Hematological Neoplasms with Eosinophilia

Citation: Morales-Camacho, R.M.; Caballero-Velázquez, T.; Borrero, J.J.; Bernal, R.; Prats-Martín, C. Hematological Neoplasms with Eosinophilia. Cancers 2024,16, 337. https://doi.org/10.3390/ cancers16020337 Academic Editors: Xavier Calvo, Leonor Arenillas and Anna Candoni Received: 30 November 2023 Revised: 5 January 2024 Accepted: 10 January 2024 Published: 12 January 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cancers Review Hematological Neoplasms with Eosinophilia Rosario M. Morales-Camacho 1,* , Teresa Caballero-Velázquez 1, Juan JoséBorrero 2, Ricardo Bernal 1 and Concepción Prats-Martín1,* 1 Department of Hematology, Virgen del Rocío University Hospital, Seville Biomedicine Institute (IBiS/CSIC), University of Seville, 41013 Seville, Spain; [email protected] (R.B.) 2Department of Pathology, Virgen del Rocío University Hospital, 41013 Seville, Spain; jborr[email protected] *Correspondence: [email protected] (R.M.M.-C.); [email protected] (C.P.-M.); Tel.: +34-955013261 (R.M.M.-C. & C.P.-M.) Simple Summary: The diagnostic assessment of eosinophilias is complex, requiring a multidisciplinary approach and often involving diagnostic challenges. This work aims for a better understanding of the cytomorphological features, immunophenotype, and biological activity of the eosinophil. Additionally, the concepts of peripheral and bone marrow eosinophilia and their potential causes are reviewed. Finally, the review focuses on the broad differential diagnosis of hematologic diseases that may underlie eosinophilia and how to diagnose them. Among the findings that should raise suspicion of hematologic diseases associated with eosinophilia are the presence of splenomegaly and/or lymphadenopathy or an abnormal blood count. In recent years, with advances in molecular techniques, new hematologic malignancies such as myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions are being defined, where eosinophilia can serve as a guiding sign. In these cases, an accurate diagnosis allows for the use of targeted therapy with an improvement in the quality of life and survival of patients. Abstract: Eosinophils in peripheral blood account for 0.3–5% of leukocytes, which is equivalent to 0.05–0.5 × 10 9 /L. A count above 0.5 × 10 9 /L is considered to indicate eosinophilia, while a count equal to or above 1.5 × 10 9 /L is defined as hypereosinophilia. In bone marrow aspirate, eosinophilia is considered when eosinophils make up more than 6% of the total nuclear cells. In daily clinical practice, the most common causes of reactive eosinophilia are non-hematologic, whether they are non-neoplastic (allergic diseases, drugs, infections, or immunological diseases) or neoplastic (solid tumors). Eosinophilia that is associated with a hematological malignancy may be reactive or secondary to the production of eosinophilopoietic cytokines, and this is mainly seen in lymphoid neoplasms (Hodgkin lymphoma, mature T-cell neoplasms, lymphocytic variant of hypereosinophilic syndrome, and B-acute lymphoblastic leukemia/lymphoma). Eosinophilia that is associated with a hematological malignancy may also be neoplastic or primary, derived from the malignant clone, usually in myeloid neoplasms or with its origin in stem cells (myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions, acute myeloid leukemia with core binding factor translocations, mastocytosis, myeloproliferative neoplasms, myelodysplastic/myeloproliferative neoplasms, and myelodysplastic neoplasms). There are no concrete data in standardized cytological and cytometric procedures that could predict whether eosinophilia is reactive or clonal. The verification is usually indirect, based on the categorization of the accompanying hematologic malignancy. This review focuses on the broad differential diagnosis of hematological malignancies with eosinophilia. Keywords: eosinophilia; hematological neoplasm; myeloid/lymphoid neoplasm with eosinophilia; tyrosine kinase gene fusions; acute leukemia Cancers 2024,16, 337. https://doi.org/10.3390/cancers16020337 https://www.mdpi.com/journal/cancers Cancers 2024,16, 337 2 of 28 1. Introduction 1.1. The Eosinophil Eosinophils are granular leukocytes originating in the bone marrow. The term “eosinophil” was coined by Paul Ehrlich in 1879 when he observed that the granules of these cells stained with acidic dyes, specifically with eosin Y (tetrabromofluorescein), producing a striking bright orange color [ 1 ]. However, it is highly likely that other researchers had observed this cell before Ehrlich [2]. Eosinophils should be considered tissue cells, with their presence in the blood being only circumstantial. They originate in the bone marrow from a pluripotent stem cell, which gives rise to a pluripotent myeloid progenitor cell, and this, in turn, gives rise to a specific or committed precursor in the genesis of the eosinophil granulocyte. Cytokines such as interleukin 3 (IL-3), interleukin 5 (IL-5) (the most specific to the eosinophil lineage), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin 33 (IL-33) act on this, regulating their maturation and differentiation [ 3 , 4 ]. After entering the bloodstream (hours), they migrate to various tissues. They are attracted in response to chemokines, primarily those of the eotaxin family (eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26, and RANTES/CCL5), which activate the CCR3 receptor (C-C motif chemokine receptor 3) expressed on eosinophils. These chemokines are produced by fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, T lymphocytes, and macrophages [ 5 ]. In the same way, complement C5a and lipid mediators have been linked as chemotactic factors [ 6 ]. Once in the tissues, eosinophils can survive for several weeks. They are preferentially present in the mucosa of the respiratory, digestive, and genitourinary tracts. 1.2. Eosinophil Biological Activity Eosinophils process a single type of granulation, with heterogeneous content in their primordial stage (pre-eosinophil), which later condenses and crystallizes into what is referred to as specific granulation [ 7 ]. In terms of ultrastructure, these granules possess a central electrondense crystalline core surrounded by a matrix, and they are rich in cationic proteins that are highly toxic to parasites (helminths), as well as to bacteria, fungi, and viruses. The “major basic protein” is concentrated in the central crystalline core, while the matrix contains the “eosinophil-derived neurotoxin” or ribonuclease 2, the “cationic protein of the eosinophil” or ribonuclease 3, and the “eosinophil peroxidase” that generates potent oxidants. They also contain galectin-10, or “Charcot-Leyden protein,” which can be observed when it is crystallized in bone marrow aspirates and in tissues affected by eosinophilic infiltration [ 8 ]. In addition, mediators such as cytokines, chemokines, growth factors, lipid mediators, as well as multiple receptors for these, and enzymes are found. Thus, they play an immunomodulatory role in innate and adaptive immune responses, have anti-inflammatory and antitumor activity, and mediate the repair, remodeling, and maintenance of tissues and their homeostasis [9]. However, when eosinophils undergo massive and persistent activation, they can damage the tissues and organs in which they operate due to the toxicity of their content. This is what happens in hypereosinophilic syndromes or immediate hypersensitivity reactions (type I). A proinflammatory and prothrombotic state is generated and transforming growth factor-beta and interleukins are released, promoting the proliferation and activation of fibroblasts, leading to subsequent fibrosis at the expense of the parenchyma [9]. 