|Year : 2002 | Volume
| Issue : 1 | Page : 4-10
Chronic myeloid leukemia: Cytogenetics and molecular genetics
Pratibha S Amare
Cytogenetics Laboratory, Tata Memorial Hospital, Parel, Mumbai - 400012, India
Pratibha S Amare
Chief 'Cytogenetics Laboratory', Tata Memorial Hospital, Parel, Mumbai - 400012
|How to cite this article:|
Amare PS. Chronic myeloid leukemia: Cytogenetics and molecular genetics. Indian J Hum Genet 2002;8:4-10
Chronic myelogenous leukemia (CML) is a pleuripotent stem cell disorder characterized by proliferation and accumulation of mature myeloid cells and their progenitors. It is a first human malignancy where a specific marker- the Philadelphia (Ph) chromosome was linked to pathogenetic events of leukemogenesis. Around 99% of CML cases are Ph positive which include 91%-96% of cases with standard Ph resulting from a reciprocal translocation between long arm of chromosome 9 and 22 (Rowley, 1973) [Figure - 1]; and 3%-8% of cases with variant or masked Ph (Rowley, et al . 1982; Kadam, et al . 1990; Bernstein, et al . 1984). Ph chromosome is also found in 5% of children and 15%-30% of adults with acute lymphoblastic leukemia (ALL) and around 2% - 6% of patients with acute myeloblastic leukemia (AML) (Kurzrock, et al . 1988; Specchia, et al . 1995; Kadam, et al . 1991) ). At gene level break occurs in ABL and BCR gene on chromosome 9 and 22 respectively, with the result the 22q11 ® qter segment including part of the BCR gene is translocated to ABL locus on chromosome 9 and reciprocally 3' segment of ABL gene is transposed and inserted to 5' segment of BCR gene on chromosome 22 [Figure - 2]. The resulting BCR-ABL fusion on Ph chromosome is transcribed in to a chimerical RNA and then translated in to a fusion protein of varying size-p210, p190 [Figure - 2].
In CML with variant Ph, apart from chromosomes 9 and 22 break occurs on a third, rarely on fourth chromosome which leads to formation of either standard Ph, a derivative Ph, masked Ph or derivative 9. Chromosome numbers 1, 2, 3, 4, 6, 7, 8, 12, 15, 16, 17 are commonly affected chromosomes in variant Ph (Kadam, et al . 1990; Bernstein, et al . 1984; Ishihara, et al . 1988). In situ hybridization, somatic cell hybridization and polymerase chain reaction (PCR) studies have concluded that variant or masked Ph do show BCR-ABL fusion. (Alimena, et al . 1987; Hagemeijer, et al . 1985; Groffen, et al . 1991). Our studies (Kadam, et al . 1990) along with literature survey (Bernstein, et al . 1984; Groffen, et al . 1991) revealed that variant Ph can be classified in to 5 types. In a simple variant break occurs on chromosome 22q11 and a deleted segment is translocated to a 3rd chromosome instead of chromosome 9 [Figure - 3]. In a complex variant, physical rearrangement among the 9, 22 and a 3rd/4th chromosome results in formation of a unusual derivative 9 and 22 [Figure - 4].
Masked Ph results due to translocation from a 3rd chromosome to Ph chromosome giving rise to a normal appearance of chromosome 22. [Figure - 5] (Kadam, et al . 1990; Bernstein, et al . 1984; Ishihara, et al . 1988). In 4th Ph variant, chromosomes 9 and 22 appear normal due to absence of physical chromosomal rearrangement, however transposition of ABL to BCR followed by BCR-ABL fusion is always present on chromosome 22 at locus q11 [Figure - 6].
A last Ph variant which is a rare variant. There are 6-7 reported cases including 4th from our laboratory in literature (Hegemeijer, et al . 1985; Amare (Kadam), et al . 2001). In this, metaphase FISH helped in the identification of double BCR-ABL fusion on chromosomes 9 indicating insertion of BCR to ABL and duplication of BCR-ABL on both chromosomes 9 as a secondary event [Figure 7] a & b, See color plates). Duplication of Ph or BCR-ABL fusion is a characteristic of CML blast crisis. In Ph variant BCR-ABL fusion is a primary genetic event followed by a secondary genetic event that results in formation of simple or complex variant Ph.
