Incidence

CML occurs at all ages but is seen predominantly in those aged 46 to 53 years. It represents about 20% of all cases of leukemia, is slightly more common in males than in females, and carried a mortality rate of 1.5 per 100,000 per year in the era before the development of imatinib mesylate (Gleevec). Imatinib is a tyrosine kinase inhibitor that has changed the prognosis and treatment for CML and is described in detail later.

Symptoms associated with clinical onset are usually of minimal intensity and include fatigue, decreased tolerance of exertion, anorexia, abdominal discomfort, weight loss, and symptomatic effects from splenic enlargement.

Cytogenetics of the Philadelphia Chromosome

A unique chromosome, the Philadelphia chromosome, is present in proliferating hematopoietic stem cells and their progeny in CML and must be identified to confirm the diagnosis. Although the cause of Philadelphia chromosome formation is unknown, it appears more frequently in populations exposed to ionizing radiation.13,14 In most patients, a cause cannot be identified. Appearance of the Philadelphia chromosome in donor cells after allogeneic bone marrow transplantation indicates the possibility of a transmissible agent.¹⁵ The Philadelphia chromosome was first identified as a short chromosome 22 in 1960 by Nowell and Hungerford in Philadelphia.¹⁶ In 1973 Rowley, of the University of Illinois at Chicago, discovered that the Philadelphia chromosome is a reciprocal translocation between the long arms of chromosomes 9 and 22 (see Chapter 31).¹⁷ This acquired somatic mutation specifically reflects the translocation of an ABL proto-oncogene from band q34 of chromosome 9 to the breakpoint cluster region (BCR) of band q11 of chromosome 22, which results in a unique chimeric gene, BCR/ABL.¹⁸ This new gene produces a 210-kD BCR/ABL fusion protein (p210^BCR/ABL) that expresses enhanced tyrosine kinase activity compared with its natural enzymatic counterpart.

Molecular Genetics

The t(9;22) translocation that produces the BCR/ABL1 chimeric gene has been observed in four primary molecular forms that produce three versions of the BCR/ABL chimeric protein: p190, p210, and p230 (Figure 34-1). The four genetic variations are based on the area of the BCR gene that houses the breakpoint on chromosome 22, because the breakpoint on chromosome 9 occurs in the same location. The wild-type (normal) ABL1 gene on chromosome 9 is a relatively large gene of approximately 230 kilobases (kb) containing 11 exons. The breakpoint consistently occurs 5′ of the second exon such that exons 2 to 11 are contributed to the BCR/ABL1 fusion gene.

There are four BCR genes in the human genome: BCR1, BCR2, BCR3, and BCR4. It is the BCR1 gene that is involved in the Philadelphia translocation. Wild-type (normal) BCR1 gene is approximately 100 kb with 20 exons. In 1984 Groffen et al identified the BCR on chromosome 22 as a 5-exon region involving exons 12 to 16 that was the area of breakage in the traditional t(9;22) translocation.¹⁹ This area was later termed the major BCR. Two other areas of breakage were identified on chromosome 22, one nearer the 5′ (head) of the BCR1 gene, called the minor BCR, and one in the 3′ end (tail) of the BCR1 gene termed the micro BCR.

Within the major BCR two specific breakpoints account for the t(9;22) translocation involved in the development of CML. Therefore, two areas of breakage in the major BCR combined with one in the minor BCR and one in the micro BCR produce the four versions of the BCR/ABL1 chimeric gene. Because the two breakpoints in the major BCR differ by only one exon, the chimeric protein product is essentially the same size and is designated as the p210 protein. Breakage in the BCR1 gene in the major BCR contributes exons 1 to 13 or 1 to 14, whereas the ABL1 gene contributes exons 2 to 11. Breakage in the minor BCR contributes only exon one from BCR, which joins with the same exons 2 to 11 of ABL1 to produce a p190 protein. The micro BCR breakpoint contributes exons 2 to 19 from BCR1, which fuse with ABL1 exons 2 to 11, producing the p230 protein. Therefore, the four possible BCR1 breakpoints produce four different chimeric genes, resulting in a total of three different protein products.²⁰

Pathogenetic Mechanism

To understand the aberrant function of the BCR/ABL fusion protein, it is first helpful to understand both the normal BCR and ABL proteins. The wild-type ABL protein, when in its usual location on chromosome 9, codes for p125, which exhibits normal tyrosine kinase activity. The BCR1 gene produces p160, expresses serine and threonine kinase activity, and is thought to function in the regulation of cell growth. Protein kinases are enzymes that catalyze the transfer of phosphate groups from adenosine triphosphate (ATP), guanosine triphosphate, and other phosphate donors to receiver proteins. A tyrosine kinase transfers the phosphate group to a tyrosine amino acid on the receiver protein. For the kinase activity of the ABL protein to occur, the ABL protein must first be phosphorylated. This is often accomplished through autophosphorylation. The ABL protein has three primary domains called SR1, SR2, and SR3 that together express and regulate the kinase activity. SR1 is the binding site for ATP; SR2 is the docking point for phosphate receiver proteins; and SR3 is the domain that controls the phosphorylation activity. When ATP binds to the ATP binding site, the phosphate is transferred to the SR2 region of the ABL protein, which initiates a conformational change that alters the tertiary structure of the protein and exposes the active site of the enzyme. When a second ATP binds the ATP binding site and a receiver protein docks in the SR2 domain, the phosphate group is transferred to the receiver protein. In most physiologically normal intracellular pathways, protein phosphorylation activates the receiver proteins (Figure 34-2). This phosphorylation initiates a cascade of phosphorylation events, each activating the next protein until a transcription factor becomes activated. These activation cascades, called signal transduction pathways, are designed to activate genes necessary to control cell proliferation, differentiation, and natural cell death, called apoptosis. There are several signal transduction pathways activated by the ABL tyrosine kinase that function in concert to activate these genes in a precise order and at the required level of activation to control these cellular events.^20,21

