INTRODUCTION
The myeloproliferative neoplasms (MPNs) comprise several clonal hematopoietic stem cell diseases that share a number of overlapping features including overproduction of one or more myeloid lineages with relatively unaffected maturation. As a consequence, the bone marrow is hypercellular and red blood cells, granulocytes, and/or platelets may be increased in the peripheral blood. Hematopoiesis eventually extends to other organs, such as the spleen and liver, and there is a variable rate of transformation to acute leukemia or MF. The MPNs recognized in the World Health Organization (WHO) classification of MPNs are listed in box 1-1.1
Chronic myelogenous leukemia (CML) has long been the prototype for this group of diseases and was the first MPN in which the molecular pathogenesis was defined.
The cytogenetic hallmark of CML is the Philadelphia (Ph) chromosome that results from a reciprocal t(9;22)(q34;q11.2) translocation or its variants t(V;9;22). The molecular consequence of this translocation is the formation of a BCR-ABL1 (breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1) gene fusion that encodes a BCR-ABL fusion tyrosine kinase. Whereas the native ABL (Abelson leukemia virus) protein has tightly regulated tyrosine kinase activity, the chimeric BCR-ABL oncoprotein is constitutively activated. Similar to other kinases, the BCR-ABL protein transfers phosphate from adenosine triphosphate (ATP) to tyrosine residues in specific substrate proteins. This activates multiple transduction pathways and results in deregulated cellular proliferation, reduced apoptosis and decreased adherence to the bone marrow stroma. These properties allow the leukemic cells to gradually displace the normal hematopoietic cells. This knowledge of the pathogenesis of CML led to the development of effective targeted therapy in the form of ABL tyrosine kinase inhibitors, such as imatinib.2–4
The success of imatinib as a targeted therapy for the fusion tyrosine kinase BCR-ABL in CML established a conceptual framework for identifying the underlying mutations in other MPNs, and great strides have been made over the last few years toward identifying the genetic abnormalities in BCR-ABL1 negative MPNs. In these diseases, however, it has become apparent that there is a broad and complex array of genetic abnormalities present rather than one defined abnormality as in CML. The discovery of mutations in the tyrosine kinase Janus kinase 2 (JAK2) in different MPN diseases5–9 made it clear that the model of one molecular abnormality defining one disease is not applicable in many types of MPNs.
It was a major breakthrough in MPN research in 2005 when the activating point mutation [V617F (a change of valine to phenylalanine at the 617 position)] in JAK2 was identified in approximately 95% of patients with polycythemia vera (PV) and approximately 50% of patients with primary myelofibrosis (PMF) or essential thrombocythemia (ET).10 JAK2 is a cytoplasmic tyrosine kinase that transduces signaling, especially signals triggered by hematopoietic growth factors such as erythropoietin. The JAK2 V617F mutation, as well as more recently identified additional JAK2 mutations and rearrangements,11 result in a constitutively activated tyrosine kinase that activates proliferation and survival pathways. The JAK-1/2 inhibitor ruxolitinib is available therapeutically and clinical trials have found beneficial effects, such as reduction of spleen size and constitutional symptoms in myelofibrosis (MF) patients.12,13 Identification of JAK2 mutations are helpful in diagnosing MPN and guiding therapy; however, JAK2 mutations are not disease specific and are also found in patients without MPNs.6,14 The classification of MPNs in the WHO includes the presence of JAK2 mutations in the criteria; however, JAK2 mutations are not disease-specific or required for any MPN diagnosis.
Many BCR-ABL1 negative MPN genetic alterations identified are in genes involved in cell signaling, cell cycle, and apoptosis. Recently, several mutations have also been identified in genes involved in epigenetic regulation.15,16 Similar to JAK2 mutations, most of these mutations are not specific to MPNs. Therapies are rapidly being developed to target many of these mutations. As underlying genetic abnormalities are uncovered in MPN with potential therapeutic implications, the classification of MPNs by the WHO has been adapted to include these abnormalities into the diagnostic classification scheme. In the WHO classification, molecular abnormalities can be disease-defining, such as BCR-ABL1, or as in the case of JAK2 mutations, they have been incorporated into the criteria in support of the diagnosis and to help guide therapy.1
A recent study identified that a significant portion of patients with essential thrombocythemia and primary myelofibrosis without JAK2 or myeloproliferative leukemia virus oncogene (MPL) mutations have somatic muations of the gene CALR, encoding calreticulin. Identification of CALR mutations may be helpful in the diagnosis of essential thrombocythemia and primary myelofibrosis and initial studies suggest the presence of CALR mutations may have prognostic implications.17
There are currently four chronic myeloid neoplasm entities in the WHO with defined molecular abnormalities: CML with BCR-ABL1, myeloid and lymphoid neoplasms with platelet-derived growth factor receptor-alpha (PDGFRA) rearrangement, myeloid neoplasms with platelet-derived growth factor receptor-beta (PDGFRB) rearrangement, and myeloid and lymphoid neoplasms with FGFR1 abnormalities. The last three entities have been grouped in the WHO into a category separate from the other MPNs and characteristically have a prominent eosinophilic component. Many of the other MPN genetic abnormalities, like mutations of JAK2, are useful in helping to diagnose MPN and potentially guide therapy, but at this time are not considered defining of one specific disease. Presumably, with the development of therapies targeting molecular abnormalities, there will be an ever-increasing number of molecularly-defined diseases.
As with CML, the three other molecularly-defined myeloid neoplasms have a fusion gene with an aberrant tyrosine kinase, with potential therapeutic implications. Abnormalities of both the α and β moieties of the platelet-derived growth factor receptor (PDGFRA and PDGFRB), a surface tyrosine kinase receptor, have been associated with myeloid neoplasms with eosinophilia. Chromosomal translocations involving the FGFR1 gene at 8p11 give rise to a myeloproliferative disorder characterized by eosinophilia and lymphoblastic lymphoma that frequently progresses to acute myeloid leukemia (AML).18 The presence of either the PDGFRA or PDGFRB rearrangement leads to constitutively activated tyrosine kinase and predicts responsiveness to imatinib.19 Although FGFR1 inhibitors have been developed, no effective targeted pharmaceutical therapy is available at this time for patients with myeloid and lymphoid neoplasms with FGFR1 abnormalities.20
This chapter summarizes the diagnosis of BCR-ABL1 negative MPN based on the established WHO criteria including clinical, morphologic, and genetic features. CML is not within the scope of this book, but a brief morphologic description is included here for comparison to the BCR-ABL1- negative MPNs. Subsequent chapters will review the newly defined molecular abnormalities in the BCR-ABL1- negative MPNs.
CHRONIC MYELOGENOUS LEUKEMIA
Chronic myelogenous leukemia is a chronic MPN causally related to the BCR-ABL1 fusion gene.21 The diagnosis is based on characteristic blood and bone marrow findings, and documentation of the presence of BCR-ABL1 fusion by cytogenetic and/or molecular analysis. The diagnosis is usually established during an initial, indolent or chronic phase. The major hematological finding at diagnosis is an excessive number of mature and immature granulocytes in both the peripheral blood and bone marrow. As the disease progresses, patients advance to an accelerated or blast phase. The accelerated phase often precedes the blast phase and is characterized by clinical deterioration, and changes in blood counts and morphology but without a sufficient number of blasts for diagnosis of acute leukemia. The blast phase represents overt acute leukemia characterized by the rapid expansion of myeloid or lymphoid lineage blasts.22
Microscopic Findings
The diagnosis of CML is usually strongly suggested by characteristic peripheral blood findings (Figure 1-1A) that include a neutrophilic leukocytosis. The neutrophil series in the blood is typically represented by all stages of maturation from the myeloblast to the segmented neutrophil with myelocytes and segmented stages usually the most numerous. In rare cases, mature neutrophils predominate. The neutrophils in the chronic phase of the disease do not typically show dysplastic changes. Basophils are increased in absolute number; this characteristic feature aids in distinguishing CML from a reactive neutrophilia. An eosinophilia is, also, common but lacks the diagnostic specificity of basophilia. Circulating immature basophils or eosinophils may be found. Myeloblasts are usually present but generally do not exceed 2–3%. Monocytes may be mildly increased but are usually less than 4% of the leukocyte differential.
