US Pharm. 2008;33(7)(Oncology
ABSTRACT: Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized by a translocation between chromosomes 9 and 22, forming the Philadelphia (Ph) chromosome. This and other chromosomal abnormalities can be detected with the use of cytogenetics, a branch of genetics focusing on chromosomal structure. Standard cytogenetics, fluorescence in situ hybridization (FISH), and reverse-transcriptase polymerase chain reaction (RT-PCR) are cytogenetic methods employed to diagnose CML and to monitor response to treatment. A pivotal trial demonstrating the superiority of imatinib in patients with newly diagnosed CML reported complete cytogenetic response, with no Ph-positive (Ph+) cells seen in a sample of bone marrow, as a primary endpoint.
Chronic myelogenous leukemia (CML) is a myeloproliferative disorder initiated by a genetic translocation within a pluripotent stem cell. This malignant transformation leads to unregulated growth and accumulation of myeloid cells.1,2 The disease has a triphasic course, with 80% diagnosed in the chronic phase (lasting an average of four to six years untreated), followed by the accelerated phase, and finally, the blast crisis.3 Most patients are asymptomatic when diagnosed. As the disease progresses, presentation may include the clinical picture of a myeloproliferative disorder: leukocytosis, thrombocytosis, anemia, weight loss, and splenomegaly.
New cases of CML are forecast to total 4,830 in the United States in 2008, with 450 deaths.4 The median age of onset is 67 years, although the disease occurs in all age groups.5 New treatment modalities such as tyrosine kinase inhibitors (i.e., imatinib, dasatinib, nilotinib) are prolonging survival, but the only current therapy that is potentially curative is allogeneic stem cell transplant. This is problematic given the morbidity associated with transplant and the older average age of patients with CML.5
Classification of CML
A number of classifications are in use for the determination of the disease stage. Increases in the number of blasts in the peripheral blood or bone marrow (5%-19%, depending on the classification system) are a key event declaring progression to the accelerated phase.5 Additional features may include anemia or thrombocytopenia unrelated to therapy, rapid leukocyte doubling time (<5 days), persistent thrombocytosis, splenomegaly, clonal evolution, persistent fever, or bone pain.5 An increase in blast count to above 20% to 30% marks progression to the final phase of CML--blast crisis. This final phase is rapidly fatal, with a median survival of three to six months if untreated.6 Treatment of CML is more successful in chronic rather than the later accelerated or blast phases.7
In 1960, Nowell and Hungerford developed a technique to disrupt cell mitosis and expand the cells using hypotonic solution.8 During normal cell replication, the chromosomes are tightly wound and impossible to visualize.9 Subsequent studies of the now more visible contents of the cell nuclei uncovered a chromosomal abnormality (termed the Philadelphia [Ph] chromosome) in the cells of seven patients with CML.10 This reciprocal exchange of material between the Abelson kinase domain (ABL) of chromosome 9 and the breakpoint cluster region (BCR) of chromosome 22 is seen in over 90% of patients diagnosed with CML. The full nomenclature t(9;22)(q34;q11) designates a translocation (t) between the two chromosomes at the short arm (q) of 9 (region 3, band 4) and the short arm of 22 (region 1, band 1).11 The hybrid BCR-ABL tyrosine kinase is constitutively active, dysregulating downstream pathways driving malignant cell proliferation and resistance to apoptosis.12 The pathway initiates with adenosine triphosphate (ATP) binding to BCR-ABL. The formation of the Ph chromosome is shown in Figure 1. Imatinib, a small molecule tyrosine kinase inhibitor approved for the first-line treatment of chronic phase CML in 2002, inhibits BCR-ABL signaling by binding to the active form of ABL at the ATP-binding site.8
Cytogenetics and CML
Methods to reveal chromosomal abnormalities enable the diagnosis and appropriate treatment of CML.13 Cytogenetics, a branch of genetics focusing on chromosomal structure, is used to determine the presence of genetic changes within cellular DNA. Standard cytogenetics is usually performed on a bone marrow sample due to the greater number of proliferating cells in bone marrow compared to peripheral blood.12 Cell division is induced and then chemically arrested in metaphase. Chromosomes are stained to increase visibility (Giemsa or G-banding), demonstrating alternating dark and light bands when viewed under the microscope.13
Conventional cytogenetics, or karyotyping, is employed at presentation to identify the characteristic Ph chromosome. The analysis is time consuming, typically involving the examination of 25 to 30 metaphase cells.12 This method is considered insufficiently sensitive to detect the presence of Ph-positive (Ph+) chromosomes in less than 10% of cells. The same methodology can also be used to monitor response to treatment at three-month intervals, defined by the percent of dividing cells in the sample that retain the Ph marker.14,15
FISH and RT-PCR Testing: Patients have an average of 1 x 1012 leukemic cells at presentation.13,16 After treatment, the lack of detection of Ph+ chromosomes using conventional cytogenetic analysis is considered a complete cytogenetic response (CCR). Patients in CCR have been shown to have up to 1 x 109 leukemic cells still present.
