How I investigate Eosinophilia
Abstract
Eosinophilia is typically secondary, that is, reactive, in nature and is associated with a wide variety of neoplastic and non-neoplastic disorders. Clonal eosinophilia is also seen in a wide variety of hematopoietic neoplasms, and sub-classification can be diagnostically challenging. A proper evaluation of persistent eosinophilia involves correlation of clinical history, laboratory data, cellular morphology, and ancillary testing. Knowledge of appropriate ancillary testing is necessary for a timely diagnosis. We present a review of the literature regarding eosinophilia, including the 2016 World Health Organization (WHO) update of WHO-defined eosinophilic disorders. We also present a review of eosinophilia in a case-based format including guidelines for evaluation of both routine and challenging cases. The purpose of this guideline is not to provide an in-depth discussion of each diagnosis, but rather a practical method that all pathologists can utilize to investigate eosinophilia.
1 INTRODUCTION
Eosinophils are terminally differentiated granulocytes that are produced in the bone marrow via a collection of transcription factors and cytokines including interleukin 5 (IL-5), interleukin 3 (IL-3), and granulocyte macrophage colony stimulating factor (GM-CSF).1-5 Maturation occurs in the bone marrow over a five-day period, and mature cells are released into circulation. Eosinophils circulate for approximately 18-24 hours prior to migrating into lymphoid organs and the mucosa of the gastrointestinal tract and other tissues/organs such as the uterus.1-4 Eosinophils produce and store more than 30 cytokines, chemokines, and growth factors; therefore, eosinophils are vital to the body's immune system response to infectious, immunologic, and inflammatory processes.2, 3 Eosinophilia presents in both reactive and clonal disorders, and when dysregulated, it has the potential to result in abnormal organ infiltration of the skin, heart, and lung, which can have life-threatening consequences.
2 BACKGROUND
Normal eosinophil percentage is between 1%-6% in the bone marrow and 3%-5% in the peripheral blood, equating to an absolute peripheral eosinophil count (AEC) of 0.35-0.5 × 109/L.1, 6 Eosinophilia is arbitrarily designated as mild (up to 1.5 × 109/L), moderate (1.5-5 × 109/L), and severe (>5 × 109/L).5 Workup of eosinophilia to exclude a clonal process would be warranted in patients with persistent, unexplained eosinophilia. As always, thorough review of the patient's clinical history including imaging and previously performed laboratory tests followed by morphologic evaluation should be an astute pathologist's first steps.
The WHO defines idiopathic hypereosinophilia (IHE) as eosinophilia (eosinophil count ≥1.5 × 109/L) persisting for ≥6 months for which no underlying cause can be found.7-9 When this persistent, unexplained eosinophilia is associated with signs of organ involvement and dysfunction, a diagnosis of idiopathic hypereosinophilic syndrome (IHES) is rendered. However, given modern advances in diagnosis/detection of clonal eosinophilia including identification of certain cytogenetic events, the 6-month watch-and-wait period is not as uniformly accepted.
