Approach to Acute Myeloid Leukemia with Increased Eosinophils and Basophils

There is remarkable morphologic and genetic heterogeneity in acute myeloid leukemia (AML). In a small percentage of cases of AML, increased eosinophils and/or basophils are present in the bone marrow and sometimes in the peripheral blood. This is often a puzzling diagnostic situation but also an important finding that requires special investigation. Unique chromosomal rearrangements have been correlated with an increased number of eosinophils and basophils in AML. The identification of the underlying genetic lesion that promotes eosinophilia and basophilia can dramatically change both the prognosis and the treatment of the patient. Thus, clinicians must be vigilant in searching for the cause of eosinophilia and basophilia in patients with AML, since the different causes may lead to different treatments and survival outcomes. In this article, we examine the significance of increased eosinophils and/or basophils in the context of AML, provide guidance that simplifies the differential diagnosis, and give prognostic and therapeutic information about specific subtypes of AML associated with eosinophilia and/or basophilia. Evidence supporting personalized (molecularly targeted) therapy for these patients is also presented.


Introduction
There is remarkable morphologic and genetic heterogeneity in acute myeloid leukemia (AML).Specific chromosomal abnormalities correlate with particular subtypes of AML that have characteristic morphologic and clinical features.Conversely, certain morphologic findings may serve as a basis for the identification of specific genetic subtypes.The presence of eosinophilia and/or basophilia in the context of a myeloid cancer such as AML usually indicates a clonal origin.These eosinophils and basophils are usually part of the neoplastic process, and may be morphologically abnormal with abnormal granulation, abnormal cytochemical reactions or nuclear hyposegmentation.Eosinophilia and/or basophilia are usually found in the bone-marrow aspirates of patients with AML and less frequently in the peripheral blood smears.
Although eosinophilia and/or basophilia may occur as a nonspecific finding in AML, this finding may also indicate specific subtypes of immediate therapeutic and prognostic relevance.Therefore, the presence of increased eosinophils and basophils may be an important diagnostic lead in a patient with AML.The aim of this article is to review the significance of increased eosinophils and basophils in the context of AML, putting emphasis on the appropriate diagnostic evaluation, prognostic significance, and treatment.

Physiologic Features of Eosinophils
Eosinophils are produced in the bone marrow from granulocyte-macrophage colonyforming units (CFU-GM).The latter cells differentiate first into hybrid precursors with properties of basophils and eosinophils (CFU-Bas/Eo) and then into a separate eosinophil lineage.Three cytokines-interleukin-3, interleukin-5, and granulocyte-macrophage colonystimulating factor (GM-CSF)-are particularly important in the development of eosinophil granulocytes [1,2].These cytokines are encoded by closely linked genes on chromosome 5q31 and bind to receptors that have a common beta chain (common beta [CD131] subunit) and different alpha chains.The regulation of genes that drive eosinophil differentiation is controlled by a network of transcription factors including C/EBPα, C/EBPε, GATA-1, GATA-2, FOG-1, and PU.1 [2,3].After maturing in the bone marrow, eosinophils migrate from the circulation into tissues [4,5].Eosinophils, unlike neutrophils, can survive in tissues for extended periods (perhaps weeks), depending on the cytokines in the microenvironment.
The granules of eosinophils contain a crystalloid core composed of major basic protein (MBP) and a matrix composed of eosinophil cationic protein (ECP), eosinophil peroxidase (EPX), and eosinophil-derived neurotoxin (EDN).In addition, eosinophils produce hydrolytic enzymes such as phospholipase A2 (lysophospholipase), cytokines (some of which are stored in the granules), and large amounts of lipid mediators (leukotriene C4, which is metabolized to leukotriene D4 and E4) that are generated after cellular activation.These three lipid mediators are the slow-reacting substances of anaphylaxis that increase vascular permeability and mucus secretion and are potent stimulators of smooth-muscle contraction [6,7].
Eosinophils normally account for 1-5% of peripheral-blood leukocytes, and the upper limit of the normal range is 0.5 × 10 9 /L.A marked accumulation of eosinophils (>1.5 × 10 9 /L) is referred to as hypereosinophilia.The normal range for eosinophils and their precursors in the bone marrow is 1-5%.Therefore, the cut-off value for increased bone-marrow eosinophilic granulocytes and their precursors is 6%.A definition of severe marrow eosinophilia has been proposed that requires ≥20% of marrow cells to be eosinophils, with or without peripheral-blood eosinophilia [8,9].There are many causes of reactive (non-clonal) eosinophilia [10].

Charcot-Leyden Crystals
Charcot-Leyden crystals occur in association with marked eosinophilia.Charcot-Leyden crystals were first reported in 1853 by Jean-Martin Charcot, who found tiny crystals in the cardiac blood and spleen of a patient who died from leukemia.In 1872, Ernst Viktor von Leyden also described colorless crystals in the sputum of patients with asthma [11].Following the discovery of Charcot-Leyden crystals, many conflicting reports appeared as to the chemical nature and significance of these crystals.At that time, it was thought that lysolecithin acylhydrolase, which has lysophospholipase activity, was the constituent protein of Charcot-Leyden crystals.Subsequent investigations revealed galectin-10, a lysophospholipase binding protein, as the constituent protein of Charcot-Leyden crystals.Figure 1 illustrates Charcot-Leyden crystals in association with AML.

