8 Peter F. Moore University of California, Davis, USA The term histiocyte has been used to generically describe cells of dendritic cell (DC) or macrophage lineage. Histiocytes differentiate from CD34+ stem cell precursors into macrophages and several DC lineages (Figure 8.1). Histiocytic diseases are far more prevalent in dogs than in cats (see later), and have been the subject of a recent review.1 The majority of canine histiocytic diseases involve proliferations of cells of various DC lineages. Intraepithelial DCs are also known as Langerhans cells (LCs). Canine histiocytoma and cutaneous Langerhans cell histiocytosis (LCH) originate from cells exhibiting LC differentiation (Table 8.1). Interstitial DCs occur in perivascular locations in many organs except the brain, although they do occur in the meninges and choroid plexus. The majority of histiocytic sarcomas originate from cells exhibiting interstitial DC differentiation. Furthermore, the reactive histiocytoses – cutaneous histiocytosis and systemic histiocytosis – arise from proliferation of activated interstitial DCs (Table 8.1). Dendritic cells that occur in T‐cell domains in peripheral lymphoid organs (lymph node and spleen) are known as interdigitating DCs. Interdigitating DCs in lymph nodes are composed of resident DCs and migratory DCs. The migratory DCs arrive in lymphatics from tissues and consist of LCs and interstitial DCs (Figure 8.2).2 Cytokines and growth factors that influence DC development include FLT3 ligand, GM‐CSF, TNF‐alpha, IL‐4, and TGF‐beta.2,3 Macrophage development from CD34‐positive precursors is influenced by GM‐CSF and M‐CSF. Blood monocytes can differentiate into either macrophages under influence of M‐CSF, or into DCs under the influence of GM‐CSF and IL‐4 (Figure 8.1).2,4,5 Figure 8.1 Histiocyte differentiation: development of dendritic cell (DC) lineages and macrophages. Horizontal bars indicate the key distinguishing cell markers. Macrophages commonly express CD11b or the alternate CD11d depending on tissue location. Table 8.1 Canine and feline histiocytic diseases a CD11c expected but not assessable in cats. b CD1a expected, not assessed to date. Strong expression in most cells; ± variable expression in a subpopulation of cells; (?) expression not yet assessed. Figure 8.2 Skin dendritic cells (DCs): location and migratory pathways. Skin DCs upregulate the chemokine receptor CCR7 and are directed to lymphatics where the ligand CCL21 is expressed on endothelial cells and also in the paracortex. Skin DCs join the interdigitating DCs of the paracortex. Dendritic cells are the most potent antigen‐presenting cells (APCs) for induction of immune responses in naive T cells. The development of canine‐specific monoclonal antibodies for functionally important molecules of DCs and macrophages has enabled their identification in canine tissues, especially skin.6,7 Dendritic cells in skin occur in two major locations: within the epidermis (LCs), and within the dermis, especially adjacent to postcapillary venules (dermal interstitial DCs).8 Canine DCs abundantly express CD1a molecules,6,9,10 which, together with major histocompatibility complex (MHC) class I and class II molecules, are responsible for presentation of peptides, lipids, and glycolipids to T cells. Hence, DCs are best defined by their abundant expression of molecules essential to their function as APCs. Of these, the family of CD1 proteins is largely restricted in expression to DCs in skin, while MHC classes I and II are more broadly expressed. The beta‐2 integrins (CD11/CD18) are critically important adhesion molecules, and are differentially expressed by all leukocytes. CD11/CD18 expression is highly regulated in normal canine macrophages and DCs. CD11c is expressed by LCs and interstitial DCs, whereas macrophages predominately express CD11b (or CD11d in hematopoietic environments such as the splenic red pulp and bone marrow) (Figure 8.1). A subset of dermal interstitial DCs also express CD11b.7,11,12 In diseased tissues these beta‐2 integrin expression patterns may be broadened. Langerhans cells and dermal interstitial DCs are also distinguishable by their differential expression of E‐cadherin (LCs are positive) and Thy‐1 (CD90) (dermal interstitial DCs are positive) (Figure 8.1). LCs localize within epithelia via E‐cadherin homotypic adhesion, with E‐cadherin expressed by epithelial cells.3 Migration of DCs (as veiled cells) beyond the skin to the paracortex of lymph nodes, where they join forces with interdigitating DCs, occurs following contact with antigen (Figure 8.2). Successful interaction of DCs and T cells in response to antigenic challenge also involves the orderly appearance of costimulatory molecules (B7 family – CD80 