Tumor cells in LCH, often referred to as Langerhans cells, are typically large and oval or round. These cells are notable for their grooved-to-convoluted nuclei. The cytoplasm was usually eosinophilic and moderately abundant. These features are histologically distinctive and can aid in the diagnosis of LCH. The tumor cells display fine chromatin, a delicate nuclear membrane, inconspicuous nucleoli, and minimal atypia; however, mitoses may be frequent. The tumor microenvironment (TME) in LCH predominantly consists of eosinophils, lymphocytes, and occasionally multinucleated giant cells. Eosinophils are often a prominent feature, which is a notable aspect of LCH. Neutrophils and plasma cells are present but were generally less common (Fig. 2A-D). As the disease progresses or responds to treatment, the number of LCH cells may decrease and fibrosis may increase. In cases of central nervous system Langerhans cell histiocytosis (CNS-LCH), LCH-associated neurodegenerative diseases (LCH-ND) progressively develop in up to 10% of patients. In these cases, inflammatory infiltrates have been described, composed of foamy macrophages, CD1a + cells, and perivascular infiltrates of mononuclear cells expressing the BRAF V600E protein, along with neurodegeneration or demyelination [1–3, 5, 6, 23–26].

Langerhans cell sarcoma (LCS) was characterized by pleomorphic histiocytes with high-grade cytology, indicating an aggressive tumor (Fig. 2E and F). This tumor can occur either as a primary de novo LCS or as a secondary LCS, with progression from low-grade LCH or transdifferentiation from other types of hematolymphoid neoplasms, such as follicular lymphoma or chronic lymphocytic leukemia. Tumor cells have large, atypical nuclei that are often plump or elongated, with clumped chromatin, high mitotic activity, and frequent atypical mitoses. Unlike LCH, LCS typically lacks characteristic nuclear grooves and shows variable eosinophil infiltrates [1–3, 5, 27, 28].

Indeterminate dendritic cell tumors (IDCT) are characterized by the proliferation of mononuclear histiocytes resembling Langerhans cells. These cells feature grooved nuclei and ample eosinophilic cytoplasm and express dendritic cell markers such as CD1a and S100. However, they lack Birbeck granules and do not express CD207/langerin. In skin lesions, which typically manifest as generalized skin eruptions, tumor cells infiltrate the dermis and/or subcutis, while sparing the epidermis. Inflammatory infiltrates were observed in the TME. The mixed inflammatory cells include eosinophils and lymphocytes, as well as reactive macrophages, and multinuclear giant cells [2, 3, 29, 30].

Interdigitating dendritic cell sarcoma (IDCS) is characterized by the proliferation of pleomorphic spindle-toepithelioid cells showing differentiation of interdigitating dendritic cells. The tumor cells display abundant eosinophilic cytoplasm and indistinct borders. Their nuclei range from spindle-to-ovoid shaped and may show occasional multinucleation, grooves, vesicular chromatin, and distinct nucleoli. IDCS grows in patterns of sheets, fascicles, whorls, or storiform patterns (Fig. 2G and H). In the TME, small lymphocytes and plasma cells are interspersed or aggregated throughout the tumor [1–3, 31].

Juvenile xanthogranuloma (JXG) typically manifests as non-encapsulated, well-demarcated, and localized lesions on the skin and/or mucosal surfaces. Extracutaneous or disseminated JXG has also been identified. In such cases, it may be classified into the ECD category of the L group, particularly when accompanied by MAPK-activating mutations, or classified as ALK-positive histiocytosis when carrying ALK immunoreactivity and/or ALK gene rearrangement. JXG lesions are composed of large, xanthomatous, and foamy histiocytes resembling dermal macrophages that do not show significant nuclear pleomorphism. Characteristic features included Touton giant cells, which are multinucleated histiocytes distinguished by their unique arrangement of nuclei and cytoplasm (Fig. 3A and B). JXG lesions often contain a combination of lymphocytes, eosinophils, plasma cells, neutrophils, and mast cells. Fibrosis may increase later, corresponding to the regression of the lesion [1–3, 5, 6, 32, 33].