Physiologic Features of Basophils
Basophils normally account for ≤2% of peripheral-blood leukocytes, and the upper limit of the absolute basophil count normal range is 0.1 × 10 9 /L.Hyperbasophilia occurs when the peripheral-blood basophil count is >1.0 × 10 9 /L.There are many causes of reactive (non-clonal) basophilia [12].Persistent basophilia (defined as lasting >8 months) is always suggestive of a neoplastic hematologic disorder.The normal range for basophils and their precursors in the bone marrow is 0-2%.

Physiologic Features of Basophils
Basophils normally account for ≤2% of peripheral-blood leukocytes, and the upper limit of the absolute basophil count normal range is 0.1 × 10 9 /L.Hyperbasophilia occurs when the peripheral-blood basophil count is >1.0 × 10 9 /L.There are many causes of reactive (non-clonal) basophilia [12].Persistent basophilia (defined as lasting >8 months) is always suggestive of a neoplastic hematologic disorder.The normal range for basophils and their precursors in the bone marrow is 0-2%.
Basophils, like mast cells, carry the high-affinity IgE receptor (FcεRI).Basophils, unlike mast cells, do not contain tryptase.Histamine is the most important constituent of basophilic granules [12].Their metachromatic granules are also rich in proteoglycans such as chondroitin and heparin and a variety of enzymes.They are also able to produce a variety of growth factors, such as VEGF and HGF, and inflammatory cytokines, namely interleukin-4 and interleukin-13 [17].

Metachromatic Granules
The presence of granules taking a metachromatic stain means that they are stained pink or red using blue dyes (toluidine blue or methylene blue).This is due to the large amount of proteoglycans in basophil and mast-cell granules.Toluidine blue staining, which is necessary to reveal the metachromatic character of the granules, stains both the basophilic and mastocytic granules and, therefore, it cannot differentiate between the two [18].The periodic-acid Schiff stain may aid in the differential diagnosis: basophils typically show a speckled pattern, whereas mast cells exhibit negativity or a very weak reaction [19].The normal counterpart of the metachromatic blast is thought to be the common progenitor of both the basophilic and mastocytic cell differentiation.CD123, commonly expressed in basophils, accounts for the α-subunit of the interleukin-3 receptor (IL-3Rα).Interleukin-3 is the main cytokine for basophilic differentiation and maturation.Other important regulators are GM-CSF, TGF-β, interleukin-5, and thymic stromal lymphopoietin (TSLP) [12].STAT5 is the effector of interleukin-3 signaling and induces GATA-2-dependent transcriptional activation [13].Other important transcription factors for basophil development include GATA-1 [14], IRF8 [15], and RUNX1 [16].Basophils have a short life span (<3 days).
Basophils, like mast cells, carry the high-affinity IgE receptor (FcεRI).Basophils, unlike mast cells, do not contain tryptase.Histamine is the most important constituent of basophilic granules [12].Their metachromatic granules are also rich in proteoglycans such as chondroitin and heparin and a variety of enzymes.They are also able to produce a variety of growth factors, such as VEGF and HGF, and inflammatory cytokines, namely interleukin-4 and interleukin-13 [17].

Metachromatic Granules
The presence of granules taking a metachromatic stain means that they are stained pink or red using blue dyes (toluidine blue or methylene blue).This is due to the large amount of proteoglycans in basophil and mast-cell granules.Toluidine blue staining, which is necessary to reveal the metachromatic character of the granules, stains both the basophilic and mastocytic granules and, therefore, it cannot differentiate between the two [18].The periodic-acid Schiff stain may aid in the differential diagnosis: basophils typically show a speckled pattern, whereas mast cells exhibit negativity or a very weak reaction [19].The normal counterpart of the metachromatic blast is thought to be the common progenitor of both the basophilic and mastocytic cell differentiation.

