The chronic myelogenous leukemias (CMLs) include BCR rearrangement-positive CML, chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, chronic neutrophilic leukemia, chronic eosinophilic leukemia, chronic basophilic leukemia, and possibly chronic monocytic leukemia. The term chronic, in contrast to acute, once had prognostic implications. However, although the terms remain useful for nosology, they no longer reflect an invariable difference in prognosis. For example, acute myelogenous leukemia in children and young adults has higher remission and cure rates than juvenile or chronic myelomonocytic leukemia in children or adults, respectively. BCR rearrangement-positive CML presents with anemia, exaggerated granulocytosis, a large proportion of myelocytes and mature neutrophils, absolute basophilia, normal or elevated platelet counts, and, frequently, splenomegaly. The marrow is hypercellular, and marrow cells contain the Philadelphia (Ph) chromosome in approximately 90 percent of cases by cytogenetic analysis. A rearrangement of the BCR gene on chromosome 22 is present in approximately 96 percent of cases by molecular diagnostic analysis. The disease usually responds to imatinib mesylate, a specific tyrosine kinase inhibitor, and median survival has been extended significantly. Allogeneic stem cell transplantation can cure the disease, especially if the transplantation is applied early in the chronic phase. The effect of stem cell transplantation is related in part to a robust graft-versus-leukemia effect, engendered by donor T lymphocytes. The chronic phase usually is followed by an accelerated phase that often terminates in acute leukemia (blast crisis), at which point therapy with imatinib mesylate and other agents may induce a remission in a proportion of patients, but median survival is measured in months. Blast crisis results in a myelogenous leukemic phenotype in 75 percent of cases and a lymphoblastic leukemic phenotype in approximately 25 percent of cases. Ph-chromosome–positive acute myeloblastic leukemia (AML) may appear de novo in approximately 1 percent of cases of AML, and Ph-chromosome–positive acute lymphocytic leukemia (ALL) may occur de novo in approximately 20 percent of cases of adult ALL and approximately 5 percent of childhood ALL cases. In Ph-chromosome–positive ALL, the translocation between chromosomes 9 and 22 results in the fusion gene encoding a mutant tyrosine kinase oncoprotein that may be identical in size to that in classic CML (210 kDa) in approximately one-third of cases. A smaller mutant tyrosine kinase (190 kDa) is encoded in approximately two-thirds of cases. In children, the cells in approximately 90 percent of cases contain a 190-kDa mutant tyrosine kinase. These acute leukemias may reflect (1) the presentation of CML in acute blastic transformation without a preceding chronic phase or (2) de novo cases resulting from a BCR-ABL mutation occurring in a different hematopoietic cell from the event in CML or with as yet unidentified modifying gene alterations. Chronic myelomonocytic leukemia has variable presenting features. Anemia may be accompanied by mildly or moderately elevated leukocyte counts; an elevated total monocyte count; a low, normal, or elevated platelet count; and sometimes splenomegaly. Although cytogenetic abnormalities may be present, there is no specific genetic marker of the disease. In a very small proportion of cases, a translocation involving the platelet-derived growth factor receptor-beta (PDGFR-) gene is associated with eosinophilia and is responsive to imatinib mesylate. Juvenile myelomonocytic leukemia occurs in infancy or very early childhood. Anemia, thrombocytopenia, and leukocytosis with monocytosis are usual. The disease is refractory to treatment and, even with current maximal therapy and stem cell rescue, cures are uncommon. Chronic neutrophilic leukemia presents with mild anemia and exaggerated neutrophilia, with very few immature cells in the blood. Splenomegaly is common. The disease usually occurs after age 60 years and is refractory to current treatment approaches. Chronic and juvenile myelomonocytic leukemia and chronic neutrophilic leukemia have a propensity to evolve into acute myelogenous leukemia. Prior to that evolution, morbidity and mortality are related to infection, hemorrhage, and complicating medical conditions. Chronic eosinophilic leukemia represents the major subset of the hypereosinophilic syndrome. It is a clonal disorder with a striking absolute eosinophilia, often neurologic and cardiac manifestations secondary to toxic effects of eosinophil granules, and sometimes a translocation involving the platelet-derived growth factor receptor-alpha (PDGFR-) gene that encodes a mutant tyrosine kinase, imparting sensitivity to imatinib mesylate.
Acronyms and Abbreviations
Acronyms and abbreviations that appear in this chapter include: AGP, 1-acid glycoprotein; ALL, acute lymphocytic leukemia; BCR, breakpoint cluster region; CCyR, complete cytogenetic remission; CFU-GM, colony-forming unit–granulocyte-monocyte; CHR, complete hematologic response; CLL, chronic lymphocytic leukemia; CML, chronic myelogenous leukemia; CMML, chronic myelomonocytic leukemia; DLI, donor lymphocyte infusion; FISH, fluorescence in situ hybridization; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-monocyte colony-stimulating factor; GRB2, growth factor receptor-bound protein-2; GTP, guanosine triphosphate; GTPase, guanosine triphosphatase; GVHD, graft-versus-host disease; HLA, human leukocyte antigen; HPRT, hypoxanthine phosphoribosyltransferase; HR, hematologic response; hsp, heat shock protein; HUMARA, human androgen receptor assay; IFN, interferon; IL, interleukin; JAK, Janus-associated kinase; LTC-IC, long-term culture-initiating cell; MCP, monocyte chemotactic protein; MCyR, major cytogenetic response; mCyR, minor cytogenetic response; MDS, myelodysplastic syndrome; MIP, macrophage inflammatory protein; MMR, major molecular response; mRNA, messenger RNA; NF-B, nuclear factor-B; NF1, neurofibromatosis tumor suppressor gene; NK, natural killer; NOD, nonobese diabetic; PCR, polymerase chain reaction; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; PHR, partial hematologic response; Ph, Philadelphia chromosome; PI3K, phosphatidylinositol 3′-kinase; Rb, retinoblastoma; RT-PCR, reverse transcriptase polymerase chain reaction; SCID, severe combined immunodeficiency; STAT, signal transducer and activator of transcription; TBI, total-body irradiation; TdT, terminal deoxynucleotidyl transferase; TGF, transforming growth factor; VEGF, vascular endothelial growth factor; WT, Wilms tumor.
Definition and History
Chronic myelogenous leukemia (CML) is a pluripotential stem cell disease characterized by anemia, extreme blood granulocytosis and granulocytic immaturity, basophilia, often thrombocytosis, and splenomegaly. The hematopoietic cells contain a reciprocal translocation between chromosomes 9 and 22 in more than 95 percent of patients, which leads to an overtly foreshortened long arm of one of the chromosome pair 22 (i.e., 22, 22q–) referred to as the Philadelphia (Ph) chromosome. A rearrangement of the breakpoint cluster gene on the long arm of chromosome 22 defines this form of CML and is present even in the 10 percent of patients without an overt 22q abnormality by Giemsa banding. The natural history of the disease is to undergo clonal evolution into an accelerated phase and/or a rapidly progressive phase resembling acute leukemia, which is refractory to therapy.
In 1845, Bennett1 in Scotland and Virchow2 in Germany described patients with splenic enlargement, severe anemia, and enormous concentrations of leukocytes in their blood at autopsy. Bennett initially favored an extreme pyemia as the explanation, but Virchow argued against suppuration as a cause. Additional cases were reported by Craige3 and others, and in 1847 Virchow4 introduced the designation weisses Blut and leukämie (leukemia). In 1878, Neumann5 proposed that the marrow not only was the site of normal blood cell production, but also was the site from which leukemia originated and used the term myelogene (myelogenous) leukemia. Subsequent observations amplified the clinical and laboratory features of the disease, but few fundamental insights were gained until the discovery by Nowell and Hungerford,6 who reported in 1960 that two patients with the disease had an apparent loss of the long arm of chromosome 21 or 22, an abnormality that was quickly confirmed7–9 and designated the Philadelphia chromosome.7 This observation led to a new approach to diagnosis, a marker to study the pathogenesis of the disease, and a focus for future studies of the molecular pathology of the disease. The availability of banding techniques to define the fine structure of chromosomes10,11 led to the discovery by Rowley12 that the apparent lost chromosomal material on chromosome 22 was part of a reciprocal translocation between chromosomes 9 and 22. The discovery that the cellular oncogene ABL on chromosome number 9 and a segment of chromosome 22, the breakpoint cluster region (BCR), fuse as a result of the translocation provided a basis for the study of the molecular cause of the disease.13,14 The appreciation that the fusion gene encoded a constitutively active tyrosine kinase (BCR-ABL) that was capable of inducing the disease in mice established the fusion gene product as the proximate cause of the malignant transformation. The search for, identification of, and clinical development of a small molecule inhibitor of the mutant tyrosine kinase has provided a specific agent, imatinib mesylate, with which to inhibit the molecule that incites the disease.15 Several more potent congeners have also been synthesized (see “Etiology and Pathogenesis” below). Thomas and colleagues established that allogeneic hematopoietic stem cell transplantation could cure the disease.16
|EpidemiologyCML accounts for approximately 15 percent of all cases of leukemia, or approximately 5000 new cases per year in the United States. The age-adjusted incidence rate in the United States is approximately 2.0 per 100,000 persons for men and approximately 1.1 per 100,000 persons for women. The incidence around the world varies by a factor of approximately twofold. The lowest incidence is in Sweden and China (approximately 0.7 per 100,000 persons), and the highest incidence is in Switzerland and the United States (approximately 1.5 per 100,000 persons).17 The age-specific incidence rate for CML in the United States increases logarithmically with age, from approximately 0.2 per 100,000 persons younger than 20 years to a rate of approximately 10.0 per 100,000 octogenarians per year (Fig. 90–1). Although CML occurs in children and adolescents, less than 10 percent of all cases occur in subjects between 1 and 20 years old. CML represents approximately 3 percent of all childhood leukemias. Multiple occurrences of CML in families are rare. There is no concordance of the disease between identical twins. Analytical epidemiologic evidence for a familial predisposition in CML was not found in a Swedish database.18
|Incidence of chronic myelogenous leukemia by age. Note the exponential increase in incidence with age from about teenagers to octogenarians. Rare cases occur in younger children but too few to generate an incidence rate.
Etiology and Pathogenesis
Exposure to very high doses of ionizing radiation can increase the occurrence of CML above the expected frequency in comparable populations. Three major populations—the Japanese exposed to the radiation released by the atomic bomb detonations at Nagasaki and Hiroshima,19 British patients with ankylosing spondylitis treated with spine irradiation,20,21 and women with uterine cervical carcinoma who received radiation therapy22—had a frequency of CML (as well as acute leukemia) significantly above the frequency expected in comparable unexposed groups. The median latent period was approximately 4 years in irradiated spondylitics, among whom approximately 20 percent of the leukemia cases were CML; 9 years in the uterine cervical cancer patients, of whom approximately 30 percent had CML; and 11 years in the Japanese survivors of the atomic bombs, of whom approximately 30 percent of the leukemia patients had CML.23 Chemical leukemogens, such as benzene and alkylating agents, are not causative agents of CML, although they are well established to produce a dose-dependent increase in acute myelogenous leukemia.24
Origin from a Hematopoietic Stem Cell Clone
CML results from the malignant transformation of a single multipotential hematopoietic cell. The disease is acquired (somatic mutation), given that the identical twin of patients with CML and the offspring of mothers with the disease neither carry the Ph chromosome nor develop the disease.25 The origin of CML from a single multipotential hematopoietic cell is supported by the following lines of evidence:
- Involvement of erythropoiesis, neutrophilopoiesis, eosinophilopoiesis, basophilopoiesis, monocytopoiesis, and thrombopoiesis in chronic phase CML26
- Presence of the Ph chromosome (22q–) in erythroblasts; neutrophilic, eosinophilic, and basophilic granulocytes; macrophages; and megakaryocytes27
- Presence of a single glucose-6-phosphate dehydrogenase isoenzyme in red cells, neutrophils, eosinophils, basophils, monocytes, and platelets, but not in fibroblasts or other somatic cells in women with CML who are heterozygotes for isoenzymes A and B28–30
- Presence of the Ph translocation only on a structurally anomalous chromosome 9 or 22 of each chromosome pair in every cell analyzed in occasional patients with a structurally dissimilar 9 or 22 chromosome within the pair31–33
- Presence of the Ph chromosome in one but not the other cell lineage of patients who are a mosaic for sex chromosomes, as in Turner syndrome (45X/46XX)34 and Klinefelter syndrome (46XY/47XXY)35
- Molecular studies showing variation in the breakpoint of chromosome 22 among different patients with CML but precisely the same breakpoint among cells within a single patient with CML36,37
- Combined DNA hybridization-methylation analysis of women who have restriction fragment length polymorphisms at the X-linked locus for hypoxanthine phosphoribosyltransferase (HPRT), which enables distinction of the two alleles of the HPRT gene in heterozygous females, coupled with methylation-sensitive restriction-enzyme cleavage patterns, which permits delineation of whether cells contain either the maternally derived or the paternally derived copy of the gene38
The foregoing observations place the parent cell of the clone at least at the level of the hematopoietic stem cell.
The Chronic Myelogenous Leukemia Stem Cell
Acquisition of the BCR-ABL fusion gene as a result of the t(9;22) (q34;q11.2) in a single multipotential hematopoietic cell results in the CML stem cell, necessary for the initiation and maintenance of the chronic phase of CML.39,40 The phenotype of the CML stem cell is not fully defined but they are among the CD34+CD33– fraction of CML cells. A large proportion of CML stem cells are in the Go phase of the cell cycle and are resistant to therapy with BCR-ABL inhibitors. These cells represent a pool for the regrowth of the tumor, if suppressive therapy is interrupted. The acquisition of genetic and epigenetic events in a derivative BCR-ABL–positive cell can result in evolution to accelerated phase and blastic transformation41 (see “Accelerated Phase and Blast Crisis of CML” below).