1.3. Normal and Pathological Eosinophil Morphology In blood smears that have been fixed with methanol and stained with Romanowsky stains, the normal mature eosinophil reaches a size between 14 and 16 µ m. The nucleus is bilobed, classically described as eyeglass-shaped, and sometimes trilobed. The cytoplasm, which appears translucent, is almost entirely occupied by a relatively thick, dense, orange granulation (0.5–1.5 µ m) that, unlike basophils, does not overlap the nucleus. Vacuoles are not typically observed (Figure 1A,B). Cancers 2024,16, 337 3 of 28 Cancers 2024, 16, 337 3 of 30 granulation (0.5–1.5 µm) that, unlike basophils, does not overlap the nucleus. Vacuoles are not typically observed (Figure 1A,B). Regarding cytochemical aspects, the presence of myeloperoxidase in normal eosinophils is demonstrated through enzymatic cytochemistry and an appropriate substrate. Indirectly, the “content” of myeloperoxidase can be deduced through staining with Sudan Black B (Figure 1C,D). Neutrophils and eosinophils can be distinguished by the thicker granulation of eosinophils. The eosinophil exhibits an absence of esterases and both acid and alkaline phosphatases. The Periodic Acid–Schiff (PAS) reaction is negative, or with weak intergranular positivity, in contrast to the marked positivity of segmented neutrophils. Additionally, metachromasia with Toluidine Blue, which is characteristic in basophilic granulocytes, is not observed [3]. Figure 1. (A). Bilobed eosinophil. (B). Trilobed eosinophil. (C). Eosinophil with high myeloperoxidase content. (D). Eosinophil myelocyte which is Sudan Black B-positive. Morphological alterations of the eosinophil, regardless of whether eosinophilia is reactive or malignant, can include nuclear morphological abnormalities (absence of lobulation, hypersegmentation, or ring nuclei), defects in the cytoplasm (total or partial degranulation, vacuoles, persistent basophilia, visualization of pre-eosinophilic granulation, or increased thickness of granules), size alterations (anisocytosis, gigantism, or small forms), and maturation disharmonies, which encompass previous defects such as the persistence of cytoplasmic basophilia in elements with advanced maturity [10] (Figure 2). Figure 1. (A) Bilobed eosinophil. (B) Trilobed eosinophil. (C) Eosinophil with high myeloperoxidase content. (D) Eosinophil myelocyte which is Sudan Black B-positive. Regarding cytochemical aspects, the presence of myeloperoxidase in normal eosinophils is demonstrated through enzymatic cytochemistry and an appropriate substrate. Indirectly, the “content” of myeloperoxidase can be deduced through staining with Sudan Black B (Figure 1C,D). Neutrophils and eosinophils can be distinguished by the thicker granulation of eosinophils. The eosinophil exhibits an absence of esterases and both acid and alkaline phosphatases. The Periodic Acid–Schiff (PAS) reaction is negative, or with weak intergranular positivity, in contrast to the marked positivity of segmented neutrophils. Additionally, metachromasia with Toluidine Blue, which is characteristic in basophil granulocytes, is not observed [3]. Morphological alterations of the eosinophil, regardless of whether eosinophilia is reactive or malignant, can include nuclear morphological abnormalities (absence of lobulation, hypersegmentation, or ring nuclei), defects in the cytoplasm (total or partial degranulation, vacuoles, persistent basophilia, visualization of pre-eosinophilic granulation, or increased thickness of granules), size alterations (anisocytosis, gigantism, or small forms), and maturation disharmonies, which encompass previous defects such as the persistence of cytoplasmic basophilia in elements with advanced maturity [10] (Figure 2). Cancers 2024,16, 337 4 of 28 Cancers 2024, 16, 337 4 of 30 Figure 2. Bone marrow aspirate. May–Grünwald–Giemsa (MGG), ×1000. Dysplastic eosinophils. (A). Giant form with a small non-lobulated nucleus. (B). Nuclear hypersegmentation. (C). Mature eosinophil with a round non-lobulated nucleus and hypogranularity. (D). Vacuolar images. (E). Persistence of cytoplasmic basophilia in a large band eosinophil. (F). Metamyelocyte with thick preeosinophilic granulation arranged peripherally. 1.4. Eosinophil Flow Cytometry By immunophenotype, the normal eosinophil is characterized by a pattern of elevated size (FSC) and complexity (SSC), reflecting its cytomorphological features. They originate in the bone marrow from pluripotent hematopoietic stem cells CD34+ CD117+. They express the panleukocytic marker CD45, and with differentiation, they lose the expression of CD34, CD117, and HLADR, acquiring CD11b, CD15, CD65, and cytoplasmic eosinophilic peroxidase (CyEPO). Eosinophils produce this peroxidase, which shares some homology with the myeloperoxidase that is found in neutrophil and monocyte leukocytes [11]. Other markers that are present in these cells include CD13, CD33, CD193 (CCR3), and the alpha subunit of the high-affinity IgE receptor (FcεR1α) [4,12]. In contrast to neutrophils, they exhibit slightly weaker expression of CD15 in the absence of CD16 (Figure 3) [12]. Figure 3. Representation of normal eosinophils (in orange) by flow cytometry in peripheral blood. Figure 2. Bone marrow aspirate. May–Grünwald–Giemsa (MGG), × 1000. Dysplastic eosinophils. (A) Giant form with a small non-lobulated nucleus. (B) Nuclear hypersegmentation. (C) Mature eosinophil with a round non-lobulated nucleus and hypogranularity. (D) Vacuolar images. (E) Persistence of cytoplasmic basophilia in a large band eosinophil. (F) Metamyelocyte with thick pre-eosinophilic granulation arranged peripherally. 1.4. Eosinophil Flow Cytometry By immunophenotype, the normal eosinophil is characterized by a pattern of elevated size (FSC) and complexity (SSC), reflecting its cytomorphological features. They originate in the bone marrow from pluripotent hematopoietic stem cells CD34+ CD117+. They express the panleukocytic marker CD45, and with differentiation, they lose the expression of CD34, CD117, and HLADR, acquiring CD11b, CD15, CD65, and cytoplasmic eosinophilic peroxidase (CyEPO). Eosinophils produce this peroxidase, which shares some homology with the myeloperoxidase that is found in neutrophil and monocyte leukocytes [ 11 ]. Other markers that are present in these cells include CD13, CD33, CD193 (CCR3), and the alpha subunit of the high-affinity IgE receptor (Fc ε R1 α ) [ 4 , 12 ]. In contrast to neutrophils, they exhibit slightly weaker expression of CD15 in the absence of CD16 (Figure 3) [12]. Cancers 2024, 16, 337 4 of 30 Figure 2. Bone marrow aspirate. May–Grünwald–Giemsa (MGG), ×1000. Dysplastic eosinophils. (A). Giant form with a small non-lobulated nucleus. (B). Nuclear hypersegmentation. (C). Mature eosinophil with a round non-lobulated nucleus and hypogranularity. (D). Vacuolar images. (E). Persistence of cytoplasmic basophilia in a large band eosinophil. (F). Metamyelocyte with thick preeosinophilic granulation arranged peripherally. 