In the case of CML, the BCR/ABL1 translocation occurs next to the SR3 domain of the ABL1 moiety, which is designed to control the rate and timing of phosphorylation. Therefore, the BCR/ABL tyrosine kinase has lost the ability to shut off kinase activity and is said to have constitutive tyrosine kinase activity. The BCR/ABL enzyme continuously adds phosphate groups to tyrosine residues on cytoplasmic proteins, activating several signal transduction pathways. These pathways stimulate gene expression keeping the myeloid cells proliferating, reducing differentiation, reducing adhesion of cells to bone marrow stroma, and virtually eliminating apoptosis. The result is increased clonal proliferation of myeloid cells secondary to a reduction in or loss of sensitivity to protein regulators.²² There is an increase in growth factor–independent cellular proliferation from activation of the RAS gene and a decrease in or resistance to apoptosis. New clones of stem cells vulnerable to additional genetic changes lead to the accelerated and blast phases of CML. In addition, the BCR/ABL protein localizes in the cytoplasm, rather than in the nucleus as does the normal ABL protein. The mutation affects maturation and differentiation of hematopoietic and lymphopoietic cells, whose progeny eventually dominate in the affected individual. Progeny cells that exhibit this chromosome include neutrophils, eosinophils, basophils, monocytes, nucleated erythrocytes, megakaryocytes, and B lymphocytes.^7,23

In addition, the loss of genetic segments in the 5′ end of the ABL1 gene results in an altered protein-binding affinity for F-actin, which leads to a reduction in contact binding of hematopoietic CML cells to stromal cells and causing premature release of cells into the circulation.²¹ Abnormal adhesion between stem cells and stroma may dysregulate hematopoiesis. One action of interferon-α therapy is to reverse the loss of adhesion of CML progenitor cells, which reduces the premature release of these cells into the circulation.²⁴

Apoptotic functions are lost because the BCR/ABL fusion protein has a propensity to be sequestered in the cytoplasm, which has antiapoptotic functions. The p210 is necessary for CML transformation of the hematopoietic stem cell.

The BCR/ABL1 fusion is also identified with Philadelphia chromosome–positive ALL. The chromosome appears in 20% of adults and 2% to 5% of children with this disease. The minor chimeric BCL/ABL1 gene that transcribes and translates to a p185/p190 protein is present in 50% of Philadelphia chromosome–positive ALL cases in adults and 75% of Philadelphia chromosome–positive ALL cases in children. The micro BCR, when fused with the ABL1 gene, produces a large p230 protein that is the least common version found.

Peripheral Blood and Bone Marrow

There are dramatic morphologic changes in the peripheral blood and bone marrow that reflect the expansion of the granulocyte pool, particularly in the later maturational stages. Table 34-1 lists the qualitative changes in the peripheral blood, bone marrow, and extramedullary tissues that are commonly observed at the time of diagnosis. A dramatic left shift is noted that extends down to the promyelocyte stage and occasionally even produces a few blasts in the peripheral blood. The platelet count is often elevated, reflecting the myeloproliferative nature of the disease. Extramedullary granulopoiesis may involve sinusoids and medullary cords in the spleen and sinusoids, portal tract zones, and solid areas of the liver.

Figure 34-3 illustrates a common pattern in the peripheral blood film of chronic phase CML at the time of diagnosis. Leukocytosis is readily apparent at scanning microscopic powers. Segmented neutrophils, bands, metamyelocytes, and myelocytes predominate, and immature and mature eosinophils and basophils are increased. Myeloblasts and promyelocytes are present at a rate of approximately 1% and 5%, respectively. Lymphocytes and monocytes are present and often show an absolute increase in number but a relative decrease in percentage. Nucleated red blood cells (NRBCs) are rare. Platelets are normal or increased, and some may exhibit abnormal morphology.

Bone marrow changes are illustrated in Figure 34-4. An intense hypercellularity is present due to granulopoiesis, marked by broad zones of immature granulocytes, usually perivascular or periosteal, differentiating into more centrally placed mature granulocytes. Normoblasts appear reduced in number. Megakaryocytes are normal or increased in number and, when increased, may appear in clusters and exhibit dyspoietic cytologic changes. They often appear small with reduced nuclear size (by approximately 20%) and reduced nuclear lobulations. Reticulin fibers are increased in approximately 20% of patients. Increased megakaryocyte density is associated with an increase in myelofibrosis.²⁵ The presence of pseudo-Gaucher cells (see Chapter 28) usually occurs.

Other Laboratory Findings

Hyperuricemia and uricosuria from increased cell turnover may be associated with secondary gout, urinary uric acid stones, and uric acid nephropathy.²⁶ Approximately 15% of patients exhibit total white blood cell (WBC) counts greater than 300 × 10⁹/L.²⁷ Symptoms in these patients are secondary to vascular stasis and possible intravascular consumption of oxygen by the leukocytes. Symptoms are reversible with the lowering of the total WBC count.²⁸

Progression

In the pre-imatinib era, most patients’ disease would eventually transform into acute leukemia.²⁹ Before blastic transformation, some patients proceed through an intermediate metamorphosis or accelerated phase. Disease progression is accompanied by an increase in the frequency and number of clinical symptoms, adverse changes in laboratory values, and poorer response to therapy than in the chronic phase. Additional chromosome abnormalities may appear, associated with enhanced dyshematopoietic cell maturation patterns and increases in morphologic and functional abnormalities in blood cells. There is often an increasing degree of anemia and, in the peripheral blood, fewer mature leukocytes, more basophils, and fewer platelets, with a greater proportion of abnormal platelets, micromegakaryocytes, and megakaryocytic fragments. The circulating blast count increases to 10% to 19%. This total blast percentage, or a combination of 20% blasts and promyelocytes, has been proposed as a diagnostic criterion for the accelerated phase.³⁰

Blast crisis involves the peripheral blood, bone marrow, and extramedullary tissues. By definition, blasts constitute more than 20% of total bone marrow cellularity; the peripheral blood exhibits increased blasts.²⁹ Blast crisis leukemia usually is AML or ALL, but origins from other hematopoietic clonal cells are possible. Extramedullary growth may occur as lymphocytic or myelogenous cell proliferations; the latter are often referred to as granulocytic sarcoma. Extramedullary sarcoma is observed at many sites or locations in the body and may precede a marrow blast crisis. The clinical symptoms of blast crisis mimic those of acute leukemia, including severe anemia, leukopenia of all WBCs except blasts, and thrombocytopenia. Chromosome abnormalities such as additional Philadelphia chromosome(s), isochromosome 17, trisomy 8, loss of Y chromosome, and trisomy 19 accumulate with disease progression.^31,32 These generally occurred in approximately 75% of patients in the pre-imatinib era.