Pluripotential versus Hematopoietic Stem Cell Lesion
Some patients in chronic phase CML have lymphocytes that are derived from the primordial malignant cell. Evidence for this finding includes the following: A single isoenzyme for glucose-6-phosphate dehydrogenase has been found in some T and B lymphocytes in women with CML who are heterozygous for isoenzymes A and B;42 blood cells from patients with CML induced to proliferate with Epstein-Barr virus (presumptive B lymphocytes) are of the same glucose-6-phosphate dehydrogenase isoenzyme type, have cytoplasmic immunoglobulin heavy and light chains, and contain the Ph chromosome;43 blood lymphocytes stimulated with B lymphocyte mitogens contain the Ph chromosome;44,45 purified B lymphocytes from the blood in chronic phase CML contain an abnormal, elongated phosphoprotein coded for by the chimeric gene resulting from the t(9;22);46 and fluorescence in situ hybridization (FISH) has detected the BCR-ABL fusion gene in approximately 25 percent of B lymphocytes in some, but not all, patients in chronic phase.47,48 These findings suggest that B lymphocytes are derived from the malignant clone, placing the lesion closer to, if not in, the pluripotential stem cell.42–46 Almost all studies find that the B lymphocyte pool is a mosaic, containing both Ph-chromosome– and BCR-ABL–positive cells and Ph-chromosome– or BCR-ABL–negative cells. Results of studies examining the derivation of T lymphocytes from the malignant clone are more ambiguous but indicate that T lymphocytes are derived from the malignant clone in some, but not most, patients.42,44,49–58 Natural killer (NK) cells isolated from patients with chronic phase CML do not contain the BCR-ABL fusion gene.59 It is possible that myelopoiesis is invariably clonal and lymphopoiesis is an unpredictable mosaic derived largely from normal residual stem cells. This conclusion is supported by the finding that progenitors of T, B, and NK lymphocytes contain the Ph chromosome and BCR-ABL, but most B-cell and all T-cell progenitors derived from the leukemic clone undergo apoptosis, leaving unaffected cells in the blood.60–63
The cell in which the mutation occurs may be even more primitive in that some endothelial cells generated in vitro express the BCR-ABL fusion gene, as do some cells in the patient’s vascular endothelium.64
Etiologic Role of the pH Chromosome
Early studies indicated that the Ph chromosome may appear after the initial leukemogenic event.65–68 Patients with CML have developed the Ph chromosome during the course of the disease, have experienced periods of the disease when the Ph chromosome disappeared,69 or have had Ph-chromosome–positive and Ph-chromosome–negative cells concurrently.70–74
Nearly all, if not all, patients with CML have an abnormality of chromosome 22 at a molecular level (BCR rearrangement). Thus, earlier studies indicating an absence of a Ph chromosome were not a valid measure of the normality of chromosome 22. The molecular abnormality in CML involving the ABL gene on chromosome 9 and the BCR gene on chromosome 22 has been established as being the proximate cause of the chronic phase of the disease (see “Molecular Pathology” below).
Coexistence of Normal Stem Cells
Most, if not all, patients with CML have hematopoietic stem cells that, after treatment75–77 or culture in vitro,78–80 use of special cell isolation techniques,81,82 or use of cell transfer to nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice83 do not have the Ph chromosome84,85 or the BCR-ABL fusion gene.86–90 The switch to Ph-chromosome–negative cells in vitro is associated with a loss of monoclonal glucose-6-phosphate dehydrogenase isoenzyme patterns, indicating the persistence and reemergence of normal polyclonal hematopoiesis rather than reversion to a Ph-chromosome–negative clone.91 In confirmation, BCR-ABL+, CD34+, human leukocyte antigen (HLA)-DR– cells isolated from women with early phase CML are polyclonal using the human androgen receptor assay (HUMARA) to assess X chromosome inactivation patterns.92 Very primitive hematopoietic cells, the long-term culture-initiating cells (LTC-ICs), are present in Ph-chromosome–negative cytapheresis samples collected during early recovery after chemotherapy for CML.93 These LTC-ICs are most commonly present when samples are collected within 3 months of diagnosis.94 Variable levels of BCR-ABL–negative progenitors are found in the CD34+DR– population, but low levels are found in the CD34+CD38– population.90,95 Preprogenitors for the CD34+DR– cells are predominantly BCR-ABL–negative in both marrow and blood at diagnosis.96 However, some cells with surface marker characteristics of very primitive normal hematopoietic cells do express the BCR-ABL gene.97 Both normal and leukemic SCID-repopulating cells coexist in the marrow and blood from CML patients in chronic phase, whereas only leukemic SCID-repopulating cells are detected in blast crisis.98,99
Progenitor Cell Characteristics
Progenitor Cell Dysfunction
The leukemic transformation resulting from the BCR-ABL fusion oncogene is maintained by a relatively small number of BCR-ABL stem cells that favor differentiation over self-renewal.100 This predisposition to differentiation and progenitor cell expansion is mediated by an autocrine interleukin (IL)-3–granulocyte colony-stimulating factor (G-CSF) loop.100 The earliest progenitors have the capacity to undergo marked expansion of erythroid, granulocytic, and megakaryocytic cell populations, and have a decreased sensitivity to regulation.100–102 This expansion is especially dramatic in the more mature progenitor cell compartment.100,103 The proliferative capacity of individual granulocytic progenitors is decreased compared to normal cells. Thus, the progenitor cell population in marrow and blood expands proportionately more than the increase in granulopoiesis.104,105 Moreover, the progenitors have buoyant density that is lighter than that of their normal counterparts but similar to that of hepatic fetal granulopoietic progenitors, suggesting an oncofetal pattern.104 The marked expansion of the total blood granulocyte pool results from a total expansion of granulopoiesis,103,106 with a minor contribution from prolonged intravascular circulation time.107 BCR-ABL reduces growth factor dependence of progenitor cells.
Erythroid progenitors are expanded, erythroid precursor maturation is blocked at the basophilic erythroblast stage, and the extent of erythropoiesis is inversely proportional to the total white cell count.108
Progenitor Cell Characterization
Phenotypic differences of stem and progenitor cells in CML patients compared to normal subjects have been identified.109 For example, a greater proportion of the circulating leukemic colony-forming unit–granulocyte-monocytes (CFU-GMs) express high levels of the adhesion receptor CD44110 and low levels of L-selectin111 in contrast to normal cells. Leukemic CD34+ cells overexpress the P glycoprotein that determines the multidrug resistance phenotype.112
BCR-ABL–positive progenitors survive less well in long-term culture than do their normal counterparts. Leukemic CFU-GM colonies, unlike normal colonies, decrease in long-term cultures that are deficient in KIT ligand,113 whereas their proliferation is favored in the presence of KIT ligand.114 Macrophage inflammatory protein (MIP)-1 does not inhibit growth factor-mediated proliferation of CD34+ cells from CML patients, as it does CD34+ cells from normal subjects, even though the MIP-1 receptor is expressed.115 Another chemokine, monocyte chemotactic protein (MCP)-1, unlike MIP-1, is an endogenous chemokine that cooperates with transforming growth factor beta (TGF-) to inhibit the cycling of primitive normal, but not CML, progenitors in long-term human marrow cultures.116 Leukemic progenitors are less sensitive than normal progenitors to the antiproliferative effects of TGF-.117
Effects of BCR-ABL on Cell Adhesion
Primitive progenitors and blast colony-forming cells from patients with CML have decreased adherence to marrow stromal cells.118,119 This defect is normalized if stromal cells are treated with interferon (IFN)-.119,120 As a result, BCR-ABL–negative progenitors are enriched in the adherent fraction of circulating CD34+ cells in chronic phase CML patients. The most primitive BCR-ABL–positive cells in the blood of patients with CML differ from their normal counterparts. They are increased in frequency and are activated, such that signals that block cell mitosis are bypassed.121
Ph-chromosome–positive colony-forming cells adhere less to fibronectin (and to marrow stroma) than do their normal counterparts. Adhesion is fostered as a result of restoration of cooperation between activated 1 integrins and the altered epitopes of CD44.122–124 CML granulocytes have reduced and altered binding to P-selectin because of modification in the CD15 antigens.125 BCR-ABL–induced defects in integrin function may underlie the abnormal circulation and proliferation of progenitors126,127 because growth signaling can occur through the fibronectin receptor.128 IFN- restores normal integrin-mediated inhibition of hematopoietic progenitor proliferation by the marrow microenvironment.129 There are conflicting data regarding the effects of tyrosine kinase inhibitor effects on adhesion of CML cells to stroma.130,131
BCR-ABL–encoded fusion protein p210BCR–ABL binds to actin, and several cytoskeletal proteins are thereby phosphorylated. The p210BCR–ABL interacts with actin filaments through an actin-binding domain. BCR-ABL transfection is associated with increased spontaneous motility, membrane ruffling, formation of long actin extensions (filopodia), and accelerated rate of protrusion and retraction of pseudopodia on fibronectin-coated surfaces. IFN- treatment slowly converts the abnormal motility phenotype of BCR-ABL–transformed cells toward normal.132 Integrins regulate the c-ABL–encoded tyrosine kinase activity and its cytoplasmic nuclear transport.133 The p210BCR–ABL abrogates the anchorage requirement but not the growth factor requirement for proliferation.134
In normal cells exposed to IL-3, paxillin tyrosine residues are phosphorylated. In cells transformed by p210BCR–ABL, the tyrosines of paxillin, vinculin, p125FAK, talin, and tensin are constitutively phosphorylated. Pseudopodia enriched in focal adhesion proteins134,135 are present in cells expressing p210BCR–ABL.
The sum of evidence suggests that defects in adhesion (contact and anchoring) of CML primitive cells remove them from their controlling signals normally received from microenvironmental cells via cytokine messages. These signals retain the balance among cell survival, cell death, cell proliferation, and cell differentiation. Inappropriate phosphorylation of cytoskeletal proteins, possibly independent of tyrosine kinase, is thought to be the key factor in disturbed integrin function of CML cells.
The genic disturbance became evident with the knowledge that CML was derived from a primitive cell containing a 22q– abnormality.6,11 The abnormal chromosome contained only 60 percent of the DNA in other G-group chromosomes.136 Cytogenetic analysis indicated the G-group chromosome involved was different from the extra G-group chromosome in Down syndrome, which had been assigned number 21. Thus, the former was assigned number 22—even though it proved to be slightly longer than the chromosome involved in Down syndrome.11,137 The Paris Conference on Nomenclature decided not to undo the concept that Down syndrome is trisomy 21 and assigned the Ph chromosome and its normal counterpart, 22.138 Using quinacrine (Q) and Giemsa (G) banding, Rowley12 reported in 1973 that the material missing from chromosome 22 was not lost (deleted) from the cell, but was translocated to the distal portion of the long arm of chromosome 9. The amount of material translocated to chromosome 9 was approximately equivalent to that lost from 22, and the translocation was predicted to be balanced.12 Moreover, the breaks were localized to band 34 on the long arm of 9 and band 11 on the long arm of 22. Therefore, the classic Ph chromosome is t(9;22)(q34;q11), abbreviated t(Ph) (Fig. 90–2). The Ph chromosome can develop on either the maternal or the paternal member of the pair.139
|Schematic of normal chromosome 9 showing the ABL gene between band q34 and qter of chromosome 22, which has the BCR and SIS genes between band q11 and qter. The t(9;22) is shown on the right. The ABL from chromosome 9 is transposed to the chromosome 22 M-bcr sequences, and the terminal portion of chromosome 22 is transposed to the long arm of chromosome 9. The 22q– is the Ph chromosome. bcr, breakpoint cluster region; c-SiS, cellular homologue of the viral simian sarcoma virus-transforming gene; IGL, gene for immunoglobulin light chains.(From De Klein A: Oncogene activation by chromosomal rearrangement in chronic myelocytic leukemia. Mutat Res 186:161, 1987, with permission.)
Mutation of ABL and BCR Genes
Mutations of the ABL gene on chromosome 9 and of the BCR gene on chromosome 22 are central to the development of CML (Fig. 90–3).140–142
|Schematic of the normal ABL and BCR genes and of the BCR-ABL fusion transcripts. In the upper panel of the diagram, the possible breakpoint positions in ABL are marked by vertical arrows. Note the position immediately upstream of the ABL locus of the 8604Met gene. The BCR gene contains 25 exons, including first (e1) and second (e2) exons. The positions of the three breakpoint cluster regions, m-bcr, M-bcr, and –bcr, are shown. The lower panel of the figure shows the structure of the BCR-ABL messenger RNA fusion transcripts. Breakpoints in –bcr result in BCR-ABL transcripts with an e19a2 junction. The associated number designates the exon (location) at which the break occurs in each gene.(From Verschraegen CF, Kantarjian HM, Hirsch-Ginsberg C, et al,179 with permission.)
In 1982, the human cellular homologue ABL of the transforming sequence of the Abelson murine leukemia virus was localized to human chromosome 9.143 In 1983, ABL was shown to be on the segment of chromosome 9 that is translocated to chromosome 22144 by demonstrating reaction to hybridization probes for ABL only in somatic cell hybrids of human CML cells containing 22q– but not those containing 9q+. v-abl is the viral oncogenic homologue of the normal cellular ABL gene. This gene (v-abl) can induce malignant transformation of cells in culture and can induce leukemia in susceptible mice.145
The ABL gene is rearranged and amplified in cell lines from patients with CML.146 Cell lines and fresh isolates of CML cells contain an abnormal, elongated 8-kb RNA transcript,147–150 which is transcribed from the new chimeric gene produced by the fusion of the 5′ portion of the BCR gene left on chromosome 22 with the 3′ portion of the ABL gene translocated from chromosome 9144 (Fig. 90–4). The fusion messenger RNA (mRNA) leads to the translation of a unique tyrosine phosphoprotein kinase of 210 kDa (p210BCR–ABL), which can phosphorylate tyrosine residues on cellular proteins similar to the action of the v-abl protein product.151–155 The anomalous tyrosine kinase is difficult to identify in chronic phase cells because of inhibitors in granulocytes;155 molecular variants reflect variations in the breakpoint on chromosome 22.156
|Molecular effects of the Ph chromosome translocation t(9;22)(q34;q11). The upper panel shows the physically joined 5′ BCR and the 3′ ABL regions on chromosome 22. The exons are solid (from chromosome 22, BCR) and hatched (from chromosome 9, ABL). The middle panel depicts transcription of chimeric messenger RNA. The lower panel shows the translated fusion protein with the amino-terminus derived from the BCR of chromosome 22 and the carboxy-terminus from the ABL of chromosome 9.(From De Klein A: Oncogene activation by chromosomal rearrangement in chronic myelocytic leukemia. Mutat Res 186:161, 1987, with permission.)
