Rodak's Hematology: Clinical Principles and Applications, 5th Ed.

CHAPTER 33. Myeloproliferative neoplasms

Tim R Randolph

OUTLINE

Chronic Myelogenous Leukemia

Incidence

Cytogenetics of the Philadelphia Chromosome

Molecular Genetics

Pathogenetic Mechanism

Peripheral Blood and Bone Marrow

Other Laboratory Findings

Progression

Related Diseases

Treatment

Polycythemia Vera

Pathogenic Mechanism

Diagnosis

Peripheral Blood and Bone Marrow

Clinical Presentation

Treatment and Prognosis

Essential Thrombocythemia

Incidence

Pathogenic Mechanism

Clinical Presentation

Diagnosis

Peripheral Blood and Bone Marrow

Treatment and Prognosis

Primary Myelofibrosis

Myelofibrosis

Hematopoiesisand Extramedullary Hematopoiesis

Pathogenic Mechanism

Incidence and Clinical Presentation

Peripheral Blood and Bone Marrow

Immune Response

Treatment and Prognosis

Summary of Current Therapy of Non-BCR/ABL1, Primary MPNs

Interconnection among Essential Thrombocythemia, Polycythemia Vera, and Primary Myelofibrosis

Other Myeloproliferative Neoplasms

Chronic Neutrophilic Leukemia

Chronic Eosinophilic Leukemia, Not Otherwise Specified

Mastocytosis

Myeloproliferative Neoplasm, Unclassifiable

Objectives

After completion of this chapter, the reader will be able to:

1. Define myeloproliferative neoplasms (MPNs), list the most common diseases included in the World Health Organization (WHO) classification of MPNs, and recognize their abbreviations.

2. Define chronic myelogenous leukemia (CML), and describe the cell lines involved, the clinical phases, and the expected clinical manifestations, key peripheral blood and bone marrow findings, and diagnostic criteria applicable to each stage.

3. Discuss the cytogenetics, molecular genetics, and molecular pathophysiology of CML and relate it to treatment approaches, monitoring minimal residual disease, mechanisms of drug resistance.

4. Define polycythemia vera (PV), and describe the cell lines involved, clinical manifestations, key peripheral blood and bone marrow findings, and the diagnostic criteria.

5. Discuss the JAK2 mutation and the proposed pathogenic mechanism in PV.

6. Discuss the progression of PV and treatment modalities to include JAK inhibitors.

7. Define essential thrombocythemia (ET), and describe the cell lines involved, clinical manifestations, key peripheral blood and bone marrow findings, and the diagnostic criteria.

8. Discuss common mutations, pathophysiology, and two complications that may occur in patients with ET.

9. Define primary myelofibrosis (PMF), and describe the cell lines involved, clinical manifestations, key pathologic features in peripheral blood, bone marrow, and tissues, and the diagnostic criteria.

10. Describe the mutations that occur in PMF and relate them to disease progression and current therapy.

11. Briefly discuss the potential interrelationships between the mutations and hypotheses for disease development and progression among between ET, PV, and PMF.

12. Briefly describe the other myeloproliferative disorders outlined in this chapter.

13. Given complete blood count and cytogenetic, molecular, and other laboratory results, recognize the findings consistent with each major MPN.

14. Recommend follow-up testing for suspected MPN and interpret the results of testing.

CASE STUDY

After studying the material in this chapter, the reader should be able to respond to the following case study:

A 34-year-old woman came to the physician with a 2-month history of increasing weakness, persistent nonproductive cough, fever and chills accompanied by night sweats, and a 13-pound weight loss over a 6-month period. Results of chest radiographs and purified protein derivative test (for tuberculosis) were negative. The patient was treated with ciprofloxacin and her cough improved, but she continued to grow weaker and was able to consume only small quantities of food. The patient appeared pale and cachectic. Tenderness and fullness were present in the left upper quadrant, and the spleen was palpable below the umbilicus. No hepatomegaly or peripheral adenopathy was noted. Her laboratory results were as follows:

WBCs—248 × 109/L

HGB—9.5 g/dL

HCT—26.3%

Platelets—449 × 109/L

Segmented neutrophils—44%

Band neutrophils—4%

Lymphocytes—10%

Eosinophils—3%

Basophils—7%

Myelocytes—30%

Promyelocytes—1%

Myeloblasts—1%

Nucleated RBCs—2 per 100 WBCs

Reticulocytes—3%

Leukocyte alkaline phosphatase (LAP) score—20 (reference range, 40 to 130)

Lactate dehydrogenase—692 IU (reference range, 140 to 280 IU)

Uric acid—8.1 mg/dL (reference range, 4 to 6 mg/dL)

1. What is the significance of the elevated WBC count and abnormal WBC differential?

2. How does the LAP score aid in the diagnosis?

3. Justify the use of cytogenetic studies in a patient with test results similar to those in this case study.

4. Predict the results of the cytogenetic studies.

5. Describe the molecular mutation resulting from the cytogenetic abnormality.

6. What is the usual treatment for this disorder?

7. Briefly discuss mechanisms of drug resistance.

The myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders caused by genetic mutations in the hematopoietic stem cells that result in expansion, excessive production, and accumulation of erythrocytes, granulocytes, and platelets. Myeloproliferation is due to hypersensitivity or independence of normal cytokine regulation that reduces cytokine levels through negative feedback systems normally induced by mature cells.12 Expansion occurs in varying combinations in the bone marrow, peripheral blood, and tissues.3-6 The MPNs have pathogenetic similarities, as well as common clinical and laboratory features.7

MPNs are predominantly chronic with accelerated, subacute, or acute phases. In certain patients it is difficult to make a clear delineation between subacute and chronic phases using clinical and morphologic findings.

The World Health Organization (WHO) has classified the MPNs into four predominant disorders: chronic myelogenous leukemia (CML); polycythemia vera (PV), also known as polycythemia rubra vera;essential (primary) thrombocythemia (ET); and primary myelofibrosis (PMF), also known as agnogenic myelofibrosis with myeloid metaplasia and chronic idiopathic myelofibrosis. Several other less common MPN conditions have been described and are classified as chronic neutrophilic leukemia (CNL); chronic eosinophilic leukemia (CEL), not otherwise specified; mastocytosis; and myeloproliferative disorder, unclassified.8 CML and PV are defined by their overproduction of granulocytes and erythrocytes, respectively.4910 PMF is a combination of overproduction of hematopoietic cells and stimulation of fibroblast production leading to ineffective hematopoiesis with resultant peripheral blood cytopenias.11 ET is characterized by increased megakaryocytopoiesis and peripheral blood thrombocytosis.12

MPNs present as stable chronic disorders that may transform first to a subacute, then to an aggressive cellular growth phase, such as acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL). They may manifest a depleted cellular phase, such as bone marrow hypoplasia, or exhibit clinical symptoms and morphologic patterns characteristic of a subacute followed by a more aggressive cellular expression. Familial MPNs have been described in families in which two or more members are affected.13

Chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is an MPN arising from a single genetic translocation in a pluripotential hematopoietic stem cell producing a clonal overproduction of the myeloid cell line, resulting in a preponderance of immature cells in the neutrophilic line. CML begins with a chronic clinical phase and, if untreated, progresses to an accelerated phase in 3 to 4 years and often terminates as an acute leukemia. The clinical features are frequent infection, anemia, bleeding, and splenomegaly, all secondary to massive pathologic accumulation of myeloid progenitor cells in bone marrow, peripheral blood, and extramedullary tissues. Neutrophilia with all maturational stages present, basophilia, eosinophilia, and often thrombocytosis are noted in peripheral blood. The clonal origin of hematopoietic cells in CML has been verified in studies of females heterozygous for glucose-6-phosphate dehydrogenase. Only one isoenzyme is active in affected cells, whereas two isoenzymes are active in nonaffected cells.14

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 prior 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.1516 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.17 The Philadelphia chromosome was first identified as a short chromosome 22 in 1960 by Nowell and Hungerford in Philadelphia.18 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 (Chapter 30).19 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, resulting in a unique chimeric gene, BCR-ABL1.20 This new gene produces a 210-kD BCR/ABL fusion protein (p210BCR/ABL) that expresses enhanced tyrosine kinase activity from the ABL moiety 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 33-1). 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.20

Image 

FIGURE 33-1 Molecular biology of the BCR/ABL fusion gene. A, Normal BCR1 gene on chromosome 22 and ABL gene on chromosome 9. B, Two BCR fusion gene products from the major BCR. C, Fusion gene product from the minor BCR. D, Fusion gene product from the micro BCR.