Normal eosinophils are round to oval, 10 to 15 μm in diameter, and have a nuclear:cytoplasmic ratio of 1:3; they are identified via their characteristic refractile, coarse, orange-red granules, which are typically uniform in size and generally evenly fill the cytoplasm.10 Eosinophils exhibit the same stages of development as neutrophils. In the most mature eosinophil form, the nucleus segments into two or more lobes connected by thin filaments with approximately 80% of segmented eosinophils containing a two-lobed nucleus with lobes of equal size and ovoid shape with dense chromatin (Figure 1). The remainder of segmented eosinophils will typically have three lobes, and occasionally, an eosinophil can have up to four or five lobes.7 Immature eosinophils are rarely seen in the blood, but can be seen in bone marrow smears, and may have fewer granules than the more mature forms. The eosinophilic myelocyte is the earliest recognizable eosinophilic form on light microscopy.10 Eosinophilic myelocytes typically contain orange-red secondary granules with rare primary granules. Sometimes irregular eosinophilic cytoplasmic granulation or abnormal nuclear lobation can alert one to a clonal eosinophilic abnormality or neoplastic process.6 For example, eosinophils can present with atypical/basophilic granules at any stage of maturation, but this is most often seen at the myelocyte stage. The abnormal granules resemble basophilic granules, but lack myeloperoxidase and toluidine blue reactivity. These cells are referred to as harlequin cells and are associated with clonal myeloid disorders, typically a specific type of acute myeloid leukemia (AML).10
To evaluate for a clonal or malignant eosinophilic process, flow cytometric analysis can be performed, although not traditionally utilized to evaluate eosinophilia in most clinical laboratories. Most eosinophils express moderate to bright CD45 and are positive for CD33 and CD13, similar to neutrophils. They also express CD11b, CD11c, and are dim to negative for CD38. They are negative for CD14, CD16, CD56, CD64, HLA-DR, CD34, and CD117.11 An abnormal immunophenotype alone is not specific to a clonal or malignant population, nor is it sufficient for diagnosis. Flow cytometry can also be utilized in evaluation of lymphocytic-variant hypereosinophilia (L-HES). In this entity, a clonal/aberrant T-cell population results in a reactive eosinophilia. The abnormal T-cell population can have a broad range of aberrancies including CD3-CD4+, CD3+CD4-CD8-, and CD3+CD4+CD7-. Few cases also show T-cell receptor gene rearrangements, but the phenotypically abnormal population detected via flow cytometric immunophenotyping is required for diagnosis.5
Immunohistochemistry (IHC) can aid in blast enumeration as well as additional phenotyping of populations. Immunohistochemistry for CD117 (c-KIT) is particularly important in cases of eosinophilia as a sensitive method to exclude the presence of mast cells aggregates, which may be difficult to identify on hematoxylin and eosin stain alone. Systemic mastocytosis (SM) can present with eosinophilia; therefore, evaluation for clinicopathologic features typical of SM may be warranted (ie, WHO defined major criterion of multifocal dense aggregates of ≥15 mast cells; minor criteria of elevated serum tryptase level (>20 ng/mL), expression of CD25 with or without CD2 on mast cells, activating point mutation at codon 816 of KIT, and>25% of total mast cells with atypical morphology such as spindling).12 Due to the clinicopathologic overlap, utilizing IHC to exclude SM may be necessary during the diagnostic workup of a patient with persistent eosinophilia.
Although ancillary testing as described briefly above is useful in evaluation of eosinophilia, in general, eosinophilia is a nonspecific finding, usually reactive in nature, and may be secondary to a variety of causes such as skin rash, allergies, asthma, and medication effect, as well as certain infections (parasitic/fungal). However, persistent unexplained eosinophilia should raise concern for a possible underlying hematopoietic neoplasm.13 A broad differential can be entertained initially, and initial workup should include thorough screening for secondary causes of eosinophilia. If a reactive process is identified, treatment of the underlying cause should be attempted with appropriate follow-up to verify resolution of the eosinophilia. If no secondary causes of eosinophilia can be identified, evaluation of peripheral blood and bone marrow biopsy including histomorphology, immunohistochemistry, cytogenetics (karyotype/FISH), and appropriate molecular studies should be performed. Due to the life-threatening organ damage that can occur due to eosinophil infiltration, physical examination including multiple imaging modalities, such as chest X-ray, ultrasound, and computed tomography (CT) scan/magnetic resonance imaging (MRI), should also be performed as needed.13
3 CASE SCENARIOS
The following sections include case-based presentations of reactive, clonal, and neoplastic eosinophilia. The cases will highlight key parameters that are important for narrowing the initially broad differential diagnosis. An in-depth discussion of each diagnosis is beyond the scope of this review, but a discussion of the most pertinent items necessary for diagnosis as well as focusing the differential is included.
