Chronic myelogenous leukemias (CMLs)

Summary

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 confirmed^7–9 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 chromosomes^10,11 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

Etiology and Pathogenesis

Environmental Leukemogens

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:

1. Involvement of erythropoiesis, neutrophilopoiesis, eosinophilopoiesis, basophilopoiesis, monocytopoiesis, and thrombopoiesis in chronic phase CML²⁶
2. Presence of the Ph chromosome (22q–) in erythroblasts; neutrophilic, eosinophilic, and basophilic granulocytes; macrophages; and megakaryocytes²⁷
3. 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 B^28–30
4. 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 pair^31–33
5. 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
6. 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 CML^36,37
7. 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 G_o 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 treatment^75–77 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 chromosome^84,85 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 progenitors^126,127 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 p210^BCR–ABL binds to actin, and several cytoskeletal proteins are thereby phosphorylated. The p210^BCR–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 p210^BCR–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 p210^BCR–ABL, the tyrosines of paxillin, vinculin, p125^FAK, talin, and tensin are constitutively phosphorylated. Pseudopodia enriched in focal adhesion proteins^134,135 are present in cells expressing p210^BCR–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.

Molecular Pathology

pH Chromosome

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

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

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 (p210^BCR–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

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

p210^Bcr–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, BCR^164,165 (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 (p210^BCR–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 (p190^BCR–ABL). Almost all CML cases at diagnosis that encode a p210^BCR–ABL also express BCR-ABL transcripts for p190.¹⁶⁷ The biologic or clinical significance of these dual transcripts is not known. Transgenic mice expressing p210^BCR–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 p21^rac, 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 p210^BCR–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 p210^BCR–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 p210^BCR–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 p210^BCR–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 p190^BCR–ABL develop an acute lymphocytic leukemia (ALL) lymphoma syndrome¹⁹⁰ that resembles human Ph-chromosome–positive ALL. When a p210^BCR–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 p210^BCR–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 p210^BCR–ABL has been causally linked to the development of Ph-chromosome–positive leukemia in man.^201–212 p210^BCR–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 p210^BCR–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 p210^BCR–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). p210^BCR–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 p210^BCR–ABL with various mediators of signal transduction.

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 p210^BCR–ABL, and it acts to relate p210^BCR–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 p210^BCR–ABL. The p210^BCR–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 p120^CBL and the adaptor proteins CRKL and c-CRK also link c-abl, p190^BCR–ABL, and p210^BCR–ABL to the PI3K pathway.²¹² The p120^CBL also coprecipitates with the p85 subunit of PI3K, CRKL, and c-CRK. The p210^BCR–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 p190^BCR–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 P62^DOK, a constitutively tyrosine-phosphorylated, p120^RAS 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 p210^BCR–ABL-mediated transformation.²²³ Expression of p210^BCR–ABL leads to activation of NF-B–dependent transcription via nuclear translocation.²²⁴

Cell lines that express p210^BCR–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; p210^BCR–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 Rho^228,229 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 p210^BCR–ABL also activates Jun kinase and requires Jun for transformation.²³¹ In some CML cell lines, p210^BCR–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 p210^BCR–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 p210^BCR–ABL inhibits apoptosis by delaying the G₂/M transition of the cell cycle after DNA damage.²³⁵ The p210^BCR–ABL also may exert an antiapoptotic effect in factor-dependent hematopoietic cells.^236,237

p210^BCR–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

Telomere Length

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

Clinical Features

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 elevations^249–251; left upper quadrant and left shoulder pain as a consequence of splenic infarction and perisplenitis; vasopressin-responsive diabetes insipidus^252,253; 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.

Laboratory Findings

Blood

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 erythrocytosis^263,264 or erythroid aplasia^265,266 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.

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 vivo^287,288 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

Marrow

Morphology

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

Cytogenetics

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

Molecular Probes

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 p210^BCR–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 p210^BCR–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 p210^BCR–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 antigens^345–348 to develop specific antibodies to p210^BCR–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

Chemical Abnormalities

Uric Acid

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}

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

Hyperleukocytosis

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

Differential Diagnosis

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

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

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

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

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

Imatinib Mesylate

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

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) p210^BCR-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

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

Combined Therapy

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.

Interferon–

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

Hydroxyurea

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

Cytarabine

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.

Busulfan

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

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

Immunotherapy

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

Radiotherapy

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

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-intensity conditioning regimens, had a 3-year survival and about one-third had a 3-year progression-free survival.⁷²¹ Patients no longer in first chronic phase do not fare as well.⁷²² Conditioning regimens include fludarabine and busulfan,^723,724 low-dose TBI and fludarabine, and low-dose TBI and cyclophosphamide, but no prospective randomized trials comparing regimens or comparing ablative and nonmyeloablative transplant approaches have been conducted.^723,725,726 In one case, imatinib given concurrently with nonmyeloablative stem cell transplantation did not compromise engraftment and resulted in a cytogenetic remission in a patient with CML in blast crisis.⁷²⁷

Use of Tyrosine Kinase Inhibitors after Stem Cell Transplant

In patients treated with stem cell transplantation before the wide availability of imatinib, complete cytogenetic remissions with imatinib treatment can occur if treated at relapse after allografting and after donor lymphocyte infusion (DLI) fails to give a response. Such remissions may include a molecular response.⁷²⁸ Complete responses after imatinib therapy have been noted in accelerated or blast phase CML persisting after stem cell transplantation.⁷²⁹ In another series, 45 percent of relapsed patients had a complete cytogenetic response of up to 28 months and without significant GVHD.⁷³⁰ In a series of 28 adults with relapse after allogeneic stem cell transplantation who then received imatinib, the response rate was 74 percent, and the complete cytogenetic remission rate was 35 percent. Five patients had recurrence of GVHD, and 13 had previous DLI infusions.⁷²⁸ In one series, imatinib was able to generate complete molecular remissions in 26 percent of chronic phase patients after allografting, with full donor chimerism usually observed.⁷³¹ Prophylactic administration of imatinib after transplantation has been used for patients at high risk of relapse.⁷³² Posttransplantation imatinib may also postpone the requirement for donor leukocyte infusions with its attendant risks of GVHD and marrow aplasia.⁷³³ Some have found that donor leukocyte infusions are superior to imatinib therapy in preventing relapse and increasing leukemia-free survival.⁷³⁴ Today, virtually all patients receive imatinib initially and receive transplantation only if refractory to or relapse after imatinib and a second-generation tyrosine kinase inhibitor.

Immunotherapy: Adoptive Cell Therapy for Posttransplantation Relapse

Substantial evidence indicates that the effectiveness of allografting in CML does not result solely from the eradication of the leukemic clone with high-dose chemoradiotherapy conditioning regimens, but also from adoptive immunotherapy provided by lymphocytes in the allograft, the graft-versus-leukemia effect (see “Myeloablative Allogeneic Transplants” above).⁷³⁵ This phenomenon has been recreated to produce a therapeutic response by infusing the lymphocytes from the stem cell donor after a relapse following allogeneic stem cell transplantation.^736,737 The overall response rate to DLI is approximately 75 percent. The response rate is higher when this approach is used early after detecting a relapse by PCR,⁷³⁸ compared to use after a hematologic or cytogenetic relapse. Patients with a short interval between transplantation and DLI have a higher probability of response than patients with longer intervals. Responses are the same with related versus unrelated donors.⁷³⁹ Some patients show a very rapid decline of BCR-ABL transcript levels (<6 months after DLI), whereas other patients demonstrate PCR negativity only over a longer period.⁷⁴⁰ The responses to DLI can be durable.⁷⁴¹ Molecular responses can occur in up to two-thirds of patients.⁷⁴²

The main toxicities of DLI have been the induction of GVHD and myelosuppression. Chronic GVHD can occur in up to 60 percent of cases.⁷⁴³ Attempts to diminish these toxicities have included use of CD8-depleted DLIs and infusion of smaller numbers of T cells.^744,745 Lower initial cell dose is associated with less myelosuppression, the same response rate, better survival, and less DLI-related mortality, leading to suggestions that the initial dose should not exceed 0.2 x 10⁸ mononuclear cells/kg.⁷⁴⁶ The initial doses should be lower when matched unrelated donors are used. Donor lymphocytes can also be transfected with vectors containing the herpes simplex virus genome in a replication defective form. If GVHD occurs, the lymphocytes can be eradicated with systemic ganciclovir treatment. The ultimate utility of such currently experimental approaches is still unmeasured.⁷⁴⁷ IFN after DLI may improve responses,^748,749 and imatinib may synergize with DLI to foster rapid molecular responses after relapse.⁷⁵⁰ Methods for administering more specific immune effector cells have been sought, but these approaches are not in widespread clinical use.⁷⁵¹

Course and Prognosis

Several large studies of treatment during the 1970s and early 1980s reported similar survival rates of patients with CML who were treated with standard chemotherapy, that is, busulfan or hydroxyurea, during the chronic phase.^752–760 Median survival ranged from 39 to 47 months, the 5-year survival rate was approximately 25 to 35 percent of patients, and the 8-year survival rate was 8 to 17 percent of patients. A large randomized study comparing hydroxyurea to busulfan showed a significant prolongation of chronic phase with hydroxyurea⁶⁴⁴ and a further prolongation with IFN therapy. Only a small fraction of patients remained in the chronic phase from 10 to 25 years.^761–768 The Surveillance, Epidemiology, and End Results Program of the National Cancer Institute gathers national statistics based on cancer registries in the United States. Table 90–4 lists the 5-year survival rates from these observations. Because these data are a mixture of the pre- and postimatinib therapy periods, they underestimate the 5-year life expectancy of patients with CML who are given imatinib treatment.