1.4. Eosinophil Flow Cytometry By immunophenotype, the normal eosinophil is characterized by a pattern of elevated size (FSC) and complexity (SSC), reflecting its cytomorphological features. They originate in the bone marrow from pluripotent hematopoietic stem cells CD34+ CD117+. They express the panleukocytic marker CD45, and with differentiation, they lose the expression of CD34, CD117, and HLADR, acquiring CD11b, CD15, CD65, and cytoplasmic eosinophilic peroxidase (CyEPO). Eosinophils produce this peroxidase, which shares some homology with the myeloperoxidase that is found in neutrophil and monocyte leukocytes [11]. Other markers that are present in these cells include CD13, CD33, CD193 (CCR3), and the alpha subunit of the high-affinity IgE receptor (FcεR1α) [4,12]. In contrast to neutrophils, they exhibit slightly weaker expression of CD15 in the absence of CD16 (Figure 3) [12]. Figure 3. Representation of normal eosinophils (in orange) by flow cytometry in peripheral blood. Figure 3. Representation of normal eosinophils (in orange) by flow cytometry in peripheral blood. Cancers 2024,16, 337 5 of 28 2. Eosinophilia Eosinophils in peripheral blood constitute 0.3–5% of leukocytes, equivalent to 0.05–0.5 × 10 9 /L. Eosinophilia is considered when a patient’s count is >0.5 × 10 9 /L (between 0.5 and 1.49 × 10 9 /L is also known as mild eosinophilia), and hypereosinophilia is considered when it is ≥ 1.5 × 10 9 /L. The latter has also been subdivided into moderate hypereosinophilia (1.5–5.0 ×109/L) and severe hypereosinophilia (>5.0 ×109/L) [13–15]. When hypereosinophilia is observed in association with clinical organ damage, the concept of “hypereosinophilic syndrome” (HES) is established. However, in some myeloid neoplasms such as chronic myeloid leukemia (CML) and certain variants of acute myeloid leukemia (AML), where leukocytosis reaches very high levels, absolute hypereosinophilia is observed, even though the percentage of eosinophils is low (<3%). This does not imply the presence of symptoms resulting from eosinophil-mediated organ damage. In this context, the International Cooperative Working Group on Eosinophil Disorders (ICOG-EO), during its 2021 Working Conference on Eosinophil Disorders and Syndromes, concluded that in these two hematologic malignancies, hypereosinophilia is only considered if there is an absolute blood eosinophilia ( ≥ 1.5 × 10 9 /L) and a relative proportion of ≥ 10% eosinophils in the peripheral blood leukocyte differentials [15]. In a normocellular bone marrow aspirate, eosinophilia is considered when the rate is above 6%. However, in a bone marrow biopsy, eosinophilia has been proposed when the concentration is ≥20% [13–15]. On the other hand, based on the underlying etiology and the nature of eosinophils, four categories of eosinophilias can be established: familial (hereditary), secondary (reactive), primary (clonal, neoplastic), and those of unknown significance (Table 1). Clonal eosinophilias are those associated with myeloid or stem cell neoplasms, in which the eosinophil belongs to the malignant clone [16]. Table 1. Approach to eosinophilia. Eosinophilia Categories Supportive Features Familial (hereditary) eosinophilia (with/without immunodeficiency) Age, family history, and genetic study Secondary (reactive) eosinophilia Non-hematological - Parasitosis - Infections - Allergic diseases - Drugs - Chronic inflammatory processes - Solid tumors Clinical history and work-up focused on secondary causes Hematological Lymphoid diseases Hematologic work-up * Primary (clonal, neoplastic) eosinophilia Stem cell and myeloid neoplasms Hematologic work-up * Eosinophilia of unknown significance Exclusion of previous disorders or conditions * Hematologic work-up: peripheral blood and bone marrow examination with cytology, flow cytometry, histopathology, cytogenetics/fluorescence in situ hybridization, polymerase chain reaction, and other molecular techniques. In practice, most eosinophilias are secondary (reactive), with the eosinophil being non-clonal. Among their causes are parasitosis, infections, allergic diseases, drugs, chronic inflammatory processes, etc. Solid tumors that can present with a leukemoid reaction featuring blood and/or bone marrow hypereosinophilia, mimicking a myeloproliferative neoplasm (MPN) (Figure 4), should also be included. In a similar manner, some hematological disorders (lymphoid) are associated with eosinophilia that is secondary to the release of eosinopoietic cytokines [ 16 ]. Also, some of the new antitumor immunotherapies can cause eosinophilia with dysplasia [17,18]. Cancers 2024,16, 337 6 of 28 Cancers 2024, 16, 337 5 of 30 2. Eosinophilia Eosinophils in peripheral blood constitute 0.3–5% of leukocytes, equivalent to 0.05– 0.5 × 109/L. Eosinophilia is considered when a patient’s count is >0.5 × 109/L (between 0.5 and 1.49 × 109/L is also known as mild eosinophilia), and hypereosinophilia is considered when it is ≥1.5 × 109/L. The latter has also been subdivided into moderate hypereosinophilia (1.5–5.0 × 109/L) and severe hypereosinophilia (>5.0 × 109/L) [13–15]. When hypereosinophilia is observed in association with clinical organ damage, the concept of “hypereosinophilic syndrome” (HES) is established. However, in some myeloid neoplasms such as chronic myeloid leukemia (CML) and certain variants of acute myeloid leukemia (AML), where leukocytosis reaches very high levels, absolute hypereosinophilia is observed, even though the percentage of eosinophils is low (<3%). This does not imply the presence of symptoms resulting from eosinophil-mediated organ damage. In this context, the International Cooperative Working Group on Eosinophil Disorders (ICOG-EO), during its 2021 Working Conference on Eosinophil Disorders and Syndromes, concluded that in these two hematologic malignancies, hypereosinophilia is only considered if there is an absolute blood eosinophilia (≥1.5 × 109/L) and a relative proportion of ≥10% eosinophils in the peripheral blood leukocyte differentials [15]. In a normocellular bone marrow aspirate, eosinophilia is considered when the rate is above 6%. However, in a bone marrow biopsy, eosinophilia has been proposed when the concentration is ≥20% [13–15]. On the other hand, based on the underlying etiology and the nature of eosinophils, four categories of eosinophilias can be established: familial (hereditary), secondary (reactive), primary (clonal, neoplastic), and those of unknown significance (Table 1). Clonal eosinophilias are those associated with myeloid or stem cell neoplasms, in which the eosinophil belongs to the malignant clone [16]. In practice, most eosinophilias are secondary (reactive), with the eosinophil being non-clonal. Among their causes are parasitosis, infections, allergic diseases, drugs, chronic inflammatory processes, etc. Solid tumors that can present with a leukemoid reaction featuring blood and/or bone marrow hypereosinophilia, mimicking a myeloproliferative neoplasm (MPN) (Figure 4), should also be included. In a similar manner, some hematological disorders (lymphoid) are associated with eosinophilia that is secondary to the release of eosinopoietic cytokines [16]. Also, some of the new antitumor immunotherapies can cause eosinophilia with dysplasia [17,18]. Figure 4. Leukemoid reaction with accompanying hypereosinophilia concurrently with the diagnosis and treatment of cervical cancer. (A). Peripheral blood smear showing abundant eosinophils (May–Grünwald–Giemsa [MGG], ×1000). (B). Hypercellular bone marrow aspirate with scattered eosinophils (MGG, ×1000). (C). Chart displaying elevated eosinophil counts in the blood, which sharply decrease following tumor remission. Figure 4. Leukemoid reaction with accompanying hypereosinophilia concurrently with the diagnosis and treatment of cervical cancer. (A) Peripheral blood smear showing abundant eosinophils (May–Grünwald–Giemsa [MGG], × 1000). (B) Hypercellular bone marrow aspirate with scattered eosinophils (MGG, × 1000). (C) Chart displaying elevated eosinophil counts in the blood, which sharply decrease following tumor remission. In the absence of familial and reactive causes, and with clonality ruled out, when eosinophilia is persistent and ≥ 1.5 × 10 9 /L, the diagnosis is hypereosinophilia of unknown significance. When symptoms due to organ involvement are also observed, it would