3.1 Reactive eosinophilia
A 73-year-old man with a past medical history of recently diagnosed prostate cancer presented with persistent peripheral eosinophilia for the last 3 months. A detailed medical history was obtained and was negative for allergic disorders, skin rashes, or lymphadenopathy. Constitutional symptoms were also absent including night sweats, recent weight loss, and pruritus. The patient also denied recent travel. There were no abnormalities noted on physical exam, and a bone scan revealed no metastatic lesions. An extensive laboratory panel to evaluate for allergic, infectious, and autoimmune causes revealed no abnormality. A peripheral blood smear was evaluated and revealed an absolute eosinophilia of 1.7 × 109/L without other significant findings including no circulating blasts, atypical lymphoid cells, or dysplastic changes. The eosinophils demonstrated normal morphology, and a bone marrow biopsy was performed. Eosinophils constituted 11.3% of the bone marrow aspirate cellularity without aggregation or atypia. There was background maturing trilineage hematopoiesis without significant dysplasia or increase in blasts. Flow cytometry was performed on the aspirate and revealed no atypical immunophenotype with polytypic mature B-cells and unremarkable T and NK-cells without increased blasts. Karyotyping revealed a normal male karyotype, and myelodysplastic syndrome (MDS) Fluorescence in Situ Hybridization (FISH) panel was negative. FISH also did not detect an abnormality of platelet-derived growth factor alpha (PDGFRA). The value of clinical history and thorough ancillary test utilization to exclude primary causes of eosinophilia are emphasized in this case.
Eosinophilia is secondary (reactive) in most cases, and a variety of non-neoplastic causes have been described including allergic disorders such as asthma or atopic dermatitis, dermatologic disorders, drug-effect, parasitic/fungal infections, gastrointestinal disorders, vasculitides, and respiratory disease (Table 1).13 Underlying neoplastic disorders, both hematologic and non-hematologic including prostate cancer, can elicit eosinophilia; therefore, a thorough workup and close clinical follow-up are recommended in these cases.14 If secondary causes of eosinophilia are excluded or certainty regarding the reactive nature of the eosinophilia is not achieved, then evaluation for a primary eosinophilic disorder should be performed.5 This is especially critical as certain neoplasms with clonal eosinophilia can be treated early and effectively to prevent end-organ damage.
| Infection—tissue-invasive parasites and fungi12, 13 |
| Allergic disorders—asthma, atopic dermatitis/eczema, seasonal allergic disorders12, 13 |
| Pulmonary disease—Loffler syndrome and sarcoidosis12, 13 |
| Dermatological disorders—Wells syndrome, angiolymphoid hyperplasia12, 13 |
| Drugs—including antibiotics and anticonvulsants12 |
| Collagen vascular disorders13 |
| Kimura disease13 |
| Gastrointestinal disorders—primary gastrointestinal eosinophilic disorders including eosinophilic esophagitis and chronic pancreatitis12 |
| Eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome)12 |
| Rheumatological disease—systemic lupus erythematosus, rheumatoid arthritis, and eosinophilic fasciitis (Shulman disease)12 |
| Neoplasms (non-hematologic and hematologic)—T-cell lymphomas, Hodgkin lymphoma, systemic mastocytosis,27 solid tumors14 |
| Atheroembolic disease12 |
3.2 Lymphocytic-variant hypereosinophilic syndrome
A 58-year-old man with no significant past medical history was directly admitted with complaints of fever and skin rash. His skin rash began four years prior and has been waxing and waning and pruritic involving his bilateral extremities. He also has a history of lymphadenopathy. No recent travel was noted. Physical exam revealed bilateral axillary lymphadenopathy and the scaly rash. A CT scan also revealed the bilateral axillary lymphadenopathy. A peripheral blood smear was evaluated and revealed leukocytosis with an absolute eosinophilia of 4.8 × 109/L without an increase in blasts, morphologically atypical lymphoid cells, or dysplastic changes. Bone marrow biopsy revealed maturing trilineage hematopoiesis with increased eosinophils without significant dysplasia or increase in blasts. Flow cytometry on the aspirate revealed a small CD3 and CD4 positive T-cell population with aberrant loss of CD7. Karyotyping revealed a normal male karyotype, and MDS FISH panel was negative. FISH also did not detect an abnormality of PDGFRA. Skin biopsy revealed fibrosis of papillary dermis, psoriasiform changes, and hyperkeratosis with mild perivascular chronic inflammation with no epidermotropism. An excisional lymph node biopsy was also performed, which revealed dermatopathic lymphadenitis.