The platelets are usually increased in number and platelet morphology is normal although occasional large platelets with decreased or absent granules may be present. Rarely, thrombocytosis may be the predominant feature of a patient presenting with CML with alterations in leukocytes inconspicuous or absent. A normochromic, normocytic anemia, and a small number of nucleated red blood cells are present in most patients.
The bone marrow is hypercellular and shows a marked increase in granulocytes. The neutrophils show normal maturation although a relative increase in myelocytes or a “myelocyte bulge” is often a prominent feature.
Figure 1-1: Chronic myelogenous leukemia, peripheral blood and bone marrow. A, The peripheral blood smear shows a neutrophilia with all stages of maturation represented. A basophil is also present. B, The bone marrow is markedly hypercellular with marked granulocytic proliferation. Several megakaryocytes are present in this field. They appear smaller than normal megakaryocytes.
The periosteal cuff of promyelocytes and myelocytes is widened, often 5–10 cells thick in comparison to 1–2 cells seen in normal bone marrow biopsies (Figure 1-1B).23 Basophils and eosinophils are increased and may be abundant. Blasts represent fewer than 10% of the bone marrow cells. Megakaryocytes may be normal in number but they are usually increased; they are scattered throughout the bone marrow biopsy or clustered in small groups. The megakaryocytes tend to be smaller than those found in normal bone marrow biopsies, in contrast to the large megakaryocytes seen in other chronic MPNs, such as ET. Hypolobate megakaryocytes may be observed. Erythroid precursors are often decreased in number. Macrophages containing blue-pigment debris (sea-blue histiocytes) and pseudo-Gaucher cells are found in the bone marrow in about one-third of cases. These macrophages are part of the neoplastic clone and result from increased bone marrow turnover of granulocytes.24 They are positive with stains for periodic acid-Schiff (PAS) and iron. Lymphoid aggregates are often present and may be prominent, especially following therapy.
Genetic Characteristics
The hallmark genetic abnormality of CML is a reciprocal chromosomal translocation, t(9;22)(q34;q11), that was first discovered as a small chromosome named the Ph chromosome. About 95% of patients with CML exhibit this translocation while another 5% have complex or variant translocations involving additional chromosomes with the same end result—development of the BCR-ABL oncogene by fusion of the BCR on chromosome 22 to the ABL gene on chromosome 9.25
BCR-ABL1 NEGATIVE MYELOPROLIFERATIVE NEOPLASMS POLYCYTHEMIA VERA
Polycythemia vera is a chronic MPN characterized by a clonal proliferation of myeloid cells with erythrocytosis being the most prominent manifestation. Virtually all patients with PV carry the JAK 2 V617F or other functionally similar mutation. Such mutations are absent in secondary or reactive causes of erythrocytosis. Elevations in hemoglobin (Hb) and the presence of JAK 2 V617F or other similar JAK 2 mutation serve as major criteria for the diagnosis of PV in the WHO diagnostic criteria for PV shown in box 1-2. Minor criteria for the diagnosis include characteristic bone marrow histopathology for PV, low serum erythropoietin level, and endogenous erythroid colony formation in vitro.26,27
Clinical Features
The mean age at diagnosis of PV is approximately 60 years with a slight male predominance (1.2: 1). About 7% of patients are diagnosed before the age of 40 years; PV is only rarely diagnosed in children.28 Most patients at diagnosis are symptomatic with the most common symptoms being headache, dizziness/vertigo, fatigue, and pruritus. Complaints of a prior or current thrombotic event are common. The main physical findings are a ruddy cyanosis, splenomegaly in 70%, hepatomegaly in about 33%, and hypertension in 50%.29
Laboratory Findings
The Hb/hematocrit and the red cell mass are elevated.26 If iron deficiency is present, the Hb/hematocrit may be normal or low. The platelet count is elevated in about 60% of patients and the leukocyte count is elevated in about 60% of patients.29
Figure 1-2: Polycythemia vera, peripheral blood, and bone marrow. A, The blood smear shows an erythrocytosis with crowding of the red blood cells. B, The bone marrow biopsy shows an erythroid hyperplasia with increased numbers of megakaryocytes. The megakaryocytes vary in size and are not dysplastic.
A basophilia as well as an eosinophilia may be present. Serum erythropoietin levels are usually low. A common feature of many MPNs, endogenous erythroid colony formation, is characteristic of PV, but testing for this feature is not widely available.30
Microscopic Findings
The peripheral blood smear shows an erythrocytosis (Figure 1-2A). The red cells are generally normochromic, normocytic but may be hypochromic, microcytic if the patient is iron deficient. The leukocytes are often increased because of an increased number of neutrophils; occasional immature neutrophils may be apparent. Basophilia and eosinophilia may be present. Platelets are frequently increased and platelet clumps may be observed.
The bone marrow in PV is usually mildly to markedly hypercellular with a panmyelosis that includes erythroid, granulocytic, and megakaryocytic hyperplasia (Figure 1-2B). Erythroid and megakaryocytic proliferation are typically the most prominent. Enlarged islands of erythropoiesis are present. The megakaryocytes vary in size from small to large and lack dysmorphic features. They tend to cluster and may be localized around bone marrow sinusoids or adjacent to bone trabeculae. The granulocyte series shows no shift to immaturity and no increase in blasts is present.31 Storage iron is almost always absent. Reticulin fibers are usually normal but may be mildly to moderately increased.32 Lymphoid aggregates may be found in up to 20% of patients.30
Genetic Characteristics
Cytogenetic abnormalities are identified at diagnosis in about 25% of patients with PV. The most common recurring aberrations include +8, +9, del(20q), del(13q), and 8del(1q).30 Molecular genetic studies including fluorescence in situ hybridization (FISH) have identified frequent cryptic abnormalities of chromosome 9p, site of the JAK2 gene.33 Chromosomal abnormalities are present with increasing frequency as the disease progresses and are found in about 80–90% of patients with post-polycythemic myelofibrosis (post-PVMF) and nearly 100% of patients who develop acute leukemia.30
As many as 97% of patients with PV possess the JAK2 V617F gene mutation resulting from a point mutation in exon 14.6,7,34 Loss of heterozygosity through mitotic combination is frequent in PV. In one study the patients homozygous for the mutation had higher Hb levels, higher incidence of pruritus, and a higher rate of fibrotic transformation than patients who were heterozygous.35 Other JAK2 mutations with effects similar to those of JAK2 V617F have been reported in patients negative for JAK2 V617F. These mutations are in-frame deletions or tandem point mutations in exon 12.36
Disease Progression
Patients with PV are at risk of transformation to post-PVMF or acute leukemia. The risk of transformation to post-PVMF is about 6% at 15 years; the risk of evolution to acute leukemia is similar at about 7% at 15 years.37 Duration of disease is a significant risk factor for developing post-PVMF while older age and cytotoxic therapy other than hydroxyurea are risk factors for progression to AML.38,39
Transformation of PV to post-PVMF has also been referred to as the “spent phase” of PV. Post-PVMF is characterized be a decrease in Hb, leukoerythroblastosis, and red cell poikilocytosis that often includes tear-drop shaped red cells. The bone marrow exhibits prominent fibrosis and may be hypocellular (Figure 1-3A). The fibrosis can be defined by reticulin staining (Figure 1-3B). Osteosclerosis may be present. Megakaryocytes, often in clusters, may be prominent; unlike those seen at diagnosis, they may be dysmorphic. Erythropoiesis and granulopoiesis are decreased, and intrasinusoidal hematopoiesis may be observed.