be classified as idiopathic HES. Organ damage can also occur in the context of the other three groups of eosinophilias, where HES would be, respectively, classified as familial, secondary (reactive), and primary (neoplastic). Regardless of the category, the symptoms overlap in all cases of HES. In this way, clinically relevant eosinophil-mediated organ damage can be considered fibrosis (e.g., in the lungs, heart, digestive tract), thrombosis, skin lesions, central or peripheral neuropathy, vasculitis, and involvement of other organs (liver, pancreas, kidney) [ 15 ]. In this context, the use of drugs such as corticosteroids, cytotoxic agents (hydroxyurea, methotrexate, cyclophosphamide), or immunomodulators (interferonα , cyclosporine) may be necessary. More recently, new eosinophil-targeted therapies have become available, such as monoclonal antibodies targeting IL-5 (mepolizumab and reslizumab) or its receptor (benralizumab). Other biologics in development include dupilumab (targeting the IL-4 receptor) and lirentelimab (targeting Siglec-8), as well as the novel eosinophil-targeting agent, dexpramipexole, with an unknown mechanism of action [19,20]. In clinical practice, the differentiation between reactive eosinophilia (associated or not with hematologic disorders) and neoplastic eosinophilia should be supported by family history, clinical data, laboratory analyses, and imaging tests (Table 1). Additionally, a thorough hematologic work-up, including peripheral blood and bone marrow examination with cytology, flow cytometry, histopathology, cytogenetics/fluorescence in situ hybridization (FISH), and polymerase chain reaction (PCR) should be carried out in order to rule out clonality. Other molecular techniques that can detect clonality and/or complement the aforementioned techniques are SNP-array, next-generation sequencing, nanopore sequencing, and optical genome mapping [21–23]. Among the clonality markers described in myeloid hematologic neoplasms or stem cell disorders that are associated with eosinophilia are tyrosine kinase gene rearrangements and JAK1,JAK2, and STAT5B mutations [ 24 , 25 ]. The minimum variant allele frequency (VAF) to consider a variant to be potentially pathogenic in a clonal (non-reactive) eosinophilia is 3% (>2%) [15,26]. Cancers 2024,16, 337 7 of 28 Some activating STAT5B mutations, particularly the N642H mutation, have recently been identified as recurrent in myeloid neoplasms with eosinophilia. In a study of 1715 patients with eosinophilia, 27 cases were detected, representing 1.6%. Among the myeloid neoplasms showing this mutation, the presentation as chonic eosinophilic leukemia (CEL) is notably significant, observed in 12 out of a series of 82 patients (15%). In CEL, STAT5B N642H is dominant or codominant with a high VAF, probably being a driver mutation. In CEL and other myeloid neoplasms, it has been demonstrated by fluorescenceactivated cell sorting (FACS) that this mutation is present in eosinophils. On the other hand, STAT5B mutations have also been described in T-cell lymphomas with eosinophilia, and it was demonstrated by FACS that the mutation is in the T-cell population and not in eosinophils. STAT5B N642H has also been noted in eosinophils from pediatric hypereosinophilias, with or without a presence in the T-cell population. The prognostic value of this mutation and its potential role as a targeted therapy are yet to be determined [ 25 , 27 – 31 ]. In practice, clonality is not directly determined based on purified eosinophils. Instead, the evidence is indirect and is based on the underlying pathology and identified markers of clonality. 3. Hematological Neoplasms Associated with Eosinophilia Hematological neoplasms that are associated with eosinophilia are divided into two groups: hematological neoplasms that are associated with reactive eosinophilia and hematological malignancies that are associated with neoplastic or primary eosinophilia (Table 2). Table 2. Hematological neoplasms associated with eosinophilia. Hematological neoplasms associated with reactive or secondary eosinophilia * 1. Hodgkin lymphoma 2. Mature T-cell neoplasms 3. Lymphocytic variant of hypereosinophilic syndrome 4. B-cell lymphoblastic leukemias/lymphomas 5. T-lymphoblastic leukemias/lymphomas Hematological neoplasm associated with neoplastic or primary eosinophilia ** 1. Myeloid/lymphoid neoplasms with eosinophilia and defining gene rearrangements: 1.1. Myeloid/lymphoid neoplasms with PDGFRA rearrangement 1.2. Myeloid/lymphoid neoplasms with PDGFRB rearrangement 1.3. Myeloid/lymphoid neoplasms with FGFR1 rearrangement 1.4. Myeloid/lymphoid neoplasms with JAK2 rearrangement 1.5. Myeloid/lymphoid neoplasms with FLT3 rearrangement 1.6. Myeloid/lymphoid neoplasms with ETV6::ABL1 rearrangement 1.7. Myeloid/lymphoid neoplasms with other tyrosine kinase gene fusions 2. Core binding factor acute myeloid leukemia 2.1. Acute myeloid leukemia with CBFB::MYH11 fusion 2.2. Acute myeloid leukemia with RUNX1::RUNX1T1 3. Mastocytosis 4. Myeloproliferative neoplasms 4.1. Chronic myeloid leukemia 4.2. Chronic eosinophilic leukemia 5. Myelodysplastic/Myeloproliferative neoplasms 6. Myelodysplastic neoplasms * In these disorders, eosinophilia is usually reactive to the release of eosinopoietic cytokines (polyclonal or benign). ** In these disorders, eosinophils are usually neoplastic (belonging to the malignant clone). 3.1. Hematological Neoplasms Associated with Reactive Eosinophilia 3.1.1. Classical Hodgkin Lymphoma The observation of pronounced and persistent reactive eosinophilia is an unusual finding in patients with B-cell lymphomas, except for Hodgkin lymphoma. Approximately Cancers 2024,16, 337 8 of 28 15% of these patients exhibit it, typically in a mild form. Histologically, it is better distinguished in the inflammatory infiltrate surrounding Reed–Sternberg cells (Figure 5). Inside these cells, IL-5 mRNA has been demonstrated by in situ hybridization. It is unknown whether these cells or the abundant Th2 lymphocytes that are present are the main source of eosinopoietic cytokines [32,33]. Cancers 2024, 16, 337 8 of 30 3.1. Hematological Neoplasms Associated with Reactive Eosinophilia 3.1.1. Classical Hodgkin Lymphoma The observation of pronounced and persistent reactive eosinophilia is an unusual finding in patients with B-cell lymphomas, except for Hodgkin lymphoma. Approximately 15% of these patients exhibit it, typically in a mild form. Histologically, it is better distinguished in the inflammatory infiltrate surrounding Reed–Sternberg cells (Figure 5). Inside these cells, IL-5 mRNA has been demonstrated by in situ hybridization. It is unknown whether these cells or the abundant Th2 lymphocytes that are present are the main source of eosinopoietic cytokines [32,33]. Figure 5. Bone marrow aspirate. May–Grünwald–Giemsa, ×1000. Patient with Hodgkin’s lymphoma presenting infiltrative nodules in the bone marrow with an eosinophilic component. (A). Hodgkin cell with a large irregular nucleolus. (B). Reed–Sternberg-like cell with mirror nucleus. (C). Bone marrow biopsy. Hematoxylin and eosin, ×630. Mononuclear cells with prominent nucleoli and infiltration of eosinophils. 