Based on the patient's clinical presentation, exclusion of all other causes of eosinophilia, and the immunophenotypically aberrant T-cell population, he was determined to have L-HES. This case illustrates the importance of flow cytometry and ancillary testing in the evaluation of eosinophilia. L-HES often presents with cutaneous manifestations and is a clonal expansion of phenotypically aberrant T-cells with a subsequent secondary eosinophilia due to the cytokines produced by the aberrant T-cells. Patients can present with end-organ damage including cardiac and neurologic involvement. This is a diagnosis of exclusion requiring elimination of all other possible entities including myeloid and lymphoid neoplasms associated with eosinophilia. Although there is not a current consensus on diagnostic criteria, the diagnosis is made via flow cytometry.5, 13 An abnormal T-cell population with a broad range of aberrancies including CD3-CD4+, CD3+CD4-CD8-, and CD3+CD4+CD7- can be seen.13 Few cases also show T-cell receptor gene rearrangements. Close follow-up of these patients is warranted as development of overt T-cell neoplasms is well described.
3.3 Chronic eosinophilic leukemia, not otherwise specified
A 74-year-old woman with a past medical history of allergic rhinitis and a previous transient ischemic attack presented with flu-like symptoms that began 3 months prior including fever, weakness, fatigue, night sweats, and early satiety. She denied recent travel. Physical exam revealed palpable splenomegaly without lymphadenopathy. Cardiac workup revealed a restrictive cardiomegaly and mitral valve regurgitation. A peripheral smear was evaluated and revealed a leukoerythroblastic smear with leukocytosis. Neutrophilia, including a left shift to few blasts (7%), and eosinophilia, 16.4 × 109/L, with atypical and immature forms were noted. Bone marrow biopsy demonstrated a hypercellular marrow with granulocyte hyperplasia including numerous eosinophils. Flow cytometry performed on the aspirate revealed a myeloid predominant marrow with increased basophils. Blasts were not significantly increased (3%). Karyotyping revealed a gain of chromosome 8 without t(9;22). FISH did not detect an abnormality of PDGFRA/PDGFRB/FGFR1. The diagnosis of chronic eosinophilic leukemia, not otherwise specified (CEL, NOS) was made. This case illustrates the importance of peripheral smear review, bone marrow morphologic examination, and cytogenetic/molecular studies in the evaluation of eosinophilia.
CEL, NOS is a myeloproliferative neoplasm (MPN) where the major proliferative component is a clonal population of eosinophil precursors. Absolute eosinophilia (≥ 1.5 × 109/L) in the peripheral blood and <20% blasts in the peripheral blood and bone marrow are required.12 WHO criteria for all other MPN must be excluded as well as failure to identify a rearrangement of PDGFRA, PDGRFB, or FGFR1, and no PCM1-JAK2 fusion. Diagnostic features of AML (including AML with inv(16)(p13.1q22)) must also be excluded. A clonal cytogenetic or molecular abnormality or increased blasts (≥2% of cells in the peripheral blood or ≥5% in the bone marrow) are also required for diagnosis.7 It is important to note that clonal molecular genetic abnormalities (ie, TET2, ASXL1, and DNMT3A mutations) seen in CEL, NOS can also be seen in a minority of elderly people in the absence of a hematologic malignancy; therefore, excluding all other causes of eosinophilia is required prior to making this diagnosis solely based on a molecular abnormality.7 It is necessary to exclude all other MPN that present with eosinophilia including chronic myeloid leukemia (CML). In many cases, it is difficult to prove clonality, and as long as there is no increase in blasts, the diagnosis of IHES is made when end-organ damage is present.7 A recent study demonstrated that a subset of IHES patients evaluated with targeted myeloid next-generation sequencing will demonstrate ASXL1, TET2, EZH2, and SETBP1 mutations. These IHES/next-generation sequencing positive patients had similar clinical features and bone marrow findings as CEL, NOS including an older age at presentation, anemia, dyserythropoiesis, dysgranulopoiesis, abnormal eosinophil morphology, and similar disease-free survival compared to the IHES/next-generation sequencing negative patients.15 Many neoplastic and non-neoplastic disorders can mimic CEL, NOS, and utilizing ancillary testing including the judicious use of FISH for cryptic aberrations and next-generation sequencing to narrow the differential is essential.