True Ph negative juvenile CML occurs in 1%-2% cases show normal genetic pattern. These cases closely resemble to chronic myelomonocytic leukemia (CMML) due to association with thrombocytopenia and leucocytosis and monosomy 7; however the prognostic features of true Ph negative juvenile CML cases are similar to Ph positive CML. CMML is generally associated with poor prognosis. (Fitzerald, et al . 1987).
In minority of CML patients metaphase FISH studies have shown large deletions of size mega base pairs adjacent to the translocation breakpoint on derivative 9 chromosome and/or a 3rd chromosome involved in variant Ph. These deletions seemed to influence disease progression (Sinclair, et al . 2000).
| Molecular biology of Ph chromosome|| |
In CML and around 30%-50% of adult Ph positive ALL and a small percentage (20%-30%) of childhood Ph +ve ALL, the breakpoints in the BCR gene are clustered within 5.8Kb region called M-bcr (major break point cluster) spanning exons 12-16 (b1-b5) [Figure - 2]. The ABL gene encodes a non receptor tyrosine kinase with molecular weight of 145Kd (p145ABL). It has 11 exons and spans over 230Kb. The BCR gene encodes for serine kinase with molecular wt of 160Kd (p160BCR). Almost all CML patients have breakpoints in 2.9 Kb region between BCR exons 13 and 15 (b2 and b4). In ABL, breakpoints occurs between exon 1b and A2 and this results in transposition of ABL-a2®a11 and insertion to BCR exons 5' to b2-b3 or b3-b4 which is followed by fusion BCR-ABL and that is transcribed in to b2a2 or b3a2 mRNA of size 8.5Kb, followed by translation to chimeric protein of 210Kd (p210BCR/ABL) (Kurzrock, et al . 1988) [Figure - 2]. In 5% of CML cases both b3-a2 and b2-a2 transcripts can be formed as a result of alternative splicing (Melo, 1996). The clinical features and response to treatment are similar to patients with b2-a2 and b3-a2 transcripts except patients with b3-a2 are found to have higher platelet count (Shepherd, et al . 1995). Occasionally in Ph positive CML, breakpoint in the BCR involves the ALL-associated m-bcr region, which results in the formation of p190BCR/ABL fusion protein (Selleri, et al . 1990). CML patients with p190 presents with monocytosis and have clinical features intermediate between CML and CMML (Melo, et al . 1994). A small percentage of CML patients is presented with transcript that results from a fusion between BCR 19 ( (m-bcr) (c3) and ABL exon [Figure - 2]. The fusion transcript e1-a2 encodes a fusion protein of 230KdBCR-ABL which appears to affect granulocytic differentiation with thrombocytosis and have a indolent clinical course in patients (Pane, et al . 1996). In rare Ph positive CML and also in some Ph positive ALL (5%) the BCR sequences are fused to ABL exon 3.
The BCR part of p210BCR/ABL protein contains oligomerization domain which mediates the formation of homodimers and tetramers of p210. The ABL part of p210 contains tyrosine kinase domain, the ATP-binding site for RB and actin binding domains [Figure 8]. In comparison with p210, p190 shows higher tyrosine kinase activity and it is known that Ph +ve ALL have comparatively poor prognosis.
Various structural alterations of ABL and BCR contributes in the leukemogenic process of CML. The N-terminal coiled motif of BCR increases tyrosine kinase activity and enables binding of F-actin by ABL. The serine-threonine kinase domain of BCR activates signaling pathway mediated by ABL tyrosine kinase and p210BCR/ABL (Recter, et al . 1994) which causes ABL to become constitutively active as a tyrosine phosphokinase. The anatomy of p210 allows multiple protein-protein interactions and suggests the involvement of diverse intracellular pathways. BCR domains serve to bind adaptor proteins such as growth factor receptor-bound protein 2 (GRB2), and SRC homologue 2- containing protein (SHC) (Puil, et al . 1994). The SH2 domain of GRB2 binds to a conserved tyrosine residue of BCR in p210BCR/ABL. This leads to linking of p210 to RAS, a guanine triphosphate binding protein involved in the regulation of cell proliferation and differentiation and also at the core of the important signaling pathway in the pathogenesis of CML (Sawyers, et al . 1995).