3.1.2. Mature T-Cell Neoplasms Mature T-cell neoplasms that are associated with eosinophilia mostly derive from memory CD4+ T cells, which can produce eosinophilopoietic cytokines. Sometimes it is doubtful whether eosinophilia is due to the tumor cells themselves or to locally attracted reactive Th2 lymphocytes. In any case, proliferating eosinophils are not related to the malignant clone, and can be integrated into the inflammatory context that surrounds the tumor cells. In the setting of eosinophilia, the study of the immunophenotype of T lymphocytes is relevant, with the antibody against TRBC1 JOVI-1 having recently been incorporated and validated as a useful marker in the identification by flow cytometry of clonal T populations with a TCR alphaBeta [34]. The presence of eosinophilia is estimated in 17–25% of mycosis fungoides cases, and it is described with much higher frequency in Sezary syndrome, where it is observed in 10 out of 13 patients (77%) [32,35]. In both mycosis fungoides and Sezary syndrome, clonal T lymphocytes commonly exhibit a CD4+ CD3+ phenotype in the absence of CD7 and CD26 expression. In the case of Sezary syndrome, a phenotype of central memory CD4 T lymphocytes (CD45RA− CD45RO+ CCR7+ CD27+, and CD62L+/−) is characteristic, unlike mycosis fungoides where its immunophenotype profile is that of an effector memory cell that is resident in the skin (CD45RA− CD45RO+ CCR7− CD27−, and CD62L−). Furthermore, it is accompanied by the expression of intense CD28 and CD279 [36]. In nodal T- follicular helper cell lymphoma, angioimmunoblastic-type eosinophilia is also common, occurring in 32–50% of cases. This lymphoma originates from a follicular CD4 T cell with a phenotype that is characterized by a weak or negative expression of CD3, with expression of CD10 and PD-1 (CD279) maintaining the expression of other pan-T markers such Figure 5. Bone marrow aspirate. May–Grünwald–Giemsa, × 1000. Patient with Hodgkin’s lymphoma presenting infiltrative nodules in the bone marrow with an eosinophilic component. (A) Hodgkin cell with a large irregular nucleolus. (B) Reed–Sternberg-like cell with mirror nucleus. (C) Bone marrow biopsy. Hematoxylin and eosin, × 630. Mononuclear cells with prominent nucleoli and infiltration of eosinophils. 3.1.2. Mature T-Cell Neoplasms Mature T-cell neoplasms that are associated with eosinophilia mostly derive from memory CD4+ T cells, which can produce eosinophilopoietic cytokines. Sometimes it is doubtful whether eosinophilia is due to the tumor cells themselves or to locally attracted reactive Th2 lymphocytes. In any case, proliferating eosinophils are not related to the malignant clone, and can be integrated into the inflammatory context that surrounds the tumor cells. In the setting of eosinophilia, the study of the immunophenotype of T lymphocytes is relevant, with the antibody against TRBC1 JOVI-1 having recently been incorporated and validated as a useful marker in the identification by flow cytometry of clonal T populations with a TCR alphaBeta [34]. The presence of eosinophilia is estimated in 17–25% of mycosis fungoides cases, and it is described with much higher frequency in Sezary syndrome, where it is observed in 10 out of 13 patients (77%) [ 32 , 35 ]. In both mycosis fungoides and Sezary syndrome, clonal T lymphocytes commonly exhibit a CD4+ CD3+ phenotype in the absence of CD7 and CD26 expression. In the case of Sezary syndrome, a phenotype of central memory CD4 T lymphocytes (CD45RA − CD45RO+ CCR7+ CD27+, and CD62L+/ − ) is characteristic, unlike mycosis fungoides where its immunophenotype profile is that of an effector memory cell that is resident in the skin (CD45RA − CD45RO+ CCR7 − CD27 − , and CD62L − ). Furthermore, it is accompanied by the expression of intense CD28 and CD279 [ 36 ]. In nodal T-follicular helper cell lymphoma, angioimmunoblastic-type eosinophilia is also common, occurring in 32–50% of cases. This lymphoma originates from a follicular CD4 T cell with a phenotype that is characterized by a weak or negative expression of CD3, with expression of CD10 and PD-1 (CD279) maintaining the expression of other pan-T markers such as CD5 (Figure 6) [ 37 ]. Adult T-cell leukemia/lymphoma follows in frequency, showing eosinophilia in 20% of cases. In this case, it is generally a CD4+ CD8- T lymphocyte phenotype, although to a lesser extent, they can be double-positive (CD4+ CD8+) or CD4 − CD8+. Furthermore, the expression of CD25 and CCR4 is typical, the latter being essential, as it is currently a therapeutic target [ 38 ]. More rarely, eosinophilia is found to be associated Cancers 2024,16, 337 9 of 28 with other T lymphomas, although histologically, it is frequently found with a peritumoral arrangement [32]. Cancers 2024, 16, 337 9 of 30 as CD5 (Figure 6) [37]. Adult T-cell leukemia/lymphoma follows in frequency, showing eosinophilia in 20% of cases. In this case, it is generally a CD4+ CD8- T lymphocyte phenotype, although to a lesser extent, they can be double-positive (CD4+ CD8+) or CD4- CD8+. Furthermore, the expression of CD25 and CCR4 is typical, the latter being essential, as it is currently a therapeutic target [38]. More rarely, eosinophilia is found to be associated with other T lymphomas, although histologically, it is frequently found with a peritumoral arrangement [32]. Figure 6. Characteristic immunophenotype of angioimmunoblastic T lymphoma (AITL) with eosinophilia is illustrated in a representative case: light blue, T cells; blue, abnormal T cells; orange, eosinophils; purple, plasma cells. (A). Abnormal T cells express CD5 brightly and lack CD3 or show dim expression. (B). CD4+ CD3−/+ weak subset. (C). Differentiation markers of T cells show effector memory phenotype (CD45RA- CD197−/+ weak). (D). Positive expression of CD279. (E). CD10 specific pattern of AITL. (F). Abnormal T cells show a clonal pattern according to the expression of TRBC1 (TRBC1-negative). 3.1.3. Lymphocytic Variant of Hypereosinophilic Syndrome This is a rare variant of hypereosinophilic syndrome (HES), with eosinophilia due to overproduction of eosinophilopoeitic cytokines secreted by clonal T lymphocytes with an abnormal immunophenotype. Persistent blood hypereosinophilia is related to tissue infiltration and organ damage. In a large review of 148 cases, the median age at diagnosis was 46 years, and there was no difference by gender. The most common clinical manifestations were cutaneous (81%), while cardiovascular manifestations occurred in 11.5% of cases. The most frequent immunophenotype of T cells was CD3- CD4+. Although their behavior is indolent, they have an increased risk of transformation to T lymphoma (risk of transformation at 10 years: 19.9%) [39]. 3.1.4. B-Lymphoblastic Leukemia/Lymphoma Significant eosinophilia during or prior to diagnosis is observed in less than 1% of B acute lymphoblastic leukemia/lymphoma (B-ALL). The expansion of eosinophils is considered to be reactive to the production of cytokines by lymphoblasts. Eosinophils can show dysplastic features in their morphology and by immunophenotype (Figures 7 and 8). Eosinophilia is often associated with B-ALL with IGH::IL3 fusion [according to the World Health Organization (WHO) nomenclature 2022] or B-ALL with t(5;14)(q31.1;q32.3)/IL3::IGH in accordance with the 2022 International Consensus Classification (ICC) [40]. It is a very rare subtype of B-ALL. In a review of 24 patients, this Figure 6. Characteristic immunophenotype of angioimmunoblastic T lymphoma (AITL) with eosinophilia is illustrated in a representative case: light blue, T cells; blue, abnormal T cells; orange, eosinophils; purple, plasma cells. (A) Abnormal T cells express CD5 brightly and lack CD3 or show dim expression. (B) CD4+ CD3 − /+ weak subset. (C) Differentiation markers of T cells show effector memory phenotype (CD45RA − CD197 − /+ weak). (D) Positive expression of CD279. (E) CD10 specific pattern of AITL. (F) Abnormal T cells show a clonal pattern according to the expression of TRBC1 (TRBC1-negative). 