Figure 1-3: Post-polycythemic myelofibrosis, bone marrow. A, The bone marrow is fibrotic and hypocellular. B, A reticulin stain shows marked reticulin fibrosis.
The spleen often enlarges further during this time secondary to extramedullary hematopoiesis. Specific criteria have been proposed by an international consortium for the diagnosis of post-PVMF, requiring the diagnosis of PV and bone marrow fibrosis with at least two of four additional criteria.40
The blast phase or acute leukemia that develops in PV is of myeloid origin41 and is preceded by myelodysplastic features in about 50% of cases.23
PRIMARY MYELOFIBROSIS
Primary myelofibrosis is a clonal stem cell disorder characterized by granulocytic and megakaryocytic proliferation, reactive bone marrow fibrosis, and extramedullary hematopoiesis. Other terms that have been used for this disorder include agnogenic myeloid metaplasia, myelosclerosis with myeloid metaplasia, and idiopathic myelofibrosis. This disease evolves from an initial prefibrotic stage characterized by a hypercellular bone marrow with absent or minimal fibrosis to a stage with marked bone marrow fibrosis and, also, frequently osteosclerosis. The fibrotic stage is characterized by a prominent leukoerythroblastic blood picture and the presence of tear drop shaped red blood cells. About 50% of patients with PMF exhibit the JAK2 V617F mutation. The WHO criteria for diagnosis of PMF are shown in box 1-3.26,42
Although granulocytes and megakaryocytes are the most prominent hematopoietic cells involved in PMF, erythroid precursors, monocytes/macrophages, and, occasionally, B- and T-lymphocytes are derived from the same clonal stem cell.43 The fibrosis is secondary and nonclonal; its production is stimulated by growth factors secreted by the neoplastic megakaryocytes. Megakaryocyte-derived transforming growth factor-β1 is the major cytokine implicated in the mediation of fibrosis in PMF.44,45
Clinical Features
The median age at diagnosis is about 60 years with a male to female ratio of 1.2: 1 to 1.6: 1. Less than 3% of patients present before the age of 30 years; PMF is exceedingly rare in children.45 The presenting clinical features vary widely and depend on the stage of the disease when the disease is first detected. Compared to ET or PV, most patients are symptomatic at diagnosis, but as many as one-third of patients are asymptomatic at diagnosis. When complaints are present, the most common presenting symptom is fatigue. Other symptoms may include pruritus, bone pain, cough, early satiety, abdominal pain, weight loss, fever, and night sweats.46
About 30% of patients are first detected in the prefibrotic or cellular phase. At this time, splenomegaly may be present but is only minimal to modest.47 Most patients with PMF present when bone marrow fibrosis is present. Splenomegaly secondary to extramedullary hematopoiesis is usually present at that time and may be marked with the spleen often extending 10 cm or more below the left costal margin.48
Symptoms related to splenomegaly, such as early satiety and abdominal discomfort may be prominent. Hepatomegaly is also common.
Laboratory Findings
Thrombocytosis is often the most prominent hematological manifestation in early PMF (prefibrotic or very early fibrotic lesions) with the platelet count reported to exceed 600 × 109/L in 86% of patients. Leukocytosis (51%) and anemia (38%) may also be present.49 Anemia is present in 50–70% of patients who present with fibrotic PMF; 25% manifest a severe anemia (Hb <8 g/dL). The leukocyte count is elevated in approximately 50% of patients and exceeds 30 × 109/L in about 10%. Patients can also exhibit a thrombocytopenia at diagnosis, typically with overt PMF.46
The absolute number of circulating clusters of differentiation (CD)34+ hematopoietic precursor cells in PMF is reported to be higher than normal controls and also higher than patients with other BCR-ABL1-negative chronic MPNs.50
Figure 1-4: Primary myelofibrosis, peripheral blood. This blood smear shows tear drop shaped red cells, a leukoerythroblastic reaction, and increased platelets. The platelets have variable morphology including large, hypogranular platelets.
Other abnormal laboratory results that may be observed include elevations in lactate dehydrogenase (LDH), alkaline phosphatase, uric acid, and vitamin B12.
Microscopic Findings
Blood and Bone Marrow
The characteristic features of the blood smear in PMF are increased poikilocytosis with tear drop shaped red cells (Figure 1-4), a leukoerythroblastic reaction with circulating nucleated red blood cells and granulocyte precursors, and large hypogranular platelets. Micromegakaryocytes and megakaryocyte nuclei are frequently present. These changes are secondary to release from sites of extramedullary hematopoiesis and increase as the disease progresses. These findings may be seen during the prefibrotic stage, but are usually subtle; thrombocytosis often dominates the blood picture at that time. Circulating blasts are often present during the fibrotic stage but should be less than 10% of cells. Blasts more than 10% should raise the possibility of an accelerated phase or acute leukemia.42
The bone marrow in the prefibrotic or cellular stage of PMF is hypercellular with minimal or absent reticulin fibrosis (Figures 1-5A to C). Prominent megakaryocytic proliferation is characteristic of this stage of PMF. A granulocytic hyperplasia with a shift to immaturity is typically observed; erythroid precursors are often decreased or exhibit shift to immaturity. The megakaryocytes form clusters and may be located next to bony trabeculae.
Figure 1-5: Prefibrotic primary myelofibrosis, peripheral blood, and bone marrow. A, The blood smear shows increased platelets that vary in size and include large, hypogranular platelets. B, The bone marrow is hypercellular; the megakaryocytes are increased and many are morphologically abnormal with increased nuclear/cytoplasmic ratios and hyperchromatic nuclei. C, Reticulin stain reveals a focus of slightly increased reticulin fibers.
They vary from small to giant and exhibit prominent morphologic abnormalities including hyperchromatic nuclei, hyposegmentation, irregular lobulation, and frequent bare or “naked” megakaryocytic nuclei. Dyserythropoiesis and dysgranulopoiesis are absent. Recognition of the morphologically abnormal megakaryocytes is critical to recognizing the prefibrotic stage and distinguishing it from other myeloproliferative disorders, particularly ET. Reticulin fibrosis, when present, tends to be concentrated around vessels. Vascular proliferation is increased.31
As the bone marrow progresses from the prefibrotic to the fibrotic stage, the cellularity decreases, and reticulin and even collagen fibrosis becomes more prominent. The bone marrow is not easily aspirated often resulting in a dry tap. Patches of hematopoietic tissue may be separated by fibrous tissue or fat. The bone marrow sinuses are often dilated and may contain immature hematopoietic cells. Morphologically abnormal megakaryocytes are usually the predominant cells and 13may occur in clusters or sheets; megakaryocytes may be particularly prominent within dilated sinuses.31 New bone formation and osteosclerosis often develop. Bone marrow microvessel density is increased.51 Lymphoid aggregates are also frequent in bone marrow biopsies from patients with PMF.