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. The wild-type (normal) BCR1 gene is approximately 100 kb with 20 exons. In 1984 Groffen and colleagues 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.21 This area was later termed the major BCR. Two other areas of breakage were identified on chromosome 22, one near 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. Therefore, two areas of breakage in the major BCR, one breakpoint area in the minor BCR, and one breakpoint region in the micro BCR produce four versions of the BCR gene that combine with the ABL1 gene to form four versions of the BCR/ABL1 chimeric gene.

Within the major BCR two specific breakpoints account for the t(9; 22) translocation involved in the development of CML. 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. 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 minor BCR contributes only exon one from BCR1, 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 fromBCR1, 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.22

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 SH1, SH2, and SH3 that together express and regulate the kinase activity. SH1 is the binding site for ATP; SH2 is the docking point for phosphate receiver proteins; and SH3 is the domain that controls the phosphorylation activity. When ATP binds to the ATP binding site, the phosphate is transferred to the SH2 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 kinase enzyme. When a second ATP binds the ATP binding site and a receiver protein docks in the SH2 domain, the phosphate group is transferred to the receiver protein. In most physiologically normal intracellular pathways, protein phosphorylation activates the receiver proteins (Figure 33-2). 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.2223

Image 

FIGURE 33-2 Signal transduction pathways influenced by the BCR/ABL fusion protein.

In the case of CML, the BCR/ABL1 translocation occurs next to the SH3 domain of the ABL1 moiety, which is designed to control the rate and timing of phosphorylation. Therefore, the BCR/ABL tyrosine kinase loses 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.24 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 dominates in the affected individual. Progeny cells that exhibit this chromosome include neutrophils, eosinophils, basophils, monocytes, nucleated erythrocytes, megakaryocytes, and B lymphocytes.925

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, causing premature release of cells into the circulation.23 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.26

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 gene 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/ABL1gene 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 associated with chronic neutrophilic leukemia and 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. 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. Table 33-1

TABLE 33-1

Common Morphologic Changes in Chronic Myelogenous Leukemia

Peripheral Blood

Erythrocytes

Normal or decreased

Reticulocytes

Normal

Nucleated red blood cells

Present

Total white blood cells

Increased

Lymphocytes

Normal or increased

Neutrophils

Increased

Basophils

Increased

Eosinophils

Increased

Myelocytes

Increased

Leukocyte alkaline phosphatase

Decreased

Platelets

Normal or increased

Bone Marrow

Cellularity

Increased

Granulopoiesis

Increased

Erythropoiesis

Decreased

Megakaryopoiesis

Increased or normal

Reticulin

Increased

Macrophages

Gaucherlike

Sea blue

Green-gray crystals

Increased

Megakaryocytes

Small

Increased

Extramedullary Tissue

Splenomegaly

Present

Sinusoidal

Present

Medullary

Present

Hepatomegaly

Present

Sinusoidal

Present

Portal tract

Present

Local infiltrates

Present

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.Figure 33-3

Image 

FIGURE 33-3 Peripheral blood films in the chronic phase of chronic myelogenous leukemia. A, Leukocytosis is evident at scanning power (×100). B, Bimodal population of segmented neutrophils and myelocytes (×500). C, Increased basophils and immature neutrophils (×1000).

Bone marrow changes are illustrated in . 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.Figure 33-427 The presence of pseudo-Gaucher cells (Chapter 29) usually occurs.

Image 

FIGURE 33-4 Bone marrow biopsy specimen in the chronic phase of chronic myelogenous leukemia, showing hypercellularity with increased granulocytes and megakaryocytes (hematoxylin and eosin stain, ×400).

Other laboratory findings

Hyperuricemia and uricosuria from increased cell turnover may be associated with secondary gout, urinary uric acid stones, and uric acid nephropathy.28 Approximately 15% of patients exhibit total white blood cell (WBC) counts greater than 300 × 109/L.29 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.30

In patients with the typical peripheral blood findings discussed above, the diagnosis of CML is confirmed by demonstrating the presence of the t(9; 22) translocation by cytogenetic analysis (Figure 30-1), detection of the BCR/ABL1 fusion gene using fluorescence in situ hybridization (Figures 30-2 and 30-21), and/or detecting the BCR/ABL1 fusion transcript by qualitative reverse transcriptase polymerase chain reaction (Figure 31-12).

Although molecular techniques are more commonly used to diagnose CML, initial testing of the cells for leukocyte alkaline phosphatase (LAP) enzyme activity may be useful in some setting for preliminary differentiation of CML from a leukemoid reaction due to severe infections (Chapter 29).

LAP is an enzyme found in the membranes of secondary granules of neutrophils. In the procedure a blood film is incubated with a naphthol-phosphate substrate and diazo dye at an alkaline pH. The LAP enzyme hydrolyzes the substrate, and the liberated naphthol reacts with the dye producing a colored precipitate on the granules. The slide is examined microscopically and 100 segmented neutrophils and bands are counted and rated from 0 to 4+ based on the intensity of the staining. The LAP score is calculated by multiplying each score by the number of cells, and adding the products. For example, 5 cells with 4+ staining, 5 cells with 3+, 25 cells with 2+, 45 cells with 1+, and 20 cells with 0 staining calculates to a LAP score of 130. Because scoring is subjective, the mean score of two examiners is reported, and they should agree within 10%.

A sample reference interval for the LAP score is 15 to 170, but every laboratory establishes its own. The LAP score is decreased in untreated CML, and normal or increased in leukemoid reactions. Individuals with polycythemia vera or those in the third trimester of pregnancy also have higher LAP scores.

Progression

In the pre-imatinib era, most cases of this disease would eventually transform into acute leukemia.31 Before blastic transformation, some patients proceed through an intermediate metamorphosis or acceleratedphase. 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 reflect evolution of the malignant clone and 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.32

Blast crisis involves the peripheral blood, bone marrow, and extramedullary tissues. Based on acute leukemia definitions, blasts constitute more than 20% of total bone marrow cellularity, and the peripheral blood exhibits increased blasts.31 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.3334 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.35 Similarly, chronic monocytic leukemia involves a comparable expansion of monocytes, including functional monocytes.36

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.37

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 the 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.38 Chronic myelomonocytic leukemia is discussed further with myelodysplastic syndromes in Chapter 34.

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.39-42 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,43 introduced in the late 1940s, and busulfan,44 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.45

Interferon-α stimulates a cell-mediated antitumor host response that reduces myeloid cell numbers, induces cytogenetic remissions, and increases survival.46 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/ABL1 gene, which was still detected by molecular and fluorescent methods.47 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.48

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 33-549

Image 

FIGURE 33-5 Mechanism of imatinib mesylate inhibition of BCR/ABL tyrosine kinase activity. A, Mechanism of tyrosine kinase activity of the BCR/ABL fusion protein. B, Mechanism of tyrosine kinase inhibition by imatinib mesylate.

Goals of therapy include complete hematologic, cytogenetic, and molecular remission indicated by a normalized CBC and differential, absence of Ph1 by karyotype analysis, and absence of measurable BCR/ABL transcripts, respectively. Complete remission from imatinib therapy is induced in part by the reactivation of apoptotic pathways.50 The effectiveness of imatinib therapy and stem cell transplantation is best monitored by measuring BCR/ABL transcripts using quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR). These monitoring tools are used to determine the extent of 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.51 Remission milestones indicating effective imatinib therapy are 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 usually results 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 25% to 30% of patients with newly diagnosed CML will discontinue imatinib therapy within 5 years due to lack of remission, resistance, or toxicity.52 The two major categories of imatinib resistance are primary and secondary. Primary resistance is defined as the inability to reach the remission milestones. This form of resistance accounts for most treatment failures and probably results from the presence of mutations other than the BCR/ABL1 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/ABL1 mutations and expression of point mutations in the ATP binding site. Additional BCR/ABL1 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/ABL1 mutation. A double dose of BCR/ABL1 can also be acquired from gene duplication during mitosis and accounts for 10% of secondary mutations. An additional BCR/ABL1 mutation will double the tyrosine kinase activity, making the imatinib dosage inadequate. In these cases higher doses of imatinib will restore remission in most patients (Figure 33-6). The majority of patients who do not respond to higher doses of imatinib express point mutations in the ATP binding site. Over 60 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 33-7). Three second-generation tyrosine kinase inhibitors—dasatinib (Sprycel), nilotinib (Dasigna), and bosutinib (Bosulib)—overcome the ATP binding site mutations because they have a much higher binding affinity than imatinib. All three are FDA approved for first-line therapy and are effective at rescuing patients resistant to imatinib, except patients who have developed the T315I mutation. The T315I mutation places a large, bulky isoleucine residue in the center of the ATP binding site, and all four FDA-approved tyrosine kinase inhibitors are resistant to this mutation. However, a third-generation tyrosine kinase inhibitor, ponatinib (Iclusig), inhibits the T315I mutation as well as drugs designed to bind the A loop (receiver protein binding site) will also inhibit tyrosine kinase activity and overcome the T315I mutation.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.