AML with Increased Eosinophils (Table 1)
3.1.AML with inv(16) or t (16;16) Of the chromosomal abnormalities associated with AML with increased marrow eosinophils (i.e., >5% marrow eosinophils), the best example is inv(16)(p13.1q22)or t(16;16)(p13.1;q22),resulting in the fusion of CBFB at 16q22 to MYH11 at 16p13.1 (CBFB-MYH11).MYH11 encodes for smooth-muscle heavy-chain myosin and CBFB codes for the beta subunit of core-binding factor (CBF), a heterodimeric transcription factor that controls the transcription of genes involved in hematopoiesis.The CBFB subunit normally heterodimerizes with RUNX1 (CBFA2), the gene product of RUNX1, and increases its DNA-binding affinity.The CBFB-MYH11 mutant protein does not bind RUNX1, leading to RUNX1 sequestration on cytoskeletal structures in the cytoplasm and rendering it inactive [20,21].The exact mechanism by which these alterations cause eosinophilia remains unclear [22,23].) is included in the current World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues in the category of "acute myeloid leukemia with defining genetic abnormalities" [24].It corresponds morphologically to acute myelomonocytic leukemia with eosinophilia (subtype M4Eo, according to the French-American-British [FAB] classification); rarely, cells with these features can be identified as acute myeloblastic leukemia with maturation (M2).The bone marrow usually shows an increased number of eosinophils at all stages of maturation; they usually amount to ≥5% of non-erythroid cells (Figure 2).
Most importantly, these eosinophils are abnormal, and some have, in addition to the characteristic specific eosinophilic granules, large basophilic (immature) granules and may have a single unsegmented nucleus.The basophilic granules are larger and more irregular, as well as more numerous, than those occasionally seen in immature eosinophils.The anomalous immature granules are mostly evident at the late promyelocyte and myelocyte stages; however, they tend to persist in some band and segmented forms.Unlike normal eosinophils, these cells stain with chloroacetate esterase and periodic-acid Schiff.The peripheral blood is not different from that in other cases of acute myelomonocytic leukemia; eosinophils are not increased in the peripheral blood.AML with inv(16)(p13.1q22)or t(16;16)(p13.1;q22) is associated with a high rate of complete remission and a favorable overall survival rate when treated with intensive induction and consolidation therapy.
3.1.1.Morphology AML with inv(16) or t (16;16) is included in the current World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues in the category of "acute myeloid leukemia with defining genetic abnormalities" [24].It corresponds morphologically to acute myelomonocytic leukemia with eosinophilia (subtype M4Eo, according to the French-American-British [FAB] classification); rarely, cells with these features can be identified as acute myeloblastic leukemia with maturation (M2).The bone marrow usually shows an increased number of eosinophils at all stages of maturation; they usually amount to ≥5% of non-erythroid cells (Figure 2).Most importantly, these eosinophils are abnormal, and some have, in addition to the characteristic specific eosinophilic granules, large basophilic (immature) granules and may have a single unsegmented nucleus.The basophilic granules are larger and more irregular, as well as more numerous, than those occasionally seen in immature eosinophils.The anomalous immature granules are mostly evident at the late promyelocyte and myelocyte stages; however, they tend to persist in some band and segmented forms.Unlike normal eosinophils, these cells stain with chloroacetate esterase and periodic-acid Schiff.The peripheral blood is not different from that in other cases of acute myelomonocytic leukemia; eosinophils are not increased in the peripheral blood.AML with inv(16)(p13.1q22)or t(16;16)(p13.1;q22) is associated with a high rate of complete remission and a favorable overall survival rate when treated with intensive induction and consolidation therapy.

Confirmation of Diagnosis
By conventional cytogenetic analysis, inv(16)(p13.1q22) is a subtle rearrangement that may be overlooked when metaphase preparations are suboptimal.In addition, occasionally, cytological features of AML with abnormal eosinophils may be present without karyotypic evidence of a chromosome 16 abnormality, but with CBFB-MYH11 nevertheless demonstrated by molecular studies such as a reverse-transcriptase-polymerasechain-reaction (RT-PCR) or fluorescent in situ hybridization (FISH), as shown in Figure 3. Therefore, RT-PCR and/or FISH for CBFB-MYH11 should be requested in AML cases (typically M4) with a proliferation of abnormal eosinophils.16)(p13.1q22).These micrographs, showing abnormal eosinophils containing both eosinophilic and basophilic staining granules, are representative of myelomonocytic leukemia with eosinophilia (May-Grünwald-Giemsa, ×1000).

Confirmation of Diagnosis
By conventional cytogenetic analysis, inv(16)(p13.1q22) is a subtle rearrangement that may be overlooked when metaphase preparations are suboptimal.In addition, occasionally, cytological features of AML with abnormal eosinophils may be present without karyotypic evidence of a chromosome 16 abnormality, but with CBFB-MYH11 nevertheless demonstrated by molecular studies such as a reverse-transcriptase-polymerase-chain-reaction (RT-PCR) or fluorescent in situ hybridization (FISH), as shown in Figure 3. Therefore, RT-PCR and/or FISH for CBFB-MYH11 should be requested in AML cases (typically M4) with a proliferation of abnormal eosinophils.

AML with t(8;21)
AML with t(8;21)(q22;q22) translocation may also present with marrow eosinophilia.The t(8;21)(q22;q22.1)involves RUNX1 (AML1), which encodes the alpha subunit of CBF, and RUNX1T1 (ETO): RUNX1-RUNX1T1 (AML1-ETO).The CBF transcription factor is essential for hematopoiesis; transformation by RUNX1-RUNX1T1 results from the transcriptional repression of normal RUNX1 target genes via the aberrant recruitment of nuclear transcriptional co-repressor complexes.AML with t(8;21) is included in the current WHO classification in the category of "acute myeloid leukemia with defining genetic abnormalities" [24].Solid tumor manifestations, such as myeloid granulocytic sarcoma, can be present at diagnosis.Although the mechanism of eosinophilia in AML with t(8;21) remains unknown, it has been shown that cultures with leukemic cells harboring t(8;21) can proliferate and differentiate into eosinophils in the presence of IL-5 [25].

Morphology
Most cases of t(8;21) are classified as M2 and rarely as M1 or M4, according to the FAB classification.Eosinophils and eosinophil precursors are frequently increased but they lack the cytological or cytochemical abnormalities seen in AML with inv(16) or t (16;16).Thus, cases of M2 with t(8;21) and marrow eosinophilia lack the abnormal granules seen in M4 with eosinophilia (Figure 4).An excess of basophils is commonly present in AML with t(8;21).Like AML with inv(16) and t(16;16), eosinophils are not increased in the peripheral blood.AML with t(8;21) is associated with a high complete remission rate and favorable overall survival when treated with intensive induction and consolidation therapy.