At the time of diagnosis, the variables most closely associated with duration of chronic phase and thus survival are percent blasts in the blood, liver and spleen size, and total basophil plus eosinophil count. Using these variables in large numbers of patients, the population segregates into three risk groups: better risk, with a median survival of approximately 5.0 years; intermediate risk, with a median survival of 3.5 years; and poor risk, with a median survival of 2.5 years.^769–771 In the better-risk group, 40 percent are alive at 7 years; in the poor-risk group, 10 percent or fewer are alive at 7 years. These figures are based on patients who were treated principally with busulfan. Treatment of chronic phase by hydroxyurea and then by IFN extended the median survival by approximately 18 months with the former, and by approximately 36 months with the latter drug in patients treated with these agents. Generalizations to all patients with CML based on these studies are not possible because many patients, especially those older than age 65 years, could not tolerate optimal doses of IFN. Thus, the results are for the select group of patients who remained on treatment with these agents. The median survival also has been dramatically improved by imatinib treatment. One projection indicated the median survival for all patients so treated will exceed 15 years.⁷⁷² It is as yet uncertain how long patients on imatinib can remain in remission on average.^691,773 In a 5-year followup of patients receiving imatinib for newly diagnosed CML, complete cytogenetic responses were seen in approximately 70 percent by 12 months and in nearly 90 percent by 60 months. In a major study, an estimated overall survival at 84 months of followup was 86 percent.⁷⁷⁴ Resistance rates decline with each passing year, and adverse effects have not emerged over time.⁷⁷⁵ Those who require 1 year or more of imatinib therapy to attain a complete cytogenetic remission have comparable rates of molecular response, progression-free, and overall survival to those who achieve cytogenetic remission sooner.⁷⁷⁶ In patients with failure to respond or intolerance to imatinib the estimated 3-year survival rate was approximately 70 percent for patients in chronic phase. Survival in chronic phase was better when subsequent therapy was nilotinib or dasatinib as compared to allogeneic hematopoietic stem cell transplantation or to others agents. This result was at a median followup of 2 years.⁷⁷⁷ Older patients have lower response rates when treated in late chronic phase, but if they attain a complete cytogenetic response, no difference was found in the level of molecular response.⁷⁷⁶

Studies linking the precise (3′) location of the breakpoint in the BCR gene with shortened duration of chronic phase⁷⁷⁸ have not been confirmed.⁷⁷⁹ Prior to the introduction of imatinib therapy, most patients died as a result of conversion from the chronic to the accelerated phase of the disease or blast crisis.⁷⁸⁰ It is too soon to determine the cause of death in patients treated with inhibitors of the BCR-ABL kinase. Very rare spontaneous cytogenetic, but not molecular, remissions of CML have been reported,^781,782 but the disease may reappear in that case.⁷⁸³

Several other ancillary factors are associated with poor prognosis in CML, including marrow angiogenesis and marrow fibrosis⁷⁸⁴; telomere length shortening⁷⁸⁵; cellular VEGF expression⁷⁸⁶; large deletions at the t(9;22) breakpoint⁷⁸⁷; derivative chromosome 9 deletions^788,789; increased marrow fiber content and reduction of medullary erythropoiesis⁷⁹⁰; higher CD7 expression by CD34-positive cells⁷⁹¹; absence of cadherin 13 expression⁷⁹²; and persistence of malignant hematopoietic progenitors in CML in complete cytogenetic remission after imatinib.⁷⁹³ Deletion of the 5′ abl region on der(9), present in approximately 9 percent of patients with CML, occurs at the time of formation of the t(9;22) translocation and may be associated with a slightly worse prognosis.⁷⁹⁴ The prognosis may not be worse with imatinib therapy. The type of BCR breakpoint (5′ or 3′) does not affect the course of chronic phase CML.^779,795 CML that occurs after treatment of other cancers appears to have comparable clinical and survival characteristics as de novo CML.⁷⁹⁶ Histamine levels during treatment with imatinib have been found to be of prognostic importance.⁷⁹⁷

Several prognostic scales have been proposed in CML, including the Sokal and Hasford systems for patients at the time of diagnosis and the European Bone Marrow Transplantation Consortium Risk Score for patients undergoing allogeneic stem cell transplantation,⁷⁹⁸ in which performance status is added to the five original variables (age, spleen size, blast cell count, basophil and eosinophil count, and platelet count [Hasford score]), which has been validated with good discrimination for survival.⁷⁹⁹ The Sokal score based on age, spleen size, platelet count, and percent myeloblasts was developed much earlier during the busulfan era of treatment and was less accurate in patients treated with IFN. A simple prognostic scale that includes donor type, stage of disease at time of transplantation, age of recipient, sex of donor and recipient, and interval between diagnosis and transplantation has been proposed to predict outcome of allogeneic stem cell transplantation.⁸⁰⁰ These prognostic scales require revalidation given the dramatic impact of conversion to universal imatinib therapy. There is evidence that a patient with chronic phase CML and a favorable Sokal Score at the time of diagnosis has a higher proportion of hematologic and cytogenetic responses than other patients.⁸⁰¹ Low neutrophil count and poor cytogenetic response at 3 months of imatinib therapy may predict a poor overall outcome,⁸⁰² but this observation requires validation.⁸⁰³ Cytogenetic response as a surrogate marker for survival appears to be useful in patients undergoing imatinib therapy.⁸⁰⁴

Detection of Minimal Residual Disease

Detection of minimal residual disease by molecular probes makes possible the identification of approximately 1 cell in 1,000,000 that is derived from the CML clone.⁸⁰⁵ Techniques used to monitor residual disease have been reviewed.^806–808 PCR permits observation of regression or persistence of subclinical disease following therapy and of progression of subclinical disease prior to the disease becoming overt, and it is therefore critical for monitoring responses to CML treatment.^809,810 The stable persistence of subclinical disease does not invariably predict early relapse.^811,812

The risk of misinterpreting negative results of RT-PCR is increased when very small numbers of transcripts are present.⁸⁰⁶ The dilution threshold for reproducible amplification is 250,000 cells. mRNA^BCR–ABL can also be detected in single progenitor colonies after culture.⁸¹³ A good correlation has been found between the proportion of Ph-chromosome–positive metaphase cells and levels of mRNA^BCR–ABL.^814–816 Efforts are underway to standardize the technique for measuring and reporting real-time RT-PCR,⁸¹⁷ and serial measurements are required for treatment decisions based on rises in transcript numbers.⁸¹⁸ Some studies show that marrow values tend to be higher than blood in real-time RT-PCR assays, but both follow a similar trend during treatment.⁸¹⁹ Interchanging these may lead to misinterpretation of disease status.⁸¹⁹ In patients on imatinib, who have major molecular responses, confirmed by real-time quantitative PCR, no marrow cell cytogenetic abnormalities were found, indicating that patients with major molecular responses do not require regular marrow examinations for cytogenetics.

Through utilization of quantitative PCR, an increase of mRNA^BCR–ABL expression has been found to precede disease progression. This increase was detected up to 16 months before laboratory or clinical parameters showed phenotypic transformation of the malignant clone.⁸²⁰ The technique of detecting minimal residual disease is highly sensitive and is subject to false-positive reactions. Nested, competitive RT-PCR is more sensitive than RT-PCR, but RT-PCR, when normalized for the total ABL transcripts, can be used to monitor CML patients during therapy.⁸²¹ Patients who achieve a major molecular remission (expressed as a 3-log reduction from median baseline value) at the time of achieving a complete cytogenetic response have been found to have longer cytogenetic remissions than those without this magnitude of molecular response.⁸²² The achievement of a 2-log molecular response at the time of a complete cytogenetic response or a 3-log response anytime thereafter is an independent prognostic marker of progression-free survival.⁸²³ Other studies, however, show that patients who achieve complete cytogenetic response do not derive additional benefit from a complete molecular response.⁸²⁴

Interphase FISH may have a false-positive rate of 5 to 10 percent.^825,826 FISH is not standardized, but a large number of cells can be rapidly analyzed (100–500). With D-FISH probes, which flank the breakpoints of the BCR and ABL genes, the false-positive rate is only approximately 0.2 percent.⁸²⁵ Fixation, specimen preparation, and hybridization conditions may account for differing false-positive ranges and scoring criteria.⁸²⁷ In CML patients treated with imatinib, FISH for BCR-ABL on interphase blood neutrophils, but not unselected white cells, correlates with marrow cytogenetics.⁸²⁸ FISH and RT-PCR can be useful complementary techniques,⁸²⁹ but FISH is generally not suitable for monitoring minimal residual disease.⁸³⁰

For patients who are undergoing allogeneic stem cell transplantation, the kinetics of minimal residual disease in either standard or nonmyeloablative transplants differ. BCR-ABL/ABL ratios were 0.2 percent with reduced-intensity transplants versus 0.01 percent in transplantation patients with traditional conditioning regimens in the first 3 months. By 12 months, however, 20 percent of patients who received standard transplants and 50 percent of patients who received reduced-intensity transplants had reached a level less than 0.01 percent, supporting the concept of different kinetics of disease eradication between the two transplantation modalities.⁸³¹ Patients who relapse after allografting have reappearance and/or rising levels of BCR-ABL transcripts.⁸³² Use of quantitative RT-PCR early (3–5 months) after stem cell transplantation can project long-term outcomes.⁸³³ When RT-PCR was negative, the 3-year risk of relapse was 16.7 percent; when RT-PCR was positive at a ratio of less than 0.02 percent, the relapse rate was 42.9 percent; and when RT-PCR was positive at a level greater than 0.02 percent, the relapse rate was 86.5 percent. Another group found that detection of blood BCR-ABL at 18 or more months after transplantation was associated with a highly significant risk of relapse and that patients who had a positive test result but failed to relapse generally had only one positive test result at a low copy number.⁸³⁴ Performance of qualitative PCR at regular intervals after allogeneic transplant (every 2–4 months in the first year and every 6 months thereafter) is appropriate. If the PCR results are persistently positive or become positive, quantitative PCR should be performed at monthly or shorter intervals. Molecular relapse is defined as a 10-fold increase of PCR positivity without any signs of cytogenetic relapse.⁸³⁵ Detection of increasing recipient chimerism by FISH for the male chromosome in sex-mismatched donor–recipient pairs or variable number of tandem repeats after allogeneic transplantation or after DLI infusion also is usually associated with a relapse.^836,837

Imatinib therapy is associated with a rapid decrease in BCR-ABL transcript levels. Nested PCR transcript levels parallel cytogenetic response, and imatinib is superior to IFN or cytarabine in terms of the speed and degree of molecular responses, but residual disease is rarely eliminated.⁸³⁸ When quantitative RT-PCR is utilized for patients undergoing imatinib therapy, almost all patients have evidence of residual disease.^839,840 Disadvantages of quantitative RT-PCR in monitoring imatinib response include lack of standardization, inability to detect clonal evolution, and current lack of widespread availability.⁸²⁵ The use of random pentadecamer primers may improve detection of BCR-ABL transcripts in CML,⁸⁴¹ and flow cytometric assays of phosphotyrosine levels may be valuable for serial evaluation of disease response.⁸⁴² Table 90–5 outlines suggested monitoring for chronic phase patients undergoing imatinib therapy.