ECD is a unique type of non-Langerhans cell histiocytosis, primarily characterized by multi-organ infiltration with distinctive histiocytes. Widespread involvement can result in various clinical manifestations. Therefore, bilateral symmetric involvement of the long bones, detected in almost all patients, is a hallmark of ECD. Additionally, cardiopulmonary involvement is frequent and infiltration of the perinephric, periaortic, CNS, and skin regions represent other characteristic clinical features. Diagnosis is based on these clinical and radiological features complemented by ECD histology. The histiocytes in ECD are bland, foamy, and lipid-laden and are often accompanied by small mononuclear histiocytes. The tumor microenvironment frequently contains giant Touton cells, along with small lymphocytes, plasma cells, and neutrophils. Such histology can resemble xanthogranuloma, making differentiation between these conditions challenging based solely on histological examination (Fig. 3C and D). Fibrosis is a common and sometimes predominant feature of ECD, potentially leading to the misinterpretation of the lesion as a reactive fibroinflammatory process rather than as a neoplastic condition. LCH may be admixed on the same biopsy [1–3, 5, 6, 34].

Rosai–Dorfman disease (RDD) is characterized by large histiocytes with round nuclei, prominent nucleoli, and abundant pale cytoplasm. Pleomorphisms, mitoses, and multinucleation were rarely observed. A notable feature of RDD is the marked sinus expansion in the lymph nodes, which is a key diagnostic criterion. Another hallmark is emperipolesis, which refers to the presence of intact hematopoietic cells within the cytoplasm of histiocytes. These cells include lymphocytes, plasma cells, neutrophils, and erythrocytes. In RDD, the tumor microenvironment is characterized by an abundance of polyclonal plasma cells accompanied by lymphocytes, neutrophils, and occasionally neutrophilic microabscesses. Capsular and stromal fibrosis, often extending into the perinodal soft tissue, is a characteristic of RDD. RDD frequently features an increased number of IgG4-positive plasma cells; therefore, differential diagnosis of IgG4-related disease is sometimes necessary. In cases of extranodal RDD, such as those involving the skin, bone, CNS, and head and neck, histiocytic clusters and a lymphoplasmacytic microenvironment created a light and dark pattern at low magnification (Fig. 3E and F). Emperipolesis can be subtle and a mix of foamy histiocytes and stromal fibrosis is often present, making diagnosis difficult. The presence of perivascular plasma cells is a helpful diagnostic indicator for extranodal RDD [1–3, 5, 6, 35, 36].

ALK-positive histiocytosis is a relatively rare subtype of istiocytosis, which are rare disorders characterized by the accumulation of histiocytes in various tissues. The histological features of this condition vary among cases. Its histopathology is characterized by large, oval, foamy, and spindle-cell histiocytes in variable proportions. Large oval histiocytes displayed irregularly folded nuclei and abundant eosinophilic cytoplasm. Multinucleation and emperipolesis are occasionally observed. Foamy histiocytes showed vacuolated cytoplasm with irregularly folded nuclei, sometimes admixed with Touton giant cells. Spindle histiocytes are characterized by fascicular or storiform growth patterns, elongated nuclei, and eosinophilic cytoplasm. As a diagnostic criterion, high-grade cytological atypia, including features such as pleomorphisms and mitoses, was lacking in the histiocytes (Fig. 3G and H). The tumor microenvironment predominantly comprises small lymphocytes and plasma cells [3, 6, 33, 37].

Histiocytic sarcoma is characterized by non-cohesive large cells with abundant eosinophilic cytoplasm, which is a hallmark of this tumor. For the definition of malignant histiocytes belonging to the M group, cytological atypia and prominent mitotic activity, including atypical mitoses, are considered critical diagnostic criteria. These cells grew in patterns of diffuse sheets with a sinusoidal distribution. The nuclei of the tumor cells were oval, grooved, and irregularly folded, featuring vesicular chromatin and distinct nucleoli (Fig. 3I-L). Variable numbers of reactive inflammatory and stromal cells are often admixed [1–3, 5].