Confirmation of Diagnosis
Usually, t(8;21)(q22;q22) is readily detected by means of conventional cytogenetic analysis.RT-PCR and FISH may allow for an earlier detection of RUNX1-RUNX1T1 in cases of AML with an eosinophilic component without abnormal granules.

AML with ETV6-ABL1 (ΤΕL-ABL1)
The t(9;12)(q34;p13) involves the ETV6 (TEL) gene at 12p13, a transcription factor frequently rearranged in myeloid and lymphoid leukemias, and ABL1 at 9q34.The ETV6-ABL1 fusion gene leads to the activation of ABL1 kinase.ETV6-ABL1 is a notable rearrangement involved in AML with eosinophilia [26][27][28].AML with ETV6-ABL1 is included in the current WHO classification in the category of "myeloid/lymphoid neoplasms with An excess of basophils is commonly present in AML with t(8;21).Like AML with inv(16) and t(16;16), eosinophils are not increased in the peripheral blood.AML with t(8;21) is associated with a high complete remission rate and favorable overall survival when treated with intensive induction and consolidation therapy.

Confirmation of Diagnosis
Usually, t(8;21)(q22;q22) is readily detected by means of conventional cytogenetic analysis.RT-PCR and FISH may allow for an earlier detection of RUNX1-RUNX1T1 in cases of AML with an eosinophilic component without abnormal granules.
AML with ETV6-ABL1 is more common in men than women and occurs predominantly between the ages of 20 and 50 years.It generally has an adverse prognosis with a poor response to traditional AML therapy or imatinib [29,30].Recent reports suggest higher response rates and improved survival outcomes with second-generation tyrosine-kinaseinhibitor therapy, such as dasatinib and nilotinib, followed by allogeneic hematopoietic-cell transplantation [30,31].

Morphology
The ETV6-ABL1 fusion gene seems to have BCR-ABL1-like activity and, therefore, patients usually present with a picture that resembles atypical chronic myeloid leukemia (aCML) with marked peripheral-blood eosinophilia.However, this translocation has also been reported in AML (usually M1 or M2) with peripheral eosinophilia [27,32].The presence of peripheral eosinophilia and abnormal bone-marrow eosinophils with coarse eosinophilic and/or basophilic granules is the morphologic hallmark of this type of AML.

Confirmation of Diagnosis
The t(9;12)(q34;p13) translocation is very difficult to be identified by conventional cytogenetics since it might result from cryptic translocation or complex chromosomal translocations involving more than two chromosomes at more than two breakpoints, and usually requires FISH with a combination of ETV6 and ABL1 probes.However, there are cases in which FISH is not diagnostic (ETV6-ABL1 occult fusion) and that require targeted RT-PCR or next-generation sequencing techniques (e.g., RNA sequencing [RNAseq] or whole-genome sequencing [WGS] for the detection of cryptic rearrangements) to be identified [27].Other techniques such as spectral karyotyping (SKY) have also been used to detect abnormalities involving ETV6 otherwise missed by conventional karyotyping [33,34].

AML with PDGFRA, PDGFRB, and FGFR1 Rearrangements
These translocations create fusion tyrosine kinases with ligand-independent tyrosinekinase activity, leading to uncontrolled cell proliferation and the stimulation of downstream signaling pathways, including those involving phosphatidylinositol 3-kinase and mitogenactivated protein kinases that promote proliferation and survival.The clinical and hematological features are influenced by the partner gene involved.These disorders typically present as chronic myeloproliferative neoplasms with high-grade peripheral eosinophilia, but may also present as de novo AML.Cases of AML with PDGFRA, PDGFRB, and FGFR1 rearrangements are included in the current WHO classification in the broad category of "myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions" [24].