Accelerated Phase and Blast Crisis of CML

Definition

In all patients with chronic phase CML, the disease has the potential to evolve into a more aggressive, more symptomatic, and troublesome phase, which is poorly responsive to the therapy that formerly controlled the chronic phase. The failure of therapy to restore or maintain near-normal red cell and white cell counts, increased spleen size, increased numbers of marrow blasts and blood basophils, loss of the sense of well-being, and appearance of extramedullary tumors are the most consistent clinical hallmarks of the metamorphosis of the chronic to the accelerated phase of CML. The most objective findings are a blood blast percentage greater than 10, a platelet count less than 100,000/L (100 x 10⁹/L), blood basophils greater than 20 percent, and new clonal cytogenetic abnormalities accompanying the Philadelphia chromosome.⁸⁴³

The terminology used has included accelerated phase, acute phase, acute transformation, or, in its most dramatic expression, blast crisis, but the metamorphosis, which can be acute, that is, myeloblastic or lymphoblastic crisis, often is more gradual and manifested by severe dysmorphic hematopoiesis, refractory splenomegaly, and extramedullary tumor masses, hence the preference for transformation or accelerated phase to describe this transition from a controllable to a poorly controlled malignancy. Blast crisis is the most severe manifestation of the accelerated phase and can occur abruptly or after a period of worsening disease. Blast crisis is in effect the evolution to overt acute leukemia.

Pathogenesis

Effect of Tyrosine Kinase Inhibitors in Rate of Progression

The advent of tyrosine kinase inhibitor therapy for CML has resulted in a marked increase in the duration of a subclinical chronic phase, with normal blood counts and spleen size, often with the loss of identifiable Ph chromosome-bearing cells in blood and marrow, and sometimes with the loss of laboratory evidence of the BCR-ABL oncogene as judged by PCR. This therapeutic advance has greatly delayed the evolution to accelerated phase and blast crisis, but the risk for such a conversion exists since under experimental conditions CML stem cells do not undergo apoptosis when exposed to BCR-ABL tyrosine kinase inhibitors and BCR-ABL-positive cells return in virtually all patients if tyrosine kinase therapy is interrupted.

Blast Crisis Stem Cells

The onset of accelerated phase is thought to occur in a BCR-ABL-bearing granulocyte-monocyte progenitor. Experimental^844,845 and theoretical⁸⁴⁶ evidence supports this concept. This locale for clonal evolution also could explain the reversion to chronic phase in some patients in whom the suppression of the advanced phase of the disease is achieved. Thus, during chronic phase there is the interplay between polyclonal normal hematopoietic stem cells and CML stem cells (see Chap. 85, Fig. 85–4) and in the acute phase a third stem cell pool (second cancer stem cell pool) is added that initiates and sustains accelerated phase and blast crisis.

Molecular and Genetic Alterations

The transformation of chronic phase CML to accelerated phase and then blast crisis or directly to blast crisis is thought to be the result of seven or more molecular processes: (1) maturation arrest, (2) failure of genome surveillance, (3) failure of adequate DNA repair, (4) development of a mutator phenotype, (5) telomere shortening, (6) loss of tumor-suppressor function, and (7) unknown factors.^847,848

Progression of chronic phase to accelerated phase is marked by an increase in BCR-ABL expression.^849,850 Superimposed on the increased transcription of mRNA^BCR–ABL are additional cytogenetic abnormalities that are added to the persistent Ph chromosome in approximately 50 to 65 percent of patients.^{843,850–853} In lymphoid blast crisis, in which the blast cells have a lymphocytic phenotype, acquisition of p16/ARF mutations occurs in approximately 50 percent of cases, and RB gene mutations occurs in approximately 20 percent of cases. In myeloid blast crisis, in which blast cells have a myeloid phenotype, approximately 25 percent of cases have cells containing a p53 mutation.⁸⁵⁴ The possible role of loss of p53 function in fostering transformation of a human chronic phase CML clone has been demonstrated in transgenic mice in which p53 function was abrogated.⁸⁵⁵ p51/p63 is a member of the p53 gene family that is not mutated in chronic phase but is mutated in approximately 8 percent of patients in blast crisis.⁸⁵⁶

Progression of the clone to a more malignant clone is reflected in a more disordered growth and maturation pattern of progenitor cells in culture, ultimately mimicking the growth failure of acute leukemia,⁸⁵¹ and in increased morphologic and functional abnormalities of blood cells,^857,858 eventuating in a block in maturation and replacement of blood and marrow by blast cells.

Approximately 65 percent of patients have cytogenetic abnormalities in addition to the Ph chromosome. A double Ph chromosome, trisomy 8, and isochromosome 17p are the secondary changes most commonly seen.^854,859 Because the frequency of trisomy 8 was greater after treatment with busulfan compared to hydroxyurea, the frequency of secondary chromosomal changes may be quite different after imatinib mesylate therapy.⁸⁵⁴ Clonal instability has also been found in cases of lymphoid blast crisis. Clones distinct from those identified later may be detected before overt lymphoid transformation. Identification of these abortive clones suggests clonal instability before the onset of transformation, which might have prognostic value.⁸⁶⁰ FISH has been used to determine which cells have secondary cytogenetic abnormalities, and these cells often are not the blast cells. This finding suggests that some chromosomal abnormalities merely denote genomic instability.⁸⁶¹ The abnormal mRNA and protein product p210^BCR–ABL are present in the marrow and blood cells of patients who have transformed to acute leukemia.^862–864

Although the breakpoint site on M-bcr was thought to be correlated with the time of the onset of the accelerated phase,⁸⁶⁵ subsequent studies have not indicated a correlation between length of chronic phase and the specific site of the BCR-ABL fusion.⁸⁶⁶ Rare cases have displayed deletion of the BCR-ABL fusion gene, loss of transcription of the message, and loss of expression of the p210 tyrosine kinase after transformation, the latter finding indicating the abnormal protein kinase may not always play a unique role in sustaining the acute state.⁸⁶⁷ In contrast, the frequent response, albeit temporary, to imatinib suggests that the mutant BCR-ABL product usually plays a role at this stage of the disease.

Numerous molecular changes identified in the cells of patients with acute transformation that might contribute to the increased malignant behavior of the CML clone, include activation of the N-RAS gene,^868,869 rearrangement of the p53 gene,^869–872 hypermethylation of the calcitonin gene,⁸⁷³ and methylation of the ABL1 gene.⁸⁷⁴ One report described p53 mutations in 17 percent of blast crisis patients. An association between the failure of CML cells to express the retinoblastoma 1 gene product and acute blast crisis with a megakaryoblastic phenotype has been reported.⁸⁷⁵ Homozygous deletions of the p16 tumor-suppressor gene are associated with lymphoid transformation of CML,⁸⁷⁶ but such deletions are not seen in the chronic phase and in myeloid blast crisis. p16 is also known as the cyclin-dependent kinase 4 inhibitor gene and is located on chromosome 9p21.^877,878 This gene inhibits the kinase CDK-4, which regulates a cell-cycle checkpoint prior to commitment to DNA synthesis. The WT gene on chromosome 11p13 encodes a zinc finger motif-containing transcription factor found in CML patients only after progression to blast crisis.⁸⁷⁹ Overexpression of the EVI-1 gene has also been found in CML blast crisis.^880,881 Microsatellite instability has not been found to be involved with progression to blast crisis.⁸⁸²BCL-2, c-MYC, and various other genes have also been implicated in the evolution of CML.^883–886

Approximately 50 genes have been identified that could play a role in the progression to accelerated phase or blast crisis,⁸⁴⁷ including genes identified by expression profiling that are dysregulated in accelerated phase compared to chronic phase.^887,888 These include the WNT–catenin and JunB pathways.

Clinical Features

Signs and Symptoms

The features that might signal the conversion of the chronic to the accelerated phase include unexplained fever, bone pain, weakness, night sweats, weight loss, loss of sense of well-being, arthralgia, and left upper quadrant pain related to splenic enlargement or infarcts. These features may occur weeks in advance of laboratory evidence of the accelerated phase. Localized or diffuse lymphadenopathy or enlarging masses in extralymphatic and extramedullary sites containing BCR-ABL–positive myeloblasts or lymphoblasts may develop. A poor response of blood cell counts and splenic enlargement despite previously effective therapy may be evident.^{843,854,889–891} Symptoms caused by histamine excess in basophilic crisis can be present.⁸⁹²

Several of these changes may occur in series or in parallel. The time of onset of transformation and the appearance of a blastic crisis and its clinical expression are unpredictable.