To detect further log reductions in disease burden requires tests of greater sensitivity. Fluorescence in situ hybridization (FISH) involves the incubation of fluorescence labeled DNA probes with denatured (single-strand) DNA. Chromosomes have a distinct pattern of light absorption, related to the density of the guanine-rich DNA. Traditional FISH, or dual-FISH analysis, uses one colored probe fluorescing from the BCR region and a second different colored probe fluorescing from the ABL region. In CML, the two regions are no longer on different chromosomes but are superimposed, fluorescing the blended color. These traditional FISH analyses have a false positive rate of up to 10%. Newer methods report false positives below 1%.12 In hypermetaphase FISH, up to 500 cells are analyzed, yielding results with much greater accuracy than conventional cytogenetics. This technique can be used to analyze samples from blood, bone marrow, or tissue in any stage of cell replication.13 FISH allows increased resolution of genetic aberrations not detailed with standard cytogenetics and without the time needed to culture cells.17 FISH analysis is used at some centers to monitor maintenance of CCR.
Molecular studies with reverse-transcriptase polymerase chain reaction (RT-PCR) begin with single strands of DNA sequences specific to a disease. Subsequent cycles with a DNA polymerase result in exponential multiplication of the target sequence.13 RT-PCR is the most sensitive of all monitoring tools with the ability to pick up one CML cell in a population of 100,000 or more cells. In CML, quantitative RT-PCR results report the ratio of the target BCR-ABL sequence to a reference gene (BCR or ABL). RT-PCR results can be available faster than FISH, but have lower specificity. False positive results can result from contamination of a BCR-ABL negative sample by a positive sample. The rapid duplication of DNA sequences (up to 30 cycles) will amplify any contamination that might be introduced.12
FISH and RT-PCR are valuable tools in the identification of individuals with Ph-negative BCR-ABL-positive CML. Of the 5% to 10% of CML patients not demonstrating the Ph chromosome with conventional cytogenetics, some may have submicroscopic BCR-ABL aberrations, or more complex translocations in addition to the classic breakpoints of chromosomes 9 and 22. Atypical Ph translocations are termed simple (chromosome 22 plus a chromosome other than 9) or complex (9, 22, and other chromosomes).18 Thirty to fifty percent of these cases of "masked" CML are readily identifiable with the more sensitive molecular methods.13 The remaining half of those Ph negative by cytogenetics and molecular methods are termed atypical CML and have a poor prognosis.6 Molecular methods are also necessary for detection of low levels of Ph+ cells, minimal residual disease, or 1 x 105 leukemic cells.16
Chromosomal Changes and Mutations: Conventional cytogenetic analysis is the only technique that allows visualization of all chromosomes. This analysis is used for monitoring patients for acquisition of additional chromosomal aberrations, which signal transition from chronic to accelerated phase.14 Blood or marrow samples from patients converting to accelerated or blast phase often demonstrate chromosomal abnormalities in addition to the Ph chromosome, termed clonal evolution. BCR-ABL down regulates the DNA repair process, enabling additional mutations.8 The most frequently seen chromosomal changes include trisomy 8, isochrome 17, duplicate Ph chromosome, loss of 17p and BCR-ABL1. Other changes seen less frequently include trisomy 19, trisomy 21, trisomy 17, and deletion 7.19 For patients in CCR, RT-PCR monitoring is recommended at three-month intervals, with conventional cytogenetics repeated annually.15
Mutations in the BCR-ABL gene can occur as well. Determination of the specific mutation is clinically important as it may dictate a change in therapy. Changes in the structure of BCR-ABL can interfere with drugs designed to inhibit downstream signaling. Over 40 different mutations involving amino acid substitutions have been reported, most often found in the area where ATP binds to BCR-ABL. This area has been termed the P loop. G250E, Q252H, Y253F, and E255K mutations in the P loop are not involved in the binding of imatinib, but have been linked with poor prognosis and a survival of four to five months.19 The "gatekeeper" mutation T315I interferes with imatinib binding, predicting resistance to imatinib and other tyrosine kinase inhibitors (i.e., dasatinib and nilotinib). Sequencing of the BCR-ABL gene, specifically the ABL kinase domain, to look for mutations may be performed if there is suboptimal response to initial therapy, loss of response to therapy, or progression to a more advanced stage of CML.
Targeted Therapies: Tyrosine Kinase Inhibitors
Currently, response to therapy in CML is defined on three levels. Hematologic response describes counts of cells in peripheral blood samples. Complete hematological response (CHR) equates to normalization of the blood counts. Cytogenetic response is determined by the percent of Ph+ chromosomes remaining in the bone marrow (complete = no Ph+ cells; partial = up to 34% Ph+ cells; minor = 35%-90% Ph+ cells).5 In addition, a major molecular response is reflective of a three-log reduction in the number of CML-defining, BCR-ABL genes.20,21
As previously described, formation of the tyrosine kinase Ph chromosome in CML results in unchecked growth of malignant cells. Tyrosine kinase inhibitors are a class of agents that interfere with the downstream signaling initiated by these abnormal proteins. These molecules block the transfer of phosphate from adenosine triphosphate, interfering with tyrosine kinase mediated signaling pathways and the subsequent proliferation of leukemic cells. The three tyrosine kinase inhibitors currently available for treatment in the U.S. are imatinib, dasatinib, and nilotinib (Table 1).