The ABL locus contains at least two alleles, one having a 500-bp deletion.157 In normal cells, the ABL protooncogene codes for a tyrosine kinase of molecular weight 145,000, which is translated only in trace quantities and lacks any in vitro kinase activity.152 The fusion product expressed by the BCR-ABL gene is hypothesized to lead to malignant transformation because of the abnormally regulated enzymatic activity of the chimeric tyrosine protein kinase.153,154,158,159 Construction of BCR-ABL fusion genes indicated that BCR sequences could also activate a microfilament-binding function, but the tyrosine kinase and microfilament-binding functions were not linked. Nevertheless, tyrosine kinase modification of actin filament function has been proposed as a step in leukemogenesis.160
p210Bcr–Abl Fusion Protein
The breakpoints on chromosome 9 are not narrowly clustered, ranging from approximately 15 to more than 40 kb upstream from the most proximate region (first exon) of the ABL gene.143,144,161 The breakpoints on chromosome 22 occur over a very short, approximately 5 to 6 kb, stretch of DNA referred to as the breakpoint cluster region (M-bcr),162,163 which is part of a much longer breakpoint cluster region gene, BCR164,165 (see Fig. 90–4). Three main breakpoint cluster regions have been characterized on chromosome 22: major (M-bcr), minor (m-bcr), and micro (-bcr). The three different breakpoints result in a p210, p190, and p230 fusion protein, respectively (see Fig. 90–3). The overwhelming majority of CML patients have a BCR-ABL fusion gene that encodes a fusion protein of 210 kDa (p210BCR–ABL), for which mRNA transcripts have e14a2 or a e13a2 fusion junction (see Fig. 90–3).166 The “e” represents the BCR exon and “a” the ABL exon sites involved in the translocation. A BCR-ABL with an e1a2 type of junction has been identified in approximately 50 percent of the Ph chromosome–positive acute lymphoblastic leukemia cases and results in the production of a BCR-ABL protein of 190 kDa (p190BCR–ABL). Almost all CML cases at diagnosis that encode a p210BCR–ABL also express BCR-ABL transcripts for p190.167 The biologic or clinical significance of these dual transcripts is not known. Transgenic mice expressing p210BCR–ABL develop acute lymphoblastic leukemia in the founder mice, but all transgenic progeny have a myeloproliferative disorder resembling CML.168
The BCR gene encodes a 160-kDa serine-threonine kinase, which, when it oligomerizes, autophosphorylates and transphosphorylates several protein substrates.169 Aberrant methylation of the M-bcr in CML occurs.166 The first exon sequences of the BCR gene potentiate the tyrosine kinase of ABL when they fuse as a result of the translocation.170 The central portion of BCR has homology to DBL, a gene involved in the control of cell division after the S phase of the cell cycle. The C-terminus of BCR has a guanosine triphosphatase (GTPase)-activating protein for p21rac, a member of the RAS family of guanosine triphosphate (GTP)-binding proteins.171 A reciprocal hybrid gene ABL-BCR is formed on chromosome 9q+ when BCR-ABL fuses on chromosome 22. The ABL-BCR fusion gene actively transcribes in most patients with CML.172
Variations in breakpoints involving smaller stretches of chromosome 9 and rearrangements outside the M-bcr of chromosome 22 can occur.37 In a few cases of CML with no evident elongation of chromosome 9, molecular probes have shown that ABL still is translocated to chromosome 22.173 In occasional patients with Ph-chromosome–positive CML, the break in chromosome 22 is outside the M-bcr, and transcription of a fusion RNA of the usual type fails or a fusion RNA is transcribed that does not hybridize with the classic M-bcr complementary DNA (cDNA) probe.174
In cases in which the Ph chromosome is not found, BCR-ABL still may be located on chromosome 9 (a masked Ph chromosome).175 The BCR gene can recombine with genomically distinct sites on band 11q13 in complex translocations in a region rich in Alu repeat elements.176 ETV6/ABL fusion genes have also been found in BCR-ABL–negative CML.177
The BCR breakpoint site has been examined as a factor in disease prognosis. Some studies have shown no correlation between CML chronicity and breakpoint site, although thrombocytosis may be more common with 3′ breakpoint sites and basophilia with 5′ breakpoint sites.178 No difference in response to IFN- therapy was noted, and survival was not significantly different, although patients with 3′ deletions tended to have shorter survival.179 Others have observed a better response to IFN- in patients with a 3′ rearrangement, which is being examined with imatinib mesylate therapy.180
CML patients with m-bcr breakpoints develop a blast crisis with monocytosis and an absence of splenomegaly and basophilia.181 The p230 (e19a2 RNA junction) encoded by –bcr is rarely expressed but has been associated with neutrophilic CML or thrombocytosis (see “Special Clinical Features” below). Other rare breakpoints have been described.182 For example, a case with a 12-bp insert between BCR1 and ABL1 resulted in a BCR-ABL–negative (false-negative), Ph-chromosome–positive CML with thrombocythemia.183 Another novel BCR-ABL fusion gene (e6a2) in a patient with Ph-chromosome–negative CML encoded an oncoprotein of 185 kDa.184 Typical CML also has been associated with an e19a2 junction BCR-ABL transcript.185
Experimental support for the hypothesis that p210BCR–ABL tyrosine phosphoprotein kinase is transforming is provided by a retroviral gene transfer system that permits expression of the protein. Mouse marrow cells transfected with BCR-ABL develop clonal outgrowths of immature cells expressing the p210BCR–ABL tyrosine kinase. Some clones progress to a malignant phenotype, can be transplanted, and can induce tumors in syngeneic mice.186 Similar studies suggest that the p210BCR–ABL can transform 3T3 murine fibroblasts if the gag gene sequence from a helper virus cooperates.187 The BCR-ABL gene from a retroviral vector has been expressed in an IL-3–dependent cell line. Clones derived from the infected line transform over months to IL-3 independency, are capable of increased proliferation, and develop chromosomal abnormalities.188
A series of mouse models in which the BCR-ABL was used to induce leukemogenesis have been described.189–197 Lethally irradiated mice have been reconstituted with marrow enriched for cycling stem cells infected with a BCR-ABL–bearing retrovirus. Fatal diseases with abnormal accumulations of macrophagic, erythroid, mast, and lymphoid cells develop.188 Classic CML did not occur, and complete transformation was not documented. The cell lines from spleen and marrow from mice with a BCR-ABL retrovirus infection were predominantly mast cells; however, in some cases these cell lines spontaneously switched to either erythroid and megakaryocytic, erythroid, or granulocytic lineages displaying maturation. They were transplantable (transformed) and contained the same proviral inserts as the original mast cell line.198 Murine marrow also has been infected with a retrovirus encoding p210BCR–ABL and transplanted into irradiated syngeneic recipients.189 Although several types of hematologic malignancies developed, a syndrome mimicking human CML also occurred. Mice transgenic for a p190BCR–ABL develop an acute lymphocytic leukemia (ALL) lymphoma syndrome190 that resembles human Ph-chromosome–positive ALL. When a p210BCR–ABL transcript is introduced into a mouse germ line (one-cell fertilized eggs), the p210 founder and progeny transgenic animals developed leukemia of B or T lymphoid or of myeloid origin after a relatively long latency period. In contrast, p190 transgenic mice exclusively developed leukemia of B-cell origin, with a relatively short period of latency. This finding was believed to be consistent with the apparent indolent nature of human CML during the chronic phase.191 When transgenic mice express p210BCR–ABL, the transgenes develop ALL, whereas the progeny develop a myeloproliferative disorder.192
Mouse models remain important for exploring the pathogenesis of the acute and chronic BCR-ABL–mediated leukemias in vivo and in examining the potential effects of new drugs targeted at BCR-ABL.199
BCR-ABL in Healthy Subjects
BCR-ABL fusion genes can be found in the leukocytes of some normal individuals using a two-step reverse transcriptase polymerase chain reaction assay. Thus, although BCR-ABL may be expressed relatively frequently at very low levels in hematopoietic cells, only infrequently do the cells acquire the additional changes necessary to produce leukemia. This may be a dosage effect.200
BCR-ABL and Signal Transduction
The tyrosine phosphoprotein kinase activity of p210BCR–ABL has been causally linked to the development of Ph-chromosome–positive leukemia in man.201–212 p210BCR–ABL is, unlike the ABL protein that is located principally in the nucleus, located in the cytoplasm making it accessible to a large number of interactions, especially components of signal transduction pathways.205,206,213 It binds and/or phosphorylates more than 20 cellular proteins in its role as an oncoprotein.206 A subunit of phosphatidylinositol 3′-kinase (PI3K) associates with p210BCR–ABL; this interaction is required for the proliferation of BCR-ABL–dependent cell lines and primary CML cells. Wortmannin, a nonspecific inhibitor of the p110 subunit of the kinase, inhibits growth of these cells.207
The pathways and interactions invoked by BCR-ABL acting on mitogen-activated protein kinases are multiple and complex.214,215
An RAF-encoded serine-threonine kinase activity is regulated by p210BCR–ABL. Downregulation of RAF expression inhibits both BCR-ABL–dependent growth of CML cells and growth factor–dependent proliferation of normal hematopoietic progenitors.208
The efficiency of cell transformation by BCR-ABL is affected by an adaptor protein that can relate tyrosine kinase signals to RAS. This involves growth factor receptor-bound protein-2 (GRB2). p210BCR–ABL also activates multiple alternative pathways of RAS.209 PI3K is constitutively activated by BCR-ABL, generates inositol lipids, and is dysregulated by the downregulation by BCR-ABL of polyinositol phosphate tumor suppressors, such as PTEN and SHIP1.213 Figure 90–5 demonstrates interaction of p210BCR–ABL with various mediators of signal transduction.
|Major intracellular signaling events associated with BCR/ABL. Constitutive activation of ABL protein tyrosine kinase (PTK) induces phosphorylation of the tyrosine moiety of various substrates, including autophosphorylation of BCR/ABL and complex formation of BCR/ABL with adaptor proteins. This process subsequently activates multiple intracellular signaling pathways, including RAS activation and phosphatidylinositol 3′-kinase (PI3-K) activation pathways. BCR/ABL also activates the c-MYC pathway, which involves ABL-SH2 domain. BCR/ABL inhibits apoptosis, possibly in part through upregulation of Bcl-2, and alters cellular adhesive properties, possibly by interacting with focal adhesion proteins and the actin cytomatrix. Broken lines indicate hypothetical pathways. ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; JNK, Jun N-terminal kinase; MEKK, MEK kinase; Sos, Son-of-sevenless; STAT, signal transducer and activator of transcription.(From Gotoh A, Broxmeyer HE: The function of BCR/ABL and related proto-oncogenes. Curr Opin Hematol 4:3, 1997, with permission.)
Reactive oxygen species are increased in BCR-ABL–transformed cells and may act as a second messenger to modulate enzymes regulated by the redox equilibrium. An increase in these reactive oxygen products is postulated to play a role in the acquisition of additional mutations as a result of production of reactive oxygen species through the chronic phase, contributing to the progression to accelerated phase.213,216
The adaptor molecule CRKL is a major in vivo substrate for p210BCR–ABL, and it acts to relate p210BCR–ABL to downstream effectors. CRKL is a linker protein that has homology to the v-crk oncogene product. Antibodies to CRKL can immunoprecipitate paxillin. Paxillin is a focal adhesion protein210 that is phosphorylated by p210BCR–ABL. The p210BCR–ABL may be physically linked to paxillin by CRKL. CRKL binds to CBL, an oncogene product that induces B cell and myeloid leukemias in mice.211 The Src homology 3 domains of CRKL do not bind to CBL, but they do bind BCR-ABL. Therefore, CRKL mediates the oncogenic signal of BCR-ABL to CBL. The p120CBL and the adaptor proteins CRKL and c-CRK also link c-abl, p190BCR–ABL, and p210BCR–ABL to the PI3K pathway.212 The p120CBL also coprecipitates with the p85 subunit of PI3K, CRKL, and c-CRK. The p210BCR–ABL may, therefore, induce the formation of multimeric complexes of signaling proteins.217 These complexes contain paxillin and talin and may explain some of the adhesive defects of CML cells.218
Hef2 also binds to CRKL in leukemic tissues of p190BCR–ABL transgenic mice. Hef2 is involved in the integrin signaling pathway219 and encodes a protein that accelerates GTP hydrolysis of RAS-encoded proteins and neurofibromin. The latter negatively regulates granulocyte-monocyte colony-stimulating factor (GM-CSF) signaling through RAS in hematopoietic cells.220 P62DOK, a constitutively tyrosine-phosphorylated, p120RAS GAP-associated protein, which is rapidly tyrosine phosphorylated upon activation of the c-kit receptor,221 is also associated with ABL.222
Nuclear factor (NF)-B activation is also required for p210BCR–ABL-mediated transformation.223 Expression of p210BCR–ABL leads to activation of NF-B–dependent transcription via nuclear translocation.224
Cell lines that express p210BCR–ABL also demonstrate constitutive activation of Janus-associated kinases (JAKs) and signal transducers and activators of transcription (STATs), usually STAT5.225 STAT5 is also activated in primary mouse marrow cells acutely transformed by the BCR-ABL226; p210BCR–ABL coimmunoprecipitates with and constitutively phosphorylates the common subunit of the IL-3 and GM-CSF receptors and JAK2.227 Both ABL and BCR are also multifunctional regulators of the GTP-binding protein family Rho228,229 and the growth factor-binding protein GRB2, which links tyrosine kinases to RAS and forms a complex with BCR-ABL and the nucleotide exchange factor Sos that leads to activation of RAS.230
The p210BCR–ABL also activates Jun kinase and requires Jun for transformation.231 In some CML cell lines, p210BCR–ABL is associated with the retinoblastoma (Rb) protein.232 Loss of the neurofibromatosis (NF1) tumor-suppressor gene, a RAS GTPase-activating protein, also is sufficient to produce a myeloproliferative syndrome in mice akin to human CML resulting from RAS-mediated hypersensitivity to GM-CSF.233
Effects of BCR-ABL on Apoptosis
Whether p210BCR–ABL influences the expansion of the malignant clone in CML by inhibiting apoptosis is uncertain. In one study, the survival of normal and CML progenitors was the same after in vitro incubation in serum-deprived conditions and after treatment with X-irradiation or glucocorticoids.234 p210BCR–ABL inhibits apoptosis by delaying the G2/M transition of the cell cycle after DNA damage.235 The p210BCR–ABL also may exert an antiapoptotic effect in factor-dependent hematopoietic cells.236,237
p210BCR–ABL does not prevent apoptotic death induced by human NK or lymphokine-activated killer cells directed against CML or normal cells.238 In accelerated and blast phases, apoptosis rates were lower in CML neutrophils. G-CSF and GM-CSF considerably decreased the rate of apoptosis in CML neutrophils.239
Patients with CML present with a somewhat shortened mean telomere length in granulocytic cells but not blood T lymphocytes at diagnosis, but considerable overlap exists in the distribution of telomere length with healthy individuals.240–242 The rate of shortening of telomere length during the chronic phase is correlated with a more rapid onset of accelerated phase.240,242 Telomerase reverse transcriptase (TERT) is the catalytic subunit, expression of which is closely correlated with telomerase activity. In CML CD34+ cells containing BCR-ABL, the expression of TERT is significantly lower than in normal CD34+ cells, consistent with accelerated shortening of telomeres in CML cells.243 A further significant decrease in telomere length occurs in the accelerated phase of CML. Telomerase activity is increased in the accelerated phase.244 When therapy permits restoration of Ph-negative cells in the blood, these cells have telomere length comparable to that in matched healthy controls.245
Signs and Symptoms Chronic myelogenous leukemias (CMLs)
In the 70 percent of patients who are symptomatic at diagnosis, the most frequent complaints include easy fatigability, loss of sense of well-being, decreased tolerance to exertion, anorexia, abdominal discomfort, early satiety (related to splenic enlargement), weight loss, and excessive sweating.246–248 The symptoms are vague, nonspecific, and gradual in onset (weeks to months). A physical examination may detect pallor and splenomegaly. The latter was present in approximately 90 percent of patients at diagnosis, but with medical care being sought earlier, the presence of splenomegaly at the time of diagnosis is decreasing in frequency.247 Sternal tenderness, especially the lower portion, is common; occasionally, patients notice it themselves.