Mature plasmacytoid dendritic cell proliferation associated with myeloid neoplasms (MPDCP) has been newly categorized in the 5th edition of the World Health Organization (WHO) classification, following recent studies that have demonstrated the clonal proliferation and neoplastic nature of plasmacytoid dendritic cells (pDCs). MPDCP is characterized by the accumulation of mature cells that exhibit a plasmacytoid morphology and is associated with an underlying myeloid neoplasm. Chronic myelomonocytic leukemia (CMML) is the most frequently associated underlying myeloid neoplasm, followed by acute myeloid leukemia (AML), myelodysplastic neoplasms, and myeloproliferative neoplasms. MPDCP is present in approximately 5% of the AML cases. These cells displayed round or oval nuclei with moderate amounts of amphophilic cytoplasm. A notable feature was the absence of mitotic figures, suggesting that the cells were not actively dividing. This characteristic aligns with the “mature” aspect of these cells. Within the bone marrow, MPDCP cells may be aggregated or interstitially scattered [3, 38–40].

Blastic plasmacytoid dendritic cell neoplasms (BPDCN) were newly classified within the category of histiocytic and dendritic cell neoplasms in the 5th edition of the WHO classification. This reclassification follows the recent concept that these tumors are derived from pDCs. BPDCN is characterized by the presence of immature blastic cells that exhibit plasmacytoid dendritic cell differentiation. Tumor cells exhibited cytological features of medium-sized immature blastic cells with scanty cytoplasm, eccentric round or irregular nuclei, fine chromatin, and one to several inconspicuous nucleoli. Mitotic figures vary and necrosis may be present. In the skin, which is one of the most commonly involved sites, the neoplastic infiltrate was typically centered in the dermis and extended to the subcutaneous tissue while sparing the epidermis and adnexal structures (Fig. 4A and B). In the lymph nodes, BPDCN predominantly involves the medullary and interfollicular areas. In the bone marrow, tumor cells infiltrate in diffuse and/or interstitial patterns [1–3, 41].

In follicular dendritic cell sarcoma (FDCS), tumor cells exhibit a spindle, ovoid, or epithelioid morphology, characterized by moderate amounts of eosinophilic cytoplasm and indistinct cell borders, contributing to their typical syncytial appearance. The nuclei were elongated with vesicular chromatin, thin nuclear membranes, and distinct nucleoli. Binucleated cells reminiscent of follicular dendritic cells may be present. The tumor cells grow in whorls, fascicles, syncytial sheets, and storiform arrangements. Occasionally, tumor cells exhibited prominent cytologic atypia characterized by multinucleation and pleomorphic nuclei (Fig. 4C and D). In TME, small lymphocytes and plasma cells are interspersed within the tumor or form aggregates [1–3, 42].

EBV-positive inflammatory follicular dendritic cell sarcoma (EBV + inflammatory FDCS) occurs almost exclusively in the liver and spleen. This tumor is characterized by the proliferation of spindle-shaped to oval cells, which show follicular dendritic cell differentiation, along with a rich lymphoplasmacytic infiltrate and a consistent association with EBV. The tumor cells displayed indistinct cell borders, scanty to moderate amounts of cytoplasm, and vesicular nuclei with small, centrally located, and distinct nucleoli. Binucleated cells were also observed. The nuclear atypia varied significantly. Typically, bland-looking spindle cells are admixed with overtly pleomorphic cells that exhibit enlarged, irregularly folded, or hyperchromatic nuclei. Occasionally, tumor cells mimicked Hodgkin Reed-Sternberg cells (Fig. 4E and F). Mitotic figures were rare, and necrosis and hemorrhage were often observed. Tumor cells are either dispersed or form loose, whorled fascicles within the tumor microenvironment, which are marked by a prominent lymphoplasmacytic infiltrate [1–3, 43].