3.4 Myeloid neoplasm with abnormality of PDGFRA
A 48-year-old man with a past medical history of diabetes and hypertension presented with complaints of syncope, melena, and hematochezia for the past 2 months. He also had recent unintentional weight loss but denied recent travel. Previous laboratory testing has shown eosinophilia and a leukocytosis. Physical exam and CT scan revealed splenomegaly without lymphadenopathy. A peripheral smear revealed a myelophthisic smear, anemia, thrombocytopenia, and atypical eosinophilia, 6.1 × 109/L, including hypogranular forms. Bone marrow biopsy demonstrated a hypercellular marrow with marked eosinophilia, mild megakaryocytic atypia including pyknotic forms and forms with atypical nuclear to cytoplasmic ratios, and erythroid hypoplasia. There was no increase in blasts, but mild diffuse marrow fibrosis was present. Flow cytometry performed on the aspirate revealed no increase in blasts or other aberrancies. Karyotyping revealed a normal male karyotype. Acute myeloid leukemia/MDS FISH panel was normal, and FISH for t(9;22) was negative. Eosinophilia FISH panel revealed a CHIC2 deletion, but was negative for abnormalities of PDGFRB and FGFR1 (Figure 2). Next-generation sequencing panel revealed no abnormalities. The findings were diagnostic of a myeloid/lymphoid neoplasm with eosinophilia and abnormality PDGFRA. This case illustrates the importance FISH analysis in making certain diagnoses due to cryptic deletions as seen in the 4q12 deletion (CHIC2), which may be missed by conventional cytogenetic testing (karyotype).
“Myeloid and lymphoid neoplasms with eosinophilia and abnormalities of platelet-derived growth factor receptor α (PDGFRA), platelet-derived growth factor receptor β (PDGFRB), or fibroblast growth factor receptor 1 (FGFR)1” is an umbrella term, which was first officially recognized in the 2008 World Health Organization (WHO) Classification of Haematopoietic and Lymphoid Tissues.5, 16-18 In these entities, aberrant tyrosine kinase activity due to fusion abnormalities leads to uncontrolled eosinophil proliferation with eosinophils possibly demonstrating evidence of activation including expression of CD23, CD25, and/or CD69.16, 19 Mast cells may be increased and are frequently CD2 negative and CD25 positive, but can be positive for both or negative for both.16 However, cases usually do not meet WHO criteria for SM. In 2016, the WHO revised the major category to include the provisional entity myeloid/lymphoid neoplasms with PCM1-JAK2.5, 16, 17 Patient presentation with these entities is highly variable.5, 12, 16
Specifically, PDGFRA abnormalities commonly present with findings similar to CEL, NOS while PDGFRB abnormalities commonly present with findings similar to chronic myelomonocytic leukemia (CMML) with eosinophilia. More heterogeneous presentation is seen with abnormalities of FGFR1 and PCM1-JAK2 .5, 12, 16 In patients with abnormalities of PDGFRA, eosinophilic atypia may be present, but is variable and not required.5, 20, 21 Bone marrow evaluation typically shows a hypercellular marrow with granulocyte hyperplasia and eosinophilia often with accompanying fibrosis.16, 20, 21 In general, conventional karyotyping can identify abnormalities of PDGFRB, FGFR1, and PCM1-JAK2, but a cryptic 4q12 deletion (CHIC2) is usually seen with abnormalities of PDGFRA, requiring FISH analysis for diagnosis.5, 12, 16, 20, 21 Identifying patients with abnormalities of PDGFRA and PDGFRB is vital for treatment and patient prognosis. Specifically, treatment with imatinib results in an excellent response in these patients and can lead to complete hematologic recovery and long-term molecular remission.5, 12, 16, 20-23
4 OTHER NEOPLASMS ASSOCIATED WITH EOSINOPHILIA
4.1 Chronic myeloid leukemia, BCR-ABL1-positive