Uncommon presenting symptoms include those of dramatic hypermetabolism (night sweats, heat intolerance, weight loss) simulating thyrotoxicosis; acute gouty arthritis, presumably related in part to hyperuricemia; priapism, tinnitus, or stupor from the leukostasis associated with greatly exaggerated blood leukocyte count elevations249–251; left upper quadrant and left shoulder pain as a consequence of splenic infarction and perisplenitis; vasopressin-responsive diabetes insipidus252,253; and acne urticata associated with hyperhistaminemia.254 Acute febrile neutrophilic dermatosis (Sweet syndrome), a perivascular infiltrate of neutrophils in the dermis, can occur. In the latter situation, fever accompanied by painful maculonodular violaceous lesions on the trunk, arms, legs, and face are characteristic.255,256 Spontaneous rupture of the spleen is a rare event.257,258 Digital necrosis has been reported as a rare paraneoplastic event.259,260
In an increasing proportion of patients, the disease is discovered, coincidentally, when blood cell counts are measured at a periodic medical examination.
The presumptive diagnosis of CML can be made from the results of the blood cell counts and examination of the blood film.26,246,247 The blood hemoglobin concentration is decreased in most patients at the time of diagnosis. Red cells usually are only slightly altered, with an increase in variation from small to large size and only occasional misshapen (elliptical or irregular) erythrocytes. Small numbers of nucleated red cells are commonly present. The reticulocyte count is normal or slightly elevated, but clinically significant hemolysis is rare.246,261,262 Rare cases of mild erythrocytosis263,264 or erythroid aplasia265,266 have been documented.
The total leukocyte count is always elevated at the time of diagnosis and is nearly always greater than 25,000/L (25 x 109/L); at least half the patients have total white counts greater than 100,000/L (100 x 109/L) (Fig. 90–6).26,246,247 The total leukocyte count rises progressively in untreated patients. Rare patients may have dramatic cyclic variations in white cell counts as much as an order of magnitude with cycle intervals of approximately 60 days.267,268 Granulocytes at all stages of development are present in the blood and are generally normal in appearance (Fig. 90–7). The mean blast cell prevalence is approximately 3 percent but can range from 0 to 10 percent; progranulocyte prevalence is approximately 4 percent; myelocytes, metamyelocytes, and bands account for approximately 40 percent; and segmented neutrophils account for approximately 35 percent of total leukocytes (Table 90–1). Often, there is a “myelocyte bulge” in which the differential count shows an exaggerated proportion of myelocytes compared to the proportion observed in normal persons. Hypersegmented neutrophils are commonly present.
|Table 90–1. White Blood Cell Differential Count at the Time of Diagnosis in 90 Cases of pH-Chromosome–Positive Chronic Myelogenous Leukemia
||Percent of Total Leukocytes (Mean Values)
|Nucleated red cells
|NOTE: In these 90 patients, the mean hematocrit was 31 mL/dL, mean total white cell count was 160 x 109/L, and mean platelet count was 442 x 109/L at the time of diagnosis.SOURCE: Hematology Unit, University of Rochester Medical Center.
Neutrophil alkaline phosphatase activity is low or absent in more than 90 percent of patients with CML.269–271 The mRNA for alkaline phosphatase is undetectable in neutrophils of patients with CML.272 The activity increases toward or to normal in the presence of intense inflammation or infection and when the total leukocytic count is decreased to or near normal with treatment.271,273 CML neutrophils regain alkaline phosphatase activity after infusion into leukopenic recipients, suggesting the effect of regulators or factors extrinsic to the neutrophils.274 In vitro, a monocyte-derived soluble mediator is capable of inducing increased alkaline phosphatase activity in neutrophils from CML patients.275 Neutrophil alkaline phosphate is decreased sporadically in a variety of disorders and conditions,276 but is decreased markedly and consistently in paroxysmal nocturnal hemoglobinuria,276 in hypophosphatasia,277 in approximately one-fourth of patients with idiopathic myelofibrosis, and in patients using androgens. Neutrophil alkaline phosphatase is increased in polycythemia vera, in 25 percent of patients with idiopathic myelofibrosis, in pregnant women, and in subjects with inflammatory disorders or infections. With the advent of specific markers, BCR-ABL in CML and JAK2 mutations in polycythemia, leukocyte alkaline phosphatase is of limited diagnostic use.
The proportion of eosinophils usually is not increased, but the absolute eosinophil count nearly always is increased. Rarely, eosinophils are so prominent that they dominate the granulocytic cells and lead to the designation Ph-positive eosinophilic CML. An absolute increase in the basophil concentration is present in almost all patients, and this finding can be useful in preliminary consideration of the differential diagnosis.26,278 Basophilic progenitor cells are increased in the blood.279 The proportion of basophils usually is not greater than 10 to 15 percent during the chronic phase but may, in rare patients, represent 30 to 80 percent of the total leukocyte count during chronic phase and lead to the designation of Ph-chromosome–positive basophilic CML.280 Flow cytometry using anti-CD203c provides very accurate assessment of the basophil frequency. Basophils may be hypogranulated or have an immature phenotype and may be left uncounted in an optical differential count. Anti-CD203c recognizes these cells as basophils.281 Granules of basophils in patients with CML, unlike normal basophils, contain mast cell -tryptase.281,282 Granulocytes containing both eosinophilic and basophilic granules (mixed granulation) are commonly present.283
The total absolute lymphocyte count is increased (mean: approximately 15 x 109/L) in patients with CML at the time of diagnosis284 as a result of the balanced increase in T-helper and T-suppressor cells.285 B lymphocytes are not increased.288 T lymphocytes also are increased in the spleen.286 NK cell activity is defective in CML patients as a result of decreased maturation of these cells in vivo287,288 and a decrease in the absolute number of circulating NK cells in patients with CML. The latter change can perhaps be related to increased apoptosis.289 The CD56 bright subset of NK cells is particularly decreased. These cells are reduced more as CML progresses, and they respond less to stimuli that recruit clonogenic NK cells compared to NK cells from normal subjects.290
The platelet count is elevated in approximately 50 percent of patients at the time of diagnosis and is normal in most of the rest.291 The median value in patients at diagnosis is approximately 400,000 cells/L (400 x 109 cells/L). The platelet count may increase during the course of the chronic phase. Platelet counts greater than 1,000,000/L (1000 x 109/L) are not unusual, and platelet counts as high as 5,000,000 to 7,000,000/L (5000–7000 x 109/L) have occurred. Thrombohemorrhagic complications of thrombocytosis are infrequent. Occasionally, the platelet count may be below normal at the time of diagnosis, but this finding usually signals an impending progression to the accelerated phase of the disease (see “Accelerated Phase and Blast Crisis of CML” below).
Functional abnormalities of neutrophils (adhesion, emigration, phagocytosis) are mild; are compensated for by high neutrophil concentrations; and do not predispose patients in chronic phase to infections by either the usual or opportunistic organisms.292–294 Platelet dysfunction can occur but is not associated with spontaneous or exaggerated bleeding. A decrease in the second wave of epinephrine-induced platelet aggregation is the most common abnormality and is associated with a deficiency of adenine nucleotides in the storage pool.295,296
The marrow is markedly hypercellular, and hematopoietic tissue takes up 75 to 90 percent of the marrow volume, with fat markedly reduced (see Fig. 90–7).297,298 Granulopoiesis is dominant, with a granulocytic-to-erythroid ratio between 10:1 and 30:1, rather than the normal 2:1 to 4:1. Erythropoiesis usually is decreased, and megakaryocytes are normal or increased in number. Eosinophils and basophils may be increased, usually in proportion to their increase in the blood. Mitotic figures are increased in number. Uncommonly a juxtamembrane domain mutant of KIT coincides with BCR-ABL in CML.299 Rare reports of marrow mastocytosis have been explained by a KIT mutation as an additional genetic abnormality or by dual clones in the marrow.300,301 Macrophages that mimic Gaucher cells in appearance are sometimes seen. This finding is a result of the inability of normal cellular glucocerebrosidase activity to degrade the increased glucocerebroside load associated with markedly increased cell turnover.302 Macrophages also can become engorged with lipids, which, when oxidized and polymerized, yield ceroid pigment. This pigment imparts a granular and bluish cast to the cells after polychrome staining; such cells have been referred to as sea-blue histiocytes.302
Collagen type III (reticulin fibrosis), which takes the silver impregnation stain, is commonly increased at the time of diagnosis in nearly half the patients,303 and is correlated with the proportion of megakaryocytes in the marrow.304,305 Increased fibrosis also is correlated with larger spleen size, more severe anemia, and a higher proportion of marrow and blood blast cells.
The marrows of CML patients have a mean doubling of microvessel density compared to healthy controls and have more angiogenesis in marrow than other forms of leukemia.306–308 This increased marrow vascularity decreases to normal after treatment.309
The marrow cells of approximately 50 percent of patients express cancer testis antigens, especially those encoded by HAGE genes.310
Progenitor Cell Growth
Cells that form colonies of neutrophils and macrophages or eosinophils (CFUs) are increased in the marrow and blood. The increase in CFUs in marrow is approximately 20-fold normal and in blood approximately 500-fold normal. The CFUs are of lighter buoyant density than those in normal marrow.95 More primitive progenitors that can initiate long-term cultures of hematopoiesis also are markedly increased.311 Spontaneous blood-derived granulocyte-macrophage colony growth is common, although CFUs also respond to growth factor stimulation.105
The marrow and nucleated blood cells of more than 90 percent of patients with clinical and laboratory signs that fall within the criteria for the diagnosis of CML contain the Ph chromosome (22q–) as measured by G-banding, and virtually all patients have the t(9;22)(q34;q11)(BCR-ABL) by fluorescence in situ hybridization. The Ph chromosome is present in all blood cell lineages (erythroblasts, granulocytes, monocytes, megakaryocytes, T- and B-cell progenitors) but is not present in the majority of blood B lymphocytes or in most T lymphocytes.49,51 Approximately 70 percent of patients in the chronic phase have the classic Ph chromosome in their cells.312 The remaining 20 percent also have a missing Y chromosome [t(Ph),–Y]; an additional C-group chromosome, usually number 8 [t(Ph),+8]; an additional chromosome 22q– but without the 9q+ [t(Ph), 22q–]; or t(Ph) plus either another stable translocation or another minor clone.75 These variations have not been shown to affect the duration of the chronic phase. Deletion of the Y chromosome occurs in approximately 10 percent of healthy men older than 60 years.313,314
Variant Ph chromosome translocations occur in approximately 5 percent of subjects with CML and involve complex rearrangements (three chromosomes), and every chromosome except the Y chromosome can be involved.315–319 The Ph chromosome, that is, 22q–, is present, but the gross exchange of chromosomal material involves a chromosome other than 9 (simple variant) or involves exchange of material among chromosomes 9 and 22 and a third or more chromosomes (complex variant; see Fig. 90–8). High-resolution techniques have indicated that 9q34-qter is transposed to 22q11 in simple and in complex translocations.320,321 Thus, the fusion of 9q34 with 22q11 seems to occur in the cells of most patients with CML.323 Complex translocations involving chromosome 3 have been notable.322–324 In rare cases, a reciprocal translocation with a chromosome other than 9 to chromosome 22 is larger than usual, and the posttranslocation shortening of the long arms of 22 is not apparent. This circumstance has been referred to as a masked Ph chromosome or masked translocation because the 22q– is not evident by microscopic examination,325,326 although t(9;22) may occur as judged by banding techniques or molecular probes.327Approximately 10 percent of patients have a deletion of the derivative 9 chromosome adjacent to the chromosome breakpoint. Although this deletion is thought to be an important factor in resistance to drug effects with IFN therapy, it does not appear to be significant with the use of imatinib.214
In a small proportion of patients with a clinical disease analogous to CML, cytogenetic studies do not disclose a classic, variant, or masked Ph chromosome. In these cases, use of a panel of restriction enzymes and Southern blot analyses with a molecular probe for the breakpoint cluster region on chromosome 22 nearly always detects rearrangement of fragments. This finding has led to the conclusion that almost all cases of CML have an abnormality of the long arm of chromosome number 22 (BCR rearrangement).328–332 Ph-chromosome–negative CML cells with BCR rearrangement can express p210BCR–ABL, and such patients have a clinical course similar to Ph-chromosome–positive CML.328,333–336
The ability to identify the molecular consequences of the t(9;22), that is, BCR rearrangement, mRNA transcripts of the mutant fusion gene, and p210BCR–ABL, has resulted in diagnostic tests supplementary to cytogenetic analysis.332 These tests include Southern blot analysis of BCR rearrangement,334–338 polymerase chain reaction (PCR) amplification of the abnormal mRNA,339 and a less complex variation on the latter, a hybridization protection assay.340
PCR can achieve a sensitivity of one positive cell in approximately 500,000 to 1 million cells. This extreme sensitivity requires special care in analysis and the inclusion of negative controls.341–344 Immunodiagnosis of CML by identification of p210BCR–ABL also is possible. This tumor-specific protein for CML is unique, based on the amino acids at the junction between the ABL and BCR sequences. Oligopeptides corresponding to the junctional amino acids have been synthesized and used as antigens345–348 to develop specific antibodies to p210BCR–ABL.
A multicolor FISH method to detect the BCR-ABL fusion in patients with CML is a rapid and sensitive alternative to Southern blot and PCR-dependent methods.349 For diagnostic purposes, FISH is simple, accurate, and sensitive, and can detect the various molecular fusions (e.g., e13a2, e14a2, e1a2).350–354 Interphase FISH is faster and more sensitive than cytogenetics in identifying the Ph chromosome. If the concentration of CML cells is very low, interphase FISH may not detect BCR-ABL, so it has limited use for detecting minimal residual disease.355 Hypermetaphase FISH allows analysis of up to 500 metaphases per sample in 1 day. Several factors influence the false-positive and false-negative rates of FISH identification of BCR-ABL, including definition of a fusion signal, nuclear size, and the genomic position of the ABL breakpoint.356 Double BCR-ABL fusion signals (double-fusion [D]-FISH) have been proposed as being more accurate than the fusion signal used in dual color (single-fusion) S-FISH, because in the latter case a small percentage of the normal BCR and ABL signals overlap.357
The frequency of cytogenetic analysis can be reduced if patients are monitored by molecular methods such as quantitative Southern blotting, FISH, quantitative Western blotting, or competitive reverse transcriptase (RT)-PCR. Molecular analyses can be performed on blood samples and therefore are much easier to use than cytogenetic analysis of marrow cell metaphases. Quantitative RT-PCR is the method of choice for monitoring patients for residual disease or reappearance of disease after marrow transplantation and for following response to tyrosine kinase inhibitors once routine cytogenetics and FISH are negative for the Philadelphia chromosome. Competitive PCR can detect reappearance of or increasing levels of RNA BCR-ABL transcripts prior to clinical relapse in patients after transplantation.358–360
An increased production of uric acid with hyperuricemia and hyperuricosuria occurs in untreated CML.361 Uric acid excretion often is two to three times normal in patients with CML. If aggressive therapy leads to rapid cell lysis, excretion of the additional purine load may produce urinary tract blockage from uric acid precipitates. Formation of urinary urate stones is common in patients with CML, and some patients with latent gout may develop acute gouty arthritis or uric acid nephropathy.362 The likelihood of complications from urate overproduction is greatly increased by starvation, acidosis, renal disease, or diuretic drug therapy.