and CD86) on DCs, and their ligands (CD28 and CTLA‐4) on T cells.13,14 In situ DCs have low expression of MHC II and costimulatory molecules and are more receptive to antigen uptake. Migratory DCs upregulate MHC II and B7 family members and become more adept at antigen presentation to T cells.14 Aspects of the developmental and migratory program of DCs are recapitulated in the section on Canine histiocytic diseases. Defective interaction of DCs and T cells may contribute to the development of reactive histiocytoses (cutaneous or systemic), which are related interstitial DC disorders that likely occur in the context of disordered immune regulation.15 The distant migratory potential of DCs is of immense clinical significance in the adverse prognosis of histiocytic sarcomas, which largely originate in interstitial DCs and rapidly disseminate via metastasis. To classify hematopoietic and lymphoid neoplasia according to the World Health Organization (WHO) system as applied to the domestic species, it is important to have access to markers for immunohistochemical (IHC) analysis.16,17 Determination of cell lineage is a critical problem in classification of leukocytic neoplasia. There are many useful antibodies available for immunophenotyping of leukocytic diseases in formalin‐fixed, paraffin‐embedded (FFPE) tissue sections (Table 8.2). Many other important cellular differentiation molecules are only detectable in frozen sections and unfixed cell smears or imprints. In the instance of histiocytic disorders, some definitive cell differentiation markers, such as CD1a, CD11c, CD11b, CD4, CD80, and CD86 are not assessable in formalin‐fixed tissue sections. Advanced planning is necessary to assess the expression of these molecules. Fresh unfixed tissue samples must be snap‐frozen in an appropriate way so that high‐quality frozen sections can be obtained for IHC. Alternatively, a sufficient number of unfixed cytological preparations could be made and stored desiccated or better in heat‐sealed tubing, and frozen at −80 °C until needed for IHC. These preparations can last indefinitely under these conditions (months to years). Table 8.2 Cell markers useful for diagnosis of leukocytic proliferative diseases in formalin‐fixed tissues of dogs and cats BD, BD‐Pharmingen; EB, EBioscience; D, Dako; LV, Labvision; S, AbD‐Serotec; SB, Spring Bioscience; T, Transgenic; UCD, Leukocyte Antigen Biology Laboratory, UC Davis; W, Wako; PC, polyclonal antibody. The importance of the macrophage scavenger receptors (CD163 and CD204) and ionized calcium‐binding adapter molecule 1 (Iba1) in the identification of histiocytes in proliferative diseases has been the subject of recent papers.18–20 Fortunately, these cell lineage markers are cross‐reactive in dogs and cats and are assessable in FFPE sections. Flow cytometry has been used to establish the cell lineage in canine leukemia, including dendritic cell leukemia.21,22 It is being increasingly used to determine cell lineage in lymphomas via fine‐needle aspirate biopsies. This technique is powerful (i.e., information rich), especially if combined with careful cytological or parallel histological assessment. There are several well‐defined histiocytic proliferative diseases that have been recognized in dogs. Canine cutaneous histiocytomas exhibit immunophenotypic features of LC differentiation, usually occur as solitary lesions in young dogs, and spontaneously regress. Cutaneous LCH covers a spectrum of disease, which may manifest with multiple cutaneous lesions, and the possibility of further spread to draining lymph nodes and internal organs. The metastatic variants of cutaneous LCH most often result in euthanasia. Individual skin lesions otherwise resemble those in histiocytoma. Hence cutaneous histiocytoma and cutaneous LCH could be viewed as members of a spectrum of a single disease with diverse biological behavior. The reactive histiocytoses – cutaneous histiocytosis and systemic histiocytosis – are related non‐neoplastic disorders arising from activated interstitial DCs. Reactive histiocytoses are interstitial DC and lymphocyte‐rich inflammatory disorders associated with a degree of immune dysregulation, which promotes lesion persistence. Lesions of the histiocytic sarcoma complex also arise from interstitial DCs, and occur as localized lesions in spleen, lymph nodes, lung, bone marrow, skin and subcutis, brain, and articular tissues of appendicular joints. Histiocytic sarcomas may occur as multiple lesions in single organs (especially spleen and skin), and rapidly disseminate to involve multiple organs. The disseminated form of the disease was formerly called malignant histiocytosis, which was believed to have a multicentric origin. Since it was