Laboratory Features

Blood Findings^{854,889–891}

Anemia may worsen and be associated with increasing poikilocytosis, anisocytosis, and anisochromia. The number of nucleated red cells in the blood may increase. These red cell changes may be accentuated further if advancing marrow fibrosis is a feature of the disease.

The total leukocyte count may fall without treatment. The proportion of blasts increases to greater than 10 percent in blood and marrow in the accelerated phase and when blast crisis ensues represents 20 to 90 percent of the cells. The morphology of the blast cells may be lymphoid or myeloid. Myelocytes decrease in number. Hyposegmented neutrophils (Pelger-Huët cells) and other dysmorphic changes may become evident. Basophils increase and often represent 20 to 80 percent of the total blood leukocytes. A decrease of the platelet count to less than 100,000/L (<100 x 10⁹/L) develops. Giant platelets, micromegakaryocytes, and megakaryocyte fragments may enter the blood. Decreased progenitor cell growth in culture is present, akin to that in acute leukemia.

Marrow Findings^{854,889–891}

The marrow findings are widely variable. Marked dysmorphic changes in one, two, or three of the major cell lineages; an increase in blast count to greater than 10 percent; marrow morphology simulating subacute myelomonocytic leukemia; or, in the extreme, florid blastic transformation with blast counts greater than 30 percent can occur. Reticulin fibers may increase in prominence, and occasionally severe reticulin and collagen fibrosis develop. Additional clonal cytogenetic abnormalities develop in as many as half the patients in accelerated phase (see “Cytogenetic Studies” below).

Extramedullary Blast Crisis

A variety of symptoms or signs may occur as a result of the specific effects of new extramedullary blastic tumors, referred to as extramedullary blast crisis.^893–896 Extramedullary blast crisis is the first manifestation of accelerated phase in approximately 10 percent of patients with CML. Lymph nodes,^894,895 serosal surfaces,^897,898 skin and soft tissue,^893–896 breast,^896,899 gastrointestinal or genitourinary tract,^894,896 bone,^{894,896,900–903} and central nervous system^{894,904–906} are among the principal areas involved. Isolated or diffuse lymphadenopathy may occur. Bone involvement may lead to severe pain, tenderness, and pathologic fracture, and may be evident on imaging of the involved area. Central nervous system involvement usually is meningeal and may be preceded by headache, vomiting, stupor, cranial nerve palsies, and papilledema and is associated with an increase in cells, protein, and the presence of blasts in the spinal fluid.^{896,904–906}

Appropriate histochemical and immunologic tests are required to determine if the extramedullary disease is composed of phenotypic myeloblasts or lymphoblasts. Because the tumor cells may have features of lymphoma cells, the terms myeloid or granulocytic sarcoma, chloroma, and myeloblastoma can be misnomers, and the term extramedullary blast crisis is used for this circumstance in CML.^{905,907–909} The lymphoblasts, like the myeloblasts, are Ph chromosome-positive. A combination of morphology, histochemistry (e.g., peroxidase, lysozyme), terminal deoxynucleotidyl transferase assay, and monoclonal antibodies specific for lymphoid or myeloid cells can be used to classify the extramedullary blast cells. Older reports (1930s–1960s) of concurrent lymphoma and CML probably were, in many cases, examples of extramedullary lymphoblast crisis in lymph nodes or other sites.

Marrow Blast Crisis

Approximately half of patients with CML enter the accelerated phase by developing acute leukemia. The onset of blast crisis can develop from days^910–912 to decades after diagnosis of CML. The signs and symptoms may include fever, hemorrhage, bone pain, and lymphadenopathy.^{848,910–912} The morphology of the acute leukemia usually is myeloblastic or myelomonocytic.^848,913 A substantial proportion of myeloid leukemia in this setting may not have myeloperoxidase demonstrable by cytochemistry.⁹¹⁴ The proportion of cases classified as erythroblastic leukemia is approximately 10 percent, based on morphologic features,⁹¹⁵ but may be as high as 20 percent if expression of glycophorin-A is used as the determinant.⁹¹⁶ Occasional cases have megakaryoblastic transformation.^875,917 These cases may be difficult to identify by light microscopy because the megakaryoblasts may be mistaken for lymphoid cells or undifferentiated blasts. Myelofibrosis is a feature of this variant. Antiplatelet glycoprotein antibodies and other monoclonal antiplatelet antibodies now are available as reagents to identify megakaryoblasts without the need for ultrastructural studies.⁹¹⁷ Promyelocytic^918–920 and eosinophilic⁹²¹ blast crises also can occur. Basophilic leukemia is a known variant of CML.⁹²² Patients with promyelocytic crisis often have t(15;17) in addition to the Ph chromosome, and some have presented with disseminated intravascular coagulation.⁹²³

CML may transform into acute lymphoblastic leukemia in approximately 30 percent of CML patients in blastic crisis.^{843,924–928} The lymphoid cells generally express terminal deoxynucleotidyl transferase (TdT)^924,925 and are of the B cell lineage,^928–930 as judged by antiimmunoglobulin staining. TdT is a DNA polymerase that adds deoxynucleoside monophosphates from triphosphate substrates to single-stranded DNA by end addition, differing in the latter respect from replicative polymerases.⁹³¹ The enzyme is present in normal immature thymocytes and in the blast cells of nearly all patients with acute lymphoblastic leukemia. Rare patients have blasts with a T-lymphocyte phenotype.^{907,908,932,934} Some cases are biphenotypic; the blasts have both lymphoid and myeloid markers.^{913,935–937} Some cases may have myeloperoxidase activity in blast cells and express CD33 or CD13. Myeloid to lymphoid clonal succession following autologous transplantation in the second chronic phase has been described.⁹³⁸ Patients with lymphoid blast crisis seldom have an intermediate accelerated phase, have less splenomegaly and basophilia, and usually have a higher degree of marrow blast infiltration. With non–tyrosine-kinase-inhibitor therapy, remission rate and survival were somewhat longer in cases of lymphoid than in myeloid blast crisis.⁹³⁹

Cytogenetic Studies

Most large studies have shown seven recurrent changes in patients’ cells prior to, or during, the accelerated phase: trisomy 8 (33% of cases), additional 22q– (30% of cases), isochromosome 17 (20% of cases), trisomy 19 (12% of cases), loss of Y chromosome (8% of males), trisomy 21 (7% of cases), and monosomy 7 (5% of cases).^940–943 In addition, a large number of other chromosome abnormalities have been described.^944–948 In one study, 46 (63%) of 73 blast crisis patients had secondary cytogenetic abnormalities. These abnormalities were more common in myeloid blast crisis and were associated with shorter remission.⁸⁶⁰ The changes may be features of myeloid blast crisis compared to lymphoid crisis.^941,946 Some abnormalities, such as inv16, are associated with early transformation to AML.^942,946,949 A significant proportion (50%) of patients in the accelerated phase or blast crisis have no additional cytogenetic abnormalities beyond t(9;22)(q34;q11) after banding and multicolor FISH analysis.⁹⁴⁸ In cases where the blastic transformation is in extramedullary sites, such as lymph nodes or spleen, the additional cytogenetic abnormalities may be in the cells at those sites but not in cells in the blood or marrow.⁹⁵⁰

Treatment

Optimal treatment is allogeneic stem cell transplantation if the patient is eligible because of the patient’s age and donor availability. The role of stem cell transplantation is also evolving as tyrosine kinase inhibitor use in these phases of diseases becomes better defined.

Tyrosine Kinase Inhibitors in Accelerated and Blast Crisis

The initial dose of imatinib in accelerated phase is 600 mg/day.⁹⁵¹ Imatinib, dasatinib, and nilotinib have been utilized as bridging therapies to permit allogeneic stem cell transplantation in accelerated phase.⁹⁵² Dasatinib and nilotinib can achieve a better molecular response, and thus the role for and timing of transplantation in accelerated phase CML is being redefined. Combination therapies are being explored in the accelerated phase of disease, including dasatinib plus imatinib.⁹⁵³ Imatinib can be combined with mitoxantrone plus etoposide or cytarabine for patients in myeloid blast crisis.⁹⁵⁴ Imatinib has produced complete hematologic remissions in approximately 20 percent of patients.^955,956 However, complete cytogenetic responses are uncommon. Central nervous system and other extramedullary blast crisis can occur during imatinib therapy for accelerated phase disease,^957,958 and all types of blast crisis, including promyelocytic blast crisis,⁹⁵⁹ can occur during imatinib therapy. Compared to historical controls in which various combinations of chemotherapy were utilized, imatinib used alone results in comparable outcomes (6-month median survival of patients in blast crisis).⁹⁶⁰ Although dasatinib therapy can result in complete cytogenetic responses in 29 percent of blast crisis CML, and nilotinib can result in complete cytogenetic response in 27 percent of myeloid blast crisis and in 43 percent of lymphoid blast crisis, these responses are rarely durable, so in a patient of appropriate age with an acceptable donor, transplantation options should be considered with the second-line tyrosine kinase inhibitors utilized as a bridge to transplantation therapy.^961,962

Chemotherapy

The treatment approach is predicated on the phenotype of the blast cells in CML patients with blast crisis and is rarely used in accelerated phase. In patients with myeloid phenotypes, the approach has been similar to that used for acute myelogenous leukemia: combinations of an anthracycline antibiotic, such as idarubicin or daunorubicin, with high-dose cytosine arabinoside and sometimes etoposide.⁹⁶³ Because this approach produces few remissions that are of short duration (median survival approximately 6 months), a variety of other drug combinations incorporating 5-azacytidine, busulfan, cladribine, fludarabine, clofarabine, high-dose cytosine, arabinoside, decitabine, etoposide, farnesyl transferase inhibitors, hydroxyurea, methotrexate, mitoxantrone, plicamycin, tiazofurin, or troxacitabine have been used, but with no significant improvement in outcome.⁹⁶⁰