Serum Vitamin B12-Binding Proteins and Vitamin B12
Neutrophils contain vitamin B12-binding proteins, including transcobalamin I and III (synonym: R-type B12-binding protein or cobalophilin).363–366 Patients with myeloproliferative diseases have an increased serum level of B12-binding capacity, and the source of the protein is principally mature neutrophilic granulocytes.363,364 The increase in transcobalamin level and the resultant increase in vitamin B12 concentration are particularly notable in CML, although any increase in the number of neutrophilic granulocytes, as in leukemoid reactions, can be accompanied by an increase in serum B12-binding protein levels and vitamin B12 concentration.366 The serum B12 level in CML patients is increased on average to more than 10 times normal.367 The increase is proportional to the total leukocyte count in untreated patients and falls toward normal levels with treatment, although increased B12 levels commonly persist even after the white cell count is lowered to near normal with therapy.
Pernicious anemia and CML may rarely coexist. In this situation, the tissues are vitamin B12 deficient, but the serum vitamin B12 level may be normal because of the elevated level of transcobalamin I, a binder with a very high affinity for vitamin B12.367
Whole Blood Histamine
Mean histamine levels are markedly increased in patients in chronic phase (median: ~5000 ng/mL) compared to healthy individuals (median: ~50 ng/mL); and, this elevation is correlated with the blood basophil count.368 Cases of exaggerated basophilia and disabling pruritus, urticaria, and gastric hyperacidity have occurred, associated with enormous increases (several hundred-fold) of blood histamine concentration.369,370
Serum Lactic Dehydrogenase, Potassium, Calcium, and Cholesterol
The level of serum lactic acid dehydrogenase (LDH) is elevated in CML.371 Pseudohyperkalemia resulting from the release of potassium from white cells during clotting372 and spurious hypoxemia or pseudohypoglycemia from in vitro utilization of oxygen or glucose by granulocytes can occur. Hypercalcemia373 or hypokalemia374 has occurred during the chronic phase of the disease, but such complications are very rare until the disorder transforms to acute leukemia. Elevated serum and urinary lysozyme levels are features of leukemia with greater monocytic components and are not features of CML.375 Serum cholesterol is decreased in patients with CML.376,377
Serum Angiogenic Factors
Angiogenin, endoglin (CD105), vascular endothelial growth factor (VEGF), -fibroblast growth factor, and hepatocyte growth factor are increased strikingly in the serum of CML patients.307,308,378,379
|Special Clinical FeaturesBCR-ABL–Positive ThrombocythemiaEither of two syndromes—thrombocythemia with the Ph chromosome and BCR-ABL rearrangement or thrombocythemia without a Ph chromosome but with the BCR-ABL rearrangement—may precede the overt signs of CML or its accelerated phase.380–386 In general, the disease closely mimics classic thrombocythemia initially: marked platelet elevation, extreme megakaryocytic hyperplasia, normal or mildly elevated white cell count, no or very slight myeloid immaturity in the blood, and minimal anemia. Minor bleeding, such as epistaxis, erythromelalgia, or signs of thrombosis, such as cerebral or limb ischemia, are occasionally present.387 In some cases, the absolute basophil count is mildly elevated. Using immunostaining, Ph-positive thrombocythemia is proposed to be distinctive from Ph-negative thrombocythemia by small megakaryocytes in the former and by large clusters of megakaryocytes in the latter.387 However, this distinction was not found in other studies,388 has to be validated, and is difficult to use as a discriminator. In two studies, approximately 5 percent of patients with apparent essential thrombocythemia had a Ph chromosome.382,389 In another study, 2 of 121 patients with essential thrombocythemia had BCR-ABL transcripts, and 1 of these patients also had a Ph chromosome in the marrow cells,390 whereas in a different study, 4 of 32 patients with thrombocythemia had low levels of BCR-ABL transcripts in blood cells.391 Approximately 1 in 20 patients with CML present with the features of essential thrombocythemia.383,384 Evolution to blast crisis may occur.381,392,393 Thus, the frequency of Ph-chromosome–negative, BCR-ABL–positive thrombocythemia ranges from approximately 1.6 to 13 percent of patients, which may reflect in part the range of sensitivity of the detection method.389–391,394,395Neutrophilic CMLA rare variant of BCR-ABL–positive CML has been described in which the elevated white cell count is composed principally of mature neutrophils.396,397 The white cell count is lower on average (30,000–50,000/L) at the time of diagnosis than is the case with classic CML (median, 100,000–150,000/L). Moreover, patients with neutrophilic CML usually do not have basophilia, notable myeloid immaturity in the blood, prominent splenomegaly, or low leukocyte alkaline phosphatase scores. The cells of these patients have the Ph chromosome but have an unusual BCR-ABL fusion gene in that the breakpoint in the BCR gene is between exons 19 and 20. This breakpoint location results in fusion of most of the BCR gene with ABL (e19a2 type BCR-ABL), which leads to a larger fusion protein (230 kDa) compared to the fusion protein in classic CML (210 kDa; see Fig. 90–3). This correlation between genotype and phenotype has not been observed in all cases.398 This variant usually has an indolent course, which may be the result of very low levels of mRNA for p230 and the undetectable or barely detectable p230 protein in cells.399
Minor-BCR Breakpoint–Positive CML
A small proportion of patients with BCR-ABL–positive CML have the breakpoint on the BCR gene in the first intron (m-bcr), resulting in a 190-kDa fusion protein instead of the classic 210-kDa protein observed in most patients with CML (see Fig. 90–3). The m-bcr molecular lesion is similar to that observed in approximately 60 percent of patients with BCR rearrangement-positive ALL. In patients with m-bcr CML, monocytes are more prominent, the white cell count is lower on average, and basophilia and splenomegaly are less prominent than in disease with classic BCR breakpoint (M-bcr). The few reported cases had a short interval before either myeloid or lymphoid blast transformation developed.400,401
Approximately 15 percent of patients present with symptoms or signs referable to leukostasis as a result of the intravascular flow-impeding effects of white cell counts greater than 300,000/L (300 x 109/L).249 Hyperleukocytosis is more prevalent in children with Ph-chromosome–positive CML.250 The effects of total leukocyte counts from 300,000 to 800,000/L (300–800 x 109/L) include impaired circulation of the lung, central nervous system, special sensory organs, and penis, resulting in some combination of tachypnea, dyspnea, cyanosis, dizziness, slurred speech, delirium, stupor, visual blurring, diplopia, retinal vein distention, retinal hemorrhages, papilledema, tinnitus, impaired hearing, and priapism.251 In asymptomatic patients with hyperleukocytosis, initial treatment with hydration and hydroxyurea usually can be used to decrease the white cell count. Hydroxyurea treatment should be designed to accomplish a gradual decrease in white cell count over a few days so as to avoid the tumor lysis syndrome. If signs of hyperleukocytosis are present, hydration, leukapheresis, and hydroxyurea can be used simultaneously; hydroxyurea dose should be selected to avoid exaggerated tumor lysis.
Concurrence of Lymphoid Malignancies
CML has an association with lymphoproliferation that can take four principal forms. (1) Patients may develop CML years after irradiation treatment of non-Hodgkin or Hodgkin lymphoma. (2) Approximately one-third of CML patients enter the accelerated phase of the disease by evolution and dedifferentiation of the CML clone into one that supports lymphoblastic proliferation (acute lymphoblastic transformation). (3) Patients may have concurrent lymphoproliferative or plasmacytic malignancies and CML. Lymphoma or lymphoblastic leukemia,402–408 essential monoclonal gammopathy,409,410 myeloma,411–413 and Waldenström macroglobulinemia414 have occurred in association with CML. Several cases of CML emergence in patients with established chronic lymphocytic leukemia (CLL) have been reported.415–417 A few patients have presented with simultaneous occurrence of the two diseases.418,419 A single case of lymphocytic leukemoid reaction simulating CLL that regressed as CML emerged has been reported.420 In some cases, the CLL lymphocytes did not contain the Ph chromosome, whereas the CML cells did, suggesting the presence of two independent clonal disorders.415,416,421,422 In other cases, the Ph chromosome was present in the myeloid and lymphoid cells, indicating a common origin.419 (4) Patients may present with Ph-chromosome–positive acute lymphoblastic leukemia and, following chemotherapy-induced remission, develop the features of typical CML.420
Diseases Mimicking CML
The diagnosis of CML is made based on the characteristic granulocytosis, white cell differential count, increased absolute basophil count, and splenomegaly coupled with the presence of the Ph chromosome or its variants (90% of patients) or a BCR rearrangement on chromosome 22 (>95% of patients).
Patients with other chronic hematopoietic stem cell diseases, such as polycythemia vera, essential thrombocythemia, or primary myelofibrosis, only occasionally have closely overlapping features. For example, the total white cell count is greater than 30 x 109/L in more than 90 percent of patients with CML and increases inexorably over weeks or months of observation, whereas the total white cell count is less than 30 x 109/L in more than 90 percent of patients with the three other classic chronic clonal myeloid diseases and usually does not change significantly over months to years. Polycythemia vera is associated with increased red cell mass and hemoglobin concentration and displays clinical signs of plethora; CML does not have these features. Patients with primary myelofibrosis invariably have marked teardrop poikilocytes and other severe red cell shape, size, and chromicity changes, as well as prominent nucleated red cells in the blood; CML rarely has these features. Patients with essential thrombocythemia have a platelet count greater than 450,000/L (450 x 109/L) and usually only mild neutrophilia (<20,000/L); the slight neutrophilia distinguishes it from the proportion (~25%) of CML patients with platelet counts greater than 450,000/L (450 x 109/L), who at the time of diagnosis have white cell counts above 25,000/L. In addition, patients with the clinical features of polycythemia vera or primary myelofibrosis do not have the Ph chromosome or BCR rearrangement in their blood and marrow cells, except in extremely rare cases. A very small proportion of patients with apparent essential thrombocythemia have BCR-ABL transcripts in their marrow and blood cells, and occasionally a Ph chromosome and may represent an atypical initial phase of CML (see “BCR-ABL–Positive Thrombocythemia” above). The presence of a mutation in the JAK2 gene in approximately 95 percent of patients with polycythemia vera and in approximately 40 to 50 percent of patients with primary myelofibrosis or essential thrombocythemia by current assay techniques is a very useful distinguishing feature when present. More sensitive assays for JAK2 may make this marker even more useful in discrimination of these myeloproliferative diseases from CML.423
Increased awareness of the features of related disorders, such as chronic myelomonocytic leukemia (CMML) and chronic neutrophilic leukemia, and an appreciation that older patients are prone to atypical clonal myeloid diseases, have minimized the inappropriate diagnosis of Ph-chromosome–negative CML, which should be avoided unless the clinical features are characteristic of classic CML and a masked Ph chromosome or BCR rearrangement is not found.
Reactive leukocytosis can occur with absolute neutrophil counts of 30,000 to 100,000/L (30–100 x 109/L). Usually these leukemoid reactions occur in the setting of an overt inflammatory disease (e.g., pancreatitis), cancer (e.g., lung), or infection (e.g., pneumococcal pneumonia). If the incitant is not apparent, the absence of granulocytic immaturity, basophilia, splenomegaly, and decreased neutrophil alkaline phosphatase activity argue against CML. The absence of a cytogenetic or molecular abnormality in chromosome 22 virtually eliminates classic CML as a consideration.
The precise diagnosis of CML is helpful in estimating the patient’s prognosis, determining the potential response to tyrosine kinase inhibitors, and assessing the timing of special therapies, such as allogeneic hematopoietic stem cell transplantation.
pH-Chromosome–Positive Clonal Myeloid Diseases and Aplastic Anemia
The Ph chromosome has been found rarely in patients with apparent polycythemia vera,27,424 polycythemia vera that later evolves into Ph-chromosome–positive CML,425–427 idiopathic myelofibrosis,428,429 and myelodysplastic syndrome (MDS).430,431 Molecular studies to determine the presence of the BCR-ABL were not performed in cases reported before 1985. Primary (essential) thrombocythemia with a Ph chromosome and/or BCR-ABL rearrangement in blood cells was discussed earlier (see “Special Clinical Features” above). Rare cases of aplastic anemia have presented with BCR-ABL–positive cells or have evolved into BCR-ABL CML.432,433 The frequency of this association is uncertain because of the inability to have sufficient cells for a cytogenetic analysis during the period of aplasia and the unavailability in past years of FISH or PCR to test for the BCR-ABL gene in patients with this clinical presentation.
Chronic myelogenous leukemias (CMLs)Therapy
Hyperuricemia and hyperuricosuria are frequent features of CML at diagnosis or in relapse.434 The need for treatment of hyperuricemia is a function of the elevated pretreatment serum uric acid concentration, blood white cell concentration, spleen size, and dose of chemotherapy planned. If these variables suggest a high risk for a significant amount of cell lysis, allopurinol 300 mg/day orally and adequate hydration to maintain a good urine flow should be instituted prior to therapy. Allopurinol is associated with a high frequency of allergic skin reactions and should be discontinued after the blood leukocyte count and spleen size have d ecreased and the risk of exaggerated cell lysis has passed. If hyperuricemia is extreme, usually over 9 mg/dL, alkalinization of urine can be achieved with sodium bicarbonate, and rasburicase can be administered.435 Rasburicase is a recombinant urate oxidase that converts uric acid to allantoin. Rasburicase, unlike allopurinol, reduces the uric acid pool very rapidly, does not result in the accumulation of xanthine or hypoxanthine, and does not require alkalinization of urine facilitating phosphate excretion.436 Although the manufacturer recommends a dose every day for 5 days, several reports have indicated that one injection will produce a rapid and sustained decrease in serum uric acid, significantly decreasing the cost of therapy.437 Another alternative is to use allopurinol for a few days after one injection of rasburicase. A dose of 0.2 mg/kg of ideal body weight of rasburicase intravenously has been used.438
Initial Cytoreduction Therapy
Imatinib mesylate (imatinib) is now used as initial therapy in almost all patients with CML presenting in the chronic phase. In cases where the white cell count is markedly elevated, hydroxyurea can be used prior to or in conjunction with imatinib. If rapid cytoreduction is required because of signs of the hyperleukocytic syndrome, leukapheresis and hydroxyurea often are combined.