Fibroblastic reticular cell tumors (FRCTs) consist of spindle-to-ovoid cells arranged in whorls, fascicles, or sheets with interspersed small lymphocytes. Nuclear pleomorphism varies and multinucleation may occur. FRCT shows overlapping morphological features with follicular dendritic cell sarcoma and interdigitating dendritic cell sarcoma, with spindle-to-oval cell shapes, indistinct cell borders, and vesicular nuclei. Although not pathognomonic, the presence of intercellular collagen fibrils is characteristic [1–3, 44].

LCH cells are typically positive for CD1a and show membranous staining. They also express the S100 protein in both the nucleus and cytoplasm and CD207/langerin, which presents as granular cytoplasmic staining. For differential LCH from other types such as indeterminate dendritic cell tumors, immunostaining for CD1a and CD207 should be included. In particular, CD207/langerin immunohistochemical staining is used as a surrogate marker for Birbeck granules, which are the ultrastructural hallmarks of LCH and are observed as cytoplasmic structures in electron microscopy. CD68 often exhibits Golgi dot-like staining. The Ki-67 proliferation index typically varies with indices of < 10%. BRAF-VE1, indicating BRAF V600E mutation, may be positive, and both cyclin D1 and p-ERK may be expressed in LCH cells as downstream markers of MAPK activation [1–3, 5, 45–49].

LCS displayed markers similar to those of LCH, including positivity for CD1a, S100, and CD207/langerin. These markers may exhibit focal expression, and the interpretation of immunohistochemistry should focus on the high-grade cells [1–3, 5, 45].

The tumor typically shows positivity for S-100 protein and CD1a but is negative for CD207/langerin [1–3, 5, 29, 30].

The tumor cells in this sarcoma were positive for S-100 protein, which highlights dendritic cell processes. Additionally, they express one or more hematolymphoid markers, including CD45, CD4, and CD43. Tumor cells also express fascin, CD68, and lysozyme. They are negative for Langerhans cell markers such as CD1a and langerin, follicular dendritic cell markers like CD21, CD23, and CD35, as well as other markers specific to certain neoplasms or melanoma [1–3, 31, 45].

JXG cells are typically positive for CD68, CD163, CD4, CD14, factor XIIIa, and fascin. The expression of factor XIIIa and fascin may indicate the origin of dermal macrophages. In a minority of cases, S100 protein may be expressed. CD1a, CD207/langerin, and ALK are negative in JXG. If CD163-positive macrophages show the BRAF VE1 mutation, adult patients should be evaluated for ECD. In children with BRAF V600E mutant LCH, this could indicate a lineage switch related to the primary disease [1–3, 6].

Cells in ECD typically express CD163, CD68, CD14, factor XIIIa, and fascin, and less commonly express S100 protein. These cells are negative for CD1a and CD207/langerin but may express p-ERK. Diffuse, strong cytoplasmic expression of the BRAF-VE1 suggests a BRAF V600E mutation, which should be confirmed with molecular studies [1–4, 6, 50].

RDD is characterized by cells positive for S100 protein, highlighting emperipolesis. The expressions of OCT2 and cyclin D1 are useful for diagnosis. The histiocytic markers CD68 and CD163 were also expressed. Tumor cells typically lack CD1a, CD207/langerin, and ALK. The expression of the BRAF V600E mutated protein is rare. For the differential diagnosis of RDD associated with IgG4-related disease, immunostaining for both IgG4 and IgG may be necessary [3, 5, 35, 51, 52].

Under this condition, the tumor histiocytes are, by definition, positive with a cytoplasmic pattern and rarely exhibit membranous or Golgi dot patterns, avoiding nuclear patterns. ALK expression may be weak or focal. At least two histiocytic markers, namely CD68, CD163, CD4, CD14, and lysozyme, are typically expressed. Other markers, such as fascin, factor XIIIa, S100 protein, cyclin D1, and OCT2, may also be positive. These cells usually lack CD1a, CD207/langerin, CD30, and the BRAF V600E mutated protein [3, 37].

