Chronic Myelogenous Leukemia (CML): Laboratory Diagnosis from the past to the future
Chronic myelogenous (or myeloid) leukemia (CML), also known as chronic granulocytic leukemia (CGL), is a form of leukemia characterized by the increased and unregulated growth of predominantly myeloid cells in the bone marow and the accumulation of these cells in the blood. CML is a bone marrow stem cell disorder in which proliferation of mature granulocytes ( neutrophills, eosinophils, and basophils) and their precursors is the main finding. It is a type of myeloproliferative disease associated with a characteristic chromosomal translocation called the Philadephia chromosome.
Laboratory diagnosis of CML is often suspected on the basis on the complete blood count, which shows increased granulocytes of all types, typically including mature myeloid cells. Basophils and eosinophils are almost universally increased; this feature may help differentiate CML from a leukemoid reaction. A bone marrow biopsy is often performed as part of the evaluation for CML, but bone marrow morphology alone is insufficient to diagnose CML. Ultimately, CML is diagnosed by detecting thePhiladephia chromosome. This characteristic chromosomal abnormality can be detected by routine cytogenetics, by fluorescent in situ hybridization, or by PCR for the bcr-abl fusion gene.
WHO Criteria for diagnosis of accelerated and blastic phase
The diagnosis of accelerated phase CML may be made when one or more of the following are present:
1.Blasts 10-19% of WBCs in peripheral blood and/or of nucleated bone marrow cells
2.Peripheral blood basophils > or = to 20%
3.Persistent thrombocytopaenia (<100 x 10/9/L) unrelated to therapy or persistent thrombocytosis (>1000 x 10/9/l) unresponsive to therapy.
4.Increasing spleen size and increasing WBC unresponsive to therapy.
5.Cytogenetic evidence of clonal evolution.
6.Blast phase may be diagnosed if one or more of the following are present:
Blasts > or = to 20%
Extramedullary blast proliferation
Large foci or clusters of blasts in the bone marrow biopsy
Laboratory testing for diagnosis of CML
1.Blood cell counts and blood cell examination
The complete blood count (CBC) is a test that measures the levels of different cells, such as red blood cells, white blood cells, and platelets, in the blood. The leukocytosis rang from 20.00/µl to more than 500,000/µl,with a mean range of 134,000 to 225,000/µl in most studies. The CBC often includes a differential (diff), which is a count of the different types of white blood cells in the blood sample. In a blood smear, the most important find a neutrophilic leukocytosis, with all stages of neutrophilic maturation represented, from myeloblast to segmented neutrophil and basophilia. An absolute basophilia is invariably present and of critical importance. There may be an eosinophilia as well, but its presence does not carry the diagnostic significance of the hybrid cells with mixed basophil-eosinophil granulation or mixed basophilic-mast cell granulation are found. The marked leukocytosis in cases of CML typically is associated with an absolute monocytosis but relative monocytopenia. The platlets may vary in appearance.Most patients have a normochromic normocytic anemia.Sometimes CML patients have low numbers of red blood cells or blood platelets. Even though these findings may suggest leukemia, this diagnosis usually needs to be confirmed with a bone marrow test.
2. Bone Marrow examination
Marrow examination can be useful in distinguishing CML from other CMPDs and reactive processes. The bone marrow is markedly hypercellular, predominantly because of a proliferation of neutrophilic precursors from myeloblasts to segmented neutrophils . The maturation sequence and morphology at each stage are normal, although the relative increase in myelocytes seen in the peripheral blood is also seen in the bone marrow. Myeloblasts do not usually exceed 5% of the marrow elements. The myeloid precursors usually are located in a periosteal location as seen in normal marrow. Increased numbers of basophils, eosinophils, hybrid cells, and their precursors as seen in the peripheral blood are also present.
Megakaryocytes are typically increased in number and occasionally clustered in groups of three or more in central intertrabecular regions. The megakaryocyte clustering is not as pronounced as it is in ET. The megakaryocytes of CML are slightly smaller than normal megakaryocytes, and occasional micromegakaryocytes are present. Some cases of CML present with a decreased number of megakaryocytes, and some authors propose a subdivision of CML based on the number of megakaryocytes. Common or granulocytic CML has a decreased, normal, or slightly increased number of megakaryocytes, whereas a marked increase in megakaryocytes may be called megakaryocytic CML. The clinical significance of this division has not been demonstrated.
