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Chronic Myeloid Leukemia Tests

Chronic myeloid leukemia (CML) is a myeloproliferative disorder comprising approximately 15-20% of adult leukemias. CML results from clonal expansion of a malignant hematopoietic stem cell carrying the Philadelphia chromosome (Ph). Ph chromosome is produced by a reciprocal translocation between chromosomes 9 and 22 that fuses the 5' exons of the BCR gene from chromosome 22 with ABL gene exons from chromosome 9. The resulting BCR-ABL gene codes for an abnormal tyrosine kinase enzyme that is responsible for white blood cell proliferation. It also is a therapeutic target for tyrosine kinase inhibitor (TKI) drugs.

Break-points in ABL occur in the first and second introns. Break-points in BCR occur in one of three regions: major break-point cluster region (M-bcr) involving exons b2 or b3, minor break-point region (m-bcr) involving exon e1 or micro break-point region (u-bcr) involving exon e19. Because of these different BCR break-points distinct fusion mRNAs of variable length including p190, p210 or p230 are generated.Most CML patients have p210 mRNA. High levels of p190 mRNA are primarily detected in acute lymphoblastic leukemia or the blast phase of CML. Low levels of p190 mRNA have been recently reported in chronic-phase CML.Historically, diagnosis and monitoring of CML required cytogenetic studies to detect the Ph chromosome. Newer and more sensitive techniques are now available for diagnosis and post therapy monitoring. A brief overview of the various techniques available for detection of these fusion mRNAs follows.

Cytogenetic testing requires culture and harvesting of bone marrow cells in metaphase (dividing cells) for diagnosis and enumeration of Ph chromosome positive cells. Conventional metaphase cytogenetic examination is still the gold standard of early TKI response, and cytogenetic analysis has the strongest prognostic value. Advantages 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 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.