CML,BCR-ABL1-positive is a MPN where the major proliferative component is granulocytes and is characterized by the chromosomal translocation t(9;22)(q34.1;q11.2) that results in formation of the Philadelphia chromosome (BCR-ABL1 fusion gene).24, 25 Peripheral absolute basophilia and eosinophilia as well as granulocyte left shift are commonly seen.24 In addition to the aforementioned findings, in the marrow, small megakaryocytes with hypolobated nuclei (ie, “dwarf megakaryocytes”) are commonly found in association with megakaryocyte hyperplasia, myeloid hyperplasia, and myelocyte “bulge”.24, 26 It is necessary to identify the chromosomal translocation in this entity to exclude other MPN that present with eosinophilia.
4.2 Systemic mastocytosis
Systemic mastocytosis has well-defined consensus criteria for diagnosis, which include a major criterion and four minor criteria. The WHO defines the major criterion as “multifocal dense infiltrates of mast cells (≥15 mast cells in aggregate) detected in sections of bone marrow and/or other extracutaneous organ(s),” and the minor criteria as follows: (a) In biopsy sections of bone marrow or other extracutaneous organs, >25% of the mast cells in the infiltrate are spindle-shaped or have atypical morphology or >25% of all mast cells in bone marrow aspirate smears are immature or atypical, (b) Detection of an activating point mutation at codon 816 of KIT in the bone marrow, blood, or another extracutaneous organ, (c) Mast cells in bone marrow, blood, or another extracutaneous organ express CD25, with or without CD2, in addition to normal mast cell markers, and (d) Serum total tryptase is persistently >20 ng/mL, unless there is an associated myeloid neoplasm, in which case this parameter is not valid.”27 In order to make the diagnosis, the major criterion and at least one minor criterion must be present, or at least three of the minor criteria.27 SM and neoplasms associated with eosinophilia can present with similar clinicopathologic features.5, 12, 27 Due to the clinicopathologic overlap, utilizing IHC to include CD117 and/or mast cell tryptase to exclude SM via detection of dense aggregates of mast cells is necessary during the diagnostic workup of a patient with persistent eosinophilia. It is important to note that CD25 mast cell positivity can be seen in myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB, or FGFR1, or with PCM1-JAK2, but in general the mast cells are negative for CD2. The mast cells in SM, by comparison, are almost always CD25 positive and CD2 positive in two thirds of cases.16
4.3 Acute myeloid leukemia
Acute myeloid leukemia with inv(16)(p13.1q22) or t(16;16)(p13.1;q22);CBFB-MYH11 may also present with eosinophilia .5, 13, 28 Unlike most cases of AML, a blast count of 20% is not required to render this diagnosis.28 However, increased blasts with monocytic differentiation will usually be encountered. Unique to this entity are harlequin cells, which are eosinophils with unusual basophilic granules that are larger than those present in immature eosinophils, are purple-violet in color, and can be so dense that they obscure the cellular nuclear morphology .28 The CBFB-MYH11 cytogenetic aberration may be cryptic by karyotyping, necessitating FISH analysis for diagnosis .28, 29
4.4 Classic Hodgkin lymphoma
Classic Hodgkin lymphoma (CHL) is a B-cell neoplasm composed of mononuclear Hodgkin cells and classic Reed-Sternberg cells with a mixed background of reactive inflammatory cells including eosinophils.30 Classic Hodgkin lymphoma is typically associated with lymphadenopathy, and not infrequently presents with reactive, non-clonal peripheral eosinophilia. Moreover, if disease is present in the marrow, concordant fibrosis is common. This diagnosis can generally be differentiated from other causes of eosinophilia as Hodgkin cells have a unique morphology and immunophenotype, typically expressing CD30, CD15, and PAX5 (reduced).30 Utilizing IHC to diagnose CHL is necessary when a reactive eosinophilia is in the differential.