Related Diseases

Several diseases exist that are clinically similar to CML but do not exhibit the Philadelphia chromosome and express only a few pseudo-Gaucher cells. Chronic neutrophilic leukemia is another MPN that manifests with peripheral blood, bone marrow, and extramedullary infiltrative patterns similar to those of CML, except that only neutrophilic granulocytes are present and fewer than 10% of peripheral blood neutrophils are immature.³³ Similarly, chronic monocytic leukemia involves a comparable expansion of monocytes, including functional monocytes.³⁴

Juvenile myelomonocytic leukemia and adult chronic myelomonocytic leukemia are classified by the WHO as myelodysplastic/myeloproliferative diseases because of the overlap in clinical, laboratory, or morphologic findings. Juvenile myelomonocytic leukemia is observed in children younger than 4 years of age and is accompanied by an expansion in the number of monocytes and granulocytes, including immature granulocytes, and manifestations of dyserythropoiesis.³⁵

The peripheral blood of adults with chronic myelomonocytic leukemia may have characteristics similar to those seen in the refractory anemias, such as oval macrocytes and reticulocytopenia. The peripheral WBC concentration may reach 100 × 10⁹/L. According to WHO criteria, absolute monocytosis (more than 1 × 10⁹ monocytes/L) must be present to make the diagnosis. Clinical features include prominent splenomegaly, symptoms of anemia, fever, bleeding, and infection. Before presence of the Philadelphia chromosome was established as a requirement for the diagnosis of CML, some cases that were classified as Philadelphia chromosome–negative CML likely represented misdiagnoses of chronic myelomonocytic leukemia.³⁶ Chronic myelomonocytic leukemia is discussed further with myelodysplastic syndromes in Chapter 35.

A puzzling group of patients exhibit Philadelphia chromosome–positive acute leukemia. Studies reveal that 2% of patients with AML exhibit Philadelphia chromosome in a significant proportion of blasts. Further, 5% of patients with childhood-onset ALL and 20% of those with adult-onset ALL test positive for Philadelphia chromosome.37–40 The proper alignment of these cases within the spectrum of CML is speculative. It is understood that some of these cases likely represent undiagnosed CML that rapidly progressed to an acute leukemia prior to diagnosis. However, because rapidly dividing malignant cells are more prone to genetic mutation, the presence of the Philadelphia chromosome in acute leukemias may reflect a late-stage mutation that contributed little to acute leukemia leukemogenesis.

Treatment

Early treatment approaches for CML were unable to produce remission, so the goal of therapy became the reduction of tumor burden. The first forms of therapy for CML included alkylating agents such as nitrogen mustard,⁴¹ introduced in the late 1940s, and busulfan,⁴² which came into use in the early 1950s. Later, busulfan in combination with 6-thioguanine was used to achieve the goal of tumor burden reduction. Other drugs like hydroxyurea and 6-mercaptopurine were introduced later and found to improve patient survival. The discovery of interferon-α in 1983 dramatically improved outcomes of patients with CML by inducing the suppression of the Philadelphia chromosome, reducing the rate of cellular progression to blast cells, and increasing the frequency of long-term patient survival.⁴³

Interferon-α stimulates a cell-mediated antitumor host response that reduces myeloid cell numbers, induces cytogenetic remissions, and increases survival.⁴⁴ It improves the frequency and duration of hematologic remission and reduces the frequency of detection of the Philadelphia chromosome. In some patients, a complete cytogenetic remission is achieved for a time.

In 1997 it was discovered that cytarabine given with interferon-α improved the frequency of hematologic remissions but did not eliminate the BCR/ABL gene, which was still detected by molecular and fluorescent methods.⁴⁵ Also, in some patients the side effects of therapy became severe, drug resistance appeared, and relapse rates were not improved compared with other chemotherapies.

Bone marrow and stem cell transplantation with either autologous or allogeneic hematopoietic stem cells have been reported as curative, especially in patients younger than age 55. Relapses occur, but long-term, disease-free survival is possible. Optimal survival occurs when the patient is treated during the chronic phase within 1 year of diagnosis and is younger than age 50. Treatment requires ablative chemotherapy followed by transplantation of mobilized normal progenitor cells that exhibit CD34⁺ surface markers. Allogeneic bone marrow transplants are more successful in patients up to age 55 when donors are matched for HLA antigens A, B, and DR. Donor-matched lymphocyte infusions after allogeneic transplantation of marrow from a sibling donor may assist in producing complete remissions.⁴⁶

Modern therapies involve the use of synthetic proteins that bind the abnormal BCR/ABL protein, blocking the constitutive tyrosine kinase activity and reducing signal transduction activation. Imatinib mesylate is a synthetic tyrosine kinase inhibitor designed to selectively bind the ATP binding site and thus inhibit the tyrosine kinase activity of the BCR/ABL fusion protein. When imatinib binds the ATP binding site, ATP is unable to bind to provide the phosphate group necessary for kinase activity. Imatinib binds the BCR/ABL protein in the inactive conformation, which precedes the autophosphorylation necessary to generate the kinase active site (Figure 34-5).⁴⁷

Goals of therapy include complete hematologic remission and cytogenetic remission induced in part by the reactivation of apoptotic pathways.⁴⁸ The effectiveness of imatinib therapy and stem cell transplantation is best monitored using quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). These monitoring tools are used to determine complete cytogenetic and molecular remission. The most sensitive measure of the effectiveness of imatinib therapy is the number of log reductions of BCR/ABL transcripts using real-time RT-PCR.⁴⁹ Expected therapeutic response to imatinib is complete hematologic remission in 3 to 6 months, complete cytogenetic response in 6 months to 1 year, and a 2 to 3 log reduction in BCR/ABL transcripts. When real-time RT-PCR is used, the greatest log reduction possible is a more than 4 log reduction, which represents the maximum sensitivity of the assay. However, discontinuation of imatinib therapy in patients who achieve a more than 4 log reduction will result in relapse.