In patients with lymphoid phenotypes, vincristine sulfate 1.4 mg/m² (not to exceed 2 mg/dose) given intravenously once per week and prednisone 60 mg/m² per day given orally are the mainstay of treatment. A minimum of two cycles of treatment (2 weeks) should be given to judge responsivity. Approximately one-third of patients with lymphoid blast transformation reenter the chronic phase after such treatment. However, because only about one-third of patients have lymphoid blasts, this number represents a remission rate of only approximately 10 percent of patients who enter blast crisis using this approach. Some relapsed patients become TdT-negative (myeloblastic relapse). However, even if relapsed patients remain TdT-positive, they are not likely to respond to a second treatment. Some therapists argue for a more intensive induction regimen for patients with lymphoblastic crisis, akin to regimens for de novo adult ALL or high-risk childhood ALL, and report somewhat better results: higher remission rates and longer remissions. The benefit of such an intensive approach has been small because remission durations have been modest. TdT-positive, CD10 (CALLA)-positive lymphoblasts may be the lymphoblast phenotype most responsive to vincristine and prednisone.⁹²⁷ mTOR inhibitors may be useful in lymphoid blast crisis.⁹⁶⁴

Allogeneic Stem Cell Transplantation Accelerated Phase and Blast Crisis of CML

Stem cell transplantation from an identical twin or sibling has been used in some patients after entry into the blastic phase. Occasional patients have had long-term survival. The 3-year survival rate is approximately 15 to 20 percent,^965–967 unlike transplantation in the chronic phase, in which the 3-year survival rate is 50 to 60 percent. However, for patients who present in blast crisis, who develop blast crisis in the first year of the chronic phase, or who delay transplantation for other reasons, transplantation remains the best hope for long-term survival if a histocompatible donor is available.^904,905 Relapse of accelerated phase after allogeneic stem cell transplantation has responded to infusion of donor cytotoxic T lymphocytes.⁹⁶⁸ The utilization of agents such as imatinib before allogeneic stem cell transplantation for patients in advanced phases of disease may favorably improve transplantation outcomes, especially when major cytogenetic response occurs before transplantation.⁹⁶⁹

Autologous Stem Cell Transplantation Accelerated Phase and Blast Crisis of CML

Autografting in the accelerated phase or blast crisis, either with stem cells collected during chronic phase or with mobilized Ph-chromosome–negative progenitor cells collected upon cell rebound after intensive chemotherapy, has resulted in apparent prolonged remission in some patients, but this procedure is rarely used in advanced disease because of the high rate of relapse.⁹⁷⁰ Whether Ph-chromosome–negative cells collected during imatinib therapy have the same potential is unknown.

Splenectomy

Splenectomy may be performed for palliation of painful splenic infarctions or hemorrhage. However, the complication rates are high, and the procedure performed in this setting should be avoided if possible.⁹⁷¹

Course and Prognosis

The accelerated phase of CML generally is very poorly responsive or refractory to treatment and is a morbid state that can be fatal in weeks to months in all but a few patients who undergo a successful stem cell transplant from a histocompatible donor. Patients with myeloid blast crisis have a median survival of approximately 6 months, whereas patients with lymphoid blast crisis have a median survival of approximately 12 months.^960–972 In the earliest study of imatinib mesylate in blast crisis, patients with myeloblastic crisis had a slightly longer median survival (about 5 months) than patients with lymphoid blast crisis plus Ph-chromosome–positive ALL (3 months). The results are poor in either case. The median survival after evidence of clonal evolution in patients in the chronic phase is approximately 15 months. A worse survival was seen with abnormalities of chromosome 17, other superimposed translocations, or a high percentage of abnormal metaphases.⁹⁷³ Severe cytopenias from repeated courses of cytotoxic therapy contribute to infections, hemorrhage, and organ dysfunction, especially liver and kidney dysfunction. Opportunistic infections with herpes viruses, cytomegalovirus, or fungi often supervene. The addition of imatinib mesylate therapy has made only a small difference in long-term outcome, although formal studies of combinations of chemotherapy and imatinib mesylate at a higher dose (600 mg/day) have not been completed, and the results of trials with second-generation tyrosine kinase inhibitors are anticipated.

Related Clonal Myeloid Diseases Without the BCR Rearrangement

Chronic Myelomonocytic Leukemia

This leukemia is part of the spectrum of clonal myeloid diseases that may have findings that simulate CML. In the past, when rigorous criteria for the diagnosis of CML were not applied, CMML was among a heterogenous group of related diseases that sometimes were referred to as Ph-chromosome–negative CML. These diseases share the feature of originating in the clonal expansion of a primitive multipotential hematopoietic cell.⁹⁷⁴ (Table 90–6)

Epidemiology

Most patients with CMML are older than age 50 years, and approximately 75 percent of patients are older than age 60 years at the time of diagnosis. The median age at diagnosis is approximately 70 years. Occasional cases have been reported in older children and younger adults. Men are affected somewhat more frequently than women (approximately 1.4:1).^975,976

Clinical Findings

Signs and Symptoms

The onset usually is insidious, and weakness, infection, or exaggerated bleeding may bring patients to medical attention.^975,976 Hepatomegaly and splenomegaly occur in approximately 50 percent of patients. Leukemia cutis occurs in a small proportion of patients and usually has a monocytic phenotype: CD45, CD68, and lysozyme positive by immunostaining.⁹⁷⁷ Immune manifestations, such as vasculitis, pyoderma gangrenosum, immune cytopenias, and connective tissue diseases, may occur in coincidence with CMML.⁹⁷⁸

Blood and Marrow Findings

The disease is characterized by anemia and blood monocytosis greater than 1000/L (1 x 10⁹/L).⁹⁷⁹ The white cell count may be slightly decreased, normal, or moderately elevated. Occasional patients may have hyperleukocytosis with total white cell counts of 250,000 to 300,000/L (250–300 x 10⁹/L) associated with respiratory insufficiency resulting from pulmonary leukostasis.⁹⁸⁰ Immature granulocytes may be present in the blood. Blood myeloblasts may be absent or, when present, do not exceed 10 percent of total white cells. Most patients have thrombocytopenia, but normal or elevated platelet counts may occur. Eosinophilia may be so prominent in occasional cases that the designation chronic eosinophilic leukemia may be appropriate.^975,976,981

The marrow is hypercellular as a result of granulomonocytic hyperplasia; the dominant cells are early myelocytes. The proportion of progranulocytes is increased. Promonocytes also are increased in number. Distinction between poorly granulated myelocytes and promonocytes with primary granules can be difficult. Macronormoblasts and hypersegmented or hyposegmented, often bilobed (acquired Pelger-Huët anomaly), neutrophils are frequent. Despite thrombocytopenia, megakaryocytes usually are present in the marrow. Microvessel density is increased in the marrow, and myelomonocytic cells contain cytoplasmic mRNA for VEGF and membrane VEGF receptors.^981,982 In vitro colony studies suggest that autocrine stimulation of cell growth by VEGF may occur. “Spontaneous” cluster/colony growth of granulocyte-monocyte colony-forming cells occurs in vitro. The spontaneous growth may result from autocrine or paracrine production of GM-CSF, based on anti–GM-CSF inhibition of colony growth.⁹⁸⁴

Cytogenetic Findings

Patients with CMML have an approximately 35 percent frequency of chromosomal abnormalities. Monosomy 7 and trisomy 8 are the most prevalent findings. Approximately 35 percent of patients have point mutations of the K-RAS or N-RAS gene. The RAS gene also may be involved in the transforming events. Abnormal methylation of p15^INK4B is a common finding in CMML.⁹⁸⁵ Translocation between the gene PDGFR- on chromosome 5(q33) and four partner genes—TEL at 12(p13), HIP-1 at 7(q11.2), H4 at 10(q22), and Rabaptin-5 at 17(p13)—occur in a very small proportion of patients (approximately 3–4%).^{975,986–989} This mutation juxtaposes the gene encoding the PDGFR- with a partner gene, which results in the encoding of a mutant tyrosine kinase that is constitutively activated and sensitive to inhibition by imatinib mesylate⁹⁹⁰ (see “Treatment” below). The cases with PDGFR- translocations are more likely to be accompanied by eosinophilia than are cases with other cytogenetic abnormalities.

Serum and Urine Findings

Plasma and urine lysozyme concentrations nearly always are elevated. Plasma levels of VEGF, hepatocyte growth factor, and tumor necrosis factor alpha are elevated. Serum B₁₂, ₂-microglobulin, and LDH levels often are elevated.^975,976

Treatment Chronic Myelomonocytic Leukemia

Treatment of most patients with CMML has been unsatisfactory, and remissions of any duration are uncommon. The age and performance status of the patient are considered in determining the intensity of treatment. Cytarabine, either standard or low-dose, etoposide, hydroxyurea, and other approaches used for the oligoblastic myelogenous leukemias have been attempted, but with little success (see Chap. 88). Decitabine and 5-azacytidine have been useful in a small proportion of patients.^975,976 Unfortunately, although a particular approach may confer significant benefit in a small proportion of cases, determining which patients will respond is not possible, except by trial and error. An exception is the patient with a translocation involving PDGFR-, which itself occurs in only a small percentage of patients. In the case of PDGFR- fusion genes with several of the partner genes, imatinib mesylate 400 mg/day has resulted in normal blood counts, cytogenetic remissions, and, occasionally, molecular remission.^{985–987,991,992} These fortunate patients probably will benefit from this treatment, as evidenced by prolonged remissions and survival, compared to other drug options, but the number of patients and duration of followup do not permit quantitative estimates at this point. Allogeneic stem cell transplantation is an option for the small proportion of younger patients with an appropriate matched-related or unrelated donor.⁹⁹³

Course and Prognosis

Median survival in CMML is approximately 12 months, with a range from approximately 1 to more than 60 months. Approximately 20 percent of patients progress to frank AML. Arbitrary stratification of CMML into types 1 and 2 based on the height of the blast count has been proposed, but distinguishing patients by whether they have 8 or 12 percent blasts on a marrow examination is useless for patient care. It is well established that blast percentage usually is a significant correlate with outcome in any patient with a clonal myeloid disease, and this factor among several others should guide therapy. Clusters of prognostic variables have been used to stratify patients into risk groups for survival duration. In general, the severity of the anemia and the height of the blast percentage are the most important. Other variables that may confer a shorter life expectancy are height of the absolute lymphocyte count, high spontaneous rates of myelomonocytic colony growth, higher total leukocyte counts, higher LDH level, and larger spleen size.^994–996 Unfortunately, at this time, unless the patient is a candidate for imatinib therapy or stem cell transplantation, long-term salutary therapeutic effects are uncommon.