4.5 Chronic myelomonocytic leukemia
Chronic myelomonocytic leukemia has features of both a MPN and a MDS. It presents with an absolute monocytosis (≥1 × 109/L), but can be associated with peripheral eosinophilia as well.31, 32 In patients with CMML and prominent eosinophilia, abnormalities of PDGFRB must be excluded. This is especially critical given the sensitivity of this neoplasm to treatment with imatinib.
5 APPROACH TO EOSINOPHILIA
5.1 Initial steps to investigating eosinophilia
- A detailed medical history should be taken to include possible reactive causes of eosinophilia as well as a thorough review of systems including skin rashes, lymphadenopathy, cardiorespiratory symptoms, and gastrointestinal symptoms. Duration of the eosinophilia as well as associated B-symptoms including fever, night sweats, and unintentional weight loss should be included. A detailed travel and medication history are also important in excluding potential reactive causes.
- Complete physical exam to evaluate rash, lymphadenopathy, and organomegaly is crucial.
- Assessment of end-organ damage including chest radiography, echocardiogram, serum troponins, and oxygen saturation may be necessary.
- Laboratory evaluation to include complete blood count (CBC) with differential, complete metabolic panel, serum tryptase levels, erythrocyte sedimentation rate and/or C-reactive protein, and vitamin B12 may be needed. Moreover, comparison of prior CBC data, if available, is suggested to evaluate duration and degree of eosinophilia. Furthermore, specialized laboratory testing based on pertinent medical history may be needed such as IgE levels and/or allergy testing.
- Once you confirm eosinophilia with CBC and peripheral smear review, evaluation of other CBC parameters and peripheral blood morphologic review is crucial. Specifically, an isolated eosinophilia is more likely to be reactive. However, if cytopenias or other abnormalities are present such as basophilia, circulating blasts, and/or leukoerythroblastosis, further testing may be necessary. In smear review, particular attention should be paid to:
- o Eosinophil morphology: the presence of atypia including hypogranulation, atypical segmentation, mature eosinophils with atypical basophilic granules, and/or immature forms should raise concern for a neoplasm.
- o Granulocyte morphology: the finding of dysplasia such as abnormal nuclear segmentation or hypogranular cytoplasm should raise concern for a neoplasm.
- o Red cell morphology: identification of significant anisocytosis and poikilocytosis, including dacrocytes and/or circulating nucleated red blood cells, may require further evaluation.
- o Platelet morphology: abnormal changes in platelet number, granularity, and size may indicate further testing is needed.
- Next integration of the aforementioned clinical history including radiologic studies, pertinent laboratory data, and physical exam with any prior pathology and current peripheral blood morphologic findings is needed.
5.2 Next steps to take
If an immediate reactive cause of eosinophilia is not identified, or findings are concerning for a hematologic neoplasm, then further workup including bone marrow biopsy with ancillary testing to potentially include flow cytometry, immunohistochemistry, cytogenetic testing (including FISH for cryptic aberrations associated with eosinophilia), and potentially molecular testing, such as T-cell gene rearrangement studies, when an aberrant T-cell population is detected, or next-generation sequencing panel, when a myeloid neoplasm is suspected, should be performed (Figure 3).
5.3 Bone marrow biopsy
Morphologic evaluation of the bone marrow biopsy and aspirate are helpful in detecting hematologic malignancies, metastatic disease, and potentially infections. A careful blast count is needed as an increase in blasts is one of the diagnostic criteria for CEL, NOS. Moreover, aspirate can be utilized for the below ancillary testing as needed.
