At a Glance

Chronic myelogenous leukemia (CML) accounts for 15% of all adult leukemias, with an estimated 4,870 new cases in the United States in 2010. The median age of onset is 67 years of age, although CML can occur in all age groups, including children.

CML is an acquired hematologic stem cell malignancy that falls in the general category of myeloproliferative neoplasms. CML is characterized by the reciprocal translocation t(9;22)(q34;q11), the altered chromosome 22, of which is termed the Philadelphia chromosome. This translocation produces a fusion gene, BCR-ABL1, which is pathognomonic for CML and results in deregulation and constitutive activation of the ABL1 kinase domain. The activity of this kinase creates an abnormal proliferation of myeloid precursor cells in the bone marrow, which is manifest in the peripheral blood as a leukocytosis with a disproportionate number of intermediate myeloid precursors.

Patients are also anemic, but may have a normal or even increased platelet count. Nearly 20-40% of patients are asymptomatic at presentation, and the diagnosis is only pursued after an abnormal leukocytosis is incidentally noted on complete blood count (CBC). However, signs of fatigue, splenomegaly, weight loss, or night sweats may bring a patient to clinical attention as well.

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Three phases of disease can be seen in CML. Approximately 80% of patients are diagnosed in chronic phase. Only a minority are diagnosed in accelerated phase or blast crisis, when the disease transforms to an acute myeloid leukemia (2/3 of cases) or an acute lymphoblastic leukemia (1/3 of cases).

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

Diagnosis is confirmed by the identification of a t(9;22), by metaphase cytogenetics, fluorescent in situ hybridization (FISH), or molecular studies (quantitative or qualitative reverse transcription polymerase chain reaction; RT-PCR).

A typical work-up is initiated by the findings of an atypical leukocytosis with a predominance of myeloid cells at all stages of maturation, with a characteristic “bulge” or relative increase in the number of intermediate myeloid forms (myelocytes and metamyelocytes). This peripheral blood finding is often associated with a basophilia, eosinophilia, anemia, and, occasionally, a thrombocytosis. Unlike an accelerated phase or blast crisis, in chronic phase CML, blasts are typically not increased in the peripheral blood or marrow.

These findings should prompt a complete history and physical exam, including assessment of spleen size by palpation (cm below the costal margin). A complete chemistry profile should also be obtained.

Establishment of the presence of the t(9;22) is required to make the diagnosis of CML. Testing for t(9;22) should be performed on the bone marrow whenever possible. Only if a bone marrow biopsy cannot be obtained (or may not be obtained for a long time) should peripheral blood be tested for the translocation. Although metaphase cytogenetics, fluorescence in situ hybridization (FISH), or RQ-PCR may be used to establish the presence of the t(9;22) in this situation, bone marrow cytogenetics and RQ-PCR are preferred.

A bone marrow biopsy, with appropriate ancillary testing of the bone marrow aspirate, is required to stage and baseline the disease. Although most patients are diagnosed in chronic phase, many can harbor more advanced disease in their marrows than what is evident in the peripheral blood, so a bone marrow biopsy is necessary to definitely assign the stage of disease. More advanced disease is associated with marrow findings of an increased blast or basophil percentage, marrow fibrosis, and atypical megakaryocytic aggregates.

In addition, certain cytogenetic changes, such as duplication of the Philadelphia chromosome, isochromosome 17, trisomy 8, are also harbingers of accelerated disease and are required for staging. Lastly, a baseline RQ-PCR evaluation of the BCR-ABL1 fusion transcript levels is required. This should be performed on both the peripheral blood and bone marrow, since both of these sample types will be monitored during tyrosine kinase inhibitor (TKI) therapy and the 2 samples are not interchangeable.

In the current testing guidelines published by the National Comprehensive Cancer Network (NCCN), older tests, such as leukocyte alkaline phosphatase enzyme activity scoring, are no longer recommended.

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

There are 3 common translocation breakpoints in the BCR gene when it rearranges to fuse with ABL1. These are the major breakpoint region (MBR, leading to the most common protein product of 210 kD found in almost all cases of CML, the p210 product, produced by either the b2a2 (e13a2) or b3a2 (e14a2) gene fusions), minor cluster region (mcr, p190, produced by the e1a2 fusion, found more commonly in Philadelphia chromosome positive precursor B lymphoblastic leukemia and in rare CMLs with monocytic features), and µ breakpoint region (µ-BCR, p230, produced by the e19a2 fusion, found in an uncommon subtype of CML with neutrophilia). The rearrangements can be identified by metaphase cytogenetics, FISH, or RQ-PCR. Because of differences in clinical and analytical sensitivity, these 3 assays differ in their ability to detect low levels of disease or the less common breakpoints.