Although imatinib has proven to be a successful form of therapy, a major limitation is the development of imatinib resistance resulting in relapse. Approximately 4% of patients with newly diagnosed CML will become imatinib resistant. There are two major categories of imatinib resistance, primary and secondary. Primary resistance is defined as the inability to reach the remission milestones. This form of resistance is rare and probably results from the presence of mutations other than the BCR/ABL mutation at the time of diagnosis. Secondary resistance involves the loss of a previous response and occurs at a rate of 16% at 42 months. The majority of cases of imatinib resistance result from two primary causes: acquisition of additional BCR/ABL mutations and expression of point mutations in the ATP binding site. Additional BCR/ABL mutations can occur through the usual translocation of the remaining unaffected chromosomes 9 and 22, which converts the hematopoietic stem cell from heterozygous to homozygous for the BCR/ABL mutation. A double dose of BCR/ABL can also be acquired from gene duplication during mitosis and accounts for 10% of secondary mutations. An additional BCR/ABL mutation will double the tyrosine kinase activity, making the imatinib dosage inadequate. In these cases higher dosages of imatinib will restore remission in most patients (Figure 34-6). The majority of patients who do not respond to higher dosages of imatinib express point mutations in the ATP binding site. Over 50 mutations have been identified in the ATP binding site, and these account for the remaining 50% to 90% of secondary mutations. Mutations in the ATP binding site reduce the binding affinity of imatinib, producing some level of resistance (Figure 34-7). Second- and third-generation tyrosine kinase inhibitors like dasatinib (Sprycel), nilotinib (Dasigna), and bosutinib (still in clinical trials at the time of publication) overcome the ATP binding site mutations except the T315I mutation. However, drugs designed to bind the A loop (receiver protein binding site) will inhibit tyrosine kinase activity and overcome the T315I mutation.^50,51 Currently, studies are under way to evaluate modification of the dosage of imatinib used, to identify and develop other tyrosine kinase inhibitors, and to discover new classes of inhibitors that may be more effective than currently known tyrosine kinase inhibitors.

The development of a care plan for treating a patient with newly diagnosed CML requires not only the formulation of alternative approaches to achieve complete cellular remission, but also the establishment of parameters to follow that confirm long-term success of therapy. Previous chemotherapies have provided cellular remission but usually do not suppress or delay clinical transitions to accelerated or blast phases. Bone marrow transplantation for patients who qualify is likely the preferred choice, but the long-term success (cure) rate remains at 50% to 70%. For a patient to qualify for transplantation, the patient must be younger than 50 years of age, must be in the first year of the disease, and must have CML that is still in the chronic phase, and a histocompatible donor must be available. Today, imatinib is considered first-line therapy for all patients with newly diagnosed CML. For the small subset of patients who qualify for hematopoietic stem cell transplantation, imatinib is used to induce hematologic remission prior to transplantation. For all other CML patients, imatinib is used as first-line therapy unless remission is not achieved (primary resistance) or until relapse occurs following remission (secondary resistance). Once the cause of relapse has been determined by cytogenetic and molecular testing, either a higher dosage of imatinib can be given (for an additional BCR/ABL mutation) or a second- or third-generation tyrosine kinase inhibitor (dasatinib, nilotinib, bosatinib) can be prescribed unless the mutation is the T315I mutation in the ATP binding site. If the T315I mutation is detected, the patient can be given an A-loop inhibitor (ONO12380) or other drugs like MK 0457 or BIRB-796 that inhibit the T315I mutation.⁵¹

Pathogenic Mechanism

In PV, neoplastic clonal stem cells are hypersensitive to, or function independently of, erythropoietin for cell growth. Trace levels of erythropoietin in serum stimulate the growth of erythroid progenitor cells in in vitro colony-forming growth systems. There is preservation of hypersensitive and normosensitive erythroid colony-forming units, however, which indicates some level of normal hematopoiesis.⁵³ Adverse clinical progression seems to correlate with the propagation of the erythropoietin-sensitive colony-forming units.⁵⁴ Understanding of the pathologic mechanism explaining this phenomenon in PV was significantly advanced in 2005 with the discovery of a consistent mutation in the JAK2 gene. The specific JAK2 mutation, JAK2 V617F, is detected in 90% to 97% of patients with PV. Shortly after the JAK2 V617F mutation was reported, several groups corroborated the finding using other approaches and showed that the mutation is acquired, clonal, present in the hematopoietic stem cell, constitutively active, and capable of activating the erythropoietic signal transduction pathway in the absence of erythropoietin. The point mutation changes the amino acid at position 617 from valine to phenylalanine, which unlocks the inhibition of the tyrosine kinase and converts it to the active conformation. More specifically, the phenylalanine mutation in the kinase domain is unable to bind the corresponding amino acid in the pseudokinase domain, as can the wild-type counterpart valine, which prevents the protein from folding and remaining inactive (Figure 34-8). Normally, erythropoietin is released from the kidney into the blood in response to hypoxia and binds to erythropoietin receptors on the surface of erythroid precursor cells. The resulting conformational change in the erythropoietin receptor causes two erythropoietin receptors to dimerize. This produces a docking point for inactive JAK2, which becomes activated through phosphorylation. Phosphorylated JAK2 undergoes a conformational change, and the valine releases from the pseudokinase domain, which converts it to an active tyrosine kinase. JAK2 phosphorylates several cytoplasmic proteins, but the signal transducer and activator of transcription (STAT) proteins are the main target. A cascade of phosphorylations produce activated transcription factors that activate a host of genes designed to drive and control cell proliferation and differentiation while also initiating apoptosis (Figure 34-9). Constitutive tyrosine kinase activity of the JAK2 protein causes continuous activation of several signal transduction pathways that are normally activated following erythropoietin stimulation via the erythropoietic receptor. Active JAK2 will phosphorylate STAT proteins in the absence of erythropoietin or will overphosphorylate in its presence (Figure 34-10). Hematopoietic stem cells that bear the JAK2 mutation are resistant to erythropoietin-deprivation apoptosis by upregulation of BCL-X, an antiapoptotic protein. PV progenitor cells do not divide more rapidly but accumulate because they do not die normally.^55–57