Extramedullary Findings
Many of the abnormalities observed in the peripheral blood are due to release of cells from sites of extramedullary hematopoiesis into the circulation. Extramedullary hematopoiesis can occur in any organ but is most prominent in spleen and liver. The red pulp of the spleen is expanded by erythroid, granulocytic and megakaryocytic cells located primarily in the sinuses. Megakaryocytes are often the most prominent. Fibrosis of the red pulp cords may be present. The hepatic sinuses may also show extramedullary hematopoiesis; fibrosis and cirrhosis may also occur.52
Genetic Characteristics
Conventional cytogenetic analysis reveals clonal abnormalities in about 50% of patients with PMF. The most frequent abnormalities include del(20)(q11;q13), del(13)(q12;q22), trisomy 8, trisomy 9, del(12) (p11;p13), monosomy, or long-arm deletions of chromosome 7 and partial trisomy 1q. None of these abnormalities is specific for PMF. An adverse prognosis has been linked to the following abnormalities: 12p-, +8, and −7/7q-;45 der(6)t(1;6)(q21–23;p21–23) has been reported to be specific for PMF although it is an uncommon finding.53 There is no BCR-ABL1 fusion gene. Comparative genomic hybridization studies have documented gains of chromosome 9p as the most frequent genetic abnormality, occurring in about 50% of patients.54
The reported frequency of the JAK2 V617F mutation in PMF ranges from 35% to 57%. Homozygosity for this mutation occurs in 9–29% of cases and has been shown to be the result of mitotic recombination. No prognostic significance for the mutation has been found although the presence of the mutation appears to be associated with older age and history of thrombosis or pruritis.45 Mutations in myeloproliferative leukemia virus oncogene (MPL), receptor for thrombopoietin, have also been described in about 5% of patients with PMF. The clinical significance of this finding is under investigation.55
A recent study has also identified mutation of the CALR gene in a significant portion of patients without JAK2 or MPL mutations, and CALR mutations may be associated with a better prognosis.17
Disease Progression
The presence of 10–19% blasts in the blood or bone marrow signifies an accelerated phase and 20% or more is diagnostic of acute transformation with the WHO guidelines.42 An international working group defined acute transformation as 20% or 14more blasts in the bone marrow, or 20% or more blasts in the peripheral blood that persist for 8 weeks.56 Acute leukemia occurs in about 8–23% of patients with PMF and does not appear to be related to prior therapy. Most of the leukemic transformations have been myeloid with the most frequent subtypes being acute megakaryoblastic and acute myeloid with varying degrees of maturation. Clonal karyotypic abnormalities are present in most patients with acute leukemia and evidence for clonal evolution is common. Leukemic transformation is associated with a poor prognosis with the median survival of 2.6 months in one series of 91 patients.57
Differential Diagnosis
The prefibrotic phase of PMF can be very difficult to distinguish from ET since isolated thrombocytosis is commonly seen in prefibrotic PMF. Careful examination of the peripheral blood and bone marrow biopsy is critical in distinguishing between these two disorders. A leukoerythroblastic reaction or atypical platelet morphology favors a diagnosis of PMF. The megakaryocytes in PMF are often clustered and variably sized with atypical morphology, while the megakaryocytes in ET tend to be scattered or only loosely aggregated, large to giant, and manifest deeply lobulated nuclei and abundant mature cytoplasm. Left-shifted granulocytic hyperplasia is characteristic of prefibrotic PMF but is usually absent in ET. A significant increase in reticulin fibrosis argues against the diagnosis of ET.
The main disorders in the differential diagnosis of the fibrotic stage of PMF are those that cause prominent bone marrow fibrosis. Any of the myeloproliferative disorders can be associated with bone marrow fibrosis. For example, CML with prominent fibrosis and megakaryocyte proliferation can closely resemble PMF. One helpful histological feature that aids in this distinction is that the megakaryocytes in CML are smaller and lack the bizarre morphology characteristic of PMF. Documentation of the BCR-ABL1 fusion gene will establish the diagnosis of CML. The distinction between the PMF and the fibrotic phase of PV or ET can be very difficult, and requires clinical and hematological history. MF can also occur in myelodysplastic syndrome (MDS), but, in contrast to PMF, MDS with fibrosis typically exhibit prominent dysplasia in one or more cell lines. In addition, the megakaryocytes lack the pleomorphism of those in PMF; they are often small with monolobate nuclei.
Fibrosis can also be associated with acute leukemia. Acute panmyelosis with MF is a rare form of AML in which patients present with prominent constitutional symptoms, little or no splenomegaly and pancytopenia. Unlike PMF, red cell poikilocytosis is minimal and dysplastic changes in the neutrophils are frequent. The bone marrow is variably cellular with a panmyeloid proliferation and predominance of immature cells; blasts often exceed 20% of the cells. Distinction from PMF that has progressed to AML may not be possible.
Fibrosis also occurs in a wide variety of other neoplastic and nonneoplastic disorders that must be considered in the differential diagnosis of PMF including lymphoproliferative disorders, such as hairy cell leukemia, metastatic tumor, inflammatory processes or toxic processes, autoimmune disorders,58 hyper parathyroidism, and osteopetrosis.
ESSENTIAL THROMBOCYTHEMIA
Essential thrombocythemia is a chronic MPN characterized by persistent thrombocytosis and a proliferation of enlarged, mature megakaryocytes. Reactive causes of thrombocytosis and other myeloproliferative disorders or MDS associated with thrombocytosis must be excluded in order to establish a diagnosis of ET. About 50% of patients with ET exhibit JAK2 V617F. The modified WHO diagnostic criteria for ET are shown in box 1-4.26,59
Clinical Features
Epidemiologic studies of ET suggest an annual incidence rate of 2.5 cases per 100,000 population per year60 with a slight female preponderance.61 The etiology of ET is unknown.
The mean age at diagnosis is about 55 years.62 ET is extremely rare in children.63 About one half of patients may be asymptomatic at presentation. However, complications related to microvascular disturbances, such as headaches, lightheadedness, erythromelalgia, and acral paresthesias are relatively common, reported in about 35% of patients.61,62
Figure 1-6: Essential thrombocythemia, peripheral blood and bone marrow. A, The blood smear exhibits increased platelets with platelet clumping and normal platelet morphology. B, The bone marrow has increased megakaryocytes including giant forms with hyperlobulated nuclei
Thrombosis and hemorrhage are also common complications of ET. Major thrombosis, primarily arterial, is present in 11–25% of patients at diagnosis and occurs in 11–22% during the course of the disease. Thrombosis accounts for 13–27% of deaths in ET.64 Bleeding manifestations also occur with major hemorrhage occurring in 2–5% of patients at diagnosis and 1–7% during later follow-up.64 The risk of hemorrhage is significantly associated with extreme thrombocytosis (platelet count >1 million).64 First-trimester miscarriages occur in 30–40% of pregnant women with ET.65 Modest splenomegaly is present in about 35% of patients at diagnosis.66
Laboratory Findings
The platelet count is elevated within a wide range but according to the modified WHO criteria must be greater than 450 × 109/L.26 Extremely elevated platelets exceeding 1,000 × 109/L may be seen.67 The Hb and hematocrit are within reference ranges and below the levels defined for the diagnosis of PV.26 The leukocyte count is usually normal but may be mildly elevated.68 Serum thrombopoietin levels are normal or elevated, but this measure is not widely available.69
Microscopic Findings
The peripheral blood smear shows an increased number of platelets and platelet clumps may be present (Figure 1-6A). Occasional large platelets may be observed but atypical or dysplastic platelets are not present. The erythrocytes are normochromic, normocytic. The leukocytes are morphologically unremarkable with no dysplastic features and leukoerythroblastosis is typically absent.