Histiocytic sarcoma cells are positive for two or more histiocytic markers such as CD163, CD68, and lysozyme. S-100 protein may be expressed in a focal, patchy manner. They are negative for CD1a, CD207/langerin (Langerhans cell markers), CD21, CD35 (follicular dendritic cell markers), ALK, and a range of myeloid, melanocytic, epithelial, vascular, and specific mature B-/T- cell markers [1–3, 45].

In flow cytometry, MPDCP is detected when cells constitute ≥ 2% of non-erythroid nucleated cells in bone marrow and/or peripheral blood. MPDCP cells show positivity for CD123 (interleukin-3 receptor α-chain) and/or other mature plasmacytoid dendritic cell markers, such as TCF4, TCL1, CD303, and CD304, although aberrant loss of these markers may be detected. Additionally, aberrant expression of CD34, CD56, and TdT was observed in MPDCP cells. Notably, CD56 may be absent, expressed at low levels, or partially expressed. The Ki-67 proliferation index is usually low, particularly when MPDCP is associated with chronic myelomonocytic leukemia [3, 39, 40, 53, 54].

This neoplasm expresses CD123 and at least one other plasmacytoid dendritic cell marker such as TCF4, TCL1, CD303, or CD304, in addition to CD4 and/or CD56. Alternatively, it may express any of the three plasmacytoid dendritic cell markers in the absence of the expected negative markers of the lymphoid or myeloid lineage, such as CD3, CD14, CD19, CD34, lysozyme, and myeloperoxidase. CD34 is typically negative, and the Ki-67 proliferation index is high [1–3, 54–57].

The cells were positive for two or more FDC markers, including CD21, CD23, CD35, clusterin, CXCL13, and D2-40 (podoplanin). They are negative for S100 protein and lymphocyte-specific markers [1–3, 45, 58].

These cells express FDC markers, such as CD21, CD35, CD23, CXCL13, and D2-40, or, rarely, fibroblastic/myoid markers, such as smooth muscle actin. They are consistently positive for EBV-encoded small RNA (EBER), characterized by an elongated, bland-looking nuclear pattern and/or a large pleomorphic nuclear pattern [2, 3, 59].

The tumor was positive for cytokeratin, actin, and desmin, highlighting delicate cellular processes. It is negative for follicular dendritic cell markers (CD21, CD23, CD35, and CXCL13) and interdigitating dendritic cell markers (S100) [2, 3, 44].

LCH is primarily marked by mutations in MAPK pathway genes, most notably BRAF V600E as the primary mutation, followed by MAP2K1. The MAPK pathway plays a key role in cell division, differentiation, and survival. BRAF is a protein involved in the MAP cell signaling pathway, and BRAF V600E mutation leads to constitutive activation. Through the MAP pathway, phosphorylation and nuclear translocation of ERK occur, acting as a transcription factor for cell proliferation and survival. Mutations in MAPK pathway genes are present in approximately 85% of LCH cases, with the BRAF V600E mutation occurring in about 50–60% of cases [4, 26, 46, 65–68]. BRAF mutations are also correlated with high-risk LCH [46, 69]. MAP2K1 (or MEK1), the downstream kinase of BRAF, mutations are detected in approximately 20% of the cases and occur in a mutually exclusive manner with BRAF mutations [68, 70]. BRAF mutations can occur at sites other than the V600 residue and may also be found in other RAF kinases, such as ARAF. Additionally, alterations can occur in other components of the MAP signaling pathway upstream of BRAF, including NRAS, KRAS, and HRAS. These somatic activating mutations in the genes of the MAPK pathway have been detected in a mutually exclusive manner [4, 65, 71].

The “misguided myeloid differentiation model” for LCH ontogeny, which is associated with MAPK activation, proposes a differentiation in the origin of LCH based on the level of risk and systemic involvement. According to this model, high-risk multisystem LCH (multisystem with risk-organ involvement, MS-RO +) is believed to originate from driver mutations in multipotent hematopoietic progenitor cells existing in the bone marrow. These cells can differentiate into various myeloid cells, including dendritic cells and monocytes/macrophages. In contrast, low-risk LCH, both multisystem without risk organ involvement (MS-RO-) and single-system LCH, is thought to arise from driver mutations in circulating or tissue-resident DC-committed precursors [46, 72]. The BRAF V600E mutation plays a crucial role in LCH pathophysiology by disrupting cell migration, inhibiting apoptosis, and inducing oncogene-related senescence [3, 5, 66, 73, 74].