Image 

FIGURE 33-6 Mechanism of imatinib mesylate resistance due to an increased copy number of BCR/ABL genes. The increased copies produce more BCR/ABL fusion proteins, which results in an increased tyrosine kinase activity requiring a higher dosage of imatinib to restore remission.

Image 

FIGURE 33-7 Mechanism of imatinib mesylate resistance due to point mutations in the adenosine triphosphate (ATP) binding site. The mutations reduce the binding affinity of imatinib, allowing ATP to bind; this restores the increased tyrosine kinase activity that drives the phenotype and pathogenesis of the disease.

The development of a care plan for treating a patient with newly diagnosed CML is an ongoing commitment requiring not only the formulation of alternative approaches to achieve and maintain complete cellular remission but also the establishment of laboratory monitoring parameters to follow that confirm long-term success of therapy. Historically, chemotherapy has provided cellular remission but usually has not prevented clinical progression 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%, and most patients will not qualify. For a patient to qualify for transplantation, the patient must be younger than 50 years of age, in the first year of the disease, and have CML that is still in the chronic phase, and a histocompatible donor must be available. For the past 15 years, imatinib has been 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 has been 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/ABL1 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 Ponatinib or an A-loop inhibitor (ONO12380) or other drugs like Omacetaxine, MK 0457, or BIRB-796 that inhibit the T315I mutation.54 Physicians are beginning to prescribe dasatinib, nilotinib and bosutinib as first-line therapy to replace imatinib in hopes that tyrosine kinase inhibitors with higher binding affinities will extend remissions by reducing the rate of mutation-induced relapses. Among the second-generation TKIs, bosutinib shows the most promise because it demonstrates high potency, has the ability to overcome most P-loop mutations (except T315I), and shows fewer side effects like neutropenia, thrombocytopenia, cardiotoxicity, and pancreatitis compared to nilotinib and dasatinib. Ponatinib and A-loop inhibitors can be used to rescue patients treated with second-generation tyrosine kinase inhibitors, particularly those who develop the T315I mutation.52

Polycythemia vera

Polycythemia vera (PV) is a neoplastic clonal myeloproliferative disorder that commonly manifests with panmyelosis in the bone marrow and increases in erythrocytes, granulocytes, and platelets in the peripheral blood.2 Splenomegaly is common. The disease arises in a hematopoietic stem cell. The hypothesis of a clonal origin for PV is supported by studies of X-linked restriction fragment-length deoxyribonucleic acid (DNA) polymorphisms that demonstrate monoclonal X chromosome inactivation in all blood cells.56

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.57 Adverse clinical progression seems to correlate with the propagation of the erythropoietin-sensitive colony-forming units.58

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,JAK2V617F, 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.5960 The point mutation replaces guanine with thymine at exon 14 of the gene, which changes the amino acid at position 617 from valine to phenylalanine. This one amino acid change prevents the inhibition conformation of the tyrosinekinase, causing it to remain in 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 valine in the wild-type counterpart, which prevents the protein from folding into the inactive conformation (Figure 33-8).6162

Image 

FIGURE 33-8 Normal regulation of JAK2 function and loss of regulation from the JAK2 V617F mutation. A, Normal function of the JAK2 protein and regulation of phosphorylation by JAK2intrachain folding and the binding of JAK2 inhibitors SOCS1 and JAB proteins. B, The JAK2 V617F mutation and the loss of normal JAK2 folding and inhibitor binding resulting in phosphorylation, activation, and the stimulation of STAT, MAP kinase, and PI3K/AKT signal transduction pathways.

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 the head of the inactive JAK2 protein at a domain known as FERM (Band-4.1, ezrin, radixin, and moesin). Docking of JAK2 stimulates a phosphorylation event, causing a conformational change, and the valine releases from the pseudokinase domain, converting it to an active tyrosine kinase.JAK2 can also bind to several other receptors to include MPL (myeloproliferative leukemia, aka TPO-R [thrombopoietin receptor]), GCSF-R (granulocyte colony stimulating factor receptor), prolactin receptor, growth hormone receptor, GM-CSF-R (granulocyte/monocyte colony stimulating factor receptor), IL-3-R (interleukin-3 receptor), IL-5-R (interleukin-5 receptor), and INF-γ2-R (interferon gamma 2 receptor).62The diversity in ligand receptor binding explains the range of myeloid proliferation observed in PV (erythroid), ET (thrombopoietic), and neutrophilic (PMF). Once activated, JAK2 phosphorylates several cytoplasmic proteins, but the STAT (signal transducer and activator of transcription) proteins are the main targets. A cascade of phosphorylation reactions through the STAT proteins produce activated transcription factors that activate a host of genes designed to drive and control cell proliferation and differentiation while also initiating apoptosis (Figure 33-9). Constitutive tyrosine kinase activity of the JAK2protein 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 33-10). 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.63-65 In addition to the role of mutated JAK2 in the abrogation of STAT signaling, it has also been shown to influence chromatin structure6667 and to decrease methyltransferase activity.68 Lastly, homozygosity of JAK2 V617F occurs more commonly in PV, whereas JAK2heterozygosity occurs more commonly in ET. Therefore, disease progression from ET to PV may partially be explained by the dosage effect of JAK2 mutations.62

Image 

FIGURE 33-9 Normal erythropoiesis involving erythropoietin binding to erythropoietin receptors and stimulation of the JAK/STAT pathway via the normal JAK2 protein.

Image 

FIGURE 33-10 Stimulation of erythropoiesis in the absence of erythropoietin that is driven by a constitutively phosphorylated and activated JAK2 protein resulting from the JAK2 V617F mutation.

Because approximately 5% of PV patients do not possess the JAK2 V617F mutation and because PV has a familial predisposition, it is thought that other mutations must be involved in the pathogenesis of PV and some must precede and possibly predispose the JAK2 V617F mutation. Since the original discovery of a mutation in the thrombopoietic receptor gene MPL in 2006,69 several gain-of-function mutations have been identified. Most MPL mutations occur in exon 10 where tryptophan 515 is substituted for a leucine, lysine, asparagine, or alanine.69-72 Tryptophan 515 is located on the cytosolic side of the membrane and is key in transducing the signal that thrombopoietin (TPO) has bound to the receptor. These mutations cause the MPL receptor to be hypersensitive to TPO and in some cases to assume the active conformation in the absence of TPO. A similar mutation, MPL S505N, was initially described in familial PV but has since been found in sporadic MPN.6972 These types of MPL mutations have since been identified in up to 15% ofJAK2V617F–negative ET and PMF patients.73

In 2007 a second type of JAK2 mutation was identified in exon 12, usually between amino acid residues 536 and 547, that also resulted in a gain of function similar to the JAK2 V617F.74 Exon 12 is not located in the pseudokinase domain, but it is hypothesized that the mutation can modify the structure of the JH2 domain, rendering the protein incapable of forming the inactive conformation.62 JAK2 exon 12 mutations have been found in 3% of patients with PV and are not associated with ET or PMF but can be found in patients who progress to secondary myelofibrosis.7475

Experts hypothesize that mutations in signaling molecules alone are insufficient to initiate MPNs, suggesting that other mutations are necessary prior to JK2 V617F to induce disease and later to drive progression. Four lines of evidence support this hypothesis: familial MPN expresses a classic PV or ET phenotype in the absence of JAK2 V617F or MPL W515L mutations and transmits in an autosomal dominant fashion; in some ET and PV clones that were erythropoietin independent, JAK2 V617F was identified in a minority of cells, indicating that a pre– JAK2 mutation drove the disease; approximately 50% of patients who developed acute leukemia from a JAK2 V617F form of MPN expressed wild-type JAK2, suggesting a line of clonal evolution independent of JAK2; and in patients with PV and ET at diagnosis, theJAK2 V617F allele burden in HSCs was low compared to the allele burden in later stages of hematopoiesis, suggesting that JAK2 V617F confers a weak proliferative advantage to HSCs.62

Three reports in 2010 identified a germline haplotype block that predisposes patients to JAK2 mutations. This haplotype block was identified as a single nucleotide polymorphism (rs10974944) located in intron 12 of the JAK2 gene, increasing the development of MPN by three- to fourfold.61