AML with PDGFRA Rearrangements
PDGFRA-related disorders usually present as hypereosinophilic syndrome (HES) of chronic eosinophilic leukemia (CEL).Rarely, they may present as AML with peripheralblood eosinophilia.The most common abnormality is a fusion of the Fip1-like 1 (FIP1L1) gene to the platelet-derived growth factor receptor α (PDGFRA) gene generated by an interstitial deletion on chromosome 4q12 between the two genes known as CHIC2 locus or LNX locus, with a resultant juxtaposition of FIP1L1 and PDGFRA [35].FIP1L1-PDGFRA encodes a constitutively activated tyrosine kinase that transforms hematopoietic cells.Because the deletion is small (~800 kb), it is not visible by conventional karyotype and should be sought after by FISH for CHIC2 or LNX deletion, as shown in Figure 5, or RT-PCR for the identification of FIP1L1-PDGFRA transcripts.Most patients with FIP1L1-PDGFRA are men (male/female ratio, 9:1) [9].PDGFRA translocations may rarely occur with other partners such as BCR and ETV6 [36].
blood eosinophilia.The most common abnormality is a fusion of the Fip1-like 1 (FIP1L1) gene to the platelet-derived growth factor receptor α (PDGFRA) gene generated by an interstitial deletion on chromosome 4q12 between the two genes known as CHIC2 locus or LNX locus, with a resultant juxtaposition of FIP1L1 and PDGFRA [35].FIP1L1-PDGFRΑ encodes a constitutively activated tyrosine kinase that transforms hematopoietic cells.Because the deletion is small (~800 kb), it is not visible by conventional karyotype and should be sought after by FISH for CHIC2 or LNX deletion, as shown in Figure 5, or RT-PCR for the identification of FIP1L1-PDGFRΑ transcripts.Most patients with FIP1L1-PDGFRΑ are men (male/female ratio, 9:1) [9].PDGFRA translocations may rarely occur with other partners such as BCR and ETV6 [36].Peripheral-blood morphology is useful in FIP1L1-PDGFRA myeloid neoplasms because it may show many abnormal eosinophils [10], including the following:
Recognizing AML with eosinophilia due to PDGFRΑ and PDGFRΒ rearrangement is important, because the aberrant tyrosine kinase activity can make the disease responsive to tyrosine kinase inhibitors such as imatinib.Therefore, we suggest that molecular genetic analysis (FISH and RT-PCR) for PDGFRΑ and PDGFRΒ should be carried out in cases of AML with peripheral eosinophilia.
Imatinib can be used effectively to treat patients carrying PDGFRA and PDGFRB translocations.The dosage ranges from 100 mg (for PDGFRA translocations) to 400 mg (for PDGFRB translocations) per day.The study by Metzgeroth and colleagues [49] reported encouraging results with imatinib in 17 patients with AML carrying these translo-
Recognizing AML with eosinophilia due to PDGFRA and PDGFRB rearrangement is important, because the aberrant tyrosine kinase activity can make the disease responsive to tyrosine kinase inhibitors such as imatinib.Therefore, we suggest that molecular genetic analysis (FISH and RT-PCR) for PDGFRA and PDGFRB should be carried out in cases of AML with peripheral eosinophilia.
Imatinib can be used effectively to treat patients carrying PDGFRA and PDGFRB translocations.The dosage ranges from 100 mg (for PDGFRA translocations) to 400 mg (for PDGFRB translocations) per day.The study by Metzgeroth and colleagues [49] reported encouraging results with imatinib in 17 patients with AML carrying these translocations: complete remission was achieved in all patients and the median overall survival was 88% at 65 months.Complete molecular remissions were achieved after a median of 5 months of imatinib treatment (range: .Other studies, however, have shown worse outcomes in AML with PDGFRA and PDGFRB translocations treated with imatinib, particularly in patients with additional cytogenetic (e.g., complex karyotype) or molecular abnormalities, who had a median overall survival of 12.5 months (range, 2-20) [35,[50][51][52].Treatmentemergent imatinib resistance was mediated by mutations in the PDGFRα or PDGFRβ kinase domain.Treatment of AML with PDGFRA and PDGFRB rearrangement should involve a combination of imatinib plus chemotherapy, with the consideration of allogeneic stemcell transplantation.In patients with high-grade peripheral eosinophilia, glucocorticoids should be used for 7-10 days in order to avoid rapid eosinophil degranulation and an associated risk of inflammatory cardiac injury after the initiation of imatinib, especially in those with a history of cardiovascular disease and/or elevated cardiac troponin levels [36].
The treatment of AML with FGFR1 rearrangement is far from standardized, owing to the poor results obtained with present-day therapy.First-and second-generation tyrosine kinase inhibitors have been used with suboptimal results [53].Recently, pemigatinib, an FGFR2 inhibitor used in patients with FGFR2-rearranged cholangiocarcinoma, received FDA approval for relapsed/refractory myeloid and/or lymphoid neoplasms with FGFR1 rearrangements.Allogeneic hematopoietic-cell transplantation is indicated for these patients.
These translocations may be identified by conventional cytogenetic analysis, but cryptic rearrangements require FISH or sequencing assays.The presence of +9/+9p chromosomal abnormalities in the context of AML with eosinophilia may be a clue for cryptic JAK2 structural rearrangements.Cases of AML with JAK2 rearrangements, e.g., PCM1-JAK2 resulting from t(8;9)(p22;p24.1),may respond to JAK inhibitors such as ruxolitinib.There have even been reports of treatment-free remission with ruxolitinib [54].Cases with FLT3 rearrangement may respond to FLT3 inhibitors.Allogeneic hematopoietic-cell transplantation seems to be the best choice for these patients.

The Authors' Recommendation for a Practical Approach to AML with Increased Eosinophils
It is not uncommon to find increased marrow eosinophils in patients with AML in the absence of peripheral-blood eosinophilia, particularly in patients with the myelomonocytic subtype.In many cases, this is a non-specific finding occurring in association with nonspecific cytogenetic lesions such as 7q deletion or normal karyotypes, but this finding may also indicate recurrent translocations with immediate therapeutic and prognostic relevance, such as CBFB-MYH11 or RUNX1-RUNX1T1.Of note, the likelihood of AML with CBFB-MYH11 is increased in the presence of abnormal eosinophils containing both eosinophilic and basophilic staining granules.Also, the myeloid blast phase of CML or BCR-ABL1-positive AML with an eosinophilic component should always be borne in mind, since about 5% of CML cases are diagnosed in the blast phase without a recognized chronic phase.Obviously, the identification of BCR-ABL1 has important therapeutic implications.Therefore, we recommend RT-PCR (or FISH) testing for CBFB-MYH11, RUNX1-RUNX1T1, and BCR-ABL1 in all cases of AML with increased eosinophils.
If these translocations are negative, there is peripheral eosinophilia, or there is a history of leukocytosis with eosinophilia, we recommend FISH for ETV6-ABL1, FIP1L1-PDGFRA, PDGFRB, or FGFR1 rearrangements.If FISH testing is negative but clinical suspicion is high, we recommend RT-PCR for FIP1L1-PDGFRA and ETV6-ABL1.In cases in which none of the tests described above are positive, additional testing with JAK2 FISH, FLT3 FISH, and RNAseq for cryptic translocations (if available) may be considered.

