33%), followed by an additional Ph chromosome (30%), i(17q) (20%), and +19 (12%); loss of the Y chromosome (8% in males), trisomy 21 (7%) and loss of chromosome 7 (5%). They may occur individually or in combination. In certain situations, the appearance of additional abnormalities, such as +8 and i(17q), is a strong indicator of an occult disease progression, often several months earlier than morphologic evidence on bone marrow examination or clinical symptoms.

The blast phase is about two thirds in myeloid and one third in lymphoid lineages. In addition to those common karyotypic evolutions described above, additional chromosome aberrations, specific for acute myeloid leukemia, can be observed such as t(8;21), and inv(16) in CML in myeloid blasts with abnormal eosinophilia, or even t(15;17) in CML with acute promyelocytic blasts. The t(3;21) and inv(3q)/t(3;3) that result in the overexpression of the EVI1 gene at 3q26.2 are also one of the recurring abnormalities in CML in transformation.

Fluorescence in situ hybridization (FISH) technique is a molecular cytogenetic tool that complements conventional chromosome analysis. Because of its high sensitivity and easy use in both metaphase and interphase cells, FISH can be used to rapidly confirm the BCR/ABL1 fusion in diagnosis and in disease follow-up and in monitoring treatment response. FISH is also very helpful in clarifying a variant, cryptic, or complex karyotype in CML patients. At the time of diagnosis of CML, FISH can be used in parallel with cytogenetic analysis to confirm the t(9;22) and determine fusion signal patterns in bone marrow or peripheral blood samples. It is particularly important if the sample was suboptimal, or there were only few dividing cells in the cultured cells. FISH is also a very sensitive tool in monitoring treatment response and disease course by comparing the percentages of the BCR/ABL1 fusion positive leukemia cells because 200 or more interphase cells are examined as compared with 20 metaphase cells in conventional cytogenetic analysis.

There are several commercial FISH probes available from various vendors. A dual fusion probe set is preferred with large genomic bacterial artificial chromosome probes covering the entire or a part of the genomic breakpoint regions of the BCR and ABL1 genes labeled in different colors. It has a very low cutoff value (around 0%–1%) for a positive result and, thus, is particularly useful in monitoring the treatment response. In certain situations, a BCR–ABL1 fusion probe with an extra signal pattern is helpful in distinguishing the major and minor breakpoints in the BCR gene. Another probe strategy is to use an internal control probe on chromosome 9q34 to detect a partial deletion of the ABL1–BCR reciprocal fusion on the derivative chromosome 9 (see below).

A combined conventional cytogenetics and FISH analysis is important in CML for diagnosis. Although it is at a low-resolution level, chromosome analysis provides a complete picture of all metaphase chromosome changes. It is particularly helpful to identify a variant of t(9;22) and clonal evolution, which is informative of disease status. FISH not only confirms the t(9;22) but it establishes a reference FISH signal pattern as well for monitoring disease status and treatment response. FISH on metaphase cells helps to identify complex karyotypes and clarify unusual chromosome aberrations, such as 3-way translocations of the t(9;22), a cryptic translocation, or a partial deletion of the genomic fusion on the derivative chromosome 9. In most patients, the percentages of leukemia cells with t(9;22) is comparable in both tests, with a little bit lower percentage seen in the FISH analysis.^15,16 Once the FISH pattern has been identified at diagnosis, FISH can be used to monitor treatment response in bone marrow or peripheral blood samples at appropriate intervals, without the need to perform standard cytogenetic analysis. However, before the patient enters hematologic and cytogenetic remission, karyotyping should be done every 3 to 6 months to avoid missing additional chromosome aberrations associated with blast crisis.