Also in 2010, mutations were discovered in the adapter protein LNK (aka Src homology 2 B3–SH2B3), which downregulates JAK-STAT signaling pathways by regulating JAK2 activation. Approximately 3% to 6% of patients with MPN bear an LNK mutation,7980 with approximately 13% of mutations appearing in the blast phase versus the chronic phase of the disease.81 Following the binding of the corresponding ligand to its receptor, LNK binds to erythropoietin receptor (EPO-R), thrombopoietin receptor (MPL), and JAK2 to downregulate the JAK-STAT pathway as a negative modulator. Mutations in LNK produce a loss of function that removes a level of inhibitory control, increasing the proliferation of erythrocytes and thrombocytes. This loss of function mutation is accentuated in the presence of JAK2 V617F and MPLW515L mutations, resulting in the PV and ET phenotypes, respectively.61 More recently, somatic mutations have been identified in genes that control DNA methylation in patients with PV and other MPNs. The most notable are TET2IDH1, and IDH2TET2 (Ten Eleven Translocation 2) is one of three members of the TET family of genes (TET1 and TET3) and the only one identified with sequence alterations. TET2appears to be highly mutagenic for three reasons: mutations have been identified in all types of myeloid disorders to include MPNs, MDSs, and AMLs; mutations have been found in all coding regions of the gene; and mutations are often biallelic (homozygous).82 TET2 catalyzes the reaction that oxidizes the 5-methyl group of cytosine (5-mC) to 5-hydroxymethycytosine (5-hmC).83 It is hypothesized that 5-hmC serves as an intermediate base in the demethylation of DNA. Methylation of histones serves to silence genes. Therefore, TET2 mutations produce a loss of function effect, resulting in hypermethylation and a loss of gene activation (inactivation of tumor suppressor genes).61 In addition, it appears that TET2 mutations precede JAK2 mutations based on three observations: TET2 mutations are expressed in CD34+ hematopoietic stem cells (HSC); TET2 mutations have been identified in all forms of myeloid disorders; and all patients with both TET2 and JAK2 mutations produced clones with both mutations and clones that were TET2 positive and JAK2 negative but none that were JAK2 positive and TET2 negative. Therefore, TET2 mutations may create abnormal clones that predispose to JAK2 mutations.82 TET2 mutations have been identified in 9.8% to 16% of PV, 4.4% to 5% of ET, and 7.7% to 17% of PMF patients.61

Mutations in the genes that code for the citric acid cycle enzymes isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) have been associated with hypermethylation of DNA in patients with MPN and AML.84 Normally these enzymes function to convert isocitrate to α-ketoglutarate, requiring the reduction of NAD(P)+ to NADPH as the energy source. In patients with MPNs, mutations in IDH1/2 occur most frequently at the IDH2 R140 residue, the IDH1 R132 residue, and the IDA2 R172 residue.85 These mutations are thought to alter enzyme function, causing the conversion of α-ketoglutarate to 2-hydroxyglutarate (2-HG).86 It is hypothesized that because TET2 is dependent on α-ketoglutarate, the IDH1/2 mutations would result in less α-ketoglutarate, thus impairing the function of TET2 and exacerbating the hypermethylation function of mutated TET2 protein products.84 IDH1/2 mutations occur most frequently (21.6% of patients) in late-stage PV and ET patients during blast transformation compared to an incidence of 1% to 5% in patients with early PV (1.9%), ET (0.8%), and PMF (4.2%).61 As expected, IDH1/2 mutations are associated with adverse overall survival, raising the potential of usingIDH1/2 mutational analysis and/or 2-HG detection as poor prognostic indicators.

Mutations in two additional genes involved in epigenetic modification, EZH2 and ASXL1, have been implicated in MPN, MDS, and MDS/MPN and, along with TET2, may precede JAK2 V617F mutations.EZH2and EZH1 function as two of several proteins that form the polychrome repressive complex 2 (PRC2) that regulates chromatin structure. More specifically, EZH1 and EZH2 provide the functional domain for the PRC2 complex to methylate histone H3 at lysine 27.87 The array of mutations noted in EZH2 to date function to either eliminate protein production or abrogate methyltransferase activity. Mutations inEZH2 have been identified in 3% of PV patients, 13% of PMF patients, 12.3% of MDS/MPN patients, and 5.8% to 23% of MDS patients.8889 ASXL1 (Additional Sex Combs–like 1) mutations have been identified in most myeloid malignancies and at a frequency similar to TET2.62 ASXL1 normally functions in conjunction with ASXL2 and ASXL3 to deubiquinate histone H2 to balance the activity of PRC1 to monoubiquinate target genes to modify chromatin structure.9091 Ubiquination tags proteins for natural removal, thus regulating their function. The function of ASXL1 in hematopoiesis is poorly understood, but the loss of function mutations have been identified in less than 7% of PV and ET and from 19% to 40% in PMF.9293

Disease progression to blast crisis occurs in less than 10% of PV and ET patients, but several genetic mutation are implicated in this transformation.94-96 In addition to those that modulate epigenetic changes previously discussed (IDH1/2 and TET2), mutations in TP53 and RUNX1 are involved in blast transformation. TP53 produces the P53 protein that is known to be a tumor suppressor gene. P53 controls cell cycle checkpoints and apoptosis, and loss-of-function mutations are implicated in a host of cancers to include disease progression in the classic MPNs. TP53 mutations have not been identified in MPNs in the chronic phase but have been found in 20% of patients with MPNs who have progressed to AML.97-99 The protein product of the RUNX1/AML1 gene is a transcription factor that is important in hematopoiesis. RUNX1mutations were observed in 30% of post–MPN-AML patients, making it a candidate for the most frequent mutation involved in MPN transformation to AML.62

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 (> 18.5 g/dL in men and > 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.8 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 × 109platelets/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.100101 Recent research indicates that the JAK2 V617F mutation can be expected in more than 90% to 95% of cases.8 The WHO criteria for the diagnosis of PV are summarized in Box 33-1.

BOX 33-1

World Health Organization Criteria for the Diagnosis of Polycythemia Vera

Diagnosis requires the presence of both major criteria and one minor criterion or the presence of the first major criterion together with two minor criteria.

Major criteria

1. Hemoglobin > 18.5 g/dL in men, > 16.5 g/dL in women or other evidence of increased red blood cell volume*

2. Presence of JAK2 V617F or other functionally similar mutation such as JAK2 exon 12 mutation

Minor criteria

1. Bone marrow biopsy specimen showing hypercellularity for age with trilineage growth (panmyelosis) with prominent erythroid, granulocytic, and megakaryocytic proliferation

2. Serum erythropoietin level below the reference range for normal

3. Endogenous erythroid colony formation in vitro

*Hemoglobin or hematocrit > 99th percentile of method-specific reference range for age, sex, altitude, or residence, or hemoglobin > 17 g/dL in men, > 15 g/dL in women if associated with a documented and sustained increase of at least 2 g/dL from an individual’s baseline value that cannot be attributed to correction of iron deficiency, or elevated red cell mass > 25% above mean normal predicted value.

From Vardiman JW, Thiele J, Arber DA, et al: The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood114:937-951, 2009.

It is not always easy to assign an early diagnosis of PV. Erythrocytosis secondary to hypoxia or erythropoietin-producing neoplasms are 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 33-2Figures 33-11 and 33-12 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.28 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 of 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.

Image 

FIGURE 33-11 Peripheral blood film in stable phase polycythemia vera with essentially normocytic, normochromic erythrocytes (×500).

Image 

FIGURE 33-12 Bone marrow biopsy specimen in stable phase polycythemia vera showing panmyelosis (hematoxylin and eosin stain, ×400).

TABLE 33-2

Common Morphologic Changes in Polycythemia Vera

Peripheral Blood

Hemoglobin

Increased

Hematocrit

Increased

Red blood cell volume

Increased

Erythrocyte morphology

Normocytic/Normochromic

Total white blood cells

Increased

Granulocytes

Increased

Platelets

Increased

Leukocyte alkaline phosphatase

Normal or increased

Bone Marrow

Normoblasts

Increased

Granulocytes

Increased

Megakaryocytes

Increased

Reticulin

Increased

Extramedullary Tissue

Splenomegaly

Present

Sinusoidal

Present

Medullary

Present

Hepatomegaly

Present

Sinusoidal

Present

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.102 Myelofibrosis occurs within the bone marrow and may come to occupy a significant proportion of bone marrow volume, with subsequent ineffective hematopoiesis.103

Treatment and prognosis

The treatment of choice for PV is therapeutic phlebotomy at a frequency necessary to maintain the hematocrit at less than 45%. Low-dose aspirin has been shown efficacious to minimize thrombosis in all risk categories.104 The alkylating agent hydroxyuria is recommended in high-risk patients with PV and can be substituted for INF-γ in younger patients105106 and busulfan in older patients who develop intolerance or resistance to hydroxyuria.107 Prognosis for patients with PV is good, with a median survival exceeding 15 to 20 years.108 However, 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.101 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.

