Chronic Myelogenous Leukemia (CML): Laboratory Diagnosis from the past to the future

•August 19, 2009 • Leave a Comment

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

Cytogenetic abnormality in acute myeloid leukemia: translocation(8;21) in AML-M2

•August 19, 2009 • Leave a Comment

Cytogenetic abnormality in acute myeloid leukemia:    translocation(8;21) in AML-M2

Acute myeloid leukemia (AML) is a heterogenous disease, with individual cases showing variability in clinical presentation. This heterogeneity extends to the molecular genetic lesions underlying the pathogenesis of AML. Although nonrandom clonal chromosomal aberrations are present in the majority of cases, each abnormality affects only a limited subset of cases. One of the most frequent cytogenetic abnormalities in AML is t(8;21)(q22;q22) that found in approximately 10±15% of AML cases. Patients with this subtype of AML typically present with FAB AML-M2 morphology, in which the leukemic blasts have prominent Auer rods, strong myeloperoxidase positivity, homogenous salmon coloured granules, cytoplasmic vacuolization, and prominent bone marrow eosinophilia.

The 8;21 translocation was shown to rearrange the AML1 gene (also referred to as CBFA2 or PEBP2aB) on chromosome 21q22 and the ETO gene (also referred to as MTG8) on chromosome 8q22 (Fig 1A). The AML1 gene encodes the DNA-binding subunit of the AML1/CBFb core binding factor transcription complex, whereas ETO encodes the mammalian homologue of the Drosophila protein Nervy. Although t(8;21) which encodes an AML1-ETO fusion protein is the critical genetic lesion. The chromosomal breakpoints resulting from the t(8;21) cluster within a single intron of both AML1 and ETO and generate similar AML1-ETO chimeric genes in every case. The encoded fusion protein consists of the N-terminal 177 amino acids of AML1 fused in frame to almost the complete ETO protein (Fig 1B).

Fig1

Fig 1.                            British Journal of Haematology, 1999, 106, 296-308

The AML1/CBFβ transcription factor complex: normal function

AML1 is the DNA-binding subunit of the core-binding transcription factor (CBF) and binds to the enhancer core sequence TGTGGT, and its affinity for DNA binding is increased through heterodimerization through the RHD with a second non-DNA-binding subunit CBFβ (Fig 2)

Fig

(Fig 2) Haematologica 1997; 82:364-370

AML1/CBFβ has been shown to function as a transcriptional activator that is critical for the tissue-specific expression of a number of haemopoietic specific genes, including those for myeloperoxidase(MPO), subunits of the T-cell antigen receptor (TCR), the cytokines IL-3 , GM-CSF and others. Although binding of AML1/CBFβ to the core enhancer sequence is important for the haemopoietic specific expression of these genes, their expression is also dependent on the presence of adjacent binding sites for lineage-restricted transcription factors.

The ETO: a putative transcriptional regulator

ETO is the mammalian homologue of the Drosophila gene nervy, and subsequent work has identified two other mammalian members of this family, MTGR1 and MTG16. The  ETO is expressed as a nuclear phosphoprotein in brain and CD34 + haemopoietic progenitors, whereas the related family member MTGR1 is ubiquitously expressed. Although ETO is a nuclear zinc-finger-containing protein, there is no experimental evidence to suggest that it can bind directly to DNA. Nevertheless, the structure of ETO suggests that it is likely to function as a regulator of transcription. The  ETO can directly interact with the nuclear co-repressors N-CoR and Sin3A, and through these interactions can recruit an active histone deacetylase  (HDAC) (Fig 3)aml3

Fig. 3               British Journal of Haematology, 1999, 106, 296-308

The AML1-ETO chimaeric protein: mechanisms of cell transformation

The AML1-ETO fusion protein retains many of the important functional domains of both AML1 and ETO, including the RHD of AML1, and the ETO sequences that mediate homo- and heterodimerization with ETO/MTG family members and interaction with nuclear co-repressors. The  AML1-ETO continues to bind the core enhancer DNA sequence and to heterodimerize with CBFβ (Fig 4). Similarly, like the wild-type AML1 protein, AML1-ETO is a nuclear phosphoprotein that regulates the nuclear accumulation of CBFβ; however, several of its critical functional properties differ from those of wild-type AML1. First, the transcriptional activation domains of AML1 are deleted and replaced by ETO sequences known to interact with nuclear co-repressors, suggests that the chimaeric protein should function not as a transcriptional activator, but instead as a transcriptional repressor.

Second, AML1-ETO binds CBFβ more avidly than AML1, and therefore accumulates CBFb more efficiently in the nucleus than the wild-type protein. Finally, AML1-ETO associates only weakly with the nuclear matrix and directly inhibits wild-type AML1 from binding to this site, thereby inhibiting its ability to stimulate DNA replication.

The ability of AML1-ETO to repress transcription is dependent on both the RHD of AML1 and the HHR and zinc fingers of ETO. Transcriptional repression requires direct DNA binding by the AML1-ETO chimaeric protein through the core enhancer sequence and appears to be mediated through the recruitment of thenuclear co-repressor complex by ETO (Fig 4).aml4

(Fig 4)             British Journal of Haematology, 1999, 106, 296±308

Mutation within the Zn-fingers of ETO that eliminate N-CoR binding abrogate the ability of the chimaeric protein to repress AML1-mediated transcription. Repression is an active process with the AML1-ETO/N-CoR/Sin3A/HDAC complex leading to the deacetylation of histones and the alteration of the chromatin structure of AML1 responsive genes such that transcription is inhibited. In addition, the recruitment of these nuclear co-repressors by AML1-ETO may also induce transcriptional repression through a HDAC independent mechanism.

AML-ETO induces haemopoietic cell transformation by (1) actively repressing  AML1-mediated transcriptional activation thereby blocking the normal activity of AML1; (2) repressing transcription by other AML1 family members; (3) repressing the activity of transcription factors other than AML1, such as C/EBP-α ; (4) interfering with the normal functions of ETO and other ETO/MTG family members expressed in haemopoietic cells; and (5) aberrantly activating the transcription of AML1-regulated and novel AML1-ETO-specific target genes. A critical step in understanding the mechanism of AML1-ETO-mediated transformation will be to identify the genes whose transcription is altered by expression of this chimaeric oncoprotein. In addition, AML1/CBFβ may either directly or indirectly regulate transcription of the p53 tumour suppressor. Inhibition of p53 expression by the AML1-ETO fusion protein would be predicted to contribute directly to cell transformation. Interestingly, p53 inactivating mutations are exceeding rare in cases of AML with alterations in the CBF complex. Thus,inhibition of p53 induction by the CBF fusion proteins may serve as an alternative mechanism of p53 inactivation.

Reference

1. Francesco Lo Coco, Simona Pisegna, Daniela Diverio, The AML1 gene : A transcription factor involved in the pathogenesis of myeloid and lymphoid leukemias, Haematologica 1997; 82:364-370

2. The AML1-ETO chimaeric transcription factor in Acute Myeloid Leukemia:Biology and clinical significance,British Journal of Haematology, 1999, 106, 296-308

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•August 13, 2009 • 1 Comment

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