difficult to demonstrate simultaneous sites of origin, malignant histiocytosis is now more appropriately termed disseminated histiocytic sarcoma. Histiocytic sarcomas mostly arise from interstitial DCs. However, the hemophagocytic form of histiocytic sarcoma is a distinctive clinicopathologic entity that arises from macrophages in splenic red pulp, and often simultaneously in bone marrow. Histiocytoma is a common, benign, cutaneous neoplasm of the dog. Histiocytomas usually occur as solitary lesions, and usually undergo spontaneous regression. The age‐specific incidence rate for histiocytomas drops precipitously after 3 years, although histiocytomas do occur in dogs of all ages.23 Epidermal invasion in histiocytoma, or presence of simultaneous multiple histiocytomas, especially in aged dogs, can present a diagnostic dilemma. Distinction from epitheliotropic and non‐epitheliotropic cutaneous T‐cell lymphoma using IHC for CD3 and CD18 is advisable, especially when epitheliotropism is marked (see Table 8.2 for interpretational issues).9,24,25 Histiocytomas most often occur as solitary cutaneous nodules with a distinctive dome‐like projection above the surrounding epidermis. Regression of solitary histiocytomas is a consistent feature. The factors that determine the onset of regression are unknown, but presumably involve migration of tumor‐infiltrating DCs to skin‐draining lymph nodes. These DCs would likely activate naive CD4+ T cells, which would assist in recruitment of CD8+ cytotoxic T cells to the cutaneous tumor site. Since massive CD8+ T‐cell infiltration is observed in all instances of advanced histiocytoma regression, therapeutic intervention with the aim of immunosuppression should be avoided once a definitive diagnosis of histiocytoma has been reached, in order to avoid interference with cytotoxic T‐cell function.9,26 The occurrence of a solitary histiocytoma, in which neoplastic histiocytes migrated to draining lymph nodes resulting in lymphadenomegaly and partial to complete obliteration, has been reported.9 Invasion of lymphatic vessels is usually observed within the cutaneous lesions and the lymph node infiltrate has the cytological properties of the cutaneous lesion. A total of seven histiocytomas that exhibited this behavior have been recognized (P.F. Moore, unpublished data). Complete regression of lesions occurred in a 1–2 month time frame in 4 of 5 dogs for which clinical follow‐up was available. Histiocytomas have a “top heavy” or epidermal focus. The histiocytic infiltrate is localized to the superficial and deep dermis, and may extend to the subcutis. Tumor histiocytes may display a range of cytological features. Nuclei may be round to oval, indented or complexly folded (convoluted) (Figure 8.3 inset). The mitotic count in histiocytomas is highly variable, but is often considerable. The cytoplasm is usually abundant and eosinophilic. Invasion of the epidermis by individual histiocytes or nests of histiocytes occurred in >60% of lesions in one case study (Figure 8.3).9 In a larger context, epidermal invasion is not quite as prevalent as in that report, but it is a diagnostically useful feature and should be carefully evaluated. Invasion of the epithelium of hair follicles does occur, but is much less common. Confusion of histiocytomas with cutaneous histiocytosis should not occur, since morphologically, histiocytomas are consistently epidermotropic and commonly epidermally invasive. These are not features of cutaneous histiocytosis, which is not focused on the epidermis. Instead the bulk of the lesion is formed by coalescence of perivascular infiltrates in the deep dermis and subcutis. Figure 8.3 Canine cutaneous histiocytoma. Histiocytes possess round to ovoid, or indented to twisted and folded nuclei. The cytoplasm is moderately abundant and mitotic figures are numerous despite typical benign behavior. Histiocytes form intraepidermal nests, which are observed to a degree (extreme in this example) in many histiocytomas. Epitheliotropic T‐cell lymphoma must be considered when nests like these occur. Human LCs characteristically possess Birbeck’s granules, an ultrastructural cytoplasmic structure that is associated with LC differentiation. Electron microscopic evaluation of histiocytomas has provided evidence of histiocytic lineage with ultrastructural features most consistent with DCs.27–29 However, Birbeck’s granules have not been demonstrated in canine histiocytomas, and are not present in normal canine LCs.30 Birbeck’s granules are present in LCs of cats, and were demonstrated in the infiltrating histiocytes in feline pulmonary LCH.31,32 Birbeck’s granules are formed by a process involving internalization of the cell surface C‐type lectin, langerin (CD207). The C‐type carbohydrate recognition domain (CRD) has conserved amino acid residues that create the principal calcium‐binding site, which is necessary for mannose binding. Variations in the amino acid sequence in the CRD alter its conformation and negatively impact mannose binding.33 There are several structural issues with canine langerin that may negatively affect ligand binding.1 Hence, the lack of Birbeck’s granules in canine LCs and in canine LC disorders may be traceable to a nonfunctional langerin molecule. This hypothesis awaits experimental verification. Histiocytomas in regression may have very few recognizable tumor histiocytes (best demonstrated by E‐cadherin staining) amidst a marked lymphocytic infiltrate dominated by T cells and reactive histiocytes (mostly interstitial DCs and macrophages). This has led to misdiagnosis of non‐epitheliotropic T‐cell lymphoma in some instances. Molecular clonality analysis of the T‐cell receptor locus (TRG) would be expected to resolve this issue. However, clonal T‐cell expansions have been documented in association with the cytotoxic T‐cell response to histiocytomas (about 50% incidence) (B. Gericota, V. Affolter, and P.F. Moore, unpublished observations). Clinical history and follow‐up are very useful in resolving this dilemma. Histiocytoma is usually diagnosed from routine cytologic or histologic slides. If there is concern that a lesion may not be a typical histiocytoma based on clinical or pathologic evidence, then IHC is indicated. Definitive diagnosis of histiocytoma can be strengthened by IHC, which is best performed on frozen sections of tumor or cytological preparations. Histiocytomas are readily distinguishable from other histiocytic disorders and cutaneous lymphoma with the aid of IHC. Histiocytomas have the immunophenotype of epidermal LCs and are considered a localized epidermal LC tumor.9 They express CD1a, MHC II, CD11c, CD18, and often E‐cadherin (Figure 8.4). The latter two molecules are assessable in sections of formalin‐fixed tissue (Table 8.2). Among skin leukocytes, E‐cadherin expression is largely restricted to LCs. Langerhans cells utilize E‐cadherin to localize in the epidermis via homotypic interaction with E‐cadherin expressed by keratinocytes. E‐cadherin expression has only rarely been observed in histiocytic sarcoma in canine skin and subcutis. A recent report challenges the specificity of E‐cadherin for diagnosis of cutaneous round cell tumors.34 However, if membranous expression of E‐cadherin is observed with or without cytoplasmic expression, then the specificity is much improved (P.F. Moore, unpublished observations). Figure 8.4 Canine cutaneous histiocytoma. Keratinocytes and histiocytes express E‐cadherin. E‐cadherin expression is noticeably membranous in keratinocytes and histiocytes. The significance of cytoplasmic E‐cadherin expression without membranous expression is dubious. Simply stated, E‐cadherin is a lineage‐associated marker but not a lineage‐specific marker for LCs. The staining pattern of E‐cadherin in many lesions is not uniform; E‐cadherin expression may be limited to the histiocytic infiltrate immediately adjacent to the epidermis. Histiocytomas lack expression of CD90 (Thy‐1), which is a marker of interstitial DCs in the dermis. They also lack expression of CD4 (frozen sections only), which is upregulated by activated normal DCs. In contrast to histiocytes in histiocytoma, histiocytes in cutaneous histiocytosis and systemic histiocytosis express both CD4 and CD90 and have the phenotype of activated interstitial DCs. The vast majority of histiocytomas lack expression of the macrophage scavenger receptors (CD163 and CD204).20 All histiocytomas assessed expressed Iba1, which is expressed by macrophages and DCs.19 The occurrence of multiple lesions with morphological characteristics of histiocytoma is uncommon. This spectrum of disease best fits under the umbrella of cutaneous LCH, since skin is invariably involved. Cutaneous LCH is well documented in humans.35 Dogs may have literally hundreds of cutaneous lesions ranging from nodules to masses, which elevate the epidermis and may be accompanied by redness and alopecia (Figure 8.5). Large lesions commonly ulcerate. Lesions at mucocutaneous junctions and in tissues of the oral cavity may also occur; these sites are also commonly involved in cutaneous lymphoma. Lesions may be initially limited to skin, or involve skin and draining lymph nodes. Rarely, internal organ involvement also occurs. Figure 8.5 Canine cutaneous Langerhans cell histiocytosis. At initial presentation there were red raised plaques that were present for 18 months. Lesions later coalesced and grew rapidly. (Image courtesy of Jessica Larson.) Cutaneous LCH