Treatment with modern JAK inhibitors has provided important benefits to patients with PV. A total of 34 patients intolerant or refractive to hydroxyuria were enrolled in the phase II study and treated with INCB018424 (ruxolitinib) at a dose of 10 mg b.i.d. Of the 34 patients, 15 (45%) had a complete remission.109 In addition, 97% (32 patients) achieved phlebotomy independence, and 80% (27 patients) achieved a 50% decrease in spleen size, as well as a reduction in pruritus, bone pain, and night sweats.110 CEP-701 (Lestaurtinib) was studied on 27 PV and 12 ET patients refractory or intolerant to hydroxyuria, and the outcomes were less promising. In 15 of the 39 patients who completed 18 weeks of treatment, 83% (15/18) achieved some degree of spleen size reduction, 60% (3/5) had a reduction in phlebotomy requirements, 20% (3/15) had a 15% decrease in JAK2 allele burden, and 15% developed thrombosis; gastrointestinal events were frequent.111

Essential thrombocythemia

Essential thrombocythemia (ET) is a clonal MPN with increased megakaryopoiesis and thrombocytosis, usually with a count greater than 600 × 109/L and sometimes with a count greater than 1000 × 109/L.112However, WHO criteria require a sustained thrombocytosis with a platelet count of 450 × 109/L or greater. Over the years, ET has been known as primary thrombocytosis, idiopathic thrombocytosis, and hemorrhagic thrombocythaemia.3113

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.113

Pathogenic mechanism

Most of the mutations described in PV also occur in ET but usually at a lower frequency. The JAK2 V617F occurs in approximately 55% of patients with ET.114 MPL exon 10 mutations (MPL W515L/K) are observed in 3% of ET patients, as well as several other mutations previously discussed to include TET2 (4.4% to 5%), ASXL1 (5.6%), LNK (3% to 6%), and IDH1/2 (0.8%).61 The manner in which these mutations alter normal cellular functions is similar to PV, as previously described.

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.113

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.115

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.113 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.113

The JAK2 V617F mutation is found in 50% to 60% of ET patients and supports the diagnosis of ET.116117 WHO diagnostic criteria were modified to include a minimum platelet count of 450 × 109/L when theJAK2mutation is present.118 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 3% of ET cases and is also used to exclude a diagnosis of reactive thrombocytosis.114 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.113

Peripheral blood and bone marrow

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. Figure 33-13

Image 

FIGURE 33-13 Peripheral blood film in stable phase essential thrombocythemia showing increased numbers of platelets and mature neutrophils (×500).

Early-phase bone marrow shows marked megakaryocytic hypercellularity, clustering of megakaryocytes, and increased megakaryocyte diameter with nuclear hyperlobulation and density (). Special studies reveal increased numbers of smaller and less mature megakaryocytes.Figure 33-14119 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 33-3.

Image 

FIGURE 33-14 Source: Bone marrow biopsy specimen in essential thrombocythemia showing marked megakaryocytic hypercellularity (hematoxylin and eosin stain, ×400).

TABLE 33-3

Common Morphologic Changes in Essential Thrombocythemia

Peripheral Blood

Hemoglobin

Slightly decreased

Hematocrit

Slightly decreased

Red blood cell volume

Normal

Total white blood cells

Normal or slightly increased

Neutrophils

Normal or slightly increased

Platelets

Increased

Platelet function

Decreased

Bone Marrow

Normoblasts

Normal or increased

Granulocytes

Normal or slightly increased

Megakaryocytes

 

Clusters

Present

Large

Present

Hyperlobulated

Present

Dense nuclei

Present

Variability in size

Increased

Reticulin

Normal or slightly increased

Extramedullary Tissue

Splenomegaly

Present

Sinusoidal

Present

Medullary

Present

Megakaryocytic proliferation

Present

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).113

Treatment and prognosis

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 an alkylating agent like hydroxyuria. 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%.114 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. For patients who develop intolerance or resistance to hydroxyuria, cytoreduction can be achieved with interferon-α in younger patients105 and busulfan in older patients.120 Low-dose aspirin is also recommended to prevent thrombosis.114

JAK2 inhibitors are being investigated in ET patients who are refractory or intolerant to hydroxyuria or are otherwise high risk. INCB018424 (ruxolitinib) was studied in 39 patients with ET at a dose of 25 mg b.i.d. In all 39 patients, the median platelet count reduced from 884 to 558 × 109/L, and the 11 patients who had leukocytosis achieved a normal WBC count after 6 months of treatment. Four patients who demonstrated splenomegaly showed spleen size reduction; 40% to 75% of patients had a 50% or greater improvement in one or more of the following: pruritus, bone pain, night sweats, and peripheral tingling/numbness. Only 13% (5 patients), achieved complete remission. However, a follow-up report at 10.4 months of treatment showed that 92% were still participating in the study, no grade 3 or 4 hematologic complications were noted, and although cytopenias were observed in 10% to 20%, they were grade 2 (mild).110 CEP701 (Lestaurtinib) was studied in 27 patients with PV and 12 patients with ET who were refractory or intolerant to hydroxyuria. In 15 of the 39 patients who completed 18 weeks of treatment, 83% (15/18) achieved some degree of spleen size reduction, 20% (3/15) had a 15% decrease in JAK2allele burden, and 15% developed thrombosis and had frequent gastrointestinal events.111

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 (Chapter 40).

The median survival for patients with ET is 20 years, including cases in which the process arises in younger patients.121 However, some patients may develop post-ET myelofibrosis, which reduces survival. Patients whose cells manifest chromosome abnormalities may have a poorer prognosis.39

Primary myelofibrosis

Primary myelofibrosis (PMF), previously known as chronic idiopathic myelofibrosis, agnogenic myelofibrosis, and myelofibrosis with myeloid metaplasia, is a clonal MPN6 in which there is splenomegaly and ineffective hematopoiesis associated with areas of marrow hypercellularity, fibrosis, and increased megakaryocytes. Megakaryocytes are enlarged with pleomorphic nuclei, coarse segmentation, and areas of hypochromia. The peripheral blood film exhibits immature granulocytes and normoblasts, dacryocytes (teardrop-shaped RBCs), and other bizarre RBC shapes.

PMF clonality was manifest in studies in which cytogenetic abnormalities were detected in normoblasts, neutrophils, macrophages, basophils, and megakaryocytes. Female patients heterozygous for glucose-6-phosphate dehydrogenase isoenzymes have PMF cells of a single enzyme isotype, whereas tissue cells, including marrow fibroblasts, contain both enzyme isotypes.6

Myelofibrosis

The myelofibrosis in this disease consists of 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.122 In approximately 30% of patients, biopsy specimens show no fibrosis.39Increases 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.123

Hematopoiesis and extramedullary hematopoiesis

Extramedullary hematopoiesis, clinically recognized as hepatomegaly or splenomegaly, seems to originate from release of clonal stem cells into the circulation.124 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.125 B and T cells may be involved.126 There is an increase in circulating unilineage and multilineage hematopoietic progenitor cells,127 and the number of CD34+ cells may be 300 times normal.128 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.129

Pathogenetic mechanism

As with PV and ET, the JAK2 V617F mutation is involved in the pathogenesis and is found in 65% of PMF patients.114 The MPL W515L/K occurs in an additional 10% of patients, along with most of the other mutations previously discussed, to include CBL (6%),130 TET2 (7.7% to 17%), ASXL1 (13% to 23%), LNK (3% to 6%), EZH2 (13%), and IDH1/2 (4.2%).61

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 33-4

TABLE 33-4

Common Morphologic Changes in Primary Myelofibrosis

Peripheral Blood

Hemoglobin

Normal or decreased

Anisocytosis

Present

Poikilocytosis

Present

Teardrop-shaped erythrocytes

Present

Nucleated red blood cells

Present

Polychromasia

Normal or increased

Total white blood cells

Normal, decreased, or increased

Immature granulocytes

Increased

Blasts

Present

Basophils

Present

Leukocyte anomaly

Present

Leukocyte alkaline phosphatase

Increased, normal, or decreased

Platelets

Increased, normal, or decreased

Abnormal platelets

Present

Megakaryocytes

Present

Bone Marrow

Cellularity

Increased

Granulopoiesis

Increased

Megakaryocytes

Increased

Erythropoiesis

Normal or increased

Myelofibrosis

Increased

Sinuses

Increased

Dysmegakaryopoiesis

Present

Dysgranulopoiesis

Present

Extramedullary Tissue

Splenomegaly

Present

Sinusoidal

Present

Medullary

Present

Hepatomegaly

Present

Sinusoidal

Present

Portal tract

Present

Local infiltrates

Present

Other tissues

Present

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 (). Micromegakaryocytes may be observed (Figure 33-15Figure 33-16).

Image 

FIGURE 33-15 Peripheral blood film in primary myelofibrosis showing nucleated red blood cells, giant platelets, and immature myeloid cells (×1000).