Diagnosis

Based on the WHO standards, the diagnosis of PV requires that two major criteria and one minor criterion be met or that the first major criterion listed and two minor criteria be met. The two major criteria are an elevated hemoglobin (Hb) level (greater than 18.5 g/dL men and greater than 16.5 g/dL in women) and the identification of the JAK2 V617F mutation, the JAK2 exon 12 mutation, or a similar JAK2 mutation. The three minor criteria are panmyelosis in the bone marrow, low serum erythropoietin levels, and autonomous, in vitro erythroid colony formation.⁶ Additional diagnostic features of PV include an increased RBC mass of 36 mL/kg or greater in males and 32 mL/kg or greater in females, an arterial oxygen saturation of 92% (normal) or greater, and splenomegaly. Other features of PV are thrombocytosis of greater than 400 × 10⁹ platelets/L; leukocytosis of greater than 12 × 10⁹ cells/L without fever or infection; and increases in leukocyte alkaline phosphatase (LAP), serum vitamin B₁₂, or unbound vitamin B₁₂ binding capacity.^58,59 Recent research indicates that the JAK2 V617F mutation can be expected in more than 90% to 95% of cases.⁶ The WHO criteria for the diagnosis of PV are summarized in Box 34-1.

It is not always easy to assign an early diagnosis of PV. Erythrocytosis secondary to hypoxia or erythropoietin-producing neoplasms is the most difficult to diagnose correctly. In individuals with these conditions, the bone marrow exhibits erythroid hyperplasia without granulocytic or megakaryocytic hyperplasia. Patients with stress or spurious erythrocytosis exhibit increased hemoglobin and hematocrit (Hct) without increased erythrocyte mass or splenomegaly.

Peripheral Blood and Bone Marrow

Common peripheral blood, bone marrow, and tissue findings in the early or proliferative phase of PV are listed in Table 34-2. Figures 34-11 and 34-12 show common morphologic patterns in peripheral blood and bone marrow morphologic and cellular changes. Not only are quantitative changes seen, but bone marrow normoblasts may collect in large clusters, megakaryocytes are enlarged and exhibit lobulated nuclei, and bone marrow sinuses are enlarged without fibrosis. Pseudo-Gaucher cells are rare.²⁶ Approximately 80% of patients manifest bone marrow panmyelosis, and 100% of bone marrow volume may exhibit hematopoietic cellularity. Although the bone marrow pattern may mimic that in other MPNs, the peripheral blood cells appear normal, with normocytic, normochromic erythrocytes; mature granulocytes; and normal-sized, granulated platelets. The other 20% of patients exhibit lesser degrees of cellularity in the bone marrow and peripheral blood. Splenomegaly, hepatomegaly, generalized vascular engorgement, and circulatory disturbances increase the risk of hemorrhage, tissue infarction, and thrombosis.

Clinical Presentation

PV initially manifests in a proliferative phase independent of normal regulatory mechanisms. PV is always associated with increased RBC mass. This is the stable phase of PV, which progresses to a spent phase in a few patients. In the spent phase, patients experience progressive splenomegaly (palpable spleen) or hypersplenism (large spleen with bone marrow hyperplasia and peripheral blood cytopenias) and pancytopenia. They may also exhibit the triad of bone marrow fibrosis, splenomegaly, and anemia with teardrop-shaped poikilocytes. The latter pattern is called postpolycythemic myeloid metaplasia, and its morphologic features are similar to those of PMF. Peripheral WBC and RBC counts vary, and nucleated erythrocytes, immature granulocytes, and large platelets are present. Usually, splenomegaly is secondary to extramedullary hematopoiesis.⁶⁰ Myelofibrosis occurs within the bone marrow and may come to occupy a significant proportion of bone marrow volume, with subsequent ineffective hematopoiesis.⁶¹

Treatment and Prognosis

The disease progresses to acute leukemia in 15% of patients. The use of myelosuppressive therapy such as phosphorus P 32 (³²P) or alkylating agents seems to increase the risk.⁵⁹ Only 1% to 2% of patients treated with phlebotomy alone experience leukemic transformation. However, the risk of thrombosis and bleeding is increased in patients treated with phlebotomy alone, so the use of alkylating myelosuppressive agents may be required to control these complications. Some patients may manifest a temporary disease pattern similar to myelodysplasia, and the cell morphology in transformation to acute leukemia may be difficult to classify. Patients with both early and advanced PV may show clinical, peripheral blood, bone marrow, and extramedullary features that mimic those of other MPNs.

Incidence

In the absence of a well-defined diagnostic algorithm, determining the true incidence of ET has been difficult. When the diagnostic system developed by the Polycythemia Vera Study Group (PVSG) is applied, however, the incidence is estimated to be between 0.6 and 2.5 cases per 100,000 persons per year. The majority of cases occur in individuals between the ages of 50 and 60 years, but a second peak occurs primarily in women in the childbearing years, approximately 30 years of age.⁶³

Clinical Presentation

In more than one half of the patients diagnosed with ET, the disorder is discovered in the laboratory by virtue of an unexpectedly elevated platelet count on a routine complete blood count; the remaining patients see a physician due to vascular occlusion or hemorrhage. Vascular occlusions are often the result of microvascular thromboses in the digits or thromboses in major arteries and veins that occur in a variety of organ systems, including splenic or hepatic veins as in Budd-Chiari syndrome. Bleeding occurs most frequently from mucous membranes in the gastrointestinal and upper respiratory tracts. Splenomegaly is observed at presentation in 50% of patients when the PVSG diagnostic criteria are used but at a much lower frequency when the WHO standards are applied. This difference is largely due to the elimination of the diagnosis of ET in patients who meet the criteria for PMF in the prefibrotic stage.⁶³