Leukapheresis can control CML only temporarily. For this reason, it is rarely used in chronic phase CML and is useful in only two types of patients: the hyperleukocytic patient in whom rapid cytoreduction can reverse symptoms and signs of leukostasis (e.g., stupor, hypoxia, tinnitus, papilledema, priapism),249–251 and in the pregnant patient with CML who can be controlled by leukapheresis treatment without other therapy either during the early months of pregnancy when therapy poses a higher risk to the fetus or, in some cases, throughout the pregnancy.439,440 Because of the large body burden of leukocytes in marrow, blood, and spleen, and the high proliferative rate in CML, leukocyte reduction by apheresis is less efficient than in other types of leukemias.249,251 Leukapheresis reduces the burden of tumor cells subject to chemotherapeutically induced cytolysis and thus the production and the excretion of uric acid. In hyperleukocytic nonpregnant patients, leukapheresis is best used in conjunction with hydroxyurea to ensure rapid and optimal reduction in white cell count.
Hydroxyurea 1 to 6 g/day orally, depending on the height of the white cell count, can be used to initiate elective therapy.441 Urgent treatment of extraordinary total white cell counts may require higher doses. The dose of hydroxyurea should be decreased as the total white cell count decreases and usually is given at 1 to 2 g/day when the total white cell count reaches 20,000/L (20 x 109/L). The drug should be temporarily discontinued if the white cell count drops below 5000/L (5 x 109/L). If hydroxyurea is being used in combination with imatinib, the hydroxyurea usually is tapered and discontinued once a hematologic response to imatinib is observed.
Anagrelide can be used for platelet reduction in patients who present with elevated platelet counts. This agent acts directly to decrease megakaryocyte mass, and it can lead to a precipitous fall in platelet counts. In occasional patients who still have significant thrombocythemia after imatinib is initiated, the combination of imatinib and anagrelide is associated with a normalization of platelet counts.442
Tyrosine Kinase Inhibitor Therapy
Patients with newly diagnosed chronic phase CML should be started on imatinib, 400 mg/day by mouth. Imatinib is easier to use, induces a higher frequency of hematologic remission, a higher frequency of complete cytogenetic remission, and greater suppression of the CML clone (molecular remission) than therapy with interferon (INF)-. The goal of imatinib therapy is to decrease the cells bearing the t(9;22) translocation (leukemic cells) to the lowest levels possible, under which conditions normal (polyclonal) hematopoiesis is restored. The efficacy of imatinib is judged by measuring the three benchmarks: hematologic response, cytogenetic response, and molecular response (Table 90–2).443,444 These are used to determine its maximal effect. The time to achieve a maximal effect is variable and can range from months to years. Thus, as long as a patient is having a continued reduction in the size of the leukemic clone as judged by cytogenetic or PCR measurements, the drug is continued at 400 mg/day. If the patient stops responding before a complete cytogenetic remission or complete molecular remission is achieved, the dose can be increased to 600 mg/day or to 800 mg/day (400 mg every 12 hours), if tolerated. About two-thirds of patients who do not have a significant hematologic response or who relapse while receiving imatinib at a dose of 400 mg/day achieve a complete or partial hematologic response with higher doses, but few cytogenetic responses occur.445 Some patients without a cytogenetic response can enter a partial or complete cytogenetic response with higher doses of imatinib. Unfortunately, the responses to higher doses of imatinib in patients lacking a hematologic or cytogenetic response at 400 mg/day usually are transient.446,447
|Table 90–2. Criteria for Extent of Imatinib Treatment Response
||White cell count <10 x 109/L, platelet count <450 x 109/L, no immature myeloid cells in the blood, and disappearance of all signs and symptoms related to leukemia (including palpable splenomegaly) lasting for at least 4 weeks.
|Major cytogenetic response
||Less than 35% of cells containing the Ph chromosome by cytogenetic analysis of marrow cells.
|Complete cytogenetic response
||No cells containing the Ph chromosome by cytogenetic analysis of marrow cells.
|Major molecular response
||Blood cell BCR-ABL/ABL ratio <0.05% (3-log reduction in PCR signal from mean pretreatment baseline value).
|Complete molecular response
||Blood cell BCR-ABL levels undetectable (usually by nested RT-PCR method).
Patients with newly diagnosed chronic phase CML treated with imatinib, 800 mg/day, administered in two 400 mg doses every 12 hours had a frequency of 90 percent complete cytogenetic responses and 96 percent had at least a major cytogenetic response. At a median of 15 months, no patients had progressed and 63 percent showed blood BCR-ABL/ABL percentage ratios of less than 0.05 percent. Twenty-eight percent of patients had undetectable BCR-ABL blood levels.448 Despite these reports the current starting dose is customarily 400 mg/day, balancing both effectiveness and tolerability in newly diagnosed patients. Moreover, the more rapid response with higher doses of imatinib may not translate into a better long-term survival.
Doses of imatinib lower than 400 mg/day result in fewer complete cytogenetic responses and a shorter duration of complete cytogenetic response. Patients who are older and who have lower body weight may only tolerate a lower dose but they are less likely to achieve a complete cytogenetic response.449 If however, a patient is on a lower dose (e.g., 300 mg/day) for a special reason (body size or tolerance level) and achieves a complete hematologic and cytogenetic response within 12 months of onset of therapy, acceptable outcomes without excess toxicity may result.450
After 5 years of experience with imatinib, the proportion of patients achieving a complete molecular response continues to increase. Imatinib has been shown to be safe and well tolerated in the majority of patients during this period of observation.451 Some patients have been studied for up to 7 years; the proportion with an undetectable BCR-ABL level in blood cells increased from 7 percent at 36 months to 52 percent at 84 months. During this time a major molecular response was lost in approximately 25 percent of patients with a detectable blood cell BCR-ABL signal. No patients with an undetectable blood cell BCR-ABL signal lost their major molecular response status after a median followup of 33 months.452
At 5 years, of the patients treated, the cumulative incidence of complete cytogenetic response was reached in more than 75 percent and of major molecular response was reached in more than 50 percent. The estimated overall survival and progression-free survival was approximately 80 percent of patients treated with imatinib at 400 mg/day for that period. By 5 years, 25 percent had discontinued imatinib treatment because of an unsatisfactory response or toxicity. The 5-year probability of remaining in major cytogenetic response while still receiving imatinib was approximately 60 percent. Achieving a complete cytogenetic response correlated with progression-free survival, but achieving a major molecular response conferred no further survival benefit.453
Use of Imatinib in Patients with Variant Chromosomal Translocations or Breakpoints
Patients with variant Ph chromosome translocations have a similar prognosis to that of patients with classic Ph chromosome translocations who are treated with imatinib.454 (See Fig. 90–3 for a diagram of breakpoints.) Patients with the e13a2 (formerly designated b2a2) p210BCR-ABL translocation respond well to imatinib, with similar rates of complete cytogenetic remission.455 The e13a2 transcript may be more sensitive to imatinib than the e14a2 (formerly designated b3a2) transcript.456 In a patient with both e1a2 and e14a2 fusion transcripts, only the p210 e14a2 transcript disappeared, whereas the e1a2 transcript persisted during progression to blast phase. No mutation in the kinase domain of ABL was found.457 This finding indicates that different clones in an individual patient may have a different sensitivity to imatinib.
Response to Imatinib in Children and Older Patients
More than 80 percent of children with chronic phase CML who are treated with imatinib, 260 to 570 mg/m2, enter a complete cytogenetic remission. Weight gain is the most common side effect of imatinib.458 In patients who were older than age 60 years, similar cytogenetic response rates and survival rates were noted as in younger patients in the late chronic phase who were treated concurrently, suggesting that age is not usually a factor in response.459,460
Side Effects and Special Treatment Considerations
Imatinib is relatively well tolerated. Most adverse effects are manageable and seldom require permanent cessation of therapy. Reduction to subtherapeutic doses is not recommended; it is better to interrupt therapy for a time.461
Myelosuppression is common in CML patients, especially at treatment onset when the CML clone accounts for most of blood cells. Dose reduction to less than 300 mg/day is not advisable for myelosuppression. The drug should be stopped until blood counts recover. G-CSF and GM-CSF can prevent or treat neutropenia.462,463 Platelet transfusion may be used for severe thrombocytopenia. Patients with imatinib-induced chronic cytopenias have inferior responses.464 Myelosuppression is an independent adverse factor for achieving cytogenetic responses with imatinib.465 Severe irreversible marrow aplasia after imatinib exposure has been reported.466
The main side effects noted with imatinib include fatigue, edema, nausea, diarrhea, muscle cramps, and rash.467 Elevated hepatic transaminases can occur. Mild transaminase elevations often respond to glucocorticoid use.468 Hepatotoxicity is uncommon, occurring in approximately 3 percent of patients, usually within 6 months of onset of imatinib use. Acute liver failure has been described.469 The severe periorbital edema occasionally observed is postulated to be an effect on platelet-derived growth factor receptor (PDGFR) and KIT expressed by dermal dendrocytes. Surgical decompression of severe edema rarely has been required.470 Although no effects on spermatogenesis have been reported, women of child-bearing age are at risk of teratogenic effects on a fetus.470
Uncommon side effects include splenic rupture,471 cerebral edema and visual disturbances resulting from retinal edema,472 varicella-zoster infection,473 gynecomastia,474 immune-mediated hemolytic anemia,475 severe muscle edema,476 severe fluid retention,477 interstitial lung disease,478,479 and panniculitis.480 Hypophosphatemia481 and altered bone and mineral metabolism have occurred.482,483
Cutaneous reactions with imatinib therapy occur in approximately 15 percent of patients.484 Except for severe reactions (approximately 5% of patients), such as Stevens-Johnson syndrome, exfoliative dermatitis, and erythema multiforme, cutaneous reactions rarely require permanent discontinuation of therapy. With milder reactions, concomitant glucocorticoid therapy or brief discontinuation of imatinib with gradual reintroduction at a lower dose and then a gradual increase in dose can be accomplished.485,486 With very mild cases, concurrent treatment with antihistamine or other symptomatic therapy may be successful. Oral desensitization regimens have been described that allow some patients to continue imatinib therapy.487 Sweet syndrome with CML cell infiltration has been reported at the time of molecular remission.488 Pityriasis rosea,489 palmoplantar hyperkeratosis,490 and oral and cutaneous lichenoid reactions,491 have also been described. Hair repigmentation492 and hypopigmentation of the skin,493 probably related to the inhibition of the KIT receptor tyrosine kinase by imatinib, have been reported. Imatinib has been proposed as a therapy for vitiligo.494
Other Effects of Imatinib
Imatinib has been found to cause regression of marrow fibrosis.495 One study found that the extent of marrow fibrosis in CML is not a prognostic factor with imatinib therapy,496 whereas another study observed that although imatinib reverses marrow fibrosis in patients with CML, it does not change the unfavorable prognosis associated with fibrosis.497
Imatinib reverses exaggerated VEGF secretion in patients with CML,498 and it may reverse exaggerated marrow angiogenesis.499 It can reduce marrow cellularity and normalize morphologic features regardless of cytogenetic response.
Pharmacokinetic Considerations during Imatinib Therapy
Mean plasma trough concentration of imatinib and its metabolite CGP74588 obtained at about 1 month (presumptive steady state) was 979 ± 530 ng/mL. The rate of complete cytogenetic response and major molecular response was higher within the highest quartiles of imatinib trough levels.500 Some therapists suggest that imatinib plasma levels be checked in cases of suboptimal response in order to adjust the dose.501 Comedications and population covariates such as body weight and white cell count had no or minimal effect on imatinib clearance.502 Patients with CML on hemodialysis have been successfully treated with imatinib.503 Therapy interruptions and nonadherence with oral imatinib usage are common, and patient education and close monitoring are important to ensure compliance.504
No significant clinical responses to imatinib have been noted in patients with acute myelogenous leukemia (AML), MDS, Ph-chromosome–negative CML, or CMML without PDGFR or KIT mutations.505
Defining a Response to Imatinib
Table 90–2 contains definitions of hematologic, cytogenetic, and molecular responses. The median BCR-ABL levels for imatinib-treated patients can continue to decrease over at least 5 years. Table 90–3 lists the approximate milestones expected of patients treated with 400 mg/day of imatinib.444 There is variation in an individual patient’s time of maximal response. Consequently, if a patient has not met those precise milestones but shows a continued decrease in the proportion of Ph-chromosome–positive cells on cytogenetic examination of marrow, or if in a complete cytogenetic remission, a continued decrease in the level of the PCR signal for the BCR-ABL, imatinib should be continued. Loss of response is defined as loss of a complete hematologic or complete cytogenetic response, an increase of 30 or more percent in the number of Ph-chromosome–positive metaphases examined at 3-month intervals, development of new cytogenetic abnormalities, or an increase in the BCR-ABL/ABL ratio of one log or more on serial RT-PCR testing or into the range associated with metaphase positivity. Because of variability in PCR testing, these changes should be confirmed within 1 month. Patients who have 100 percent Ph-chromosome–positive cells after 6 months of therapy have a minimal chance of later achieving a major or complete cytogenetic response and may be offered allogeneic stem cell transplantation, if applicable.444,506
|Table 90–3. Guidelines for Response to Imatinab Mesylate443,444
|Time of Observation (months)
|CHR, complete hematologic response; CCyR, complete cytogenetic response; HR, hematologic response; mCyR, minor cytogenetic response; MCyR, major cytogenetic response; MMR, major molecular response; PHR, partial hematologic response.NOTE: Response is defined in Table 90–2. These data are applicable to therapy with imatinib, 400 mg/day, as initial therapy in chronic phase. Unsatisfactory or suboptimal implies need to consider change in treatment approach, as appropriate for that patient. Usually this change is an increase in the dose of imatinib, a shift to a second-generation tyrosine kinase inhibitor, or allogeneic stem cell transplantation, if eligible. These guidelines are approximate in that a patient showing continued response to imatinib can be continued on that therapy until a response plateau has been reached, at which time the response can be evaluated using the benchmarks noted. See text for further details.
Stopping Imatinib Therapy
Discontinuation of imatinib in 12 patients who had undetectable disease for at least 2 years resulted in 6 patients having a molecular relapse within 1 to 5 months (imatinib was reintroduced with a response) and 6 others remaining in molecular complete remission for a median of 18 months.507 There are numerous anecdotes of patients relapsing when imatinib was stopped. In patients with intolerable side effects on imatinib, the dose may be reduced in some cases without the loss of a complete molecular response.508 In occasional patients in whom imatinib is stopped, a cytogenetic response of up to 15 months has persisted.509–512 Because early, quiescent Ph-chromosome–positive cells (CD34+Lin–) are insensitive to imatinib in vitro,513 at present it is advisable to maintain treatment indefinitely until the criteria for cessation, if any, can be established in clinical trials.