Image 

FIGURE 33-16 Source: Peripheral blood film in primary myelofibrosis exhibiting increased platelets and a micromegakaryocyte (×1000).

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.131 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

A diverse spectrum of therapies has been implemented to alleviate symptoms or modify clinical problems in patients with PMF, but none has been disease-modifying, so treatment approaches have been largely palliative. Treatment has been targeted at the amelioration of anemia, hepatosplenomegaly, and constitutional symptoms. Between 34% and 54% of PMF patients present with a hemoglobin of less than 10 g/dL.132,133 Severe anemia has been treated with androgen therapy, prednisone, danazol,134 thalidomide,135136 or lenalidomide,137 and hemolytic anemia with glucocorticosteroids. Approximately 20% of patients respond with an average duration of 1 to 2 years. Thalidomide and lenalidomide must be used with caution due to the occurrence of neuropathies and myelosuppression, particularly if the patient has been identified with del(5q31).138

Splenomegaly is present in 90% of patients with PMF, and 50% show hepatomegaly.132133 The most common first-line therapy for splenomegaly is hydroxyuria, but caution must be exercised so as not to exacerbate preexisting cytopenias.139140 Splenectomy and local radiation to the spleen and liver have been used in patients refractory to hydroxyuria, but patients must be carefully monitored for postoperative thrombosis, bleeding, infections, and cytopenias.141

The most common constitutional symptoms encountered by patients with PMF include fatigue (84%), bone pain (47%), night sweats (56%), pruritus (50%), and fever (18%).142143 However, treatment to alleviate these symptoms are minimally effective.

The development and testing of JAK inhibitors were directed at PMF because the symptoms and outcomes are worse compared to those of PV and ET. Among the JAK2 inhibitors tested, the four that showed the most promise were two JAK1/2 inhibitors, INCB018424 (ruxolitinib) and CYT387; one JAK2 inhibitor, TG101348; and one non-JAK inhibitor, CEP-701 (Lestaurtinib).

INCB018424 (ruxolitinib) was evaluated in a phase I/II trial at a dose of 25 mg b.i.d. and 100 mg q.d. on 153 patients with high- and moderate-risk PMF, both with primary disease and those who progressed from PV and ET. Clinical improvement associated with splenomegaly reduction (smaller spleen without progressive disease or increase in severity of anemia, thrombocytopenia, or neutropenia) was reported in 44% (16/140), and 14% (4/28) who were transfusion dependent showed anemia improvement, while 25% (38/153) withdrew from the study due largely to grade 3 thrombocytopenia and anemia (27%) that reduced to 16% following dose reduction to 10 mg b.i.d.144 A majority of patients reported a 50% improvement in symptoms, probably due to a measurable reduction in proinflammatory cytokines. However, there was no appreciable reduction in JAK2 V617F allele burden.114143 A phase III trial (COMFORT-1) of INCB018424 (ruxolitinib) is under way.

A phase I trial of TG101348 was conducted on 59 patients with high- and moderate-risk PMF at a maximum tolerated dose (MTD) of 680 mg q.d. Low-grade, transient nausea; vomiting; and diarrhea occurred in up to 69% of subjects, and along with adverse events, 45% withdrew from the study. Increases in serum lipase, transaminases, and creatinine occurred in approximately 25% of patients without any associated symptoms. Over 50% of subjects reported improvement in early satiety, night sweats, fatigue, pruritus, and cough along with a greater than 50% reduction in spleen size among 68% of the subjects. Thrombocytosis was normalized in 90% of patients and leukocytosis was normalized in 57%. In addition, 78% of patients had a 50% reduction in JAK2 V617F burden, while 13% increased and 9% remained unchanged.145

CYT387 is in phase I trials using 36 patients with high- and moderate-risk PMF, including 28% who were previously treated with INCB018424 or TG101348. At an MTD of 300 mg/day, all subjects continued after 15 weeks of treatment. Drug-related thrombocytopenia was observed in 22% (grade 3 or 4), anemia in 3% (grade 3), first-dose effect of lightheadedness and hypotension in 36% (grade 1), and several incidences of grade 3 adverse events, including elevations in liver and pancreatic enzymes, headaches, and QTc prolongations on ECG tracings. Reduction in splenomegaly was observed in 97% of subjects, with 37% achieving a greater than 50% response. The majority of patients reported a reduction in fatigue, pruritus, night sweats, cough, bone pain, and fever, and 41% of patients with pretreatment anemia showed clinical improvement.146

CEP-701 (lestaurtinib) was evaluated in phase II clinical trials at a dose of 80 mg b.i.d. using 20 patients who were JAK2 V617F positive. Twenty patients (91%) withdrew from the study for a variety of reasons, including diarrhea (73%), nausea (50%), vomiting (27%), headache (32%), mucositis (14%), peripheral neuropathy (14%), elevated transaminases (27%), anemia (27%), and thrombocytopenia (23%). Neither JAK2V617F allele burden nor serum levels of proinflammatory cytokines were affected by therapy. However, 27% of subjects reported clinical improvement in anemia splenomegaly reduction.147 A new phase I/II trial is under way with a new formulation of CEP-701 at higher doses.148

Reduction of myelofibrosis and of marrow and tissue hypercellularity has been accomplished with busulfan hydroxyurea and, in a few patients, interferon-α and interferon-γ. Radiotherapy is considered for patients with severe splenic pain, patients with massive splenomegaly who are not clinical candidates for splenectomy, patients with ascites secondary to serosal implants (metastatic nodules), and 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.149 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.150 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%.151

Average survival from the time of diagnosis is about 5 years, but patients have lived as long as 15 years. During this time, increasing numbers and pleomorphy of megakaryocytes lead to progressive marrow failure. Marrow blasts may increase.39 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.

Summary of current therapy of non-bcr/abl1, primary mpns

JAK2 inhibitors are most effective in patients with PMF due in part to the more severe symptoms in PMF compared to PV and ET. INCB018424, TG101348, and CYT387 appear to have a significant effect on decreasing splenomegaly—one of the more serious symptoms in PMF patients—within the first cycle of therapy, which peaks in 3 months. Splenic responses are dose dependent, durable through 12 treatment cycles, and limited by concomitant myelosuppression. Splenomegaly quickly returns with cessation of JAK2 inhibitors either within days for INCB018424 or within weeks for TG101348, due largely to their respective half-lives and possibly mode of action. The same three JAK2 inhibitors improve constitutional symptoms and appear to be durable. Treatment-related anemia is associated more with some JAK1/2 inhibitors (INCB018424) than others (CYT387) and is also associated with some JAK2 inhibitors (TG101348).114

In contrast, adverse events are dissimilar across the JAK inhibitors. For example, gastrointestinal events occur more frequently with the JAK2 inhibitor (TG101348) and with the non–JAK2 inhibitor (CEP-701), which might be due to the off-target FLT3 inhibition. Acute relapse of symptoms with drug discontinuation is seen in only one particular JAK1/2 inhibitor (INCB018424), which may be due to a “cytokine flare.” Lastly, only one of the JAK1/2 inhibitors (CYT387) produces first-dose symptoms of transient hypotension, flushing, and light-headedness. Future JAK inhibitor treatment may start with an induction dose to maximize response, followed by a maintenance dose with the addition and removal of other therapies tailored to the unique symptoms of each patient. For example, treatment-related myelosuppression could be ameliorated with pomalidomide, androgens, erythropoietin, and transfusions, and constitutional symptoms can also be managed through a host of traditional therapies. Outcomes in PV and ET are not as impressive, and the need to modulate symptoms is not as critical. Nonetheless, the JAK2 inhibitor TG101348 is most useful due its ability to normalize leukocytosis and thrombocytosis.114145

Interconnection among essential thrombocythemia, polycythemia vera, and primary myelofibrosis

The discovery of the JAK2 V617F mutation has advanced our understanding of the MPNs but has also raised questions about the interconnection of three of the primary myeloproliferative conditions: ET, PV, and PMF. Why is the JAK2 mutation found in more than 90% to 95% of patients with PV but in only 50% to 60% of patients with ET and PMF, and how can the same mutation produce three distinct phenotypes?