Diagnosis

ET must be differentiated from secondary or reactive thrombocytoses and from other MPNs. Thrombocytosis may be secondary to chronic active blood loss, hemolytic anemia, chronic inflammation or infection, or nonhematogenous neoplasia. The diagnostic criteria for ET first proposed by the PVSG were intended to distinguish ET from other MPNs. These features included a platelet count of greater than 600 × 10⁹/L, a hemoglobin of less than 13 g/dL or a normal erythrocyte mass, and stainable iron in the bone marrow or a failure of iron therapy. Philadelphia chromosome negativity, absence of marrow collagen fibrosis (less than one third of a biopsy specimen is fibrous), no splenomegaly, absence of leukoerythroblastic reaction, and no known cause of reactive thrombocytosis all support the diagnosis.⁶⁴

The newest WHO group now requires the documentation of four major criteria to establish a diagnosis of ET. First, the WHO group lowered the platelet count threshold to 450 × 10⁹/L or greater to capture patients who would eventually meet diagnostic criteria but who were experiencing hemorrhage or thrombosis with platelet counts of between 450 × 10⁹/L and 600 × 10⁹/L.⁶³ Because a lower platelet threshold could lead to false-positive ET diagnoses, all the WHO criteria must be met to eliminate such patients. Second, the bone marrow must show significant megakaryopoiesis characterized by large, mature-looking megakaryocytes with no substantial increase in erythropoiesis or granulopoiesis or left shift in the neutrophil line. Third, the condition cannot meet the criteria of any other MPN, myelodysplasia, or other myeloid neoplasm. Fourth, patients must demonstrate either the JAK2 V617F or other clonal mutation or, in the absence of a clonal marker, the absence of reactive thrombocytosis. Careful analysis of the bone marrow biopsy specimen is useful in distinguishing ET from myelodysplastic syndromes (MDSs) associated with the del(5q) mutation, refractory anemia with ringed sideroblasts with thrombocytosis, and the prefibrotic phase of PMF. Likewise, the identification of the JAK2 V617F mutation excludes cases of reactive thrombocytosis.⁶³

The JAK2 V617F mutation is found in 50% to 60% of ET patients and supports the diagnosis of ET.65,66 WHO diagnostic criteria were modified to include a minimum platelet count of 450 × 10⁹/L when the JAK2 mutation is present.⁶⁷ JAK2 mutations have not been identified in the germline of any patient with MPN disorder, which supports the view that the mutation is acquired. MPL W515K/L, a mutation in the thrombopoietin receptor (MPL), has been reported in 1% of ET cases and is also used to exclude a diagnosis of reactive thrombocytosis. Other genetic mutations are uncommon but have been reported to be found in 5% to 10% of cases when the diagnostic criteria proposed by the PVSG are applied. The most commonly reported additional mutations are +8, 9q, and (del)20q.⁶³

Peripheral Blood and Bone Marrow

Figure 34-13 shows a peripheral blood film that exhibits early-phase thrombocytosis with variation in platelet diameter and shape, including giantism, agranularity, and pseudopods. Commonly, platelets are present in clusters and tend to accumulate near the thin edge of the blood film. Segmented neutrophils may be increased; basophils are not. Erythrocytes are normocytic and normochromic, unless iron deficiency is present secondary to excessive bleeding.

Early-phase bone marrow shows marked megakaryocytic hypercellularity, clustering of megakaryocytes, and increased megakaryocyte diameter with nuclear hyperlobulation and density (Figure 34-14). Special studies reveal increased numbers of smaller and less mature megakaryocytes.⁶⁸ Increased granulopoiesis and erythropoiesis may contribute to bone marrow hypercellularity, and in a few patients, reticulin fibers may be increased. The major peripheral blood, bone marrow, and extramedullary findings are listed in Table 34-3.

A diagnosis of ET is questionable in patients with a platelet count of more than 450 × 10⁹/L if certain features are observed on the bone marrow biopsy specimen. For example, increased erythropoiesis or granulopoiesis in the bone marrow is a questionable finding for ET and suggests an alternative diagnosis of PV or PMF, respectively, especially if bizarre or significantly atypical megakaryocytes are also observed. Dyserythropoiesis and/or dysgranulopoiesis suggests a myelodysplastic disorder and should prompt an investigation for (del)5q, (inv)3, and/or t(3 ; 3).⁶³

Treatment and Prognosis

Patients with ET experience relatively long survival provided they remain free of serious thromboembolic or hemorrhagic complications. Clinical symptoms associated with thromboembolic vasoocclusive events include the syndrome of erythromelalgia (throbbing and burning pain in the hands and feet, accompanied by mottled redness of areas), transient ischemic attacks, seizures, and cerebral or myocardial infarction. Other symptoms include headache, dizziness, visual disturbances, and dysesthesias (decreased sensations). Hemorrhagic complications include bleeding from oral and nasal mucous membranes or gastrointestinal mucosa and the appearance of cutaneous ecchymoses (see Chapter 43).

Treatment involves prevention or early alleviation of hemorrhagic or vasoocclusive complications that occur as the platelet count increases. The production of platelets must be reduced by suppressing marrow megakaryocyte production with ³²P or one of several alkylating agents. As observed in PV, ET patients so treated may incur an increased risk for disease transformation to acute leukemia or myelofibrosis. However, malignant transformation occurs at a frequency of less than 5%.⁶³ Hydroxyurea therapy may achieve a desired reduction of peripheral platelets without the risk of complications experienced with myelosuppressive agents. This may relate to the youth of ET patients, in whom the risk of leukemic transformation seems relatively low. More recently, cytoreduction has been achieved with interferon-α.