Metaphase cytogenetics is a global view of the chromosomal changes at a resolution of 5-10 Mb. Thus, although it recognizes all 3 breakpoint regions, it cannot distinguish between them. In addition, since cytogenetic studies typically examine only 20-30 metaphases, the analytical sensitivity is low. Metaphase cytogenetics is required to identify clonal evolution, such as in accelerated phase, however.

FISH can typically detect all 3 breakpoint regions, but may not be able to distinguish between all of them, depending on the type of FISH probe. Some do not differentiate between the breakpoints, whereas others are designated specifically to distinguish between the p210/p230 and the p190. Typically, FISH examines 200 cells and is considered a semi-quantitative method with only moderate analytical sensitivity.

RQ-PCR, conversely, is very sensitive, examining 105-107 cells, depending on the laboratory assay. Of the 3 methods, it is the only truly quantitative assay. However, since primers are designated specific to each breakpoint, 3 separate assays are required to interrogate all 3 possible translocations. Most laboratories have developed only the assay for the fusion translocation leading to the most common p210 product. Thus, at diagnosis, RQ-PCR may give a false negative if the less common translocations are involved.

Other factors that may affect laboratory results include the adequacy of the sample, especially if an inadequate bone marrow biopsy is performed that does not reflect the bone marrow contents. Although the peripheral blood will contain the t(9;22), the metaphase cytogenetic studies or blast counts may not reflect the marrow, preventing the appropriate diagnosis of accelerated phase or blast crisis.

Improper sample handling may affect the RQ-PCR reaction, since this is an RNA-based assay and requires specific handling procedures to prevent the degradation of the RNA by RNases, which are nearly ubiquitous.

What Lab Results Are Absolutely Confirmatory?

Establishment of the presence of the t(9;22) is required to make the diagnosis of CML. Again, this is ideally performed by bone marrow cytogenetics, as well as quantitated by RQ-PCR on both the bone marrow and peripheral blood at diagnosis.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

Patients with CML should be monitored by a hematologist. At this time, first line therapy for CML is a tyrosine kinase inhibitor (TKI), such as imatinib. Second line therapies include the later generation TKIs, such as dasatinib and nilotinib. BCR-ABL1 levels are checked at 3-month intervals by peripheral blood RQ-PCR after diagnosis, and bone marrow metaphase cytogenetic studies and bone marrow RQ-PCR are performed at 6-month intervals until the patient achieves a complete cytogenetic remission. Following that time, peripheral blood RQ-PCR alone can be used to monitor the patient with bone marrow studies as clinically needed. A 3-log reduction in RQ-PCR levels within a 12-month period is prognostically significant, with excellent long-term survival in those patients.

Should the patient develop resistance to 1 of the TKIs, especially imatinib, additional testing may be required, such as allele-specific testing for common mutations, such as the T315I mutation in the ABL1 kinase active site that prevent binding by the inhibitor, or sequencing of the ABL1 gene for other mutations.

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

FISH is only appropriate at diagnosis if a bone marrow biopsy cannot be obtained. Since bone marrow metaphase cytogenetic studies and RQ-PCR are required at diagnosis to stage the disease and for the establishment of a RQ-PCR baseline, FISH is typically an unnecessary and not cost-effective way to make the diagnosis, unless a bone marrow biopsy cannot be obtained.

Since patients are followed by a hematologist at regular intervals following diagnosis by RQ-PCR and bone marrow cytogenetics, at no time after diagnosis is FISH recommended by the current NCCN guidelines.

It is important to note that different laboratories use different methods for RQ-PCR of BCR-ABL1. Unfortunately, this assay is very sensitive to the reference gene used in the assay, as well as other factors, preventing comparison of results from different laboratories and different sample types (blood vs. bone marrow). Like the prothrombin time (PT) coagulation study, the results, therefore, vary tremendously from lab to lab. However, there currently is no widespread use of the proposed international scale (IS) for BCR-ABL, as is the case with the international normalized ratio (INR) for PT assays, that allows the standardization of these results. Thus, when a patient transfers to a different institution for his or her care, rebaselining of the RQ-PCR is recommended and comparison for measurement of a 3-log reduction in transcript levels should be done with the initial diagnostic laboratory.