Currently four hypotheses exist to account for this apparent discordance. One prevailing thought suggests that the resulting phenotype is dependent on the stage of differentiation of the hematopoietic stem cell. For example, if the hematopoietic stem cell has developed a predilection toward platelet development at the time of the JAK2 mutation, ET will develop. Reports have described differences in differentiation programs152153 and in JAK2 mutations among ET, PV, and PMF.154 A second hypothesis proposes that the genetic background of the patient predisposes the patient to a particular phenotype. Mutations in the erythropoietin receptor, thrombopoietin receptor (MPL), and granulocyte colony-stimulating factor receptor have all been implicated.155 The third hypothesis suggests that the phenotype depends on the level ofJAK2 tyrosine kinase activity, called the dosage effect. Patients diagnosed with PV showed greater tyrosine kinase activity than patients presenting with an ET phenotype. Experiments in which erythroid progenitors were collected from these patients and tested in colony-forming assays showed that nearly all the cell cultures developing a PV phenotype were homozygous for the JAK2 mutation, whereas the ET phenotype was observed in the vast majority of cell cultures expressing a heterozygous genotype.156157 This phenomenon was corroborated in experiments with transgenic mice.158159 The last hypothesis proposes that a pre- JAK2 mutation produces a premalignant clone, predisposing the hematopoietic stem cell to a particular phenotype, and that the JAK2 mutation drives the malignant transformation. Groups have reported mutations coexisting with the JAK2 V617F mutation, like BCR/ABL,163164 MPL mutations,165 and another version of the JAK2 mutation.166167 Familial MPN provides the strongest support for a pre–JAK2mutation.168169

The most appealing model includes all of the hypotheses previously presented and suggests that ET, PV, and PMF may represent a continuum of diseases. It seems reasonable to assume that a pre– JAK2mutation occurs in the hematopoietic stem cell most if not all of the time to create a hyperproliferative clone that predisposes to additional mutations like the JAK2 mutation. The pre– JAK2 mutation can be familial, congenital, or somatic. Because MPL is expressed in high levels on megakaryocyte precursors, one JAK2 V617F mutation (heterozygous) is sufficient to induce MPL signaling and thus stimulate megakaryocyte production. This could lead to the ET phenotype. In contrast, the erythropoietin receptor is expressed in low density on the surface of erythroid precursors, which requires the higher amount of JAK2 V617F tyrosine kinase that is produced by two JAK2 mutations (homozygous). This could lead to the PV phenotype. Because MPL stimulation begins with the first JAK2 mutation and continues with the second JAK2mutation, the MPL receptor undergoes continuous stimulation. It has been shown that excessive thrombopoietin stimulation leads to myelofibrosis, which may result in a progression to PMF.170 The identification of additional mutations in the BCR/ABL1 negative MPNs to include negative regulators of signaling pathways (LNKc-CBLSOCs), tumor suppressor genes (IZF1TP53), and epigenetic regulators (TET2IDH1/2ASXL1EZH2) combines to set the disease on a particular course. JAK2 and MPL mutations serve as the drivers for the disease, but mutations in the negative regulators of signaling pathways may synergize with the driver mutations. Mutations in the epigenetic regulator genes may be early events that precede JAK2 but can also appear late to promote progression. Tumor suppressor gene mutations tend to occur during phases of disease progression.62 More than likely, most, if not all, of the hypotheses previously described function together to drive the BCR/ABL1 negative MPNs down a particular phenotype and through the phases of clonal expansion and disease progression.

Other myeloproliferative neoplasms

Chronic neutrophilic leukemia

Chronic neutrophilic leukemia (CNL) is a clonal disorder in which a hyperproliferation of neutrophilic cells in the bone marrow produces sustained neutrophilia in the peripheral blood and hepatosplenomegaly. CNL must be differentiated from CML, based on the absence of the Philadelphia chromosome and the BCR/ABL fusion gene, as well as from both a reactive neutrophilic process and other MPNs.171

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.171

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.171

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.171

The bone marrow reflects the peripheral blood in that it is hypercellular with predominantly a proliferation of neutrophils, including myelocytes, metamyelocytes, bands, and segmented neutrophils. The myeloid-to-erythroid ratio is at least 20:1. RBCs and platelets are normal in number, and no cell line exhibits significant dysplastic morphology.171

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 thePDGFRA, PDGFRB, or FGRF1genes. Lastly, there can be no evidence of PV, PMF, ET, MDS, or MDS/MPN disorders.171

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.171

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.171

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.172

Clinical presentation

Although some patients may be asymptomatic when found to have eosinophilia, most have signs and symptoms of fever, fatigue, cough, angioedema, muscle pain, and pruritus. A more severe sequela of CEL involves the heart. Fibrosis can form in the heart (endomyocardial fibrosis), which can evolve into cardiomegaly. Within the heart, scar tissue may form in the mitral and tricuspid valves, affecting valve function and predisposing to thrombi formation. Other serious complications include peripheral neuropathy, central nervous system dysfunction, pulmonary symptoms from eosinophilic infiltrates, and rheumatologic problems.172

Peripheral blood and bone marrow

Peripheral eosinophilia must be observed, with the majority of eosinophils appearing normal. Some evidence of eosinophil abnormality is found, however, and includes the presence of eosinophilic myelocytes and metamyelocytes, hypogranulation, and vacuolization. Neutrophilia is a common finding; other features such as mild monocytosis, basophilia, and the presence of blasts are less common. The bone marrow is hypercellular owing to eosinophilic proliferation and can demonstrate Charcot-Leyden crystals. Myeloblast numbers are elevated but below the 20% threshold necessary to classify the disorder as an acute leukemia. Erythrocytes and megakaryocytes are normal in number but sometimes demonstrate dysplastic morphologic features. Bone marrow fibrosis occurs due to the release of eosinophilic basic protein and eosinophilic cationic proteins from the eosinophil granules. Bone marrow fibrosis contributes to the premature release of eosinophils into the circulation, and they deposit in a variety of tissues.172

Diagnosis

The diagnosis of CEL requires eosinophilia with a count of more than 1.5 × 109 cells/L and the presence of malignant features, and the elimination of reactive eosinophilia and other malignancies that have concomitant eosinophilia. Reactive conditions like parasitic infections, allergies, Loeffler syndrome (pulmonary disease), cyclical eosinophilia, angiolymphoid hyperplasia of the skin, collagen vascular disorders, and Kimura disease must be excluded in the differential diagnosis. Likewise, other malignancies that can produce a concomitant eosinophilia include T cell lymphoma, Hodgkin lymphoma, systemic mastocytosis, chronic myelomonocytic leukemia, atypical CML, and ALL. These disorders lead to the release of a variety of interleukins that can drive a secondary eosinophil reaction. No single genetic abnormality is specific for CEL, but CEL can be ruled out by the presence of several karyotypic abnormalities, such as PDGFRA, PDGFRB, FGFR1, and BCR/ABL. Common myeloid mutations like +8, i(17q), and JAK2 can support a diagnosis of CEL.172

Prognosis

Survival is variable, but approximately 80% of patients will live 5 years after diagnosis. Features of dysplasia, an increase in karyotype abnormalities, or an increase in blasts indicates an unfavorable prognosis.172

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.173

Incidence

Mastocytosis can occur at any age, with cutaneous mastocytosis seen most often in children. Babies can be born with cutaneous mastocytosis, and half of affected children develop the disease before 6 months of age. In contrast, systemic mastocytosis generally occurs after the second decade of life. Approximately 80% of patients with mastocytosis show skin involvement regardless of the type of mastocytosis diagnosed. Cutaneous mastocytosis occurs in the skin; systemic mastocytosis usually involves the bone marrow and other organ systems like the spleen, lymph nodes, liver, and gastrointestinal tract; and mast cell leukemia is characterized by mast cells in the peripheral blood.173

Clinical presentation

Patients present with urticarial lesions (wheel and flare) that may become activated when stroked upon physical examination. Skin lesions also tend to have melanin pigmentation. Four categories of symptom severity have been described in mastocytosis: constitutional systems like fatigue and weight loss; skin manifestations; mediator-related systemic events such as abdominal pain, gastrointestinal distress, headache, and respiratory symptoms; and musculoskeletal complaints like bone pain, arthralgias, and myalgias. Hematologic findings include anemia, leukocytosis, eosinophilia, neutropenia, and thrombocytopenia. In patients with systemic mastocytosis with associated clonal hematologic non–mast-cell-lineage disease the most common associated hematologic finding is chronic myelomonocytic leukemia, but any myeloid or lymphoid malignancy can occur, although myeloid versions predominate.173