Studies commonly show a median survival of longer than 10 years, including cases in which the process arises in younger patients. However, some patients may develop post-ET myelofibrosis, which reduces survival. Patients whose cells manifest chromosome abnormalities may have a poorer prognosis.³⁷

Myelofibrosis

The myelofibrosis in this disease comprises three of the five types of collagen: I, III, and IV. Increases in type III collagen are detected by silver impregnation techniques, increases in type I by staining with trichrome, and increases in type IV by the presence of osteosclerosis, which may be diagnosed from increased radiographic bone density.⁶⁹ In approximately 30% of patients, biopsy specimens show no fibrosis.³⁷ Increases in these collagens are not a part of the clonal proliferative process but are considered secondary to an increased release of fibroblastic growth factors, such as platelet-derived growth factor, transforming growth factor α from megakaryocyte α-granules, tumor necrosis factor-α, and interleukin-1α, and interleukin-1β. Marrow fibrosis causes expansion of marrow sinuses and vascular volume with an increased rate of blood flow. Bone marrow fibrosis is not the sole criterion for the diagnosis of PMF, because increases in marrow fibrosis may reflect a reparative response to injury from benzene or ionizing radiation, may be a consequence of immunologically mediated injury, or may represent a reactive response to other hematologic conditions.

Type IV collagen and laminin normally are discontinuous in sinusoidal membranes but appear as stromal sheets in association with neovascularization and endothelial cell proliferation in regions of fibrosis. In addition, deposition of type VII collagen is observed, and this may form a linkage between type I fibers, type III fibers, and type I plus type III fibers.⁷⁰

Hematopoiesis and Extramedullary Hematopoiesis

Extramedullary hematopoiesis, clinically recognized as hepatomegaly or splenomegaly, seems to originate from release of clonal stem cells into the circulation.⁷¹ The cells accumulate in the spleen, liver, or other organs, including adrenals, kidneys, lymph nodes, bowel, breasts, lungs, mediastinum, mesentery, skin, synovium, thymus, and lower urinary tract. The cause of extramedullary hematopoiesis is unknown. In experimental animal models, chemicals, hormones, viruses, radiation, and immunologic factors have been implicated. The disease is associated with an increase in circulating hematopoietic cells, but fibroblasts are a secondary abnormality and not clonal.⁷² B and T cells may be involved.⁷³ There is an increase in circulating unilineage and multilineage hematopoietic progenitor cells,⁷⁴ and the number of CD34⁺ cells may be 300 times normal.⁷⁵ The increase in circulating CD34⁺ cells separates PMF from other MPNs and predicts the degree of splenic involvement and risk of conversion to acute leukemia.

Body cavity effusions containing hematopoietic cells may arise from extramedullary hematopoiesis in the cranium, the intraspinal epidural space, or the serosal surfaces of pleura, pericardium, and peritoneum. Portal hypertension, with its attendant consequences of ascites, esophageal and gastric varices, gastrointestinal hemorrhage, and hepatic encephalopathy, arises from the combination of a massive increase in splenoportal blood flow and a decrease in hepatic vascular compliance secondary to fibrosis around the sinusoids and hematopoietic cells within the sinusoids.⁷⁶

Incidence and Clinical Presentation

The disease occurs in patients older than age 60 and may be asymptomatic. PMF generally presents with fatigue, weakness, shortness of breath, palpitations, weight loss, and discomfort or pain in the left upper quadrant associated with splenomegaly.

Peripheral Blood and Bone Marrow

PMF presents with a broad range of changes in laboratory test values and peripheral blood film results, but examination of the bone marrow biopsy specimen provides most of the information for diagnosis. Changes commonly observed in peripheral blood and bone marrow examinations are summarized in Table 34-4.

Abnormalities in erythrocytes noted on peripheral blood films include the presence of dacryocytes, other bizarre shapes, nucleated RBCs, and polychromatophilia. Granulocytes are increased, normal, or decreased in number and may include immature granulocytes, blasts, and cells with nuclear or cytoplasmic anomalies. Platelets may be normal, increased, or decreased in number with a mixture of normal and abnormal morphologic features (Figure 34-15). Micromegakaryocytes may be observed (Figure 34-16).

Bone marrow biopsy specimens exhibit intense fibrosis, granulocytic and megakaryocytic hypercellularity, dysmegakaryopoiesis, dysgranulopoiesis, and numerous dilated sinuses containing luminal hematopoiesis. Neutrophils may exhibit impairment of physiologic functions such as phagocytosis, oxygen consumption, and hydrogen peroxide generation, and decreased myeloperoxidase and glutathione reductase activities. Platelets show impaired aggregation in response to epinephrine, decreased adenosine diphosphate concentration in dense granules, and decreased activity of platelet lipoxygenase.

Immune Response

Humoral immune responses are altered in approximately 50% of patients and include the appearance of autoantibodies to erythrocyte antigens, nuclear proteins, gamma globulins, phospholipids, and organ-specific antigens.⁷⁷ Circulating immune complexes, increased proportions of marrow-reactive lymphocytes, and the development of amyloidosis are evidence for active immune processes. Collagen disorders coexist with PMF, which suggests that immunologic processes may stimulate marrow fibroblast activity.

Treatment and Prognosis

Average survival from the time of diagnosis is about 5 years, but patients have lived 15 years. During this time, increasing numbers and pleomorphy of megakaryocytes lead to progressive marrow failure. Marrow blasts may increase.³⁷ Adverse prognostic indicators include more severe anemia and thrombocytopenia, greater hepatomegaly, unexplained fever, and hemolysis. Mortality is associated with infection, hemorrhage, postsplenectomy complications, and transformation to acute leukemia.

A diverse spectrum of therapies has been implemented to alleviate symptoms or modify clinical problems in these patients. Severe anemia has been treated with androgen therapy, and hemolytic anemia with glucocorticosteroids. Reduction of myelofibrosis and of marrow and tissue hypercellularity has been accomplished with busulfan and other alkylating agents, hydroxyurea, and, in a few patients, interferon-α and interferon-γ. Radiotherapy is considered for patients with severe splenic pain, for patients with massive splenomegaly who are not clinical candidates for splenectomy, for patients with ascites secondary to serosal implants (metastatic nodules), and for patients with localized bone pain and localized extramedullary fibrohematopoietic masses in other areas, especially in the epidural space. Splenectomy is performed to end severe pain, excessive transfusion requirements, or severe thrombocytopenia and to correct severe portal hypertension.