In the pivotal trial comparing the efficacy of imatinib, an oral tyrosine
kinase inhibitor, to interferon alfa plus low-dose cytarabine in patients with
newly diagnosed CML, cytogenetic response was found to be associated with
event-free survival. The International Randomized Study of Interferon and
ST1571 (IRIS) trial reported that of patients demonstrating a CCR at 12
months, only 3% had progressed to accelerated or blast phase in 60 months. In
the group not achieving a major cytogenic response in 12 months, 19%
progressed to accelerated or blast phase by 60 months.22 Based on
these outcome data and availability of sensitive methods used to monitor
response to treatment, clinicians are able to change therapy if a response is
not evident by 12 months following the initiation of treatment.
Many of the definitions and time frames for response arise from experience with imatinib. Patients with suboptimal response might demonstrate normal blood counts, but have no cytogenic response at three months, greater than 35% Ph+ cells at six months, greater than 5% of Ph+ cells at 12 months, or no molecular response following 18 months of treatment. Guidelines recommend for patients exhibiting suboptimal response with standard treatment doses of imatinib (400 mg daily) that if tolerated, the dose may be escalated to 600 to 800 mg daily.23
Resistance to imatinib is defined as no hematologic response after three months of treatment, no cytogenetic response after six months of treatment, greater than 35% Ph + after 12 months, or greater than 5% Ph+ cells after 18 months of treatment. Those fitting the definition of resistance are considered to have failed imatinib, and the treatment should be changed.23 In the IRIS study, 4% of newly diagnosed CML patients became resistant yearly, with the number reducing to fewer than 2% in year 5 of the study.22 However, it is estimated that 40% of those in late chronic phase and 70% to 90% in accelerated or blast phase are resistant to imatinib.21
The IRIS study also demonstrated the prognostic value of cytogenetic changes in CML. All patients achieving a major molecular response by 12 months of imatinib treatment had progression-free survival at 60 months.21,22
Dasatinib: One treatment consideration for patients failing imatinib is the multitargeted tyrosine kinase inhibitor, dasatinib. Dasatinib inhibits BCR-ABL in both the active and inactive formation, as well as other downstream proteins, including c-KIT, platelet-derived growth factor receptor, and the Src family of kinases. The diversity of binding mechanisms available to dasatinib contributes to its ability to inhibit the constitutive activity of the Ph chromosome in patients with mutations that prevent activity of imatinib.
The Phase II START (SRC/ABL Tyrosine kinase inhibitor Activity Research Trials of dasatinib) trials tested the efficacy of dasatinib in different CML populations.24 In START-C, patients with CML in chronic phase with resistance to imatinib were treated with 70 mg of dasatinib twice daily. Most patients (90%) achieved a CHR. In the START-A arm, 107 accelerated-phase CML patients with primary or acquired resistance to imatinib (n = 99), or intolerance to imatinib therapy (n = 8), received 70 mg dasatinib twice daily. At the onset of this study, 60% of participants exhibited BCR-ABL mutations. At eight months follow-up, 87% (n = 81) achieved an overall hematologic response (major and minor hematologic responses), and 24% (n = 26) achieved a CCR. START-R was a randomized trial comparing dasatinib 70 mg twice daily to imatinib 800 mg daily in chronic-phase CML patients resistant to imatinib. A major cytogenetic response was attained in 32% of dasatinib patients and 4% of imatinib patients after 12 weeks of therapy. Of note, dasatinib was not effective in patients with the T315I mutation.24
Nilotinib: Recently approved in October 2007, nilotinib is another tyrosine kinase inhibitor effective against many imatinib-resistant mutations. In a phase II trial, 119 patients with imatinib resistant or intolerant accelerated-phase CML received 400 mg of nilotinib twice daily. Hematologic responses were achieved in 47% of patients. Major cytogenetic response was achieved in 29% of patients. Like dasatinib, nilotinib was active in most patients with mutations conferring resistance to imatinib, with the exception of the T315I mutation.25
Therapies under development to treat patients resistant to tyrosine kinase inhibitors include aurora kinase inhibitors, which are active against BCR-ABL demonstrating the T315I mutation. Farnesyl transferase inhibitors and P13-K inhibitors target the BCR-ABL pathway downstream of the ATP binding site and are unaffected by T315I mutations.21
The presence of a specific genetic abnormality, targeted drug therapies, and the availability of sensitive and specific methods to monitor response make CML a unique disease amongst hematologic malignancies. Recent advances allow clinicians to make adjustments to therapy often before overt changes in the clinical course occur. Notably, knowledge of the molecular pathways and events driving disease progression are leading to the development of novel drug therapies.
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