5.4 Flow cytometry
Blood and/or bone marrow evaluation by flow cytometry can assess blast populations as well as populations with aberrant immunophenotype. Increased blasts (but <20%) in the peripheral blood/bone marrow can be diagnostic of CEL, NOS in the appropriate setting.7 Identification of an abnormal T-cell population is necessary to make the diagnosis of L-HES.5, 7 Next evaluation for aberrant expression of CD25 and/or CD2 on mast cells may aid in the diagnosis of SM, being cognizant that CD25 positivity can be seen in mast cells of myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB, or FGFR1, or with PCM1-JAK2.
5.5 Immunohistochemistry
Immunohistochemistry can aid in blast enumeration as well as additional phenotyping of populations. Immunohistochemistry for CD117 and/or mast cell tryptase is particularly important in cases of eosinophilia as a sensitive method to exclude the presence of mast cells aggregates, which may be difficult to identify on hematoxylin and eosin stain alone.27 Immunohistochemistry can also aid in excluding other diagnoses associated with eosinophilia such as classic Hodgkin lymphoma. It is important to note that a subset of SM cases will express CD30, and stain interpretation must be correlated with clinical picture and overall cell morphology.27
5.6 Cytogenetics
Screening the peripheral blood or bone marrow aspirate for FIP1L1-PDGFRA by FISH or reverse transcriptase-polymerase chain reaction (RT-PCR), as well as conventional karyotyping analysis for reciprocal translocations involving 4q12 (PDGFRA), 5q31-q33 (PDGFRB), 8p11-12 (FGFR1), or 9p24 (JAK2) is necessary in initial evaluation of primary (ie, non-reactive) eosinophilia.5 Waiting 6 months to evaluate for persistence of eosinophilia may not be appropriate as this may delay treatment, resulting in potential end-organ damage. Although in general, conventional karyotyping can identify abnormalities of PDGFRB and FGFR1, if strong clinical suspicion persists, FISH analysis may be warranted as rare cryptic abnormalities have been reported. Moreover, AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22);CBFB-MYH11 may also be cryptic, necessitating FISH analysis for diagnosis.5, 12, 16, 20, 21, 28, 29 This is especially true when harlequin cells are seen or blasts with monocytic differentiation are appreciated. Finally, identifying an absence of other clonal abnormalities is also necessary to exclude potential CEL, NOS and confirm IHE/IHES.5, 7
5.7 Molecular testing
In patients with persistent unexplained eosinophilia without proven clonality via traditional karyotyping/FISH and without elevated blast count, molecular testing for certain aberrations via next-generation sequencing may be warranted. Specifically, TET2, ASXL1, and DNMT3A aberrations may be seen in patients with CEL, NOS. However, these aberrations in isolation are not diagnostic of a hematopoietic neoplasm, and clinicopathologic correlation is needed.
6 KEY PROBLEM AREAS
- Many reactive causes of eosinophilia exist, requiring a thorough history and laboratory workup to exclude secondary causes of eosinophilia, which are much more common than primary eosinophilia.
- Cryptic cytogenetic abnormalities including, but not limited to inv(16) and CHIC2 deletions, that cannot be identified through conventional karyotyping require FISH or RT-PCR analysis. A pathologist must be familiar with these cryptic aberrations and when to appropriately order FISH analysis.
- Lack of consensus criteria for the diagnosis of L-HES may result in diagnostic confusion and clinician uncertainty. Moreover, close follow-up of these patients is needed as they may develop overt lymphoma during follow-up necessitating more aggressive therapy.
- Clonal molecular genetic abnormalities (ie, TET2, ASXL1, and DNMT3A) seen in CEL, NOS can also be seen in a minority of elderly people in the absence of a hematologic malignancy and must be evaluated in the clinical context.
7 SUMMARY
In general, eosinophilia typically proves to be reactive. A thorough step-wise evaluation is required to exclude neoplastic processes. This requires correlation with the clinical history, physical exam, laboratory data, cellular morphology, as well as ancillary testing. Integration of all available information is required to provide an accurate diagnosis in the setting of eosinophilia.