Macrophages with coarse, granular, periodic acid-Schiff (PAS)-positive, cytoplasmic material (pseudo-Gaucher cells) are present in approximately one-third of patients. These inclusions are the result of increased lipid turnover from granulocytic membranes and are of three types:blue birefringent inclusions, the most common(Gaucher-like);blue sea-blue nonbirefringent ,sea-blue histiocytes; and gray-green with birefringent. Iron stores in macrophages as detected by Prussian blue staining are decreased in virtually all cases to amounts lower than in normal subjects.
Erythroid precursors may be present in increased, normal, or decreased numbers, although the myeloid to erythroid ratio is invariably increased. Erythroid precursors may be distributed unevenly as well, with virtually no erythroid cells in some microscopic fields and numerous cells in others.
Deposition of connective tissue as detected by reticulin or PAS stains is not noted in most cases. Nevertheless, in some cases, deposition of connective tissue ranging from an increased number and thickness of fibers to multifocal areas of acellular connective tissue deposition reminiscent of idiopathic myelosclerosis. The deposition is typically around vessels and near megakaryocytes. Connective tissue deposition is associated with larger spleen sizes, increased blast percentages in the peripheral blood, decreased hemoglobin levels, and additional karyotypic abnormalities. As a result, it is not surprising that most studies have indicated that reticulin fiber deposition is associated with a worse prognosis, although a small set of patients with marked fibrosis and early stage CML has been reported to have a prolonged course.
3.Cytochemistry
After cells from the sample are placed on microscope slides, chemical stains (dyes) that react only with certain types of cells are added. The color changes from these stains, which can be seen only under a microscope, can help the doctor determine what types of cells are in the sample. The leucocyte alkaline phosphatase (LAP) score is based on a cytochemistry test that was once often used to test blood samples of patients who were suspected of having CML. Normally the LAP score goes up as the white blood cell (WBC) count goes up. People with CML, however, tend to have high WBC counts with low LAP scores. This test isn’t often used anymore, now that there are ways to test blood for CML
4. Cytogenetic detection
Karyotypic analysis is usually best performed from the bone marrow material, although peripheral blood may be used. The finding of a simple or complex translocation between chromosome 9 and 22, generally the t(9;22)(q34;q11), confirms the diagnosis, and 5 to 10% of the cases have a variant translocation leading to rearrangement of the BCR gene. Patients with variant and classic Ph-producing translocations are clinically and hematologically identical and distinct from Ph(-) cases. Typically, the Ph chromosome remains the sole chromosomal abnormality throughout most of the chronic phase. In a small number of cases with clinical and morphologic features of CML, a t(9;22) or some variant thereof is not identified by karyotypic analysis but may be demonstrated by molecular techniques such as Southern blot or PCR.
The variant Ph chromosomes fall into two subgroups:simple and complex. In simple variant translocations, the segment from 22q is translocated onto a chromosome other than 9. Three or more chromosomes are involved in complex variant translocations. Although the disease appears identical among patients with classic and variant Ph chromosomes, there is controversy as to whether the chromosomal breakpoints and other molecular features are identical.
Although t(9;22) is the hallmark of CML, it is not exclusive to CML. ALL may be accompanied by a t(9;22) in 10 to 20% of adult and in 2 to 5% of childhood cases. In addition, a t(9;22) appears to be found in some bona fide cases od de novo AML as well as in very rare cases of lymphoma and myeloma. Recently, Ph(+) CNL has also been added to this group.
FISH
FISH makes use of differently labeled fluorescent DNA probes. In the first-generation FISH technique, two probes are utilized. One probe, specific for ABL, labeled orange, for example, hybridizes to the 39 end of the ABL breakpoint region. The other probe, specific for BCR, labeled green, for example, hybridizes to the 59 end of the BCR breakpoint. In BCR-ABL translocations, the 39 portion of ABL joins the 59 end of BCR, the orange signal overlies the green signal, and a yellow fusion signal is generated. This technique suffers from low specificity because of the random superimposition of orange and green in normal interphase nuclei. This leads to false-positive results that severely limit the use of first-generation FISH for detection of minimal residual disease. The frequency of false positivity can be 3–10%, making quantification below 10% unreliable.