The cardinal feature of the bone marrow is the presence of increased megakaryocytes in a normocellular to moderately hypercellular bone marrow. 17The megakaryocytes are usually dispersed through the bone marrow but may be occasionally found in loose clusters. The megakaryocytes are characteristically large and may be giant (Figure 1-6B), especially in contrast to those seen in CML. They exhibit abundant, mature cytoplasm and deeply lobulated nuclei. Emperipolesis of other bone marrow cells in the megakaryocyte cytoplasm may be present. Grossly atypical or dysplastic megakaryocytes are absent. Granulocytic proliferation is not significantly elevated or left-shifted. Erythroid hyperplasia may be observed if the patient has recently experienced hemorrhage. Reticulin fibrosis is usually absent.31,70 Storage iron is adequate or, if it is low, a trial of iron therapy fails to increase the Hb levels or red cell mass into the polycythemic range.68
Genetic Characteristics
Conventional cytogenetic analysis reveals clonal abnormalities in only about 5% of patients with ET; none are diagnostic. Structural abnormalities include deletions of the long arm of chromosomes 5, 7, 13, 17, and 20. Application of FISH may disclose additional abnormalities of chromosomes 8 and 9 not detected by routine cytogenetic analysis.62
About 50% of patients with ET possess the JAK2 V617F mutation.71 The presence of this mutation in ET has been associated with more advanced age at diagnosis, higher leukocyte counts and Hb levels, and higher frequency of thrombotic events than in patients without the mutation.62,72,73 Mutations of MPL have been reported in less than 5% of patients with ET.55 As in primary myelofibrosis, mutation of the CALR gene is present in a significant portion of patients without JAK2 or MPL mutations.17
The risk of progression of ET to a fibrotic bone marrow picture in one study occurred with a 10-year risk of 0.8%, and at 15 years 9.3%.74 Progression to AML in patients with ET is unusual in the first 10 years with the risk in one study at 0.7%. The risk at 15 years increased to 2.1%.74 The progression rates of ET to fibrosis or acute leukemia are significantly lower than those in early/prefibrotic PMF, emphasizing the necessity of accurate diagnosis.74
Differential Diagnosis
The first condition that must be ruled out before a diagnosis of ET is established is a reactive thrombocytosis.62 Numerous conditions can result in a reactive thrombocytosis including iron deficiency anemia, asplenia, trauma, bleeding, hemolysis and a variety of inflammatory processes.67 However, the presence of such a condition does not rule out ET if other criteria for the diagnosis are met. The identification of an acquired JAK2 mutation or a clonal cytogenetic abnormality effectively rules out a reactive thrombocytosis. A bone marrow biopsy showing the characteristically increased large, hyperlobulated megakaryocytes also supports a diagnosis of ET.
Identification of the BCR-ABL1 translocation establishes the diagnosis of CML and rules out ET. This is of particular importance in those patients who present with isolated thrombocytosis but lack other peripheral blood findings typical for CML. The presence of small megakaryocytes that are characteristic of CML may be a morphologic clue in the bone marrow aspirate and biopsy to the diagnosis of CML.
The main criteria distinguishing PV from ET is the presence of an elevated Hb/hematocrit or red cell mass in PV, and a bone marrow showing panhyperplasia with prominent erythroid and megakaryocytic proliferation. Some patients with PV may present with iron deficiency and decreased or normal Hb levels. In these cases, the blood and bone marrow may closely resemble ET, but the diagnosis of ET should not be made unless iron replacement therapy fails to increase the Hb to a polycythemic range.
The prefibrotic phase of PMF may be very difficult to distinguish from ET. Prefibrotic PMF differs from ET in that the bone marrow is characterized by both a granulocytic and megakaryocytic hyperplasia with morphologically abnormal megakaryocytes. In addition, the blood in prefibrotic PMF may exhibit a leukoerythroblastic reaction or morphologically abnormal platelets.
Therapy with growth factor may produce pronounced megakaryocytic hyperplasia and thrombocytosis that may resemble a myeloproliferative disorder.75
Most MDS are associated with thrombocytopenia although occasionally, such as in the 5q-syndrome a thrombocytosis may be observed. The presence of dysplastic morphology in one or more cell lines supports the diagnosis of a MDS rather than ET.
Familial ET is a rare disorder characterized by the excessive production of platelets. It is a heterogeneous disorder with some cases reported to have mutations in the thrombopoietin gene or in the gene for c-Mpl (the thrombopoietin receptor).76 A careful history is crucial in identifying these patients.
CHRONIC NEUTROPHILIC LEUKEMIA
Chronic neutrophilic leukemia (CNL) is a rare chronic MPN characterized by a persistent mature neutrophilic leukocytosis and a hypercellular bone marrow. Reactive causes of a neutrophilia must be excluded before the diagnosis is made. BCR-ABL1 positive CML must also be ruled out. CNL achieved formal recognition with the 2001 WHO classification; the WHO diagnostic criteria are listed in box 1-5.77
Clinical Features
Chronic neutrophilic leukemia is rare with less than 50 well documented cases published in the literature.78 The etiology is unknown. The median age at diagnosis is 65.5 years (range 15–86) with approximately equal numbers of females and males. Many patients are asymptomatic at diagnosis. Fatigue is the most common presenting symptom; other symptoms that may be present include weight loss, easy bruising, bone pain, and night sweats.
Splenomegaly is usually present at diagnosis while lymphadenopathy and hepatomegaly are unusual.78,79
Laboratory Findings
The leukocyte count is elevated with a mean leukocyte count of about 50 × 109/L (range 10–172 × 109/L).78,80 Mild anemia is often present. The platelet count is frequently normal; it may be deceased but thrombocytopenia, when present, is usually not severe.78,79
Serum vitamin B12 levels are elevated and neutrophil alkaline phosphatase (NAP) scores are usually elevated.79
Microscopic Findings
The blood smear shows a prominent neutrophilic leukocytosis. The majority of the neutrophils are mature; immature neutrophils may be observed but are usually 20less than 5% of the leukocytes. Blasts are usually absent. The neutrophils frequently exhibit pronounced toxic granulation and Döhle bodies. Dysplasia is absent. Monocytes, basophils, and eosinophils are not increased, and red cell and platelet morphology is normal.78
The bone marrow is hypercellular due to marked granulocytic proliferation. Granulocyte maturation appears normal with the majority of neutrophils at the myelocyte to segmented neutrophil stage. Blasts are not increased. Dysplasia is not present. Reticulin fibrosis is absent or only mild.78–81
Genetic Characteristics
Cytogenetic abnormalities are present in about 20% of patients at diagnosis and may also develop in a subset of patients during follow-up. The most common aberrations include 20q-, 21+, and 11q-.78 By definition, no patient has a BCR-ABL1 fusion. The JAK2 mutation is uncommon in CNL, reported in only one of six patients in one study.82
Differential Diagnosis
Reactive causes of neutrophilia must be excluded before a diagnosis of CNL is made. A prominent neutrophilia can also be seen in association with epithelial malignancies and may be secondary to tumor production of cytokines.78 Therefore, a search for an occult malignancy is warranted before a diagnosis of CNL is made. A disproportionate number of CNL have been reported in association with plasma cell myeloma. The neutrophilia in these cases is likely reactive, and are, also, possibly cytokine driven. Evidence to support this hypothesis includes reports of remissions of the CNL after treatment of the plasma cell disorder and documentation of polyclonal neutrophils in some cases.78 The WHO recommends that the bone marrow of all cases of CNL be examined for a plasma cell dyscrasia and, if present, clonality of the neutrophil proliferation supported by cytogenetic or molecular studies before a diagnosis of CNL is made.81 The diagnosis requires the absence of features characteristic of other MPNs. The rare cases of CML that are characterized by a predominance of mature and segmented neutrophils but that exhibit a BCR-ABL1 fusion gene should be regarded as CML rather than CNL.