The role of p210 in leukemogenesis has been explained on the basis of experimental evidences that have shown that p210 has the ability to assemble itself in to oligomers followed by binding to the cytoskeletal protein F-actin which results catalyzation of these proteins (Daley, et al . 1990). With the result, CML cell escapes the normal cellular mechanism of anchorage dependence for intercellular reaction. Defective adherence of immature hematopoietic CML progenitors to marrow stromal elements may facilitate their release in to the blood (Gordon, et al . 1990). Defective cytoadhesion of CML cells has been restored by preoccupation of Ph +ve cells with antisense oligonucleotides tyrosine kinase inhibitor targeted against p210 and treatment with interferon-a (Verfaillie, et al . 1997). However animal model studies have demonstrated that CML cells in addition to p210, acquire several genetic insults at molecular level and this is in combination with p210 are essential for development of fully malignant transformation (Laneuville, et al . 1992).
| Genetic aspects of blast transformation in CML|| |
As the disease evolves from CML- chronic phase to acute phase (AP) and /or blastic phase (BP) of myeloid (M) or lymphoid (L) phenotype, this progression is frequently (60%-80%) preceded or accompanied by recurring secondary chromosomal abnormalities such as +Ph, +8, +19, i (17q) (Nanjangud, et al . 1994; Swolin, et al . 1985). These abnormalities occur as sole or in combination. Less common abnormalities include monosomy 7, monosomy 17, trisomy 21, t (3;21) and loss of Y chromosome. Patients with myeloid blast-crisis frequently show occurrence of extra Ph, +8 and i (17q), whereas patients with lymphoid blast-crisis expressing generally CALLA positivity rarely show development of additional anomalies (Nanjangud, et al . 1994; Advani, et al . 1991; Derderian, et al . 1993). There are some conflicting observations concerning trisomy 8 in lymphoid blastic phase (LBP) of CML (Derderian, et al . 1993; Anastasi, et al . 1995). Studies by Anastasi et al (1995) indicated that trisomy 8 was present in subclone of monocytic and granulocytic cells and absent in lymphoblasts in a case of CML-LBP, indicating that +8 probably occurred before transformation or it was present in CP or AP predominantly in myeloid series. Sequential studies in CML-CP and CML-MBP from our laboratory alongwith literature reports have indicated correlation of i (17q) with thrombocytopenia and basophilia (Nanjangud, et al . 1994). Our findings also revealed that higher frequency of additional abnormalities increases in patients treated with busulphan (70%) than hydroxyurea (44%) indicating probably genotoxic effect of alkylating drug busulphan. In conclusion, specificity of MBP or LBP-associated aberrations indicate that some abnormalities may be directly related to the development of BP and provides clues to the involvement of certain genes responsible for the blast transformation. The corresponding genes which probably involves in blastic transformation include p53 (17p53), c-myc (8q24), RB1 (13q14), p16 (INK4A) (9p21), RAS. P53 alterations such as deletions, rearrangements, point mutations are common in CML-MBP (20%-30%) (Ahuja, et al . 1989). Abnormalities of RB1 are commonly seen in lymphoid blast crisis. Homozygous deletions of p16INK4A are also associated with CML-LBP (Still, et al . 1995).Occasionally, altered methylation has also been demonstrated within the M-BCR in chronic phase CML.
| Ph positive ALL/Lymphoid blast-crisis|| |
A controversy at the time of diagnosis whether Ph chromosome positive ALL or acute phase of CML in lymphoid transformation, is not uncommon. Although the relationship between Ph + ve ALL and CML-LBP is unresolved issue on the basis of certain clinico-hematological and genetic presentations one can distinguish between these two entities. First CML is rare in children unlike to Ph +ve ALL. CML is always presented with 100% Ph positive clone. Except Ph +ve adult ALL, p210 protein is exclusively present in CML. Additional chromosomal abnormalities are rare in Ph +ve ALL but common in blastic phase of CML. In comparison with CML-LBP, in Ph positive ALL clinical remission is commonly accompanied by elimination of the Ph +ve clone. Relapse from Ph positive ALL to blastic phase of CML indicates that the underlying disease at initial presentation was CML (Catovsky, 1979).