AML with Increased Basophils
In assessing leukemias with a prominent basophilic component, it is important to distinguish whether the basophils constitute a mature or immature population.The best example of the latter is acute basophilic leukemia, a separate subtype of AML in which the primary differentiation is to basophils.AML with t(6;9) is associated with basophilia in 42-62% of cases.Core-binding factor AML can also produce basophilia.Moreover, many myeloid cancers can transform to AML with hyperbasophilia, including CML, aCML, Ph-negative myeloproliferative neoplasms, and myelodysplastic syndromes.

Differential Diagnosis of Leukemias with Basophilic Granules (Table 2)
As mentioned, the presence of blast cells with dark basophilic granules should raise suspicion for acute basophilic leukemia.This condition should always be distinguished from the basophilic myeloid blast crisis of CML ("secondary acute basophilic leukemia"), as well as from AML with DEK-NUP214 and AML with RUNX1-RUNX1T1.It should also be differentiated from acute mast-cell leukemia.Acute promyelocytic leukemia (APL) should always be borne in mind when one sees a case of AML with blasts containing dark granules.

Basophilic Blast Phase of CML
In most cases of blast-phase CML, the blast lineage is myeloblastic, but may also be monocytic, megakaryocytic, erythroid, eosinophilic, or basophilic (or any combination thereof).Rarely, patients may present in the blast phase with a morphologic picture identical to de novo acute basophilic leukemia.Thus, RT-PCR for BCR-ABL1 should always be performed in any case of AML with a prominent basophilic component.
Basophils of the blast phase of CML may display dysplastic features such as reduced granulation [55] and tryptase production [56].The source of the basophilic blast phase of CML may be the CFU-Bas/Eo hybrid progenitor.IKZF1 mutations that produce either a loss of IKAROS or dominant negative isoforms have been described in CML lymphoblastic crises [57,58].The disruption of IKAROS activity in primitive CML cells mimics myeloid disease progression with enhanced STAT5 activation and shifted granulopoiesis to the basophilic lineage [59].4.1.2.AML with t(6;9)(p23;q34.1);DEK-NUP214 AML with t(6;9)(p23;q34.1)shows primarily myeloblastic or myelomonocytic differentiation (M2 or M4, according to the FAB classification).It is accompanied by marrow basophilia consisting of mature, dysplastic basophils in 42-62% of cases, and it is often associated with prominent multilineage dysplasia [60].It is included in the current WHO classification in the category of "acute myeloid leukemia with defining genetic abnormalities" [24].

Acute Mast-Cell Leukemia
Acute mast-cell leukemia is the leukemic variant of systemic mastocytosis in which peripheral-blood smears contain ≥10% mast cells and bone-marrow aspirates contain ≥20%.A diagnosis of aleukemic mast-cell leukemia is made if the percentage of mast cells in the peripheral-blood smear is <10% (Figure 8) [60].Unlike in indolent systemic mastocytosis, the mast cells in mast-cell leukemia are often round rather than spindleshaped.Some of these mast cells may exhibit bilobed nuclei ("promastocytes").Rare cases, in which the mast cells are mature-appearing and the clinical course less aggressive, constitute chronic mast-cell leukemia.
A strong expression of CD117 and positive staining for tryptase are of great diagnostic value in the diagnosis of acute mast-cell leukemia.Acute mast-cell leukemia may also express myeloid antigens, such as CD13 and CD33, and sometimes, CD203c, CD30, and CD38 [18].Typically, in systemic mastocytosis, the neoplastic mast cells show a dual expression of CD2 and CD25.In mast-cell leukemia, however, there is often a loss of one or both antigens: a loss of CD25 occurs in 25%, a loss of CD2 in 42%, and 30% of patients are negative for both CD2 and CD25 [64].The absence of C-KIT D816V is more common in CD2-and/or CD25-negative cases [64].Bone-marrow biopsies shows a diffuse, dense infiltration with atypical mast cells that are tryptase-positive.The majority of patients with mast-cell leukemia have no skin lesions.Acute mast-cell leukemia is an aggressive disease with poor prognosis (median overall survival is ≤12 months).Helpful features in distinguishing patients with acute basophilic leukemia from those with acute mast-cell leukemia are given in Table 3.
peripheral-blood smears contain ≥10% mast cells and bone-marrow aspirates contain ≥20%.A diagnosis of aleukemic mast-cell leukemia is made if the percentage of mast cells in the peripheral-blood smear is <10% (Figure 8) [60].Unlike in indolent systemic mastocytosis, the mast cells in mast-cell leukemia are often round rather than spindle-shaped.Some of these mast cells may exhibit bilobed nuclei ("promastocytes").Rare cases, in which the mast cells are mature-appearing and the clinical course less aggressive, constitute chronic mast-cell leukemia.The basophilic subtype of APL (subtype M3b, according to the FAB classification) was first described in 1982 as a "hyperbasophilic microgranular variant" of APL [65].It is characterized by neoplastic promyelocytes with unusual nuclear lobulation and a deeply basophilic cytoplasm containing few or no dark granules.The cytoplasm is scanty and may have cytoplasmic projections or blebs.Like the microgranular subtype of APL, CD2 is positive in the subtype with basophilic cells.In addition, APL with basophil differentiation, with blasts containing large basophilic granules, has been described [19,66].Aberrations of the 12p13 locus-in addition to t(15;17)(q22;q11-12)-have been described in M3b [19,66].Relapse of APL after all-trans retinoic acid (ATRA) therapy with M3b morphology may occur.APL with eosinophilic differentiation has also been described.Recognition of the less common, atypical forms of APL with basophilic blasts is important because of the unique response of this disease to retinoic acid and arsenic trioxide.