Use of Imatinib in Pregnancy
Imatinib is possibly teratogenic. Normal newborns have been delivered by patients who conceived and ingested imatinib during early pregnancy.514–517 In 125 women exposed to imatinib during pregnancy, 50 percent delivered normal infants, and 25 percent underwent elective terminations, three of the latter following the identification of fetal abnormalities. Twelve other infants had abnormalities.518 The majority of patients who discontinue imatinib during pregnancy lose their complete hematologic remission and their cytogenetic responses.519 One fetal fatality during pregnancy as a result of a meningocele has been reported. Males treated with imatinib have fathered healthy infants.520 Current recommendations are to practice contraception during imatinib treatment or if pregnant at the onset of the disease, to consider IFN treatment until delivery.517,521 Imatinib does appear in breast milk.522
Secondary Chromosomal Changes with Imatinib Mesylate
Clonal abnormalities in cells lacking a detectable Ph chromosome or BCR-ABL rearrangements have been detected in patients undergoing imatinib therapy who previously were treated with IFN-.523,524 These cytogenetic changes were noted in 7 patients at a median of 13 months of imatinib therapy, and trisomy 8 was the most frequent abnormality. All of these patients had major cytogenetic responses to imatinib.523 In some patients, clonal evolution may be related to imatinib resistance.525 Clonal abnormalities may be present in up to 10 percent of patients taking imatinib.526 Some of these cases may be associated with an MDS, especially in those patients with previous exposure to cytarabine and idarubicin. The antiproliferative effect of imatinib allows restoration of a polyclonal hematopoiesis in complete cytogenetic remission, which might favor the manifestation of a Ph-chromosome–negative disorder.527 Some investigators have found that, with the possible exception of +8, +Ph, and i(17), additional chromosomal abnormalities at diagnosis are not associated with an inferior outcome.528,529 In contrast, another group found that development of trisomy 8 in patients taking imatinib, while associated with pancytopenia, did not result in signs of disease progression. In a series of 34 CML patients who developed Ph-chromosome–negative clones while taking imatinib, the most common abnormalities were trisomy 8 and monosomy 7. In 11 of these patients, no archival evidence of these clones was present before imatinib therapy was initiated, and none of the patients developed myelodysplasia.530 In patients treated at diagnosis with imatinib, 9 percent developed chromosomal abnormalities in Ph-negative metaphases. These appeared at a median of 18 months, and the most common abnormalities were –Y and +8. Most were temporary and had disappeared within 5 months. Only one patient with –7 progressed to AML.531 Cytogenetic clonal evolution may not be an important impediment to achieving a major or complete cytogenetic response with imatinib, but it is an independent poor prognostic factor for survival of patients in chronic and accelerated phases of CML.532 Imatinib therapy may overcome the poor prognostic significance of derivative chromosome 9 in CML.533
Development of Imatinib Mesylate Resistance
The development of resistance to imatinib is not surprising.534–538 Its specificity and “snug fit” into the ABL-kinase pocket provide the ideal circumstance for resistance.539 Some cases demonstrate primary resistance to imatinib, and gene profiling has demonstrated differential expression of about 46 genes in responders compared to nonresponders.540 Even in patients with complete cytogenetic response, malignant progenitors at the LTC-IC stage persist. Chronic phase CML stem cells are resistant to imatinib and are genetically unstable.541 These cells have a high level of BCR-ABL transcription, and they are thought to express transporter proteins that result in abnormal imatinib flux.542 Mathematical models suggest that imatinib rapidly eliminates differentiated leukemic progenitors, but does not deplete leukemic stem cells. Such models predict the probability of developing resistant mutations and can estimate the time that resistance will emerge.543 Intermittent administration of G-CSF exposure may promote elimination of CML CD34+ cells.544,545 Imatinib upregulates CXCR4 expression, and thus might promote survival of quiescent CML stem cells by enhancing their interaction with marrow stroma.546
Several potential mechanisms of resistance include BCR-ABL amplification in the presence of imatinib,547–549 P-glycoprotein–mediated drug efflux,550,551 altered drug metabolism,538 acquisition of BCR-ABL–independent signaling characteristics,549 and point mutations in the ABL kinase domain that alter imatinib binding. There is evidence that each of these mechanisms of resistance may have clinical relevance.
Expression of the OCT-1 cellular transporter, which mediates drug influx, is thought to be important for imatinib but not dasatinib effectiveness.553,554 Many CML patients who have a suboptimal response to imatinib have low OCT-1 activity but this can be overcome with higher doses of imatinib or use of dasatinib.554,554 CML CD34+ cells overexpress the drug transporter ABCG2, but imatinib mesylate is not a substrate for this protein.555
Amplified gene expression and increased BCR-ABL protein expression are often reported in resistant patients. Duplication of the Ph-chromosome and isodicentric chromosomes are a possible mechanism of resistance to imatinib.556,557
Mutations in the ABL kinase domain may predate imatinib treatment,558 and several BCR-ABL kinase domain mutants associated with imatinib resistance remain sensitive to the drug, suggesting a need for characterization before a resistant phenotype can be attributed to the given mutation.559 The mutant clone does not always have a proliferative advantage.560 Some of these mutations may lie outside the kinase domain, and more than 40 such mutations have been described. Screening early phase CML patients for mutations before the start of imatinib therapy is not cost-effective because of their low incidence, but in patients with evidence of an increase in CML cells while on imatinib, mutation searches are indicated.561 BCR-ABL kinase domain point mutations are rare in those who have had good cytogenetic responses to imatinib, and when detected in that setting, their presence does not always predict relapse.562 Mutations in the ABL portion of the BCR-ABL oncogene are present in approximately 40 percent of patients who do not achieve a hematologic or cytogenetic response to imatinib. ABL mutations were found in those patients with both primary and acquired resistance. Amino acid substitutions in seven residues accounted for 85 percent of all mutations associated with resistance.563 The mutations most associated with resistance are Thr315ILe, Gly250Glu, Glu255Lys, and Thr253His substitutions. Few of the described mutations directly affect imatinib binding.537,564 Mutations in the ABL–ATP phosphate-binding loop (P-loop) are most closely associated with a poor prognosis,565 and these P-loop mutations predict for disease progression. Overall survival is worse for P-loop and for T315I mutations but not significantly different for other mutations.566
Second-generation BCR-ABL inhibitors (see “Dasatinib and Nilotinib” below) are able to overcome imatinib-resistant mutants, with the exception of the T315I mutations. This mutation results in steric hindrance which precludes access of some inhibitors to the ATP-binding pocket of the ABL kinase domain.567 Based on the crystal structure, the kinase domain of ABL T315I can be predicted by Aurora kinase inhibitors. The inhibitor binds in an active conformation of the kinase domain in the ATP-binding pocket.568 In a series of 27 patients with T315I mutation, survival was dependent on stage of disease, with many of the chronic phase patients described as having an indolent course.569 Agents such as IFN- and homoharringtonine have been proposed as salvage therapy for those with the T315I mutation.570
In some cases of resistance associated with imatinib, other signal pathways independent of BCR-ABL may become important in cell proliferation.571 These include heat shock protein 70,572 survivin,573 LYN kinase,574 SRC,575 and GRB2.576
Dose escalation, combination therapy, and treatment interruption have been proposed as means to overcome drug resistance.577 Combination therapy from the outset578 also has been proposed to prevent development of resistance. Treatment interruption to stop clonal selection of resistant cells has been proposed.575 Gene expression profiles may be useful to predict the clinical effectiveness of imatinib for CML treatment, thereby allowing individualized therapy from the outset.578 In patients with relapse or resistance, alternative approaches include increasing the dose of imatinib or switching to dasatinib or nilotinib.579
Dasatinib and Nilotinib
Dasatinib and nilotinib, two second-generation tyrosine kinase inhibitors, are used in cases of imatinib resistance or intolerance. Trials are underway to ascertain whether use of these agents at time of diagnosis will improve response rates and obviate later resistance.
Dasatinib is 325-fold more potent than imatinib and responses occur among all ABL mutant genotypes with the exception of T315I.580 As a dual inhibitor of SRC and ABL kinases, dasatinib is able to bind to BCR-ABL with less stringent conformational requirements.581 Dasatinib,100 mg/day, is administered in chronic phase CML.582,583 The major cytogenetic response rate is approximately 50 percent, and molecular responses occur as well.584 In patients resistant to imatinib, dasatinib, 140 mg/day (70 mg q12h), resulted in a higher proportion of major cytogenetic responses, complete cytogenetic responses, and major molecular responses than did 800 mg/day (400 mg q12h) of imatinib. Treatment failure was decreased and progression-free survival was improved with dasatinib.585 Unlike imatinib, dasatinib penetrates the blood–brain barrier.586
The major adverse event with dasatinib use is cytopenia. In addition to hematologic toxicity, fluid retention, diarrhea, and skin rash also can occur. Dasatinib may be more effective in the presence of F359I ABL mutation as compared with imatinib and nilotinib.587 Unlike the case with imatinib, dasatinib cellular uptake is not affected by OCT-1 activity, which is a substrate of the efflux proteins, ABCB1 and ABCG2.588 Resistance to dasatinib is often found with point mutations at residue 315 or 317.589
Nilotinib is an orally bioavailable, ATP-competitive inhibitor of BCR-ABL. It is FDA approved for patients in chronic phase CML resistant or who are intolerant to imatinib. It is approximately 30 times more potent than imatinib.590 Like imatinib, it does not induce apoptosis in CD34+ CML cells.591 A dose of 400 mg every 12 hours induced approximately 40 percent of patients who were resistant to or intolerant of imatinib into a major cytogenetic response, and approximately 30 percent into a complete cytogenetic response, with all resistance-inducing ABL mutations except T315I.
Adverse effects included neutropenia and minimal other side effects such as hyperbilirubinemia and hypophosphatemia.592 Nilotinib can cause electrocardiographic QT interval prolongation, so caution is required with concurrent medications that can prolong the QT interval.593 There is preclinical data to show that despite binding at the same site in the same target kinase, use of imatinib and nilotinib in combination may have additive or synergistic effects as BCR-ABL inhibitors.594
Agents that have been proposed for use in combination to improve response rates or to overcome resistance to imatinib have included IFN-, cytarabine, daunorubicin, homoharringtonine, multiagent chemotherapy, arsenic trioxide, and decitabine, with some supporting in vitro data.595–600 Combining imatinib with chemotherapeutic agents is more myelosuppressive, and final effects on response rates and survival have yet to be determined.601 Trials examining combinations of imatinib and either dasatinib or nilotinib are underway.602
Other Combinations Proposed to Overcome Imatinib Resistance
Several inhibitors of other signal transduction mediators involved in the downstream effects of BCR-ABL have been proposed for use in imatinib-resistant CML. These inhibitors include the JAK2 inhibitor AG490,603 SRC kinase inhibitors, mTOR (mammalian target of rapamycin) inhibitors, such as rapamycin,604 the proteasome inhibitor bortezomib,605,606 histone deacetylators,607,608 PI3K or MEK (mitogen-activated kinase) inhibitors, such as wortmannin and LY294002,609 and inhibitors of prenylation of RAS-related proteins downstream of BCR-ABL. These agents include the bisphosphonate zolendronate610 and farnesyltransferase inhibitors.611,612 The farnesyltransferase inhibitors SCH66336 and R115777 have shown some activity in CML.613–616 Imatinib resistance often is associated with restored activation of the BCR-ABL signal transduction pathway, suggesting that BCR-ABL remains a valid target to overcome resistance in these cases.617 BCR-ABL point mutations isolated from patients with imatinib-resistant CML remain sensitive to inhibitors of the BCR-ABL chaperone heat shock protein (hsp) 90, such as geldanamycin.618 Many of these agents have not yet entered clinical trials. Some are being used in conjunction with imatinib in resistant cases.
Disease Prognosis and Monitoring during Imatinib Therapy
Treatment failure should lead to alterations in therapeutic strategy.619 For patients treated initially with imatinib, BCR-ABL expression in cytogenetic responders and nonresponders was similar. BCR-ABL expression became significantly different 3 months after treatment and became increasingly different between responders and nonresponders with continued therapy at 6, 9, and 12 months.620
One mode of monitoring patients undergoing imatinib therapy is to measure blood counts at least once per month and to obtain marrow samples every 6 months until a complete cytogenetic remission is obtained.621 Thereafter, marrow samples are obtained yearly to monitor for other clonal abnormalities. Quantitative RT-PCR is performed every 3 months on blood or marrow. A one log increase in the level of BCR-ABL reactivity, confirmed on a repeat sample at least 1 month later, suggests a loss of response to treatment. In patients who do not have a complete hematologic response at 3 months, or a major cytogenetic response after 6 to 12 months, other therapeutic options are considered.622 The molecular response after 2 to 3 months of therapy is a strong predictor of clinical and cytogenetic response.623 Sequencing the BCR-ABL kinase domain can reveal emergence of resistant clones and is useful if there is an insufficient initial response to imatinib (see Table 90–3) or any sign of loss of response, such as relapse to Ph-positive status, a 1-log increase in BCR/ABL transcript ratio, or loss of a major molecular response (MMR).444 In patients receiving second-generation tyrosine kinase inhibitors, those who have no cytogenetic response at 3 to 6 months should be considered for allogeneic transplantation or switched to an alternative therapy in a clinical trial. After 12 months, those with a major cytogenetic response had a significant survival advantage over those with lesser responses.624
Summary of Imatinib Effects
Imatinib was first used experimentally for CML treatment in June 1998. Although it has completely altered the treatment approach to CML, its use has raised several questions. Studies require long-term followup of survival, but the degree of cytogenetic response, and degree of molecular response can be used as surrogate endpoints.625,626 The durability of cytogenetic and molecular responses in the face of persistent minimal residual disease during imatinib therapy require further followup of larger numbers of patients, especially because more than 95 percent of cases have molecular evidence of disease at 2 years.626
The success of imatinib therapy has diminished the use of allogeneic stem cell transplantation in chronic phase CML.627 Imatinib trials show a 95 percent progression-free survival at 24 months, but patients usually have evidence of ongoing molecular disease. Because stem cell transplantation is associated with high toxicity and mortality rates, deciding when and for whom to use this modality while responses to imatinib are ongoing can be difficult. For patients who lose or never achieve an imatinib-induced major cytogenic response and for whom an acceptable donor is available, allogeneic stem cell transplantation should be considered.628 Another therapeutic issue that has arisen is whether to use dasatinib or nilotinib before allogeneic stem cell transplantation in those in whom imatinib response has been absent or suboptimal.629 Some therapists would first increase the dose of imatinib, and if unsuccessful try dasatinib or nilotinib, and only if those steps were unsuccessful consider allogeneic transplantation.