3.1.3. Lymphocytic Variant of Hypereosinophilic Syndrome This is a rare variant of hypereosinophilic syndrome (HES), with eosinophilia due to overproduction of eosinophilopoeitic cytokines secreted by clonal T lymphocytes with an abnormal immunophenotype. Persistent blood hypereosinophilia is related to tissue infiltration and organ damage. In a large review of 148 cases, the median age at diagnosis was 46 years, and there was no difference by gender. The most common clinical manifestations were cutaneous (81%), while cardiovascular manifestations occurred in 11.5% of cases. The most frequent immunophenotype of T cells was CD3 − CD4 + . Although their behavior is indolent, they have an increased risk of transformation to T lymphoma (risk of transformation at 10 years: 19.9%) [39]. 3.1.4. B-Lymphoblastic Leukemia/Lymphoma Significant eosinophilia during or prior to diagnosis is observed in less than 1% of B acute lymphoblastic leukemia/lymphoma (B-ALL). The expansion of eosinophils is considered to be reactive to the production of cytokines by lymphoblasts. Eosinophils can show dysplastic features in their morphology and by immunophenotype (Figures 7and 8). Eosinophilia is often associated with B-ALL with IGH::IL3 fusion [according to the World Health Organization (WHO) nomenclature 2022] or B-ALL with t(5;14)(q31.1;q32.3)/ IL3::IGH [in accordance with the International Consensus Classification (ICC) 2022] [ 40 ]. It is a very rare subtype of B-ALL. In a review of 24 patients, this leukemia mainly affects adolescent or young adult males who suffer frequent manifestations derived from eosinophilia (neurological, thromboembolic, pulmonary, and cutaneous) [ 41 ]. The relevance of eosinophilia in the blood and bone marrow can relativize the percentage of blasts, which can be less than 20%. In this way, the diagnosis of leukemia when associated with hypereosinophilia may be difficult due to the low fraction of leukemic cells in the bone marrow, within the context of the hypereosinophilic component. In addition, eosinophilia and Cancers 2024,16, 337 16 of 28 it has been previously overlooked. There are no conclusive data on somatic mutations in this group. Because the ETV6::ABL1 rearrangement shares the constitutive activation of the same tyrosine kinase with the BCR::ABL1 rearrangement, it has the peculiarity of being the one that most closely resembles chronic myeloid leukemia (CML). The myeloid neoplasm with ETV6::ABL1 rearrangement typically presents in the chronic phase with a CML-like morphology. The presence of eosinophilia is almost constant, and basophilia is common. As in CML, there may be progression to blast crisis (myeloid or lymphoid), or rarely, patients present as AML. When the ETV6::ABL1 rearrangement presents as B-ALL, it most often corresponds to a Ph-like B-ALL. In the series by Zaliova and collaborators (own and a review of the literature) that includes 44 cases with this rearrangement, 22 cases correspond to myeloid malignancies ( 18 MPN , 4 AML); and up to 22 cases were ALL (21 being Ph-like ALL by a standardized assay). The median age in the 18 cases described in the chronic phase is 51 (range, 24–72 years), with a predominance in men [116]. In the chronic phase, these patients respond to tyrosine kinase inhibitors, such as imatinib, or second- and third-generation ones. Just like in CML, the response is poor in the blast phase [51,116,117]. 3. Myeloid/lymphoid neoplasms with other tyrosine kinase gene fusions These gene fusions affect other tyrosine kinase genes that are not included in the previous categories, have hematopoietic lineage plasticity and eosinophilia, and present similarly. Some of these rearrangements are ETV6::FGFR2 [ 118 ], ETV6::LYN [ 119 ], ETV6::NTRK3 [ 120 ], RANBP2::ALK [ 121 ], BCR::RET, and FGFR1OP::RET [ 122 ]. In these rearrangements, experimental treatment with other tyrosine kinase inhibitors could be eligible [118,119,122–124]. 3.2.2. Core Binding Factor Acute Myeloid Leukemias AML with inv(16)(p13.1q22) o t(16;16)(p13.1;q22); CBFB::MYH11 The diversity of these leukemias presents a myelomonocytic cytology, which is sometimes purely monocytic. The percentage of eosinophils in the blood is usually normal—although eosinophilia may exist, as these leukemias are usually hyperleukocytic— and eosinophils do not present relevant dysplasia. In the bone marrow examination, eosinophilia is usually clear, with immature elements. Thick and dark violet-purple granules are characteristic (Figure 10) [125,126]. Cancers 2024, 16, 337 17 of 30 Figure 10. Bone marrow aspirate. May–Grünwald–Giemsa, ×1000. Acute myeloid leukemia with inv(16)(p13.1q22). In both images, dysmorphic eosinophils with thick reddish-violet granules are evident. In the left image, the intensity of eosinophilia is observed. At times, the presence of eosinophils, less than 6%, is observed in a blastic hypercellularity environment. In our series of 13 cases, we observed blood eosinophilia in 6/13 (46%) and marrow eosinophilia in 10/13 (77%). In exceptional cases, bone marrow eosinophilia becomes massive, without expression in the blood (Figure 11). Figure 11. Bone marrow aspirate. May–Grünwald–Giemsa, ×1000. Oligoblastic acute myeloid leukemia with inv(16)(p13.1q22). (A). Cytological image with a blast containing a long Auer rod, surrounded by eosinophils. (B). Pseudo-Chédiak–Higashi inclusions. (C). The histological image clearly shows massive bone marrow eosinophilia. (D). Bone marrow karyotype: 46,XY,inv(16)(p13.1q22) [3]/46,idem,t(1;18)(p22;p11.2) [17]. Cytochemically, positivity is typical both with the PAS reaction and in the enzymatic demonstration of chloracetate esterase (Figure 12). In the immunophenotypic pattern, the presence of blasts with myeloid differentiation is common, as is another subtype of blasts with commitment to monocytes according to their markers [127]. Figure 10. Bone marrow aspirate. May–Grünwald–Giemsa, × 1000. Acute myeloid leukemia with inv(16)(p13.1q22). In both images, dysmorphic eosinophils with thick reddish-violet granules are evident. In the left image, the intensity of eosinophilia is observed. At times, the presence of eosinophils, less than 6%, is observed in a blastic hypercellularity environment. In our series of 13 cases, we observed blood eosinophilia in 6/13 (46%) and marrow eosinophilia in 10/13 (77%). In exceptional cases, bone marrow eosinophilia becomes massive, without expression in the blood (Figure 11). Cancers 2024,16, 337 17 of 28 Cancers 2024, 16, 337 17 of 30 Figure 10. Bone marrow aspirate. May–Grünwald–Giemsa, ×1000. Acute myeloid leukemia with inv(16)(p13.1q22). In both images, dysmorphic eosinophils with thick reddish-violet granules are evident. In the left image, the intensity of eosinophilia is observed. At times, the presence of eosinophils, less than 6%, is observed in a blastic hypercellularity environment. In our series of 13 cases, we observed blood eosinophilia in 6/13 (46%) and marrow eosinophilia in 10/13 (77%). In exceptional cases, bone marrow eosinophilia becomes massive, without expression in the blood (Figure 11). Figure 11. Bone marrow aspirate. May–Grünwald–Giemsa, ×1000. Oligoblastic acute myeloid leukemia with inv(16)(p13.1q22). (A). Cytological image with a blast containing a long Auer rod, surrounded by eosinophils. (B). Pseudo-Chédiak–Higashi inclusions. (C). The histological image clearly shows massive bone marrow eosinophilia. (D). Bone marrow karyotype: 46,XY,inv(16)(p13.1q22) [3]/46,idem,t(1;18)(p22;p11.2) [17]. Cytochemically, positivity is typical both with the PAS reaction and in the enzymatic demonstration of chloracetate esterase (Figure 12). In the immunophenotypic pattern, the presence of blasts with myeloid differentiation is common, as is another subtype of blasts with commitment to monocytes according to their markers [127]. Figure 11. Bone marrow