Acute Basophilic Leukemia
Although acute basophilic leukemia has long been recognized [67], it was not until the 2008 edition of the WHO classification of myeloid neoplasms that it became a distinct clinicopathologic entity classified within the category of AML, not otherwise specified.It is a very rare disease, accounting for <1% of AML cases [60].It is included in the current WHO classification in the category of "acute myeloid leukemia, defined by differentiation" [24].
The characteristic morphologic feature of acute basophilic leukemia is the presence of blasts carrying coarse basophilic granules (see Figure 9 for an example).
According to the current WHO classification [24], there are three diagnostic requirements for acute basophilic leukemia: (1) blasts and mature/immature basophils with metachromatic granules (as shown by toluidine blue stain); (2) blast cytochemistry negative for myeloperoxidase (MPO), Sudan Black B, and non-specific esterase (ANAE); and (3) the absence of strong CD117 expression.Valent and co-workers have proposed simpler diagnostic criteria for acute basophilic leukemia, including the presence of myeloid blasts and metachromatic blasts at ≥20% and basophils at ≥40% of the total nucleated bone-marrow or peripheral-blood cells.
On morphological grounds, it is not possible to make a differential diagnosis of acute basophilic leukemia from acute mast-cell leukemia, since blast cells with metachromatic granules are present in both disorders.Specific esterase (chloroacetate esterase [ChlorE]) staining is helpful in distinguishing basophilic from mastocytic granules.It reacts with mastocytic granules but does not react with basophilic granules [73].Electron microscopy, if available, is also helpful in distinguishing the lineage of the metachromatic blasts: the presence of Θ granules, i.e., electron-dense particles that carry a single fold of their membrane ("theta" character), is typical of basophilic differentiation.In contrast, mast cells carry four different types of granules (crystal-rich, with multiple membrane folds, soliddense granules, and non-dense granules) without a theta character [69].The expression of CD123 and/or CD11b indicates basophil differentiation, whereas tryptase and/or CD117 indicate a mast-cell origin.Markers of systemic mastocytosis, i.e., CD2 and CD25, are less helpful [64].
Owing to its rarity, little is known about cytogenetic lesions in acute basophilic leukemia.One abnormality occurring in male infants with acute basophilic leukemia is t(X;6)(p11;q23) [74].This translocation leads to MYB-GATA1 fusion, which disrupts the translational regulation by GATA1.Since male patients have only one copy of GATA1 on their X chromosome, GATA1-dependent cellular differentiation is completely abrogated.On the other hand, the chimeric protein shows great intracellular stability and retains MYB function.The MYB-GATA1 mutant protein is a positive transcriptional regulator of both interleukin 1 receptor-like 1 (IL1RL1) and NTRK1, which promote basophilic differentiation.Other chromosomal alterations that have been reported include t(16;21)(p11;q22), which creates the FUS-ERG fusion gene, and chromosomal aberrations of the 12p13 locus involving ETV6, e.g., 12p13 deletion [75,76].Chromosome 17 abnormalities, as well as mutations in TP53, TET2, and NPM1, have also been reported [77].
cause of the unique response of this disease to retinoic acid and arsenic trioxide.