Treatment and Prognosis
Optimal therapy for CNL has not been established. Therapy to date has mostly consisted of cytoreductive agents, such a hydroxyurea with about 75% of patients having a decrease in leukocyte count and/or splenomegaly. Successful use of α-interferon has been reported. Allogeneic stem cell transplant has also been reported. Disease progression is marked by refractoriness to therapy, marked splenomegaly, anemia and thrombocytopenia, and the appearance of immature cells in the peripheral blood. Transformation to AML can occur. The median survival 21of patients with CNL is reported as 23.5 months (range 1–106). The most common causes of death include intracranial hemorrhage and progressive disease.78,83
CHRONIC EOSINOPHILIC LEUKEMIA, NOT OTHERWISE SPECIFIED
Hypereosinophilia (≥1.5 × 109/L) can be divided into three categories: (1) reactive, (2) clonal, and (3) idiopathic. In reactive eosinophilia, the eosinophilia is a response to an underlying disorder, such as an allergic condition or a tissue invasive parasitic infection. The clonal eosinophilias are myeloid malignancies in which eosinophils are part of the neoplastic clone. Examples include AML with inv(16), BCR-ABL1 positive CML, disorders associated with JAK2 mutations or other genetic abnormalities, and the separate myeloid neoplasm categories of eosinophilia with PDGFR or FGFR1 rearrangements. In idiopathic eosinophilia, the reason for the eosinophilia is unknown despite a thorough investigation.84–86
Chronic eosinophilic leukemia, not otherwise specified (CEL-NOS) is a clonal eosinophilia in which there is an autonomous, neoplastic proliferation of eosinophils leading to a persistent eosinophilia in blood (≥1.5 × 109/L), bone marrow, and other organs (Box 1-6). This diagnosis requires not only the exclusion of reactive eosinophilias, but also the exclusion of other hematological neoplasms in which the eosinophils are part of the clone (including those with PDGFR or FGFR1 rearrangements). Abnormal T-cell populations with eosinophilia secondary to cytokine production must also be excluded. Evidence documenting clonality in CEL include the identification of a clonal genetic abnormality or an increase in blasts in the blood (>2%) or bone marrow (5–19%).87 Advances in our understanding of the molecular pathophysiology of these disorders have shifted an increasing proportion of cases from CEL-NOS to other genetically defined eosinophilic disorders.
Clinical Features
Chronic eosinophilic leukemia-NOS can occur at any age but the peak incidence is in the fourth decade.87 In about 10% of patients, the eosinophilia is discovered incidentally in otherwise asymptomatic individuals. The most common signs and symptoms at presentation include weakness and fatigue, cough, dyspnea, myalgias or angioedema, rash or fever, and rhinitis.88 The organs most commonly infiltrated by eosinophils are heart, skin, central nervous system, lungs, spleen, liver, eyes, and gastrointestinal tract.88 Organ damage results from release of cytokines, enzymes, and others proteins into the tissues by the infiltrating eosinophils.
Laboratory Findings
Persistent eosinophilia is the primary feature of CEL (≥1.5 × 109/L). The leukocyte count is usually modestly elevated in the range of 20–30 × 109/L although higher counts can occur. The percentage of eosinophils is variable but levels of 30–70% are common. Anemia may be present. The platelet count is variable although thrombocytosis is reported to be more common than thrombocytopenia.88
Microscopic Findings
The most prominent feature in the peripheral blood is eosinophilia; the majority of the eosinophils are mature although occasional immature eosinophils including myelocytes and promyelocytes may be present. The eosinophils often exhibit some morphologic abnormalities, such as hypogranularity, nuclear hypo- or hypersegmentation, and cytoplasmic vacuolization. These changes can also be seen in reactive eosinophilia and cannot be used to confirm a diagnosis of CEL. A neutrophilia may also be present. Blasts are usually absent.
The bone marrow is hypercellular with an increase in eosinophils and their precursors. Eosinophil maturation is orderly in most cases with no increase in blasts. Charcot-Leyden crystals are often present. Erythroid precursors and megakaryocytes are usually normal appearing. Dysplasia in other cell lineages or increased blasts (<20%) may be present in occasional cases. Reticulin fibrosis is also common.87
Genetic Abnormalities
A variety of genetic aberrations have been reported in CEL-NOS although the mechanism for leukemogenesis in these patients is unknown. These abnormalities include those well recognized in other myeloid neoplasms, such as trisomy 8, monosomy 7, iso(17q), and del(20)(q11q12). Loss of the Y chromosome has been reported in several patients. Other abnormalities including trisomy 10, trisomy 15, 17q+, 15q-, trisomy 21, and complex chromosomal rearrangements have also been reported.89
Differential Diagnosis
Before making a diagnosis of CEL-NOS, secondary causes of eosinophilia must be ruled out. Worldwide, the most common underlying cause of secondary eosinophilia is helminthic infection; in Europe and North America, it is allergic/atopic disorders. Other reactive causes include medications, vasculitis, collagen vascular diseases, pulmonary disorders, such as Löeffler syndrome, neoplasms including carcinomas, renal allograft rejection, and many other disorders. In these cases, the eosinophils are produced in response to cytokines, such as interleukin-3, interleukin-5, and granulocyte/macrophage colony stimulating factor, usually produced by T-helper lymphocytes.84
Reactive eosinophilia can be seen in association with hematological neoplasms, such as Hodgkin lymphoma, non-Hodgkin lymphoma, especially T-cell lymphoma, and acute lymphoblastic leukemia (LBL). Abnormal populations of T-cells, often clinically occult, can also drive an eosinophilia through production of interleukin-5. These T-cells exhibit an aberrant immunophenotype, typically CD3-CD4+ or CD3+CD4-CD8-, and are often clonal by T-cell receptor gene rearrangement. Some of these patients eventually develop an overt T-cell lymphoma.90
The diagnosis of CEL should be made only if evidence for clonality is identified. However, some hematopoietic neoplasms are associated with clonal eosinophilia and these should be excluded before the diagnosis of CEL is made. These include acute leukemias associated with eosinophilia, such as AML with inv(16)(p12q22) or t(16;16)(p13;q22), myelodysplastic neoplasms, and other MPNs. The presence of BCR-ABL1 rules out a diagnosis of CEL. The presence of rearrangements of PDGFRA, PDGFRB, and FGFR1 also rule out the diagnosis of CEL-NOS.
Treatment and Prognosis
Some patients with CEL respond to prednisone alone or in combination with hydroxyurea. Responses to imatinib alone have been reported in some patients 2.5with eosinophilia without known genetic abnormalities; therefore, some advocate a trial of this agent. Allogeneic stem cell transplant can be considered for patients refractory to drug therapy.84,91
MASTOCYTOSIS
Mastocytosis is a MPN characterized by a clonal proliferation of mast cells with infiltration of one or more organs.92,93 Mastocytosis is heterogenous and broadly divided into cutaneous mastocytosis and systemic mastocytosis. In cutaneous mastocytosis, the disease is confined to the skin. Urticaria pigmentosa is the most common form of cutaneous mastocytosis. It occurs most frequently in children and may regress spontaneously. Systemic mastocytosis occurs in adults and is a persistent disease in which there is widespread organ involvement including bone marrow, lymph node, spleen, and liver.