Chronic Eosinophilic Leukemia

History and Definition

The recognition of eosinophilic lineage prominence in myelogenous leukemia dates to a case published in 1912.⁹⁹⁷ In 1968, the term hypereosinophilic syndrome was introduced to encompass a group of disorders with (1) prolonged exaggerated eosinophilia without an apparent cause, (2) frequent cardiac and neurologic tissue damage, (3) a poor or transient response to therapy, and (4) a progressive course and a high fatality rate. Shortly thereafter, Benvenisti and Ultmann⁹⁹⁸ presented five cases of eosinophilic leukemia and reviewed the literature regarding that phenotypic designation. In 1975, Chusid and colleagues⁹⁹⁹ described 14 cases of hypereosinophilic syndrome, highlighted the frequency of secondary cardiac and neurologic disorders, and suggested the existence of a continuum of manifestations. Because some cases had clonal cytogenetic abnormalities and hematologic findings compatible with a clonal myeloid disease, the presence of eosinophilic leukemia was suspected in this apparently heterogeneous group of patients.

The relationship of blood eosinophilia to clonal myeloid diseases is complex because the former can be reactive or represent acute eosinophilic leukemia, chronic eosinophilic leukemia, or eosinophilia associated with a different category of disease, such as BCR-ABL–positive CML, idiopathic myelofibrosis, oligoblastic leukemia (MDS), or mastocytosis.¹⁰⁰⁰ Chronic eosinophilic leukemia is a BCR-ABL–negative, clonal myeloid disease with a striking eosinophilia in the blood and marrow, often with clonal cytogenetic abnormalities that have features including, when present, cytogenetic findings that usually distinguish chronic eosinophilic leukemia from other clonal myeloid diseases that may have an associated eosinophilia, such as CMML. The phenotype of the eosinophilic variant of CMML overlaps somewhat with that of chronic eosinophilic leukemia. However, the fusion gene associated with CMML involves the PDGFR- (see “Chronic Myelomonocytic Leukemia” above), whereas in chronic eosinophilic leukemia the cytogenetic findings are different and in some cases involve PDGFR-. This definition of chronic eosinophilic leukemia recognizes that, at the margins, classification may be arbitrary. Studies in cases with the FIP1L1-PDGFR- translocation were consistent with multilineage involvement, consistent with an origin in a pluripotential lymphohematopoietic cell.¹⁰⁰¹ Another report found multilineage involvement but with a presumptive lesion in a multipotential, not pluripotential, hematopoietic cell.¹⁰⁰²

Signs and Symptoms

Fever, cough, weakness, easy fatigability, dyspnea, abdominal pain, maculopapular rash, cardiac symptoms and signs of heart failure, and a variety of neurologic manifestations ranging from peripheral neuropathy to cerebral encephalomalacia may occur, ranging from mild to severe in expression. Splenomegaly often is evident.

Laboratory Findings

Eosinophilia is a constant finding (see Chap. 62, Fig. 62–3). Anemia usually but not always is present at the time of presentation. The leukocyte count may be high-normal or more often elevated. Platelet counts often are normal or mildly decreased. The marrow shows myelocytic and eosinophilic hyperplasia and occasionally Charcot-Leyden crystals. Mast cells may be increased. Megakaryocytes usually are present but may appear dysmorphic. Reticulin fibrosis is common. Immunophenotyping and PCR does not show evidence of either a clonal T-cell population or T-cell-receptor rearrangement. Pulmonary function studies may provide evidence of fibrotic (restrictive) lung disease. Echocardiography may detect mural thrombi, thickening (fibrosis) of the ventricular wall, valvular dysfunction from papillary muscle, and chordae fibrosis. Magnetic resonance imaging can detect subendocardial fibrosis, thickening of ventricles, and markedly reduced ventricular lumen volume. Serum immunoglobulin (Ig) E, vitamin B12, and tryptase levels usually are elevated. Skin biopsy of lesions uncovers intense eosinophilic infiltrates. Neural or brain biopsy may disclose eosinophilic infiltrates, often perivascular, with microthrombi, axonal degeneration, and gliosis.

Cytogenetic Findings

A wide array of cytogenetic findings have been reported in cases of chronic eosinophilic leukemia.¹⁰⁰³ Notable translocations include a high frequency of translocations involving chromosome 5, t(1;5), t(2;5), t(5;12), t(6;11), 8p11, trisomy 8, and numerous others infrequently. Chromosome 5 often is translocated at the site of the PDGFR- gene, and the phenotype usually is more compatible with CMML with eosinophilia. Chromosome 5 from band q31-35 contains several genes relevant to eosinophilopoiesis, including those encoding IL-5, IL-3, GM-CSF, and PDGFR-. A cryptic interstitial deletion on chromosome 4(q12;q12) results in the fusion gene FIL1L1-PDGFR- and in a phenotype of chronic eosinophilic leukemia, which is of particular note because, like the PDGFR- mutations in CMML with eosinophilia, of a near-universal response to treatment with imatinib mesylate.^1003–1005

Serum Tryptase Level Elevation versus Normal Levels

The elevation of serum tryptase level (>11.5 ng/mL) has been used to distinguish a subset of patients who (1) are male, (2) have marrows that are intensely hypercellular with a higher proportion of immature eosinophils and with dysmorphic mast cells with a CD117–CD25+CD2– genotype and phenotype (distinguishing these cells from classic mastocytosis, which are CD117+CD25+CD2+), (3) have dramatically higher serum B₁₂ and IgE levels, (4) are more prone to restrictive pulmonary disease and endomyocardial fibrosis, (5) have the FIP1L1-PDGFR- fusion gene, and (6) are responsive to imatinib mesylate.⁹⁴¹ Patients with normal serum tryptase levels are more prone to obstructive pulmonary restrictive disease, eosinophilic dermatitis, and gastrointestinal complaints.

Differential Diagnosis

Eosinophilia can occur for many reasons (see Chap. 62). The first step is to identify signs that may point to a clonal myeloid disease. These signs include anemia, thrombocytopenia, splenomegaly, immature eosinophils in the marrow examination, evidence of dysmorphic cells in blood or marrow, for example, atypical megakaryocytes or dysmorphic mast cells, cardiac or pulmonary manifestations, which may occur secondary to chronic eosinophilic leukemia, and markedly elevated serum tryptase or vitamin B12 level. The former signs, especially in the aggregate, are highly suggestive, but the presence of a cytogenetic abnormality in myeloid cells is diagnostic of a clonal myeloid disease (leukemia). If the latter is not evident, PCR and/or flow cytometry to search for a clonal T lymphocyte abnormality should be performed. Whether the eosinophilic leukemia is typical or represents an eosinophilia with idiopathic myelofibrosis, CMML, or MDS is less important than if it has a mutation that is imatinib mesylate sensitive (e.g., PDGFR mutation).

Therapy

Patients (nearly always men) whose cells display a FIP1L1–PDGFR- have a very high probability of responding to imatinib mesylate at a dose of 100 to 400 mg/day.^1004–1008 The tyrosine kinase activity of this fusion protein is two orders of magnitude more sensitive to imatinib than that of BCR-ABL. However, because not all patients taking 400 mg/day achieve a molecular remission, and that goal may be more likely to result in long-term remission, initial therapy remains at 400 mg/day and PCR monitoring is appropriate. Dose adjustment upward if molecular remission is not achieved can be considered. Unlike the case in CML, patients with chronic eosinophilic leukemia with significant side effects when taking imatinib mesylate, 400 mg/day, have a reasonable probability of having a good response at lower doses.¹⁰⁰⁸ Responses to dasatinib and nilotinib have also been reported.¹⁰⁰⁹

In patients with eosinophilic leukemia without an imatinib mesylate-sensitive mutation or in patients who become resistant to imatinib mesylate and unresponsive to second-generation tyrosine kinase inhibitor (e.g., dasatinib) and who are progressing, ablative or nonablative allogeneic stem cell transplantation can be considered if they are in an acceptable age range and have access to a matched-related or matched-unrelated donor.^1010,1011

In imatinib mesylate-insensitive patients without the option of transplantation, empirical treatment with glucocorticoids, hydroxyurea, or anti-IL5^1012,1013 to decrease eosinophil counts and mute the progress of eosinophil-mediated cutaneous, cardiac, pulmonary, and neurologic tissue damage should be considered. These approaches may relieve symptoms for a time, but are temporizing if therapy with drugs that might be effective in inhibiting clonal expansion or evolution is not effective (e.g., cytarabine, anthracycline antibiotic, etoposide).