aspirate. May–Grünwald–Giemsa, × 1000. Oligoblastic acute myeloid leukemia with inv(16)(p13.1q22). (A) Cytological image with a blast containing a long Auer rod, surrounded by eosinophils. (B) Pseudo-Chédiak–Higashi inclusions. (C) The histological image clearly shows massive bone marrow eosinophilia. (D) Bone marrow karyotype: 46,XY,inv(16)(p13.1q22) [3]/46,idem,t(1;18)(p22;p11.2) [17]. Cytochemically, positivity is typical both with the PAS reaction and in the enzymatic demonstration of chloracetate esterase (Figure 12). In the immunophenotypic pattern, the presence of blasts with myeloid differentiation is common, as is another subtype of blasts with commitment to monocytes according to their markers [127]. Cancers 2024, 16, 337 18 of 30 Figure 12. Bone marrow aspirate. May–Grünwald–Giemsa, ×1000. Acute myeloid leukemia with inv(16)(p13.1q22). (A). Eosinophil with thick dark pre-eosinophilic granules. (B). Positive periodic acid–Schiff (PAS) reaction in immature eosinophils, in contrast to the negativity in an immature segmented neutrophil. (C). Two immature eosinophils showing marked chloroacetate esterase activity. AML with t(8;21)(q22;q22.1); RUNX1::RUNX1T1 These leukemias are known for their peculiar morphology, with large Auer rods in the blasts and marked neutrophil dysgranulopoiesis. Eosinophilia, both in the blood and in the marrow, is much less evident than in the previous group. In a series of 165 patients collected by the Spanish Hematological Cytology Group [128], blood eosinophilia was observed in 7/148 cases (4.7%) and marrow eosinophilia in 22/137 (16%) (unpublished data). The eosinophils are less dysplastic and without thick and dark red-violet granulation (Figure 13). The chlorocetate esterase is negative, and the PAS reaction may be negative or positive [125,129,130]. In the immunophenotype, most common is the presence of immature blasts with expression of CD34 and CD117, with expression of myeloid markers, such as CD13 and CD33 and lymphoid markers, especially CD19 and CD56 [127]. Figure 13. Bone marrow aspirate. May–Grünwald–Giemsa, ×1000. Acute myeloid myeloid with t(8;21)(q22;q22.1). (A). Granulocytic dysplasia with peripheral basophilia reinforcement. (B). Dysplastic eosinophils with increased granulation thickness. (C). Eosinophils with mirror nuclei. Figure 12. Bone marrow aspirate. May–Grünwald–Giemsa, × 1000. Acute myeloid leukemia with inv(16)(p13.1q22). (A) Eosinophil with thick dark pre-eosinophilic granules. (B) Positive periodic acid– Schiff (PAS) reaction in immature eosinophils, in contrast to the negativity in an immature segmented neutrophil. (C) Two immature eosinophils showing marked chloroacetate esterase activity. AML with t(8;21)(q22;q22.1); RUNX1::RUNX1T1 These leukemias are known for their peculiar morphology, with large Auer rods in the blasts and marked neutrophil dysgranulopoiesis. Eosinophilia, both in the blood and Cancers 2024,16, 337 18 of 28 in the marrow, is much less evident than in the previous group. In a series of 165 patients collected by the Spanish Hematological Cytology Group [ 128 ], blood eosinophilia was observed in 7/148 cases (4.7%) and marrow eosinophilia in 22/137 (16%) (unpublished data). The eosinophils are less dysplastic and without thick and dark red-violet granulation (Figure 13). The chlorocetate esterase is negative, and the PAS reaction may be negative or positive [ 125 , 129 , 130 ]. In the immunophenotype, most common is the presence of immature blasts with expression of CD34 and CD117, with expression of myeloid markers, such as CD13 and CD33 and lymphoid markers, especially CD19 and CD56 [127]. Cancers 2024, 16, 337 18 of 30 Figure 12. Bone marrow aspirate. May–Grünwald–Giemsa, ×1000. Acute myeloid leukemia with inv(16)(p13.1q22). (A). Eosinophil with thick dark pre-eosinophilic granules. (B). Positive periodic acid–Schiff (PAS) reaction in immature eosinophils, in contrast to the negativity in an immature segmented neutrophil. (C). Two immature eosinophils showing marked chloroacetate esterase activity. AML with t(8;21)(q22;q22.1); RUNX1::RUNX1T1 These leukemias are known for their peculiar morphology, with large Auer rods in the blasts and marked neutrophil dysgranulopoiesis. Eosinophilia, both in the blood and in the marrow, is much less evident than in the previous group. In a series of 165 patients collected by the Spanish Hematological Cytology Group [128], blood eosinophilia was observed in 7/148 cases (4.7%) and marrow eosinophilia in 22/137 (16%) (unpublished data). The eosinophils are less dysplastic and without thick and dark red-violet granulation (Figure 13). The chlorocetate esterase is negative, and the PAS reaction may be negative or positive [125,129,130]. In the immunophenotype, most common is the presence of immature blasts with expression of CD34 and CD117, with expression of myeloid markers, such as CD13 and CD33 and lymphoid markers, especially CD19 and CD56 [127]. Figure 13. Bone marrow aspirate. May–Grünwald–Giemsa, ×1000. Acute myeloid myeloid with t(8;21)(q22;q22.1). (A). Granulocytic dysplasia with peripheral basophilia reinforcement. (B). Dysplastic eosinophils with increased granulation thickness. (C). Eosinophils with mirror nuclei. Figure 13. Bone marrow aspirate. May–Grünwald–Giemsa, × 1000. Acute myeloid myeloid with t(8;21)(q22;q22.1). (A) Granulocytic dysplasia with peripheral basophilia reinforcement. (B) Dysplastic eosinophils with increased granulation thickness. (C) Eosinophils with mirror nuclei. 3.2.3. Mastocytosis In mastocytosis, a clonal proliferation of eosinophils can be observed that would have its origin in the same neoplastic precursor (multilineage involvement due to the KIT mutation). The 2022 WHO classification includes peripheral and/or central eosinophilia among the signs of myeloproliferation and/or myelodysplasia that are part of the B findings of systemic mastocytosis (SM). However, this is only the case when no reactive cause is identified and the criteria for associated hematological neoplasia are not met [26,131]. In a series of 2350 patients with mastocytosis, eosinophilia was reported in 6.8% and hypereosinophilia in 3.1%, mainly associated with advanced forms. Eosinophilia is frequently seen in bone marrow aspirates and biopsies, even in cases without significant peripheral eosinophilia [ 132 , 133 ]. In exceptional cases, eosinophils can become phagocytosed by the mast cells of an SM, perhaps as a sign of malignancy [134]. In advanced SM with eosinophilia, a differential diagnosis must be made with M/LN- eo and defining gene rearrangements, because the coexistence of both is exceptional [ 135 ]. It should be noted that in the M/LN-eo, an increase in mast cells with an aberrant expression of CD25 with or without the expression of CD2 may occur. However, dense multifocal infiltrates do not form, and they do not present KIT mutations [ 26 ]. Another frequently described marker (80%) in SM mast cells is CD30 [ 136 , 137 ], for which no conclusive descriptions currently exist in the M/LN-eo. To diagnose the presence of both entities, it is necessary to demonstrate the rearrangement of an M/LN-eo and meet the SM criteria [ 138 ]. 