| Minimal residual disease and Prediction of disease course in CML|| |
The unique Ph marker and resultant chimeric gene BCR-ABL are considered to be pivotal for assessment of residual disease in remission. Quantitative estimation of residual disease provides assessment for individual response to effective treatment modalities such as stem cell transplantation or interferon-a or recently introduced STI 571, an tyrosine kinase inhibitor in CML (Amare (Kadam), et al . 2001; Palka, et al . 1996; Faderl, et al . 1999). Our data along with literature reports on MRD in post-BMT marrow revealed that MRD plays an important role in prediction of disease recurrence (Faderl, et al . 1999; Roth, et al . 1992). The RT-PCR and fluorescence in situ hybridization (FISH) are considered to be very efficient techniques for evaluation of MRD. Review of the literature reveals controversial results with regard to presence of residual disease in post- BMT remission marrow by PCR and its significance in the prediction of future course of disease (Faderl, et al . 1999; Gaiger, et al . 1993). However, quantification of BCR-ABL transcript by recently developed quantitative PCR has been found to be highly sensitive for detection of MRD and prediction of relapse in CML patients undergone BMT (Nagler, et al . 1993). FISH technique is another very sensitive technique of molecular diagnosis and most important is its application in the evaluation of quantitative estimation of residual leukemic cells (Amare (Kadam), et al . 2001; Bentz, et al . 1994; Bose, et al . 1998; Trachuk, et al . 1990; Dewald, et al . 1998). The ability of FISH to visualize and identify a single aberrant cell among the heterogeneous population of normal and malignant cells has shown its valuable application in the evaluation of quantitative burden (1 X 103-4) of BCR-ABL +ve cells. Recently developed BCR/ABL probes with ability to detect double BCR-ABL fusion (D-FISH) (Seong, et al . 1997) has shown that false positivity can be reduced from 1%-5% to 0.07%. Additionally, metaphase FISH is advantageous over other molecular techniques by its ability to identify various submicroscopic chromosomal rearrangements and their abnormal loci (Amare, et al . 2001; Faderl, et al . 1999; Trachuk, et al . 1990;Dewald, et al . 1998).
| Mechanism of BCR-ABL translocation|| |
The strong evidences regarding the mechanisms of underlying interchromosomal exchanges and recombinations are lacking in the litertature. One of the mechanisms has been postulated in the reciprocal translocation involving immunoglobulin (Ig) or T-cell receptor (TCR) gene and protooncogene. According to this mechanism translocation involving Ig/TCR and oncogene involves sequences with high homology to heptamer sequences of recombinase that mediate normal VDJ recombination (Rabbitts, 1994). Presence of Alu sequences in close proximity to the breakpoints may enhance the probability of illegitimate homologous recombination (Heister, et al . 1985).
The physical proximity of the involved chromosomal regions is generally accepted hypothesis for the process of chromosomal translocations. By using in situ hybridization and confocal microscopy Neves et al . (1999) group measured the distance between the BCR and the ABL in three dimensionally preserved hematopoietic cells belonging to different cell lineages at various stages of differentiation and at various stages of the cell cycle. They found significant association of ABL with BCR at the transition between S and G2. They also found higher proportion of cells with ABL-BCR were most frequently hematopoietic precursor cells. They could demonstrate that chromosomes in metaphase are arranged in a rosette fashion by size of respective chromosomes and by the geometry of metaphase plate which probably results in reducing the intergenic distances between ABL and BCR. These findings suggested that intrinsic spatial dynamics is established early in hematopoiesis and perpetuated differentially in distinct cell lineages, that may facilitate the collision of individual genes and thus reciprocal recombination between them at subsequent stages of hematopoietic differentiation.
The understanding of cytogenetic and molecular processes in CML leukemogenesis has helped in the identification of molecular targets which led to the development of targeted therapy as tyrosine kinase inhibitor STI 571, immunomodulatory therapy and autografting.
| Acknowledgement|| |
I am thankful to Ms. P. Ghule for graphical assistance.
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[Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4], [Figure - 5], [Figure - 6]
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