Course and Prognosis

If chronic eosinophilic leukemia is not imatinib sensitive, the long-term outlook is one of probable progressive cardiac and neurologic disability. Transformation to acute eosinophilic or myelogenous leukemia can occur. Allogeneic stem cell transplantation is potentially curative. In imatinib-sensitive cases, hematologic normalization, reversal of marrow fibrosis and mastocytosis, resolution of skin lesions, normalization of spleen size, and restoration of well-being occurs in the great preponderance of cases. Cardiac, neurologic, and pulmonary changes usually cannot be reversed but should be stabilized. The long-term outlook with imatinib mesylate therapy is uncertain. However, imatinib likely will decisively and dramatically improve survival compared to other prior therapy and should greatly improve the prognosis of patients with a molecular target for the drug.

Chronic Basophilic Leukemia

This type of clinical disorder, in which the patient has marrow and blood basophilia and other findings compatible with a clonal myeloid disease without evidence of the BCR-ABL translocation, is rare. Two reports of such a syndrome occurring in five patients have been published.^1014,1015 The marrow was intensely hypercellular in the three major lineages. Dysmorphic megakaryocytes were evident. Basophilia in marrow and blood was striking, although eosinophilia also was evident in two patients and increased mast cells in three patients. The clinical effects of basophilic mediator release were evident in two patients. One patient evolved to AML; another recovered after allogeneic transplantation. The cases had similar findings, leading to the suggestion they represented Ph-negative chronic basophilic leukemia. In one case, a PRKG2-PDGFR- fusion gene was evident and the patient responded to imatinib mesylate.

Chronic Monocytic Leukemia

History

In 1937, Osgood¹⁰¹⁶ reviewed his experience with monocytic leukemia and included a case that probably represented the rare disorder chronic monocytic leukemia. In 1981, approximately 28 bona fide cases had been reported, 5 cases were added, and the characteristics were reviewed.¹⁰¹⁷

Clinical Findings

Patients range in age from 30 to 80 years. Males are affected more frequently than females. Fever, fatigue, and left upper quadrant pain are the most common complaints. Splenomegaly and hepatomegaly are nearly constant findings.^1017–1019

Laboratory Findings

Anemia is mild. Anisocytosis and poikilocytosis usually are present. The leukocyte count usually is normal or low but can be elevated in a minority of patients. The percentage of monocytes is increased, but the absolute monocyte count often is normal, ranging from 300 to 1500/L (0.3–1.5 x 10⁹/L), or mildly elevated. Occasional patients have more striking monocyte counts. The platelet count may be normal or decreased. Rare nucleated red cells may be present in the blood. The monocytes in the blood contain -naphthol acetate esterase, tartrate-sensitive acid phosphatase, fluoride-sensitive naphthol AS-D acetate esterase, and peroxidase as judged by histochemical tests. The marrow is cellular, often without an increase in monocytes. The Ph chromosome is absent. The leukemic cells are similar to mature monocytes with abundant cytoplasm. Erythrophagocytosis or thrombocytophagocytosis by monocytes may be seen.

The disease often is not recognized because the total white cell count, monocyte count, and number of marrow monocytes may not be elevated until the spleen is removed, usually for diagnostic purposes.¹⁰¹⁷ Following splenectomy, a gradual leukocytosis of 3000 to 100,000/L (3–100 x 10⁹/L) may develop.¹⁰¹⁷ The absolute monocyte count increases dramatically, often from less than 1000/L (1 x 10⁹/L) to as high as 75,000/L (75 x 10⁹/L). The marrow may contain more than 50 percent mature monocytes following splenectomy.

The spleen is enlarged (300–2500 g). The red pulp is infiltrated with mononuclear cells, often obliterating sinus lumens. Erythrophagocytosis by the mononuclear cells frequently is evident. Liver biopsy may show a mononuclear infiltrate in the sinusoids. Although clinical lymph node enlargement is rare, lymph node biopsies show striking infiltration by leukemic monocytes.

Course, Prognosis, and Treatment

Median survival is approximately 25 months. Patients often die of septicemia^1017–1019 or acute monocytic leukemia.^1020,1021 Therapy has not been studied systematically, but neither intensive combination chemotherapy nor glucocorticoids have changed the course of the disease.

The World Health Organization Committee on the Lymphohematopoietic Tumors does not describe a disease called chronic monocytic leukemia and recent reports of cases are very rare. A few well-documented cases of chronic monocytosis, presumably clonal, evolving into myelogenous leukemia could also satisfy this designation (see Chap. 88).

Juvenile Myelomonocytic Leukemia

Epidemiology

Ph-chromosome–positive, adult-type CML occurring in children younger than age 15 years composes approximately 3 percent of childhood leukemias and approximately 10 percent of all cases of CML.¹⁰²² Although CML occurs in children of all ages, it is rare in children younger than age 5 years. With the exception of a propensity to present with higher total leukocyte counts and with leukostatic signs or symptoms, CML in children has the typical manifestations and course of the disorder seen in adults.

A disorder different from adult-type CML, designated juvenile myelomonocytic leukemia (juvenile CML), represents approximately 1.5 percent of childhood leukemias. It occurs most often in infants and children younger than age 4 years and is similar in some respects to adult subacute or chronic myelomonocytic leukemia because the two diseases share a prominent monocytic component in the leukemic cell population.^1023–1026

Pathogenesis

This disorder is a clonal myeloid disease that originates in an early hematopoietic multipotential cell. Evidence indicates this cell may be pluripotential (myeloid-lymphoid) in some cases and myeloid in others.^1027–1030 RAS mutations in hematopoietic cells are present in approximately 20 percent of patients.¹⁰³¹ Approximately 1 in 10 patients with juvenile myelomonocytic leukemia have mutations of NF1 and manifest type 1 neurofibromatosis. This frequency is approximately 400 times the expected occurrence in a comparable pediatric population.^1032–1034 The linkage between neurofibromin, the protein encoded by the NF1 gene, guanosine triphosphatase activity proteins, and the activation state of RAS-encoded proteins has led to a postulated sequence of events that may be triggered by the extraordinarily heightened sensitivity of the colony-forming cells in the marrow and blood of infants with the disease to the proliferative effects of GM-CSF. The latter initiates signal transduction from the cell membrane to the nucleus via RAS protein activation.^1035,1036 Mutations in the PTPN11 gene have been found in approximately one-third of children with juvenile CML, and the mutations in NF1, RAS, and PTPN11 usually do not coincide.^1035,1036 However, they each may act through a common pathway. PTPN11 encodes SHP-2, a nonreceptor tyrosine kinase, which is an upstream regulator of RAS; thus, all three mutations can contribute to deregulation of RAS signaling. As an aside, children with Noonan syndrome, which is characterized by short stature, dysmorphic facies, skeletal abnormalities, and cardiac defects, have a germ-cell mutation of PTPN1. These children may have a transient disorder that closely mimics juvenile myelomonocytic leukemia.¹⁰³⁶

Clinical Findings

Symptoms and Signs

Infants present with failure to thrive, and children present with malaise, fever, persistent infections, and exaggerated skin, oral, or nasal bleeding. Hepatomegaly can occur. Splenomegaly, sometimes massive, is present in almost all cases. Lymphadenopathy is frequent.^1023–1026 More than half of the patients have eczematoid or maculopapular skin lesions¹⁰³⁷ and xanthomatous lesions, and multiple café-au-lait spots (neurofibromatosis type 1) may occur.¹⁰²⁴ The xanthomas may be the earliest signs of neurofibromatosis.^1024,1025 Noonan syndrome (dysmorphic facies, short stature, heart disease, mental retardation, cryptorchidism, webbed neck, chest deformities, and bleeding diathesis) may coexist.¹⁰²⁵

Laboratory Findings

Anemia, thrombocytopenia, and mild to moderate leukocytosis are common. The leukocyte count usually is greater than 10,000/L (10 x 10⁹/L) with a median leukocyte count at diagnosis of about 35,000/L (35 x 10⁹/L). The blood has an increased monocyte concentration of 1000 to 100,000/L (1–100 x 10⁹/L), immature granulocytes including a small percentage of blast cells, and nucleated red cells. Fetal hemoglobin concentration is increased in approximately two-thirds of the patients. The marrow aspirate is hypercellular as a result of granulocytic hyperplasia; the number of erythroblasts and megakaryocytes usually are decreased. Monocytic cells are increased but may not be as striking as in the blood. Leukemic blast cells are present in modest proportions (<20%).

Cell culture of blood and marrow shows a striking preponderance of monocytic progenitors, even in the absence of overt monocytosis in the marrow.^1038,1039 Granulocyte-monocyte colony-forming cells show a marked tendency to spontaneous growth if adherent (monocytic) cells are not depleted from culture.¹⁰³⁹ The effect is mediated by a release of large quantities of GM-CSF by monocytes in culture.¹⁰⁴⁰

Although clonal chromosome abnormalities have been found in some cases,¹⁰⁴¹ the cytogenetic abnormalities have no consistent pattern, and more than half of the patients have normal karyotypes. The BCR-ABL fusion gene is not present.^1041–1043 The phenotype of monosomy 7 syndrome overlaps with juvenile myelomonocytic leukemia, and an abnormality of chromosome 7 (del 7, del 7q, others) is present in approximately one-fifth of patients.¹⁰²³

Course, Prognosis, and Treatment

The median survival of patients with juvenile CML has been less than 2 years.^1023,1024 Younger children (younger than 2 years) are more likely to have a protracted course.⁹⁵⁵ The disease has been refractory to most chemotherapy. In a study of 9 patients, 4 of whom were treated with a 5- or 6-drug intensive regimen, remissions were 11 to more than 27 months, compared with untreated or lightly treated patients, 4 of whom died within 7 months.¹⁰⁴⁴ Even in the treated patients, complete suppression of the disease did not occur, and treatment protocols to induce and sustain remissions were lacking.¹⁰³⁹ A program of cytosine arabinoside, etoposide, vincristine, and isotretinoin resulted in a highly favorable response in five children treated. Three patients relapsed and were treated with cytarabine by infusion and subcutaneously and with etoposide. All patients were alive, and the range of survival at the time of publication was 8 to 89 months, with a median survival of 27 months. The resistance of these cells to currently available therapy is gruesomely highlighted by the sense of success in prolonging the life of infants and young children by a few years. Intensive therapy can control disease, but curative chemotherapy has been elusive.¹⁰⁴⁵ The inclusion of isotretinoin was based on a prior report of responsiveness to the drug used alone; however, this observation has not been confirmed.¹⁰⁴⁶ The GM-CSF antagonist E21R, inhibitors of RAF-1 gene expression, blockers of RAS protein farnesylation, and angiogenesis inhibitors are among other drug approaches to the disease being studied.^1026,1047

Allogeneic stem cell transplantation is an important approach to therapy and may provide the best chance of long-term survival in selected children.^1048,1049 Hence, a rapid search for a matched-unrelated donor is important in patients without matched sibling donors. Transplantation from a histocompatible sibling or matched-unrelated donor resulted in an event-free survival at 4 years of 54 percent in one study of 27 patients that used a variety of conditioning regimens. A cytogenetic abnormality, such as monosomy 7, was a dismal prognostic finding in that study, and children transplanted before age 1 year had better results than did older children.¹⁰⁴⁸ DLI has placed a posttransplantation patient in relapse into remission,¹⁰⁵⁰ but in general this procedure is ineffective in patients who relapse after transplantation.