Systemic mastocytosis generally occurs in older adults with a similar incidence in males and females. Systemic mastocytosis is divided into indolent systemic mastocytosis, systemic mastocytosis with associated clonal hematological non-mast cell lineage disease, aggressive mastocytosis, mast cell leukemia, mast cell sarcoma, and extracutaneous mastocytoma.92,94,95 When systemic mastocytosis is preceded by skin lesions, the diagnosis is generally readily established because of the high degree of clinical suspicion. In the absence of cutaneous lesions, the diagnosis may not be recognized because of the rarity of the disorder and the lack of familiarity with the characteristic pathologic findings. The bone marrow is the most common diagnostic biopsy specimen. Diagnosis of systemic mastocytosis as defined by the WHO is the presence of the major criteria and one minor criteria, or at least three minor criteria (Box 1-7).92
Clinical Features
A wide variety of symptoms are associated with mastocytosis. They include constitutional symptoms, skin manifestations, mediator-related symptoms, and musculoskeletal symptoms. Constitutional symptoms, such as weakness, fatigue, fever, night sweats, and weight loss are common. Other symptoms frequently present are attributed to chemical mediators released by the mast cells and include generalized pruritis, urticaria, episodic flushing, headache, tachycardia, bronchospasm, nausea, vomiting, and diarrhea. Patients with aggressive disease are less likely to have skin lesions and more likely to have hepatomegaly, splenomegaly, prominent skeletal lesions and/or fractures, malabsorption, anemia, and/or thrombocytopenia.92
Laboratory Findings
Peripheral blood findings in systemic mastocytosis are variable. Eosinophilia is present in about one half of patients. Anemia, leukopenia, leukocytosis, and/or thrombocytopenia may be present. Circulating mast cells are rare. Serum tryptase levels may be elevated in systemic mastocytosis. In the absence of any other known clonal myeloid disorders, the presence of a persistently elevated tryptase over 20 ng/mL is a minor criterion for systemic mastocytosis.92 In addition, recent studies have demonstrated that the coexistent findings of increased serum β2-microglobulin levels and decreased LDH levels are closely associated with the presence of aggressive systemic mastocytosis.96,97
Microscopic Findings
The major criterion for diagnosis of systemic mastocytosis in the bone marrow is the presence of multifocal dense infiltrates of mast cells. The pattern of infiltration is either focal or diffuse. The focal lesions are paratrabecular, perivascular, or randomly distributed. They may be accompanied by a diffuse interstitial infiltrate surrounding the lesions or throughout the bone marrow. The paratrabecular lesions are sometimes associated with fibrosis and widening of the bony trabeculae. The perivascular lesions are usually associated with medial and adventitial hypertrophy of vessel walls. Dense, diffuse infiltration replacing large portions of bone marrow is rare. In most instances, the mast cell lesions in the bone marrow are polymorphous with an admixture of mast cells, lymphocytes, eosinophils, neutrophils, histiocytes, endothelial cells, and fibroblasts in varying proportions. Occasionally, focal lesions are characterized by a central core of lymphocytes surrounded by mast cells giving a “bull's eye” appearance to the lesions. Some mast cell lesions appear monomorphic, with the cellular population consisting almost entirely of mast cells. Mast cell morphology in bone marrow sections varies considerably from round with abundant cytoplasm to spindle-shaped. Atypical morphology (e.g., spindle shaped) is a minor criterion for diagnosis of systemic mastocytosis.92
Because of the fibrosis that is often associated with mast cell lesions, the bone marrow aspiration smears may not reflect the mast cell proliferation. However, mast cells are occasionally present in smears and may be normal appearing, large with abundant cytoplasm and scattered granules, or even spindle shaped.
Mast cells are difficult to identify in hematoxylin and eosin (H&E) stained sections. A particularly useful technique for detection of mast cells in sections is immunohistochemistry for tryptase. This technique is both sensitive and specific for mast cells. Mast cells are, also, positive for chloroacetate esterase but lack myeloperoxidase. Immunophenotyping is also an important aid in diagnosis of systemic mastocytosis. Mast cells express CD45, CD33, and CD117. They lack CD14, 26CD15, CD16, as well as B and T-cell markers. Aberrant expression of CD2 and/or CD25 is a minor criterion for diagnosis of systemic mastocytosis.92
Genetic Abnormalities
A point mutation in codon 816 of the c-kit proto-oncogene (usually D816V) is detected in most patients with systemic mastocytosis. This mutation results in activation of KIT (c-kit receptor tyrosine kinase) tyrosine kinase but patients are resistant to imatinib. Other mutations of exon 17 of KIT have also been identified.92 Clonal cytogenetic abnormalities have been identified in a significant proportion of patients with systemic mastocytosis. Most have been single abnormalities and include those seen in other myeloid malignancies, such as +8, 5q-, del(7), −7, +13, and del(20). In addition, recent studies have identified point mutations in multiple genes including ten-eleven-translocation 2 (TET2), DNA (cytosine-5)-methyltransferase 3 alpha (DNMT3A), additional sex combs-like protein 1 (ASXL1), and casitas B-cell lymphoma (CBL).95,98 Cytogenetic abnormalities characteristic of other myeloid neoplasias may be identified when the neoplasia is associated with a case of systemic mastocytosis.
Diagnostic Criteria
Studies subsequent to the publication of the 2008 WHO re-evaluated the WHO criteria used for diagnosis and classification of mastocytosis.96,97 These studies identified subcategories of mastocytosis in addition to those described in the WHO. These additional categories include well-differentiated systemic mastocytosis, indolent systemic mastocytosis with skin lesions, and indolent systemic mastocytosis without skin lesions. The studies also defined clonal mast cell activation disorders which do not fulfill the criteria for systemic mastocytosis.96,97 Additional diagnostic tests (serum β2-microglobulin and LDH) were proposed to further refine the diagnosis and classification of mastocytosis.96,97
MYELOPROLIFERATIVE NEOPLASM, UNCLASSIFIABLE
Some patients present with features of an MPN but cannot be readily classified into one of the defined diagnostic categories. In these instances, the designation, MPN, unclassifiable, can be used. Some cases may represent early myeloproliferative disorders lacking one or more of the diagnostic criteria. For example, some patients with abdominal vein thrombosis have been found to have a JAK2 V617F mutation even though they do not fit the criteria for either ET or PV. While some of these patients subsequently develop erythrocytosis and/or thrombosis, they represent unclassified MPN when first identified.26,99 Others may represent end stage MPN including those with prominent fibrosis in which the primary MPN cannot be discerned. Still others may represent cases that exhibit features of one or more diagnostic categories.100
MYELOID AND LYMPHOID NEOPLASMS WITH EOSINOPHILIA AND ABNORMALITIES OF PDGFRA, PDGFRB, OR FGFR1
Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR1 is a newly defined category of myeloid neoplasms in the 2008 WHO classification consisting of three disease categories all resulting from an aberrant tyrosine kinase encoded by a fusion gene.20 The presentation of all three disease entities may be as MPNs, but not invariably.20 Due to the pluripotent nature of the mutated cell of origin, the neoplasms may present as AML T-lymphoblastic lymphoma (T-LBL), or precursor B-lymphoblastic leukemia/lymphoma (pre B-ALL). Eosinophilia is a predominant feature in these disease entities, regardless of the nature of the disease presentation. Correctly diagnosing the entities in this category is critical, as the PDGFRA and PDGFRB neoplasms are responsive to imatinib.20,101–103
Myeloid and Lymphoid Neoplasms with PDGFRA Rearrangement
Myeloid and lymphoid neoplasms with PDGFRA rearrangement is defined as a MPN, AML, or LBL/lymphoma with prominent eosinophilia and presence of a FIP1L1-PDGFRA fusion gene or other variants with a PDGFRA rearrangement. BCR-ABL1 fusion gene must not be present for the diagnosis of this neoplasm.