Recent modifications, however, have greatly improved the specificity of FISH. In one of these technical modifications, two ABL probes are employed. One hybridizes to the 59 side and the other to the 39 side of the usual breakpoints in ABL. In normal cells, these two orange signals are juxtaposed, giving rise to one large orange signal. In the BCR-ABL translocation, these two orange signals are split. The 39 orange fuses with the green on chromosome 22, generating a yellow signal, and the 59 orange probe remains hybridized to chromosome 9. Therefore, in addition to the signals from normal homologs, a truly positive cell carries both a yellow and an orange signal, and a cell with random superimposition carries only a yellow signal. This modification reduces the lower limit of quantification from 9–10% to below 0.5% . Other modifications of the FISH technique with similar specificity have been reported.
FISH detects BCR-ABL in about 95% of CML cases. It is the most sensitive test for diagnosis because it detects the approximately 5% of cases with “masked” translocations that are missed by cytogenetics, and it also detects rare cases with variant breakpoints falling outside the regions covered by PCR primers. In addition, a FISH study routinely analyses 200 to 500 nuclei; thus, quantification generated by FISH is more accurate than cytogenetics, especially when few leukemic cells are present, as is frequently seen posttherapy. In one study correlating cytogenetics and FISH, FISH detected 2.5–8% BCR-ABL-positive cells in seven of nine specimens, from six patients, in which cytogenetic results were negative. Because of the added accuracy and sensitivity, FISH is being used increasingly to replace cytogenetics in monitoring of patients on IFNa and newer biological or chemotherapies.
FISH has several advantages over cytogenetics. The specificity of the newer split signal assay is high. Also, unlike cytogenetics, which requires dividing metaphase cells, FISH can be performed on interphase nuclei in peripheral blood. It therefore may bypass the requirement for a bone marrow specimen. However, the percentage of BCR-ABLpositive nuclei determined by FISH using peripheral blood specimens seems to be lower than that using bone marrow.
5. Molecular Diagnosis and Clinical Correlate
All patients with CML and a demonstrable classic Ph chromosome by cytogenetics have molecular fusion of the BCR and ABL genes. This chromosomal translocation may also be demonstrated by Southern blot analysis, or the transcripted messenger RNA (mRNA) fusion product may be detected by reverse transcriptase PCR (RT-PCR). Although Southern blot analysis and RT-PCR may not detect complex translocations, Southern blot can detect a translocation in a small minority of cases of CML reported as falsely negative using cytogenetic analysis. The clinical and hematologic features of this small cohort of cases that are falsely karyotypically normal but have BCR rearrangement detected by Southern blot are comparable with cases having karyotypically obvious Ph chromosomes. Using both cytogenetic and molecular techniques, a Ph chromosome can be demonstrated in all but approximately 1% of cases. These cases have been called Ph(-) CML or atypical CML by some. However, they probably represent another type of CMPD, so it is not surprising that these behave more aggressively than CML.
RT-PCR detects different length products corresponding to chimeric BCR-ABL proteins of 190 kd, 210 kd, and 230 kd. The breakpoint as detected by RT-PCR may be helpful in distinguishing ALL, CML, AML, and CNL. In the vast majority of cases of CML in adults and in virtually all cases in children, a p210 fusion protein is present. Cases of Ph(+) ALL are associated with the p190 protein, although rare cases of CML and AML with the smaller fusion protein have been reported. A large p230 fusion protein is present in cases of CNL. The p230 transcript has also been reported in cases of CML, but review of these reports suggests that these cases may actually represent CNL.
There are also two types of p210 transcripts: b2a2 and b3a2. Although definitive prognostic differences between these groups are controversial, patients with b3a2 transcripts are likely to have higher platelet counts. In addition, the relative frequency of b2a2 and b3a2 is different in childhood and adult CML, with two-thirds of adults having b3a2 transcripts and the overwhelming majority of children with CML having b2a2 transcripts.
Reference
1. Wang Y. Lynn et al. Chronic Myelogenous Leukemia: Laboratory Diagnosis and Monitoring. Genes, Chromosome & Cancer ,2001,32:97–111
2. Steven Le Gouill et al. Fluorescence In Situ Hybridization on Peripheral-Blood Specimens Is a Reliable Method to Evaluate Cytogenetic Response in Chronic Myeloid Leukemia. Journal of Clinical Oncology, Vol 18, No 7 (April), 2000: pp 1533-1538
