Chronic Myeloid Leukemia Tests

Chronic myeloid leukemia (CML) is a myeloproliferative disorder comprising approximately 15-20% of adult leukemias. Approximately 150,000 people in the United States and 5 million worldwide have CML. CML typically affects adults and is rare in children. 

CML results from clonal expansion of a malignant hematopoietic stem cell carrying the Philadelphia chromosome (Ph). The karyotype of Ph is t(9:22)(q34;q11). The Ph chromosome is produced by a reciprocal translocation between chromosomes 9 and 22 that fuses the  5' exons of the breakpoint cluster region gene (BCR) from chromosome 22 with the Abelson gene (ABL1) gene exons from chromosome 9. The resulting BCR-ABL1 oncogene codes for an abnormal tyrosine kinase enzyme that leads to decreased apoptosis and accumulation of CML cells that possess a survival advantage. 

Although various breakpoints within the BCR and ABL1 genes have been described, more than 95% of CMLs undergo a translocation in which either the BCR exon 13 (e13) or BCR exon 14 (e14) is fused to the ABL1 exon 2 (a2). The resulting fused oncogenes are called e13/a2 and e14/a2, respectively. They are transcribed into p210 mRNA, which is translated into 210-kDa protein (p210), that is an abnormal tyrosine kinase. 

About 1% of patients with CML have an e1/a2 and e1/a3 fusions that are transcribed into p190 mRNA and translated into a 190 kDa protein. This BCR/ABL1 protein form is found in approximately 75% of patients with childhood acute lymphoblastic leukemia (ALL) and approximately 50% of patients with adult ALL. The p190 is also the predominant fusion form in approximately 1% of patients with CML.

All patients with suspected CML should undergo cytogenetic, FISH and PCR testing at the time of initial evaluation and diagnosis. The Ph chromosome can be detected in 90 to 95% of patients with CML by conventional metaphase cytogenetic examination. Cytogenetics requires culture and harvesting of bone marrow cells in metaphase (dividing cells) for the detection and enumeration of Ph chromosome positive cells. Cytogenetic demonstration of the Ph chromosome is an important feature in the differential diagnosis of CML. The Ph chromosome is also present in approximately 20% of adults and 5% of children with acute lymphoblastic leukemia and 2% of adults with acute myeloid leukemia. The presence of the Philadelphia chromosome in acute leukemia connotes a poorer prognosis.

The major advantages cytogenetic analysis include high specificity and detection of additional cytogenetic abnormalities, if present. Disadvantages include low sensitivity (90%), longer turnaround time, and higher cost. Cytogenetic analysis generally must be performed on bone marrow rather than peripheral blood, because it requires proliferating cells. At least 20 metaphase preparations must be examined to gauge the cytogenetic response to TKI.

Fluorescence in situ hybridization (FISH) uses differently labeled fluorescent DNA probes that specifically bind to regions in BCR or ABL. Presence of a translocation results in fusion of these probes and change in color. Metaphase and interphase (nondividing) cells can be used, and thus FISH can be done on cells derived from bone marrow or peripheral blood. FISH is more sensitive than cytogenetic analysis but less sensitive than PCR. New generation probes have significantly improved the lower limit of detection from 10% to below 0.5% abnormal cells. At diagnosis, FISH is not a substitute for cytogenetic analysis, because FISH can detect the Philadelphia chromosome but not other clonal chromosomal changes that suggest advanced-phase disease. FISH should be used as a diagnostic tool only when bone marrow cannot be obtained. The major disadvantage is technical failure including loss of cells during processing. Advantages include faster turnaround time, higher sensitivity, and the ability to detect rare variants of BCR-ABL and masked break points that may be missed by cytogenetic testing.

Qualitative reverse transcriptase-polymerase chain reaction (RT-PCR) testing uses mRNA extracted from either peripheral blood or bone marrow. Disadvantages include the inability to detect rare variants, lower specificity due to RNA cross-contamination, and false positivity. Advantages are quick turnaround time and the ability to test either bone marrow or peripheral blood. 

Quantitative RT-PCR is specifically designed to quantify BCR-ABL mRNA for monitoring disease progression. Commercially available assays use real time RT-PCR.  Quantitative PCR can generally detect 1 CML cell in approximately 100,000 normal cells. A major benefit of quantitative PCR is that peripheral blood can be used rather than bone marrow, making monitoring less invasive.  The quantity of BCR-ABL mRNA is standardized against a reference housekeeping gene such as ABL. A common measure of relative CML response is the magnitude of reduction in BCR-ABL transcripts from a standardized baseline value, and the major molecular response (MMR) is defined as a 3-log or greater reduction in the BCR-ABL/control gene ratio. The major problem with quantitative PCR for BCR-ABL is a lack of standardization. Different laboratories use different control reference genes, and have different BCR-ABL/control gene ratios.

In summary, either karyotyping and FISH or qualitative RT-PCR can be used for diagnosis of CML. However, FISH is preferred due to its optimal sensitivity and ability to detect variants. For post-therapy monitoring, quantitative RT-PCR is the most sensitive test available. 

References

Jabbour E, Kantarijan, Chronic Myeloid Leukemia: A Review, JAMA, 2025;333(18):1618-1629.

Burmeister T, Reinhardt R. A multiplex PCR for improved detection of typical and atypical BCR-ABL fusion transcripts. Leuk Res. 2008;32(4):579-585.