Clinical Features
The presentation of PDGFRA neoplasms is heterogenous, but they typically present as CEL with occasional mast cell and neutrophil involvement. PDGFRA neoplasms can also present as AML or T-LBL.19 Patients often have a marked peripheral blood eosinophilia. Organ damage, such as cardiac or pulmonary damage can be present due to infiltration or the tissues by eosinophils and mast cells, and the subsequent cytokine release. Many patients have fatigue and/or pruritus and symptoms of the affected organs. It is far more common in men, and splenomegaly is common.20,104
Laboratory Findings
The most prominent laboratory finding in PDGFRA neoplasms is a markedly increased peripheral blood eosinophil count (typically ≥1.5 × 109/L). Serum tryptase is usually increased to more than 12 ng/mL and serum vitamin B12 is elevated.20
Microscopic Findings
Similar to the morphologic findings in CEL-NOS, the most prominent feature of the majority of myeloid PDGFRA neoplasms is a peripheral blood and bone marrow eosinophilia (Figures 1-7A and B). The eosinophils in the peripheral blood are typically mature, though occasional immature eosinophils can be found. Eosinophil abnormalities may be present including altered granulation and nuclear hypersegmentation or hyposegmentation.20
Figure 1-7: Myeloid neoplasm with PDGFRA rearrangement, peripheral blood and bone marrow. A, The blood smear exhibited an increase in eosinophils. They appear mature. B, The bone marrow biopsy shows a hypercellular bone marrow with a prominent eosinophilia.
Myeloid PDGFRA neoplasms may have mast cell infiltrates which can consist of subtle loose aggregates of mast cells, but most do not meet criteria for systemic mastocytosis.
Genetic Abnormalities
Platelet-derived growth factor receptor-alpha rearrangement usually occurs as a result of a small 800 kb interstitial deletion on chromosome 4q12 that results in fusion of the PDGFRA gene to the centromeric FIP1L1 gene. The protein product of this fusion gene is a constitutively activated tyrosine kinase that is inhibited by imatinib.10,20 The frequency of the FIP1L1-PDGFRA fusion gene in eosinophilic neoplasms is not known as reports have varied widely.18 The fusion is not apparent on conventional cytogenetic analysis but can be detected by real time-polymerase chain reaction (RT-PCR) or FISH. CHIC2 deletion, detected by FISH, is a surrogate assay for the FIP1L1-PDGFRA fusion gene.105 Analysis suggests that the fusion gene occurs in a pluripotent progenitor cell that may give rise to many cells including eosinophils and mast cells.106 Other fusion genes associated with similar eosinophilic disorders have also been described including BCR-PDGFRA, KIF5B-PDGFRA, and CDK5RAP2-PDGFRA. 84
Myeloid Neoplasms with PDGFRB Rearrangement
Myeloid neoplasms with PDGFRB rearrangement are defined as myeloid neoplasms occurring with rearrangement of PDGFRB at 5q31~33. These neoplasms typically present similar to chronic myelomonocytic leukemia (CMML) with eosinophilia, but can also present similar to other types of myeloid neoplasms including atypical CML, CEL, and AML.
Clinical Features
The myeloid PDGFRB neoplasms are more common in men than in women. Though the PDGFRB neoplasms can present as various types of myeloid neoplasms, the 29usual presentation is CMML. Eosinophilia is frequently found, but not always. As with the PDGFRA neoplasms, splenomegaly is common, and organ damage occurs from infiltration by eosinophils.20
Laboratory Findings
Peripheral blood eosinophilia is frequently found and serum tryptase may be elevated. Giving the heterogeneity of the presentation of the myeloid PDGFRB neoplasms, there are few definitive laboratory findings and the diagnosis is based on molecular abnormalities.20
Microscopic Findings
The morphology of myeloid PDGFRB neoplasms is heterogenous and is dependent on the presentation. In the peripheral blood there is typically a leukocytosis with increased neutrophils, eosinophils, monocytes and immature myeloid cells.20,107 The bone marrow is hypercellular and there may be an increase of mast cells.107
Genetic Abnormalities
The majority of patients with myeloid neoplasms with PDGFRB harbor the t(5;12) (q33;p13); this translocation results in fusion of the tyrosine kinase encoding region of PDGRFB on chromosome 5q33 to ETV6 on chromosome 12p13. Other rearrangements involving the PDGRFB gene have also been documented.18,91 These cytogenetic aberrations produce a MPN that is typically associated with prominent eosinophilia and sometimes monocytosis.20 Patients with PDGRFB rearrangements respond to imatinib, making it especially important to recognize patients with this abnormality.84
Myeloid and Lymphoid Neoplasms with FGFR1 Abnormalities
In the myeloid and lymphoid neoplasms with FGFR1 abnormalities, a mutation in a pluripotent stem cell results in a MPN featuring eosinophilia or lymphoblastic lymphoma, usually precursor T-cell type. The disease morphology is heterogenous, but around 90% of patients with this disease will have eosinophilia. This disorder frequently progresses to AML 1–2 years after diagnosis and is associated with poor prognosis. Also known as 8p11 myeloproliferative syndrome, multiple translocations with a chromosomal 8p11 breakpoint can give rise to the FGFR1 fusion genes. FGFR1 can have multiple fusion partners, all giving rise to the aberrantly activated tyrosine kinase.19 This neoplasm is not responsive to any imatinib or other current therapies, and the outcome is poor. In vitro studies suggest the FGFR1 inhibitor ponatinib may have efficacy in this neoplasm, but clinical trials have not been completed to test the effects on patients.108
Clinical Features
The clinical findings are heterogeneous in neoplasms with FGFR1 abnormalities. Some patients present with lymphoma and some patients present with splenomegaly 30as a result of myeloproliferation. Given the systemic nature of the neoplasms, systemic symptoms like fever, and weight loss may be present.20,104
Laboratory Findings
Peripheral blood eosinophilia is commonly found in neoplasms with FGFR1 abnormalities. Other laboratory findings are widely variable and are dependent on the type of presentation of the neoplasm.
Microscopic Findings
There are also widely variable findings in the morphology in the presentation of neoplasms with FGFR1 abnormalities.20,109 The most common feature is eosinophilia, occurring in about 90%.20 Patients presenting in both chronic phase and acute leukemic or lymphoblastic phases can have eosinophilia.20,104 Patients presenting with a lymphoma may have eosinophilic infiltration in the lymphoma.20 In the chronic form of the neoplasm, neutrophils and occasionally monocytes are also elevated in the peripheral blood. Lymphoblasts and myeloblasts will be present in the peripheral blood or bone marrow in the acute presentation of the neoplasm.20
Genetic Abnormalities
The most common translocation is t(8;13)(p11;q12). In this translocation, the FGFR1 is fused to the ZNF198 gene on chromosome 13q12 which results aberrant FGFR1 tyrosine activity.18 Other translocations include t(8;9)(p11;q33), t(6;8)(q27;p11), and t(q8;22)(p11;q22).91
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