Acute Basophilic Leukemia
Although acute basophilic leukemia has long been recognized [67], it was not until the 2008 edition of the WHO classification of myeloid neoplasms that it became a distinct clinicopathologic entity classified within the category of AML, not otherwise specified.It is a very rare disease, accounting for <1% of AML cases [60].It is included in the current WHO classification in the category of "acute myeloid leukemia, defined by differentiation" [24].
The characteristic morphologic feature of acute basophilic leukemia is the presence of blasts carrying coarse basophilic granules (see Figure 9 for an example).
Like other AML subtypes, patients with acute basophilic leukemia present with features related to bone-marrow failure and may or may not have circulating blasts.Skin involvement, hepatosplenomegaly, lytic lesions, and symptoms related to hyperhistaminemia may be present.Histamine, an autocrine and paracrine vasoactive hormone, is found in abundance in the metachromatic granules of basophils.Thus, erythematous cutaneous reactions are frequently seen in acute basophilic leukemia.Moreover, histamine promotes hydrochloric acid production in the stomach, causing peptic ulcers and gastritis.Lytic and osteoporotic lesions are also common (histamine affects osteoblasts, inducing RANKL expression, which directly activates osteoclasts [78]).Other symptoms suggestive of hyper-histaminemia include diarrhea, malabsorption, abdominal pain, marked bronchospasm, nausea, and migraine.Induction chemotherapy can worsen or elicit such symptoms as a consequence of the chemotherapy-induced cell lysis and the release of large amounts of histamine in the circulation.Serious complications of hyperhistaminemia during induction chemotherapy include anaphylactic shock, status asthmaticus, pulmonary edema, capillary leak syndrome, arrhythmias, heart failure, gastrointestinal bleeding, and hepatic and coagulation abnormalities [79][80][81][82].Whilst the anti-leukemic regimens do not differ from standard AML, it is crucial that patients with acute basophilic leukemia be given H1 and H2 inhibitors, proton-pump inhibitors (PPIs), and corticosteroids to abrogate or treat the effects or hyperhistaminemia.The cases observed have generally been associated with a poor prognosis due to both the occurrence of allergic reactions during induction chemotherapy and disease refractoriness.

Conclusions
In a small percentage of cases of AML, increased eosinophils and/or basophils are present in the cytologic material, usually in the bone marrow and sometimes in the peripheral blood.This is often a puzzling diagnostic situation that requires special investigation with RT-PCR and FISH for specific genetic abnormalities of clinical relevance.

Figure 1 .
Figure 1.Charcot-Leyden crystals in association with AML.These bizarre bipyramidal crystal structures were noted in the bone marrow aspirate smears of a 75-year-old woman with pancytopenia.

Figure 1 .
Figure 1.Charcot-Leyden crystals in association with AML.These bizarre bipyramidal crystal structures were noted in the bone marrow aspirate smears of a 75-year-old woman with pancytopenia.

Figure 3 .
Figure 3.An interphase fluorescence in situ hybridization (FISH) study with a dual-color breakapart probe set containing two probes flanking the breakpoint in the CBFB gene, showing a nucleus with inv(16)(p13.1 q22).An intact gene results in the colocalization of the two probes, producing a fusion (yellow) signal.The presence of CBFB rearrangement is indicated by separate red and green signals.

Figure 3 .
Figure 3.An interphase fluorescence in situ hybridization (FISH) study with a dual-color break-apart probe set containing two probes flanking the breakpoint in the CBFB gene, showing a nucleus with inv(16)(p13.1 q22).An intact gene results in the colocalization of the two probes, producing a fusion (yellow) signal.The presence of CBFB rearrangement is indicated by separate red and green signals.

Figure 5 .Figure 5 .
Figure 5. Detection of FIP1L1-PDGFRA in a case of AML with eosinophilia, using a three-color probe strategy.On interphase fluorescence in situ hybridization performed with the use of probes to LNX1Figure 5. Detection of FIP1L1-PDGFRA in a case of AML with eosinophilia, using a three-color probe strategy.On interphase fluorescence in situ hybridization performed with the use of probes to LNX1 (in red), FIP1L1 (in green), and PDGFRA (in aqua), the nucleus shows one chromosome with all three signals intact and another chromosome with intact FIP1L1 and PDGFRA signals but without the LNX1 signal (i.e., loss of red signal).This finding indicates a deletion of the region between FIP1L1 and PDGFRA on chromosome 4q12, consistent with the FIP1L1-PDGFRA fusion.
J. Clin.Med.2024, 13, x FOR PEER REVIEW 9 of 21 (in red), FIP1L1 (in green), and PDGFRA (in aqua), the nucleus shows one chromosome with all three signals intact and another chromosome with intact FIP1L1 and PDGFRA signals but without the LNX1 signal (i.e., loss of red signal).This finding indicates a deletion of the region between FIP1L1 and PDGFRA on chromosome 4q12, consistent with the FIP1L1-PDGFRA fusion.

Figure 7 .
Figure 7.A 70-year-old man with monocytosis and eosinophilia due to PDGFRB rearrangement.An interphase fluorescence in situ hybridization study with a dual-color break-apart probe set containing two probes flanking the sequence of PDGFRB gene showed that most cells had one fused signal and separate red and green signals, indicating disruption of the PDGFRB gene (a normal gene produces a colocalization, i.e., a yellow signal, whereas a rearranged gene results in two separate green and red signals).

Figure 7 .
Figure 7.A 70-year-old man with monocytosis and eosinophilia due to PDGFRB rearrangement.An interphase fluorescence in situ hybridization study with a dual-color break-apart probe set containing two probes flanking the sequence of PDGFRB gene showed that most cells had one fused signal and separate red and green signals, indicating disruption of the PDGFRB gene (a normal gene produces a colocalization, i.e., a yellow signal, whereas a rearranged gene results in two separate green and red signals).

Table 1 .
Subtypes of AML with increased eosinophils.

Table 2 .
Differential diagnosis of leukemias with prominent basophilic granules.

Table 3 .
Acute basophilic leukemia, as compared with acute mast-cell leukemia.