A minority of patients have a smoldering course for 2 to 4 years. Thereafter, the disease usually rapidly progresses, and patients die of infection or hemorrhage. Occasional patients have a very long survival (>10 years) despite persistence of abnormal blood counts and splenomegaly, independent of the type or intensity of therapy. Some children convert to a full-blown acute myelogenous leukemia with a rapidly fatal outcome. Cases of juvenile myelomonocytic leukemia may be associated with transformation to acute lymphoblastic leukemia.¹⁰⁵¹

Chronic Neutrophilic Leukemia

History, Pathogenesis, and Epidemiology

In 1920, Tuohey¹⁰⁵² described the first recorded case of an unusual sustained neutrophilia with splenomegaly without fever, inflammation, cancer, or other cause of a leukemoid reaction. Use of X-chromosome-linked polymorphic genes in blood cells and FISH of chromosome abnormalities have been indicative of a clonal myeloid disorder.^1053–1055 Some cases may arise in the hematopoietic multipotential cell, others in a neutrophil progenitor cell (see Chap. 85).^1053–1057 Evidence points to defective apoptotic signals accounting, in part, for the striking accumulation of segmented neutrophils in the blood.¹⁰⁵⁸ The median age at onset is approximately age 65 years. Younger patients may be affected.¹⁰⁵⁹ As in most clonal myeloid diseases, men are affected more frequently than are women.

Clinical Features

Symptoms and Signs

Patients may complain of weakness, anorexia, weight loss, abdominal pain, and easy bruising. Symptoms and signs of gouty arthritis occur in approximately one-third of cases. The spleen is enlarged in almost all cases, and the liver frequently is enlarged. Lymphadenopathy is very infrequent.¹⁰⁵⁷ A hemorrhagic tendency is present in some patients.

Laboratory Findings

Although some patients have a normal hemoglobin concentration at the time of presentation, most have mild to moderate anemia on presentation. The reticulocyte count usually is between 0.5 and 3.0 percent. The platelet count rarely is less than 125,000/L (125 x 10⁹/L) and usually is normal. Coagulation times are normal. The total leukocyte count usually is between 25,000 and 75,000/L (25 and 75 x 10⁹/L) in most cases, and only rarely is less than 20,000/L (20 x 10⁹/L) or exceeds 100,000/L (100 x 10⁹/L). Neutrophils compose 85 to 95 percent of the white cells. Although segmented cells usually dominate, occasional cases have a high proportion of band forms. Very infrequently, metamyelocytes, myelocytes, and nucleated red cells may be present in patients. Basophil and eosinophil counts are not increased. Blasts nearly always are absent from the blood. Neutrophil alkaline phosphatase activity is increased in almost all cases.

The marrow invariably shows granulocytic hyperplasia with myeloid-to-erythroid (M:E) ratios as high as 10:1. Myeloblasts are not overtly increased in number (0.5–3.0%). Megakaryocytes are either normal or slightly increased in number and have normal distribution and morphology. Erythropoiesis usually is mildly decreased. Unlike CML, reticulin fibrosis is unusual. A few cases with dysmorphic features in the marrow (acquired Pelger-Hüet anomaly, erythroid, dysplasia, micromegakaryocytes) have been reported. By definition, the Ph chromosome, BCR gene rearrangements, and BCR-ABL transcripts are absent.^1059–1062 Most patients have normal karyotypes, but approximately 25 percent of patients have nonrandom abnormalities of chromosomes.¹⁰⁶² Deletions of chromosome 20q and trisomy 21 or 9 are the most common abnormalities. Serum vitamin B12-binding protein and vitamin B₁₂ levels both are markedly increased above normal. Serum uric acid concentration is increased, and serum lactic dehydrogenase activity may be increased.

Almost every case examined postmortem had liver and splenic enlargement. Portal hepatic and splenic red pulp infiltrates of neutrophils or islands of extramedullary hematopoiesis with immature myeloid cells and megakaryocytes are characteristic.

Differential Diagnosis

Most leukemoid reactions are associated with an obvious underlying cause, such as pancreatitis, carcinoma, connective tissue disease, smoker’s neutrophilia, and chronic bacterial or fungal infection. The leukocyte alkaline phosphatase level usually is markedly elevated in chronic neutrophilic leukemia and markedly decreased in CML. More to the point, molecular studies identifying BCR gene rearrangement or the presence of BCR-ABL transcripts should distinguish chronic neutrophilic leukemia (BCR-ABL–negative) from neutrophilic CML (BCR-ABL–positive; see “Special Clinical Features” above). In the latter case, more than half of the patients have thrombocytosis and megakaryocytic hyperplasia, which are uncharacteristic of chronic neutrophilic leukemia.

Treatment

No systematic studies of treatment have been reported. Although hydroxyurea, IFN-, or cytarabine may decrease the white count and spleen size, long-term benefit is unusual.^1059–1062 Intensive therapy has led to early posttreatment deaths. Allogeneic stem cell transplantation in eligible patients may be curative.¹⁰⁶³

Course and Prognosis

The disease is fatal, with a median survival of approximately 2.5 years and a range of 0.5 to 6 years.^1059–1062 A case of spontaneous remission has been reported. The prognosis is considerably worse than the prognosis for CML despite the prevalence of mature neutrophils and the paucity of blasts. Causes of death have included (1) intracranial hemorrhage, sometimes in the presence of adequate platelet counts and coagulation times, suggesting a vascular infiltrative process; (2) severe infection; (3) transformation to acute myelogenous leukemia; and (4) the toxic effects of intensive therapy. The disease usually afflicts older subjects, and cardiac, pulmonary, and vascular diseases contribute to a fatal outcome.

A remarkable frequency of concordant essential monoclonal gammopathy or myeloma has been described.^{1055,1064–1072} In two cases, the extreme neutrophilia proved to be a polyclonal response to a plasma cell disorder.^1055,1073 Chronic neutrophilic leukemia has evolved from polycythemia vera or oligoblastic leukemia,^1074–1078 supporting its relationship to the clonal hemopathies.^{1055,1079,1080}

BCR Rearrangement-Negative Chronic Myelogenous Leukemia

A very small proportion of patients (~4%) with clinical manifestations within the limits usually applied to the diagnosis of CML have neither a Ph chromosome (classic, variant, or masked) nor evidence of rearrangement of BCR on chromosome 22. This circumstance represents BCR-negative CML. The literature describing Ph-chromosome–negative CML prior to 1987 is difficult to evaluate because many cases were not studied carefully for masked or variant translocations and for the BCR gene rearrangement. Ph-chromosome–negative CML is a clonal disease⁹⁷⁴ that has the propensity for lymphoid and myeloid transformation.^1081,1082 Although most cases of BCR rearrangement-negative CML are closer in manifestations to CMML,^{996,974,1083–1086} some cases are indistinguishable from classic CML but without the BCR-ABL after exhaustive molecular diagnostic evaluation.^1087–1091 In a report of 76 such patients, the median age was 66 years (range: 24–88), splenomegaly was present in 50 percent of cases, the median white cell count was 38,000 cells/L (38 x 109 cells/L; range: 11–296), and the median hemoglobin was 11 g/dL (range: 7 to 16), with classical morphologic features in blood and marrow.¹⁰⁹¹ As the disease progressed, patients developed severe cytopenias.¹⁰⁸⁸ Median survival was 24 months and only 7 percent survived for more than 5 years. Myeloid blast crisis occurred in one-third of those followed until their death. Occasional patients had extended complete remissions with INF- therapy.¹⁰⁹⁰ Hydroxyurea can be useful as palliative therapy.

Some patients have transposition of ABL to chromosome 22 but not the classic translocation. In such cases, including TEL-ABL translocations, transient responses to imatinib mesylate have been observed.¹⁰⁹²

Uncommon cases of coexisting BCR– and BCR+ clones have been described, and the basis for such cases is in dispute.¹⁰⁹³ One proposed explanation is that this case is an example of “field carcinogenesis” in which multiple clones coexist.¹⁰⁹³ An alternative explanation is that these cases represent the dual progeny of a single unstable clone.¹⁰⁹⁴ The long-term survival of patients with CML may permit the emergence of a drug-induced or spontaneous second malignancy, and in the former it may be notably associated with imatinib (see above “Secondary Chromosomal Changes with Imatinib Mesylate” under “Tyrosine Kinase Inhibitors”).^1095–1097