Diagnosis

The typical skin lesion is the first diagnostic clue to mastocytosis. Cutaneous mastocytosis occurs in three forms: urticaria pigmentosa, diffuse cutaneous mastocytosis, and mastocytosis of the skin, all of which occur predominantly in children. In urticaria pigmentosa, mast cells are confined to the skin and form aggregates in the dermis, whereas in diffuse cutaneous mastocytosis, mast cells are found in more than one cutaneous location. In systemic mastocytosis, mast cells are observed in one area of the bone marrow with fibrosis, other areas of the bone marrow are hypercellular with panmyelosis, and/or mast cells are identified in other extracutaneous sites. In addition to the major criteria just described, at least one of four minor criteria must be met. These include (1) more than 25% of mast cells must be immature or have atypical morphology, like a spindle shape; (2) mast cells must express a KIT mutation at codon 816; (3) mast cells must express normal markers and CD2 and/or CD25; and (4) total serum tryptase must be above20 mg/mL. Key diagnostic features distinguish the six types of systemic mastocytosis. Indolent systemic mastocytosis is characterized by a low–mast cell burden. Systemic mastocytosis with associated clonal hematologic non–mast-cell-lineage disease presents with myelodysplastic syndrome, myeloproliferative neoplasm, AML, lymphoma, or another hematopoietic neoplasm. Aggressive systemic mastocytosis usually does not manifest with skin lesions or mast cells in circulation but does have mast cells in bone marrow, dysplastic hematopoietic changes, and/or hepatosplenomegaly. Mast cell leukemia is characterized by more than 20% atypical mast cells in the bone marrow and more than 10% in the peripheral blood. Mast cell sarcoma presents as a single unifocal mast cell tumor with a high-grade pathology. Extracutaneous mastocytoma also exhibits a unifocal mast cell tumor, but it is of low-grade pathology.173

Genetics

The most common genetic mutation in patients with mastocytosis involves codon 816 in the KIT gene and occurs in about 95% of adults and 33% of children with systemic mastocytosis. This mutation replaces aspartic acid with valine, which alters the tyrosine kinase receptor activity so as to cause constitutive kinase activity in the absence of ligand. Usually the mutation is somatic, but a few cases of familial KITmutations have been reported. Additional mutations can push proliferation of hematopoietic clones, causing systemic mastocytosis with associated clonal hematologic non–mast-cell-lineage disease. These include mutations of RUNX1/RUNX1T in AML, JAK1 in MPN, and FIP1L1/PDGFRA in myeloid neoplasms with eosinophilia.173

Prognosis

Cutaneous mastocytosis in children has a favorable prognosis and may regress spontaneously around puberty. Milder versions like cutaneous mastocytosis and indolent cutaneous mastocytosis follow a benign course and are associated with a normal life span. Hematologic involvement usually evolves into the corresponding hematologic disease. Patients with aggressive systemic mastocytosis, mast cell leukemia, and mast cell sarcoma are often treated with cytoreductive chemotherapy but may survive only a few months after diagnosis. Signs and symptoms that predict a poorer prognosis include elevated lactate dehydrogenase and alkaline phosphatase, anemia, thrombocythemia, abnormal peripheral blood morphology, bone marrow hypercellularity, and hepatosplenomegaly.173

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: patients with an early stage of PV, ET, or PMF in which the criteria that define the disorders are not yet fully developed; patients presenting with features indicative of advanced disease resulting from clonal evolution that masks the potential underlying condition; and 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 condition 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.174

Summary

• MPNs are clonal hematopoietic stem cell disorders that result in excessive production and overaccumulation of erythrocytes, granulocytes, and platelets in some combination in bone marrow, peripheral blood, and body tissues.

• Within the classification of MPN, the four major conditions are CML, PV, ET, and PMF.

• In CML, there are large numbers of myeloid precursors in the bone marrow, peripheral blood, and extramedullary tissues.

• The peripheral blood exhibits leukocytosis with increased myeloid series, particularly the later maturation stages, often with increases in eosinophils and basophils.

• The LAP score is dramatically decreased in CML.

• The Philadelphia chromosome, t(9; 22), either at the chromosomal or molecular level, must be present in all cases of CML.

• In CML, the bone marrow exhibits intense hypercellularity with a predominance of myeloid precursors. Megakaryocyte numbers are normal to increased.

• Patients with CML progress from a chronic stable phase through an accelerated phase into transformation to acute leukemia.

• Bone marrow transplantation has been successful in CML, and imatinib mesylate, a tyrosine kinase inhibitor, produces remission in most cases.

• Approximately 4% of CML patients given imatinib as first-line therapy develop imatinib resistance.

• Dosage escalation or administration of second-generation tyrosine kinase inhibitors restores remission in most patients with imatinib resistance.

• PV manifests with panmyelosis in the bone marrow with increases in erythrocytes, granulocytes, and platelets.

• The clinical diagnosis of PV requires a hemoglobin of greater than 18.5 g/dL in men and greater than 16.5 g/dL in women or other evidence of increased RBC volume and the presence of JAK2 V617F or another mutation in the JAK2 gene. If only one of these major criteria is met, two of the minor criteria must be satisfied.

• The JAK2 V617F mutation is found in 90% to 95% of PV patients and contributes to the pathogenesis of the disease.

• PV is currently treated with phlebotomy, hydroxyuria, and low-dose aspirin, and then with JAK2 inhibitors in the future.

• ET involves an increase in megakaryocytes with a sustained platelet count greater than 450 × 109/L.

• Other diagnostic criteria include normal RBC mass, stainable iron in the bone marrow, absence of the Philadelphia chromosome, lack of marrow collagen fibrosis, absence of splenomegaly or leukoerythroblastic reaction, and absence of any known cause of reactive thrombocytosis.

• In the early phases of ET, peripheral blood shows increased numbers of platelets with abnormalities in size and shape. Bone marrow megakaryocytes are increased in number and in size.

• Complications of ET include thromboembolism and hemorrhage.

• The JAK2 V617F mutation is observed in 50% to 60% of patients with ET and PMF and contributes to the pathogenesis of the disorders.

• PMF manifests with ineffective hematopoiesis, sparse areas of marrow hypercellularity (especially with increased megakaryocytes), bone marrow fibrosis, splenomegaly, and hepatomegaly.

• The peripheral blood in PMF exhibits immature granulocytes and nucleated RBCs; teardrop-shaped cells are a common finding.

• Platelets may be normal, increased, or decreased in number with abnormal morphology. Micromegakaryocytes may be present.

• Immune responses are altered in about 50% of patients.

• Treatment of PMF includes a variety of approaches to include transfusions, hydroxyuria, INF-γ, busulfan, androgens, erythropoietin, and others.

• JAK inhibitors improve splenomegaly and constitutional symptoms in patients with PMF to a greater degree than in ET or PV.

• Other MPNs include CNL, CEL not otherwise specified, mastocytosis, and unclassifiable MPN.

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

1. A peripheral blood film that shows increased neutrophils, basophils, eosinophils, and platelets is highly suggestive of:

a. AML

b. CML

c. MDS

d. Multiple myeloma

2. Which of the following chromosome abnormalities is associated with CML?

a. t(15; 17)

b. t(8; 14)

c. t(9; 22)

d. Monosomy 7

3. A patient has a WBC count of 30 × 109/L and the following WBC differential:

Segmented neutrophils—38%

Bands—17%

Metamyelocytes—7%

Myelocytes—20%

Promyelocytes—10%

Eosinophils—3%

Basophils—5%

Which of the following test results would be helpful in determining whether the patient has CML?

a. Nitroblue tetrazolium reduction product increased

b. Myeloperoxidase increased

c. Periodic acid–Schiff staining decreased

d. FISH positive for BCR/ABL1 fusion

4. A patient in whom CML has previously been diagnosed has circulating blasts and promyelocytes that total 30% of leukocytes. The disease is considered to be in what phase?

a. Chronic stable phase

b. Accelerated phase

c. Transformation to acute leukemia

d. Temporary remission

5. The most common mutation found in patients with primary PV is:

a. BCR/ABL

b. Philadelphia chromosome

c. JAK2 V617F

d. t(15; 17)

6. The peripheral blood in PV typically manifests:

a. Erythrocytosis only

b. Erythrocytosis and thrombocytosis

c. Erythrocytosis, thrombocytosis, and granulocytosis

d. Anemia and thrombocytopenia

7. A patient has a platelet count of 700 × 109/L with abnormalities in the size, shape, and granularity of platelets; a WBC count of 12 × 109/L; and hemoglobin of 11 g/dL. The Philadelphia chromosome is not present. The most likely diagnosis is:

a. PV

b. ET

c. CML

d. Leukemoid reaction

8. Complications of ET include all of the following except:

a. Thrombosis

b. Hemorrhage

c. Seizures

d. Infections

9. Which of the following patterns is characteristic of the peripheral blood in patients with PMF?

a. Teardrop-shaped erythrocytes, nucleated RBCs, immature granulocytes

b. Abnormal platelets only

c. Hypochromic erythrocytes, immature granulocytes, and normal platelets

d. Spherocytes, immature granulocytes, and increased numbers of platelets

10. The myelofibrosis associated with PMF is a result of:

a. Apoptosis resistance in the fibroblasts of the bone marrow

b. Impaired production of normal collagenase by the mutated cells

c. Enhanced activity of fibroblasts owing to increased stimulatory cytokines

d. Increased numbers of fibroblasts owing to cytokine stimulation of the pluripotential stem cells

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