Prior to the approval of imatinib mesylate for upfront therapy in CML, INF- was often used as initial therapy. A complete cytogenetic response with IFN- was uncommon (13%), but 10-year survival rates in responders were approximately 70 percent.630 Cytogenetic responses to IFN- were stable and durable.631 Approximately 50 percent of complete responders become long-term survivors. Common toxicities of INF- use include fatigue, low-grade fever, weight loss, liver function test abnormalities, hematologic changes, and neuropsychiatric symptoms. Most studies have shown no benefit of high-dose IFN- compared with low-dose IFN- for chronic phase CML (5 million units/m2 per day vs. 3 million units/m2 five times per week).632 Low-dose IFN- minimizes toxicity and cost. Pegylated IFN-, which has a longer half-life, can be administered as a once-per-week injection at 6 mcg/kg per week.633 Dose-limiting toxicities are neurotoxicity, thrombocytopenia, fatigue, and liver dysfunction. In later studies, 4.5 mcg/kg per week was proposed as the optimal dose because of the toxicity at higher doses. A single weekly dose of 450 mcg pegylated IFN- has also compared favorably to the standard IFN- dose in terms of toxicity and response rates.634 Pegylated IFN- plus low-dose cytosine arabinoside administered weekly is effective but has significant toxicity in patients with CML.635
Overall survival is improved in imatinib-treated patients compared with patients treated with IFN- or IFN- plus cytarabine.636 Nevertheless, among all patients who attained a major or complete cytogenetic response at 12 months, the survival rate was comparable in either case. IFN- has also been proposed as an immune stimulant to consolidate imatinib remissions because additive effects have been noted.637,638 Conversely, those treated initially with INF- who achieve a complete cytogenetic response have an improved molecular response with imatinib.639,640 Some patients intolerant to a tyrosine kinase inhibitor may be treated successfully with INF-.
Use of Other Chemotherapeutic Agents in Chronic Phase
The major side effect of hydroxyurea is an extension of its pharmacologic effect, that is, reversible suppression of hematopoiesis, often with megaloblastic erythropoiesis. The median survival of patients with CML treated with hydroxyurea alone is approximately 5 years. Studies with high-dose hydroxyurea indicate that marrow metaphase cells in some patients lose the Ph chromosome either partially or completely after such therapy.641 The drug may be very useful in patients of advanced age, in patients with comorbid conditions, and in patients in whom imatinib and IFN cannot be tolerated or are ineffective. Hydroxyurea often is used for initial cytoreduction. Chronic use of hydroxyurea is associated with leg ulcers.642
IFN-2b combined with cytarabine (20 mg/m2 per day for 10 days per month) in the chronic phase was associated with a greater proportion of major cytogenetic response at 12 months after randomization and with greater survival prolongation than was IFN alone.643 Toxicities with these drug combinations were greater, and this combination has been replaced by tyrosine kinase inhibitor therapy.
Once the mainstay of treatment for the chronic phase, busulfan usage now is rare.644 It is used primarily as part of the preparative regimen for allografting or autografting. It may be used occasionally in older patients who do not tolerate tyrosine kinase inhibitors.
Homoharringtonine, a plant alkaloid, can induce responses, including cytogenetic responses, in patients in the late chronic phase.645
Other Cytotoxic Agents
Intensive multidrug regimens have been used in an attempt to eradicate the Ph-chromosome–positive clone and have lead to prolongation of remission or cure of the disease. This approach has not significantly increased survival.646
Other Potential Therapeutic Agents in CML
The farnesyltransferase inhibitors lonafarnib and tipifarnib have been combined with imatinib and have activity after imatinib failure.647,648 The hypomethylation agent decitabine has activity in imatinib refractory CML.649 Berbamine, a natural small molecular compound, has in vitro activity against primary CML cells.650 Adaphostin, a tyrphostin, inhibits CML cell growth, including those cell populations resistant to imatinib by inducing oxidative stress.651 INNO-406, a dual BCR-ABL/LYN inhibitor, suppresses the growth of CML cells in the central nervous system where imatinib has limited penetration.652 This agent also enhances autophagy in CML cells.653 Agents that disrupt autophagy when combined with histone deacetylase inhibitors such as suberoylanilide hydroxamic acid (SAHA) are able to overcome imatinib resistance in preclinical models.654 The SRC-ABL inhibitor, SKI-606 (bosutinib), is able to overcome most resistance-mediating ABL mutations, except T315I, and it has entered clinical trials.655 Several third-generation inhibitors are being developed for inhibition of the T315I mutation.656 MicroRNA technology may eventually play a role in CML treatment,657 and synthetic BCR-ABL siRNA (small interfering ribonucleic acid) has been used in a patient with resistant CML, postallografting with inhibition of BCR-ABL noted.658
Ribozymes targeting BCR-ABL mRNA have been used as CML treatment,659,660 and these approaches probably will have the most utility for in vitro purging of CML marrow cells before autotransplantation.661,662
Several antigens have been proposed as targets of immune therapy for CML. These antigens include BCR-ABL itself, PR1, Wilms tumor protein-1 (WT1), minor histocompatibility antigens, CML-66, CML-28, and survivin.663,664 Other targets are VEGF and hsp90.665 A BCR-ABL fusion peptide, used as a vaccine, can elicit a specific T-cell immune response.666,667 CML-derived dendritic cells can process and present endogenous BCR-ABL fusion proteins to CD4+ T lymphocytes in an HLA class II-restricted antigen presentation.668 NM23-H2, an HLA-A32 restricted tumor associated antigen aberrantly expressed in tumors such as CML, can generate reactive T cells after transplantation.669 Immunization with GM-CSF–producing tumor vaccines to enhance a vaccine antitumor effect is being studied in CML.670 Numerous peptides from the BCR-ABL fusion region have been identified as vaccine candidates.671 Peptides from the e14a2 BCR-ABL junction have been examined in vaccine trials, elicit T-cell responses, and demonstrate molecular responses of a delayed nature in those who have had a major cytogenetic response to imatinib. Randomized trials with these vaccines have not yet been reported.672
Splenic irradiation may be useful occasionally in subjects who have entered the accelerated or advanced chronic phase and are troubled with extreme splenomegaly with splenic pain, perisplenitis, and encroachment of the spleen on the gastrointestinal tract.673 Splenic irradiation may palliate symptoms for a short time.674
Radiotherapy may be useful for extramedullary tumors, which may occur occasionally in bone or soft tissue during the late chronic or accelerated phase.
Splenectomy does not prolong the chronic phase of CML, delay the onset of the accelerated phase, enhance sensitivity to standard or intensive chemotherapy, or prolong survival of patients.675 In carefully selected patients with symptomatic thrombocytopenia unresponsive to therapy, mechanical discomfort, hypercatabolic symptoms, and portal hypertension, splenectomy may be useful. Postoperative morbidity from infection, thrombosis, or hemorrhage has been high, with mortality rates up to 10 percent reported.676 Splenectomy performed before allografting has not been found to influence the severity of graft-versus-host disease (GVHD) or survival after allogeneic stem cell transplantation.677 Splenectomy may reverse poor graft function after allogeneic transplantation, but hyposplenism may trigger or worsen chronic extensive GVHD, leading to increased morbidity and mortality.678
Treatment of Chronic Phase CML during Pregnancy
Treatment of chronic phase CML during pregnancy is sometimes needed to prevent placental insufficiency from hyperleukocytosis. Imatinib use during pregnancy has the risk of a teratogenic effect.514–517 IFN can be used during pregnancy with minimum risk of teratogenicity. Eight patients treated with IFN from the first trimester have been described, and each of these pregnancies resulted in normal infants, except for one with mild neonatal thrombocytopenia. All infants had normal growth.680 Hydroxyurea may be useful during the second and third trimester but should be avoided in the first trimester.517,679 Leukapheresis in the first trimester (or longer) also can be used to avoid fetal drug exposure early in pregnancy (see “Leukapheresis” above). It is important to use imatinib after delivery to achieve the best outcome. Further observation may show it to be safe later in pregnancy. Although controversial, stopping and later restarting imatinib may not result in as favorable an outcome of therapy. 517
High-Dose Chemotherapy with Autologous Stem Cell Infusion -Chronic myelogenous leukemias (CMLs)
Since the availability of imatinib, autografting in CML is rarely used.681 Ph-chromosome–negative stem cells are present in most patients with CML at the time of diagnosis. Techniques that use these cells to reconstitute hematopoiesis after high-dose therapy have been developed.682 Ph-chromosome–negative progenitors can be mobilized with G-CSF and collected from the blood of patients who have responded to prior treatment with IFN or imatinib.683 Such cells also can be collected after recovery from chemotherapy regimens, such as after idarubicin and cytarabine, followed by G-CSF stimulation.682 G-CSF was used for at least 4 days while imatinib treatment was continued for stem cell mobilization in 58 patients with a complete cytogenetic response. The cells were collected in two cytapheresis procedures in 74 percent of patients, and the cells of 84 percent of those cytapheresis products were negative for the Ph chromosome.684
In another series, stem cells were mobilized in 32 patients in complete cytogenetic remission after imatinib, with uninterrupted imatinib therapy in 50 percent of patients and with imatinib temporarily withheld in approximately 50 percent. Blood levels of BCR-ABL were not changed by the use of G-CSF.685 In yet another series, 13 of 15 patients were successfully mobilized with G-CSF while receiving imatinib, and 28 percent of stem cell harvests were negative for BCR-ABL mRNA. No change in blood BCR-ABL transcript level was noted after stem cell mobilization as assessed by RT-PCR.686 No series of patients autografted with cells mobilized while they were receiving imatinib have been reported.687 Autografting might find a role in cases of imatinib resistance,688 or to reduce the level of residual disease in cases without a molecular response.689 Imatinib can be effective and safe in chronic phase CML patients who have previously undergone autografting,690 although increased hematologic toxicity occurs.
Allogeneic Stem Cell Transplantation -Chronic myelogenous leukemias (CMLs)
Until imatinib became available in 2000, allogeneic transplantation was used in most new patients with CML who were younger than 65 years of age and who had a suitable donor. The advent of imatinib treatment and the projected survival of patients with a complete cytogenetic remission has changed the indications for transplantation in CML.691,692 There has been a marked reduction in number of transplants performed for CML worldwide and a decline in the proportion performed in first chronic phase.693,694 Although no randomized trials of imatinib versus transplantation have been or are likely to be conducted, there is circumstantial evidence that survival is superior for populations of patients treated with drugs who have a complete cytogenetic remission as compared to transplantation.695 Allografting continues to play a prominent role in the treatment of patients with suboptimal imatinib responses, who are refractory or intolerant to tyrosine kinase inhibitors, and remains the optimal therapy in those who progress to accelerated phase or blast crisis.
Patients in the chronic phase of CML who are younger than 65 years and who have an identical twin,696 or a histocompatible sibling,697,698 or who are younger than 55 years with access to a histocompatible unrelated donor,699 can be transplanted after intensive therapy, usually with cyclophosphamide and fractionated total-body irradiation (TBI) or a combination of busulfan and cyclophosphamide. Busulfan can be administered as an intravenous preparation and as a single daily dose.700 When targeted steady-state busulfan levels are utilized, a 3-year survival rate of 86 percent and a disease-free survival rate of 78 percent with no age effect is achieved.701 With nonmyeloablative or “reduced-intensity” conditioning regimens, older patients and those with comorbidities can undergo successful allografting.
Myeloablative Allogeneic Transplants-Chronic myelogenous leukemias (CMLs)
Stem cell transplantation from HLA-compatible siblings results in engraftment and an actual or projected long-term survival in 45 to 70 percent of recipients.702–704 In patients older than age 50 years, survival rates are slightly less at 5 years. The risk of CML relapse is approximately 20 percent, with a plateau of relapse at 5 to 7 years. Transplanted T lymphocytes, especially if activated by a (mild) GVHD, may be an important factor in preventing leukemic relapse. This phenomenon, referred to as graft-versus-leukemia reaction, is thought to suppress the leukemic process through T-cell–mediated cytotoxicity.697 The relative benefit of marrow compared to mobilized blood stem cells as the source of the allograft has not been established.705,706 Mobilized blood stem cells engraft more rapidly but may be associated with more chronic GVHD. The majority of survivors have no evidence of residual leukemia.707
For younger patients who do not have a histocompatible sibling, an unrelated donor or a mismatched family member as a source of stem cells is feasible. The toxicity of this procedure is greater than that of an HLA-identical sibling donor transplant. Five-year disease-free survival is approximately 40 percent.708 Younger patients with cytomegalovirus-seronegative donors who are matched at the HLA-DRB1 allele by molecular methods fare better.709 When class I HLA genes are typed with molecular methods, an improvement in matching and better outcomes using unrelated donors are expected. When matched-unrelated donor and sibling donor transplants were compared, unrelated donor transplants had increased risk of graft failure and acute GVHD, but only a slightly poorer survival and disease-free survival. For patients who survived to 1 year, only a slightly inferior disease-free survival was observed.710 The rate of extensive chronic GVHD is up to 60 percent with unrelated donor transplants, but 63 percent disease-free survival in younger CML chronic phase patients has been reported. Cord blood stem cell transplantation from an unrelated donor has also been used in adults with CML.711
Pretransplantation imatinib is not associated with increased transplant-related morbidity or decreased survival, but those who are transplanted with suboptimal response to imatinib or loss of response to imatinib do poorer, probably related to a higher disease burden at the time of transplantation and more aggressive disease.712,713 Second-generation tyrosine kinase inhibitors do not increase transplant-related toxicity.714 Disease status after allografting can be monitored with cytogenetic studies, PCR, or FISH analysis. A positive PCR assay 3 months after allogeneic transplantation has not been found to correlate with an increased risk of relapse compared with PCR-negative patients. A positive assay at 6 months and beyond is associated with subsequent relapse. In one series, 42 percent of patients with a positive PCR assay at 6 to 12 months relapsed versus 3 percent with a negative assay.715 Paradoxically, patients who remain BCR-ABL positive more than 36 months after transplantation have little propensity for relapse.715 Serial quantitative RT-PCR analysis of blood specimens has been proposed to distinguish patients destined to relapse.716 Patients who remain in remission have undetectable, low, or falling BCR-ABL levels on sequential analysis. After 6 to 9 months, these levels are undetectable in most cases. Recognition of relapse at the molecular level may allow for early therapeutic intervention.
Killer immunoglobulin-like receptors (KIRs) are expressed by NK cells and subpopulations of T cells. NK clones from a single individual can vary substantially in the type of KIR molecules they express. The ligands for several of the inhibitory KIR have been shown to be subsets of HLA class I molecules. Missing KIR ligands in recipients lead to less relapse and increased GVHD based on NK alloreactivity.717 KIR ligand mismatch has also been found to be an important prognostic factor in achieving molecular responses after transplantation for CML.718 Increased frequency of Treg cells characterized as CD4+, CD25-high are associated with higher rates of relapse after allografting in CML.719
Nonmyeloablative Allogeneic Transplants- Chronic myelogenous leukemias (CMLs)
Nonablative regimens have been developed in an attempt to expand the indication for allogeneic transplantation to older patients. These regimens rely on immunosuppressive therapy to allow engraftment of cells that potentially will generate a graft-versus-leukemia effect. These procedures in general are associated with acceptable degrees of engraftment, less mortality, similar rates of GVHD, and possible durable effects on persistent or recurrent disease.720 Approximately 60 percent of patients, mostly in initial chronic phase (median age: 50 years) and transplanted with reduced-inten