Chemotherapy is partially successful in reducing the number of CD34⁺ cells and immature hematopoietic cells, marrow fibrosis, and splenomegaly.⁷⁸ Single-agent chemotherapy is most helpful in the early clinical phases of the disease, and agents such as busulfan, 6-thioguanine, and chlorambucil, alone or in combination with other chemotherapy, are useful. Other therapies include interferon-α, hydroxyurea, and combinations of the previously mentioned drugs.⁷⁹ The most successful treatment to date for patients younger than age 60 is allogeneic stem cell transplantation. Five-year survival approaches 50% in patients undergoing transplantation, but 1-year mortality is 27%, and graft-versus-host disease occurs in 33%.⁸⁰

Incidence

The incidence of CNL is not known, but it is a rare disorder of which about 150 cases have been reported. However, if the WHO criteria had been applied in these cases, many might have been reclassified as reactive rather than neoplastic conditions.¹⁰⁰

Clinical Presentation

Hepatosplenomegaly is the most common finding, but 25% to 30% of patients report bleeding from mucocutaneous sites like the gastrointestinal tract. Other symptoms include gout from WBC turnover and pruritus that may be associated with neutrophil infiltration of tissues and organs.¹⁰⁰

Peripheral Blood and Bone Marrow

Patients have a WBC count of more than 25 × 10⁹/L with a slight left shift. Neutrophils dominate but the increase in bands, metamyelocytes, myelocytes, and promyelocytes in combination usually comprise fewer than 5% of WBCs but can be as many as 10%. Neutrophils do not appear dysplastic, but they often contain toxic granules. RBC and platelet morphology are normal in the peripheral blood.¹⁰⁰

The bone marrow reflects the peripheral blood in that it is hypercellular with predominately a proliferation of neutrophils, including myelocytes, metamyelocytes, bands, and segmented neutrophils. The myeloid-to-erythroid ratio is at least 20 : 1 or greater. RBCs and platelets are normal in number, and no cell line exhibits significant dysplastic morphology.¹⁰⁰

Diagnosis

The WBC count must be greater than 25 × 10⁹/L with greater than 90% mature neutrophils, fewer than 10% immature neutrophilic cells, and fewer than 1% blasts in the peripheral blood. The bone marrow shows an increase in normal-appearing neutrophilic cells with fewer than 5% myeloblasts. Megakaryocytes are normal or slightly left-shifted. Splenomegaly must be present and is often accompanied by hepatomegaly. Reactive neutrophilia must be excluded by eliminating infection, inflammation, and tumors as a cause of the neutrophilia. A diagnosis of CNL can still be made in the presence of a reactive process if clonality of the myeloid line can be documented by karyotyping or molecular analysis. There must be no evidence of the Philadelphia chromosome, BCR/ABL mutation, or rearrangements of the PDGFRA, PDGFRB, or FGRF1 genes. Lastly, there can be no evidence of PV, PMF, ET, MDS, or MDS/MPN disorders.¹⁰⁰

Genetics

Approximately 90% of CNL patients have a normal karyotype, but chromosomal abnormalities are observed, particularly as the disease progresses, including +8, +9, +21, del(20q), del(11q), and del(12p). The Philadelphia chromosome or a BCR/ABL mutation cannot be expressed in CNL; otherwise a diagnosis of CML is required. JAK2 mutations have been observed but rarely.¹⁰⁰

Prognosis

CNL is a slow, smoldering condition, and patient survival ranges from as short as 6 months to longer than 20 years. The neutrophilia does progress, and some patients develop myelodysplasia and can experience transformation into AML.¹⁰⁰

Chronic Eosinophilic Leukemia, Not Otherwise Specified

Chronic eosinophilic leukemia (CEL) is a clonal proliferation of eosinophils from eosinophil precursors that dominate in the bone marrow and peripheral blood. Eosinophils are found in other peripheral tissues, including heart, lungs, central nervous system, gastrointestinal tract, and skin. Hepatosplenomegaly is observed in approximately 30% to 50% of patients. Infiltrating eosinophils degranulate to release cytokines, enzymes, and other granular proteins that damage the surrounding tissue, which results in organ dysfunction.¹⁰¹

Mastocytosis

Mastocytosis is a broad term referring to a clonal neoplastic proliferation of mast cells, which accumulate in one or more organ systems, but it can present differently and manifest in a range of severities. The WHO group has classified mastocytosis into seven subcategories: cutaneous mastocytosis, indolent systemic mastocytosis, systemic mastocytosis with associated clonal hematologic non–mast-cell-lineage disease, aggressive systemic mastocytosis, mast cell leukemia, mast cell sarcoma, and extracutaneous mastocytoma.¹⁰²

Myeloproliferative Neoplasm, Unclassifiable

The category myeloproliferative neoplasm, unclassifiable (MPN-U) is designed to capture disorders that clearly express myeloproliferative features but either fail to meet the criteria of a specific condition or have features that overlap two or more specific conditions. Most patients with MPN-U fall into one of three groups: (1) patients with an early stage of PV, ET, or PMF in which the criteria that define the disorders are not yet fully developed; (2) patients presenting with features indicative of advanced disease resulting from clonal evolution that masks the potential underlying condition; and (3) patients who have clear evidence of an MPN but who have a concomitant condition like a second neoplasm or an inflammatory condition that alters the MPN features. MPN-U may account for as many as 10% to 15% of MPN disorders, but caution should be exercised so that morphologic changes caused by the patient’s cytotoxic drug therapy or growth factor therapy, or poor collection of samples are not confused with the features of MPN-U. In patients with MPN-U in the early stages of development or with a concomitant disorder like inflammation, the MPN-U may be reclassified to a specific category of MPN once the disease begins to express typical features or the secondary conditions subsides. Likewise, in patients with an advanced MPN, the disorder may be reclassified as an acute leukemia once the blast criterion of more than 20% blasts in the bone marrow is met.¹⁰³