3.2.4. Myeloproliferative Neoplasms MPN can present with eosinophilia that requires the ruling out of associated MS or M/LN-eo. Within this group, eosinophilia is more common in CML, and especially in CEL. Cancers 2024,16, 337 19 of 28 Chronic Myeloid Leukemia In the chronic phase, eosinophilia may be present in peripheral blood and bone marrow. Unusual presentation forms have been identified, and they are recognized as “eosinophilic variants of CML” with intense eosinophilia, similar to chronic eosinophilic leukemia. Approximately six cases have been described, with a lower median age than that of CML, most without splenomegaly, and with frequent cutaneous manifestations and vascular symptoms [139]. Chronic Eosinophilic Leukemia CEL (2022 WHO), or CEL, not otherwise specified (ICC 2022) is an extremely rare disorder. CEL is accompanied by peripheral hypereosinophilia and significant infiltration of the marrow and different organs. Cases with other MPN or MPN/MDS, M/LN-eo, MDS, mastocytosis, and AML with CBF translocations are excluded. The diagnostic criteria for CEL have been updated in the 2022 WHO classification. In addition to eosinophilia (on at least two occasions over an interval of at least four weeks), the criteria include evidence of clonality and abnormal bone marrow morphology. The WHO has eliminated the increase in blasts ( ≥ 5% in the bones marrow and/or ≥ 2% in the peripheral blood) as an alternative CEL criterion to clonality. This finding is maintained along with the rest of the criteria in the ICC 2022 [26,102]. Organ involvement due to eosinophil infiltration in the absence of abnormal bone marrow morphology, blastosis, and/or genetic clonality points to idiopathic HES. In the same circumstances, if organ damage is absent, idiopathic hypereosinophilia would be considered. One of the best studied series is from the Mayo Clinic, with 17 patients who were diagnosed according to the 2016 WHO criteria (median 63 years, male 88%). Most patients presented with systemic symptoms due to digestive, cardiac, and pulmonary involvement, as well as splenomegaly and hepatomegaly. The blood count showed leukocytosis with eosinophilia (median 6.4 × 10 9 /L) and moderate anemia. Eosinophils presented dysplasia in half of the cases. In bone marrow, an increase in the M:E ratio 5:1 (47%), eosinophilia (median 43%), dysmegakaryopoiesis (41.2%), fibrosis (17%), and one case with blasts (>5%) was described. Cytogenetic alterations were described in 15/17 patients and mutations in ASXL1 in 2 patients. The prognosis was poor, with a median survival of 16 months and progression to acute leukemia in only three cases [140]. Activating mutations in JAK1 and STAT5B have also been described as markers of clonality and potentials for targeted therapies in CEL. STAT5B N462H is a recurrent mutation in LEC. So, STAT5B-mutated eosinophilia/HES should be reclassified as myeloid neoplasm or CEL [24,25,27]. 3.2.5. Myelodysplastic/Myeloproliferative Neoplasms MDS/MPN can present with eosinophilia. In these cases, an associated systemic mastocytosis must be ruled out and/or, less frequently, an M/LN-eo. 3.2.6. Myelodysplastic Neoplasms The finding of eosinophilia in MDS is unusual and requires reactive causes to be ruled out. A retrospective series of 288 patients described marrow eosinophilia in 36 (12.5%) patients, of which only 18% had peripheral eosinophilia. The usual assumption is that eosinophilia is part of the neoplastic clone. The most common cytogenetic abnormalities are alterations of chromosome 7, complex karyotypes, and isochromosome 17q. A worse survival of these patients is reported, but it could be mediated by the underlying cytogenetics [141]. Cancers 2024,16, 337 20 of 28 Table 3. Myeloid/lymphoid neoplasms with eosinophilia and defining gene rearrangements (WHO 2022). Myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions (ICC 2022). M/LN-eo with Frequency * n/Total Cases (%) Age Median or Range Median (Age Range) Male/Female Ratio Most Common Presentation Treatments Patient Series PDGFRA rearrangement 78/135 (57.8%) Range median 45.5–51 years (2–82) 17.8:1 to 43:1 CEL Imatinib mesylate Pozdnyakova et al., 2021 [48] Akiely et al., 2022 [63] Metzgeroth et al., 2023 [49] PDGFRB rearrangement 26/135 (19.3%) Range median 38–53 years (0.25–86) 2.5:1 to 10:1 MDS/MPN or MPN with eosinophilia Imatinib mesylate Metzgeroth et al., 2023 [49] Pozdnyakova et al., 2021 [48] Di Giacomo et al., 2021 [71] Jawhar et al., 2017 [69] Cheah et al., 2014 [70] FGFR1 rearrangement 9/135 (6.7%) Range median 46–62 years (0.41–84) 1.1:1 to 1.6:1 MPN with/without lymphoblastic lymphoma and eosinophilia Allogeneic stem cell transplantation Intensive chemotherapy Ponatinib Midastaurina Futibatinib Pemigatinib (NCT03011372) Patnaik et al., 2010 [142] Strati et al., 2017 [85] Hernández-Boluda et al., 2022 [86] Parasuraman et al., 2022 [87] McKeague et al., 2023 [84] JAK2 rearrangement 11/135 (8.1%) Median 47 (6–86) 3.4:1 MPN or MDS/MPN with eosinophilia Allogeneic stem cell transplantation Inh JAK1/JAK2 ruxolitinib (Phase II clinical trial with Ruxolitinib NCT03801434) Tang et al., 2019 [143] Pozdnyakova et al., 2021 [48] Kaplan et al., 2022 [103] FLT3 rearrangement Not reported Median 46.5 (0.6–80) 1.8:1 MPN or MDS/MPN with eosinophilia Allogeneic stem cell transplantation Sorafenib, sunitinib, or gilteritinib Tang et al., 2021 [106] ETV6::ABL1 rearrangement 11/135 (8.1%) Median 51 (24–72) 2.4:1 CML-like with eosinophilia Dasatinib or nilotinib Imatinib mesylate Zaliova et al., 2016 [116] Cancers 2024,16, 337 21 of 28 Table 3. Cont. M/LN-eo with Frequency * n/Total Cases (%) Age Median or Range Median (Age Range) Male/Female Ratio Most Common Presentation Treatments Patient Series Other tyrosine kinase gene fusions: FGFR2 LYN NTRK3 ALK RET Not reported Few cases Few cases, but predominates in males Variable ETV6::FGFR2: TKI (ponatinib) or FGFR inhibitors ETV6::LYN: Dasatinib ETV6::NTRK3: TRK inhibitor RANBP2::ALK: Crizotinib BCR::RET and FGFR1OP::RET: Sorafenib Isolated cases M/LN-eo: myeloid/lymphoid neoplasms with eosinophilia; CEL: chronic eosinophilic leukemia; MDS/MPN: myelodysplastic/myeloproliferative neoplasm; MPN: myeloproliferative neoplasm; CML: chronic myeloid leukemia; TKI: tyrosine-kinase inhibitors; TRK: tropomyosin receptor kinase. * Frequency within the M/LN-eo is based on the series by Metgeroth et al., 2023, with 135 cases of M/LN-eo [49]. Cancers 2024,16, 337 22 of 28 4. Conclusions Eosinophilia that is associated with a hematological malignancy can be reactive or secondary to the production of cytokines eosinophilopoietic. This type of eosinophilia is mainly observed in lymphoid neoplasms. Eosinophilia can also be neoplastic and primary, derived from the malignant clone, usually in myeloid or stem cell neoplasms. No data collected in cytological and cytometric studies can predict whether eosinophilia is reactive or clonal. The verification is indirect and supported by the categorization of the accompanying hematological neoplasm. A thorough hematologic work-up, including peripheral blood and bone marrow examination with cytology, flow cytometry, histopathology, cytogenetics/fluorescence in situ hybridization, polymerase chain reaction, and other molecular techniques, should be carried out in order to rule out clonality. Detecting clonality allows for distinguishing clonal eosinophilias that are associated with hematological malignancies from reactive eosinophilias that are associated with non-hematological disorders. The detection of clonality in eosinophilia, such as STAT5B mutations or tyrosine kinase fusion genes, could enable the use of targeted therapies, leading to an improvement in the quality of life and survival of patients. Author Contributions: All authors designed the review. All authors examined the available data. All authors wrote and reviewed the text. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflicts of interest. References 1. Ehrlich, P. Über die spezifischen Granulationen des Blutes. Arch. Anat. Physiol. 1879,1, 571–579. 2. Kay, A.B. The early history of the eosinophil. Clin. Exp. Allergy 2015,45, 575–582. [CrossRef] [PubMed] 3. 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