A subset of these cases shows mutations in the MAPK pathway, including alterations in the KRAS gene and, less frequently, the BRAF V600E mutation. In secondary LCS, clonality or molecular alterations may be shared with the original hematologic neoplasm [2, 3, 27, 28, 75].

Due to the rarity of indeterminate dendritic cell tumors and interdigitating dendritic cell sarcomas, comprehensive information specific to these tumors is limited. In indeterminate dendritic cell tumor, mutations in genes such as BRAF V600E are reported, albeit rarely [63]. Interdigitating dendritic cell sarcoma, on the other hand, is characterized by mutations in the MAPK pathway, including BRAF V600E, KRAS, NRAS, and MAP2K1 [63, 75].

JXG is characterized by alterations in the MAPK pathway, including activating mutations in MAPK21, NRAS, KRAS, CSF1R, BRAF, and NTRK1 fusions [3, 6, 68, 76].

ECD is characterized by mutations in several MAPK pathway genes, including BRAF V600E, ARAF, NRAS, KRAS, MAP2K1, and PIK3CA. The BRAF V600E mutation occurs in approximately 50–60% of ECD cases. Mutations in MAPK pathway genes, such as BRAF V600E and MAP2K1, have been detected in bone marrow progenitors, indicating that ECD ontogeny is associated with MAPK activation. The presence of these mutations in early progenitor cells highlights the significant role of MAPK pathway activation in ECD pathogenesis. Furthermore, mutations in other genes of the MAPK pathway, such as MAP2K1, ARAF, NRAS, and KRAS, have been observed in ECD, as well as in PIK3CA, which is part of the PI3K-AKT-activating pathway. Regarding prognosis, the implications of BRAF mutations in ECD have not shown differences from other alternative mutations, although BRAF mutations are associated with an increased risk of cardiovascular involvement [4–6, 60–62, 77, 78].

Although the mutation frequency is much lower than that in LCH, JXG, and ECD, RDD involves mutations in the MAPK/ERK pathway, encompassing genes such as KRAS, NRAS, MAP2K1, ARAF, CSF1R, and on rare occasions, BRAF V600E [6, 52, 62, 76].

ALK-positive histiocytosis is characterized by rearrangements in the ALK gene, with the ALK::KIF5B fusion being more commonly detected. Fusions with uncommon partners such as CLTC-ALK, TPM3-ALK, TFG-ALK, EML4-ALK, and DCTN1-ALK have also been identified. These ALK rearrangements lead to the activation of ALK kinase, which in turn activates several downstream signaling pathways crucial for cell growth and survival, including the MAPK pathway [33, 37].

Mutations commonly occur in the MAPK pathway in histiocytic sarcomas. These mutations often involve genes such as KRAS, BRAF V600E, NRAS, MAP2K1, and CSF1R [76, 79].

When associated with myeloid neoplasms, such as CMML, clonal tumor cells from MPDCP often exhibit mutations in the RAS pathway. In cases associated with AML, clonal MPDCP cells share a mutational landscape similar to that of CD34 + blasts [38, 40].

Blastic plasmacytoid dendritic cell neoplasm is characterized by the activation of the NF-κB pathway [80]. The E-box transcription factor TCF4 (known as transcription factor E2-2) is a critical regulator of BPDCN development [81].

Follicular dendritic cell sarcoma recurrently shows alterations in the NF-kB pathway and BRAF V600E mutations [82, 83]. Detailed molecular characteristics of EBV-positive inflammatory follicular dendritic cell sarcomas and fibroblastic reticular cell tumors remain limited owing to the rarity of these conditions.