Mutation detection in Chronic Myeloid Leukaemia (CML)

Introduction: Chronic myeloid leukaemia (CML) is a type of blood cancer that is caused due to the reciprocal translocation of genetic material between chromosomes 9 and 22. A piece of the long arm of chromosome 9 containing an ABL gene breaks off and fuses with the long arm of chromosome 22 containing the BCR gene. It results in a fused gene called BCR-ABL1 and the abnormal chromosome 22 called the Philadelphia chromosome. CML has three phases:

1. 1. Chronic Phase
2. 2. Accelerated Phase and
3. 3. Blast Phase

1. Chronic Phase:

Chronic is the first phase of CML (CP-CML). In this phase, progression to the next phase is prolonged. The next phase could take months or even years to develop. And this is the period that responds well to treatment (“NCCN Guidelines for Patients Chronic Myeloid Leukemia,” 2021).

2. Accelerated Phase:

The accelerated phase is the second stage of CML (AP-CML). CML cells proliferate fast during the accelerated phase. Philadelphia chromosome (Ph+) is present in all stages of CML, but in the accelerated phase, Ph+ cells may undergo new mutations (“NCCN Guidelines for Patients Chronic Myeloid Leukemia,” 2021).

3. Blast Phase:

The blast phase is the third and final stage of CML (BP-CML). It is also called a blast crisis. On reaching this stage, CML is life-threatening and difficult to treat. Therefore, the primary focus of the treatment is to prevent the blast phase. In this stage, the number of cells containing the Philadelphia chromosome is relatively high (3 of every 10 cells) (“NCCN Guidelines for Patients Chronic Myeloid Leukemia,” 2021)

Laboratory diagnosis of CML

Blood Tests: Measuring the complete blood count (CBC) is the first line for detection for CML. CML patients have many white blood cells, but high levels of white blood cells may also be due to some other illnesses than leukaemia. Therefore, more advanced tests are advised to confirm the disease condition (“NCCN Guidelines for Patients Chronic Myeloid Leukemia,” 2021).

Cytogenetics: Cytogenetical analysis (Karyotyping) allows the examination of chromosomes under the microscope for any abnormality such as the Philadelphia chromosome (Ph+). The presence of the Philadelphia chromosome (Ph+) in the patient’s blood/bone marrow sample confirms the diagnosis of CML (“NCCN Guidelines for Patients Chronic Myeloid Leukemia,” 2021). Some people with all clinical symptoms of CML do not have cytogenetically detectable Ph chromosomes. Hence, they are prescribed high sensitivity tests like qPCR.

Fluorescence In Situ Hybridization (FISH): FISH is another method that examines genes and chromosomes in the cell under a microscope. It is more sensitive than karyotyping method of chromosome analysis. BCR and ABL1 genes are labelled with different coloured probes. FISH identifies the BCR and ABL1 genes in chromosomes using colour probes that bind to DNA. FISH detects the fragment of chromosome 9 that has shifted to chromosome 22 in CML cells. The overlapping colours of the two probes show the BCR-ABL1 fusion gene.
FISH is recommended as the first-line test along with Karyotyping before proceeding to molecular methods as FISH & Karyotyping can help identify the source of chromosomal rearrangements (“NCCN Guidelines for Patients Chronic Myeloid Leukemia,” 2021).

Molecular testing: The molecular method for testing CML is through quantitative polymerase chain reaction (qPCR), which is relatively more sensitive. It is a DNA based test that detects and measures the number of BCR-ABL1 genes in blood or bone marrow samples. The number of cells containing the BCR-ABL1 gene is compared with the international Scale (IS). This test has a very low limit of detection where even traces of the BCR-ABL1 gene can be identified with high accuracy. It can identify a single abnormal cell in approximately 1 million healthy cells. This test is also used to track how well a patient responds to treatment (Qin and Huang, 2016).

Mutation Detection: If treatment resistance or disease recurrence occurs, the BCR-ABL1 kinase domain mutation analysis is to be performed to guide further treatment. This detection is done by gene sequencing. BCR-ABL1 kinase domain mutation testing is done in those patients where the tyrosine kinase inhibitor therapy does not work (Chaitanya et al., 2017; Chandrasekhar et al., 2019).

References:
Chaitanya, P.K., Kumar, K.A., Stalin, B., Sadashivudu, G., Srinivas, M.L., 2017. The Role of Mutation Testing in Patients with Chronic Myeloid Leukemia in Chronic Phase after Imatinib Failure and Their Outcomes after Treatment Modification: Single‑institutional Experience Over 13 Years. Indian Journal of Medical and Paediatric Oncology 38, 328–333. https://doi.org/10.4103/ijmpo.ijmpo_115_17 Chandrasekhar, C., Kumar, P.S., Sarma, P.V.G.K., 2019. Novel mutations in the kinase domain of the BCR-ABL gene causing imatinib resistance in chronic myeloid leukaemia patients. Sci Rep 9, 2412. https://doi.org/10.1038/s41598-019-38672-x NCCN Guidelines for Patients Chronic Myeloid Leukemia, 2021. . Chronic Myeloid Leukemia 72. Qin, Y.-Z., Huang, X.-J., 2016. Molecular Detection of BCR-ABL in Chronic Myeloid Leukemia, in: Li, S., Zhang, H. (Eds.), Chronic Myeloid Leukemia, Methods in Molecular Biology. Springer New York, New York, NY, pp. 1–15. https://doi.org/10.1007/978-1-4939-4011-0_1