limited to skin is more common in shar‐pei dogs, but can occur in any breed. Delayed regression of cutaneous LCH can occur and lesions may persist for many months before onset of regression. In about 50% of instances, dogs with cutaneous LCH are euthanized due to lack of regression of lesions and complications in management of the extensive ulcerated lesions that are often present (P.F. Moore, unpublished observations). Cutaneous LCH with lymph node metastasis has an even poorer prognosis, since spontaneous regression usually does not occur, and the vast majority of dogs with clinical follow‐up were euthanized (P.F. Moore, unpublished observations). Cutaneous LCH may spread beyond skin and draining lymph nodes to involve internal organs. This is a more serious and rapidly progressive disease. Peripheral lymph nodes and lung are consistently affected. Beyond this, metastatic disease can be widespread. This spectrum of disease manifestation and diverse clinical behavior is most like cutaneous LCH of humans.35,36 The important diagnostic features of cutaneous LCH are for the large part clinical (e.g., numerous cutaneous lesions), but in aggressive cases there is a much higher incidence of cytologic atypia. The lesions of cutaneous LCH in the skin closely resemble those of cutaneous histiocytoma. Individual lesions are nodules or masses that elevate the epidermis. The histiocytic infiltrate is heavily focused on the superficial dermis and epidermis (Figure 8.6
Canine and Feline Histiocytic Diseases
Histiocytic differentiation and canine histiocytic diseases
Disease
Species
Cell of origin
Immunophenotype (distinctive)
Histiocytoma
Dog
Langerhans cell
CD1a+, CD11c/CD18+, E‐cadherin+, CD204−, Iba1+
Cutaneous LCH
Dog
Langerhans cell
CD1a+, CD11c/CD18+, E‐cadherin+, CD204−, Iba1+
Cutaneous histiocytosis
Dog
Activated interstitial DC
CD1a+, CD4+, CD11c/CD18+, CD90+, CD204‐, Iba1+
Systemic histiocytosis
Dog
Activated interstitial DC
CD1a+, CD4+, CD11c/CD18+, CD90+, CD204(?), Iba1+
Histiocytic sarcoma
Dog, cat
Interstitial DC
CD1a+, CD11c/CD18+, CD204±, Iba1+
Histiocytic sarcoma – hemophagocytic
Dog, cat
Macrophage
CD1a±, CD11d/CD18+ (dog), CD204+, Iba1+
Feline progressive histiocytosis
Cat
Interstitial DC
CD1a+, CD11a/CD18+, CD5+ (50%), CD204±, Iba1+
Pulmonary Langerhans cell histiocytosis
Cat
Langerhans cell
CD1a+,b CD18+, E‐cadherin+, CD204(?), Iba1(?)
Immunophenotyping in hematopoietic neoplasia
Cell marker
Clone (source)
(species)
Description
CD3ε
CD3–12 (S) (UCD)
(dog and cat)
Expressed on the cell surface as a signaling component of the T‐cell antigen receptor complex in both αβ T cells and γδ T cells. Expressed in the cytoplasm of activated NK cells
CD79a
HM57 (S) (D)
(dog and cat)
Expressed on the cell surface as a signaling component of the B‐cell antigen receptor complex. Expressed by all stages of B cell differentiation. Expression is reduced in plasma cells
CD20
Rabbit PC (LV)
(dog and cat)
Surface molecule expressed at all stages of B‐cell differentiation. CD20 expression is reduced or lacking in plasma cells. Plasma cells in cats appear to retain considerable CD20 expression. CD20 is not lineage specific and expression has been observed in T‐cell lymphomas occasionally. Surface expression of CD20 is specific. Caution is advised in interpretation of diffuse cytoplasmic expression, which can occur in many cell types
Pax5
Clone 24 (BD)
(dog and cat)
Transcription factor essential for maintenance of B‐cell differentiation. Useful B‐cell marker
MUM1/IRF4
MUM1p (D)
(dog and cat)
Transcription factor essential for plasma cell differentiation. Useful plasma cell marker. MUM1 is expressed by a subset of large B‐cell lymphomas
CD11d
CA18.3C6 (UCD)
(dog)
αD subunit of β2 integrin (CD18) family. CD11d is expressed by macrophages, especially in hematopoietic environments – splenic red pulp, bone marrow, and lymph node medullary sinuses. Some T cells (especially in splenic red pulp) express CD11d. CD11d is consistently expressed in diseases emanating from splenic red pulp (LGL form of chronic lymphocytic leukemia; hepatosplenic lymphoma, and hemophagocytic histiocytic sarcoma)
CD18
CA16.3C10 UCD)
(dog)
FE3.9 F2 (UCD)
(cat)
β subunit of the β2 integrin family of leukocyte adhesion molecules. CD11a, CD11b, CD11c, or CD11d constitute the α subunits, which are expressed as heterodimers with CD18. Leukocytes express at least one form of the heterodimer. Hence CD18 is expressed on all leukocytes ‐ the expression level on myeloid cells is especially high compared to normal lymphocytes. CD18 has been used as a histiocytic marker, but this is dependent upon exclusion of lymphocyte differentiation by the use of other markers (CD3, CD20 and CD79a)
CD45
CA12.10C12 (UCD)
(dog)
Surface molecule expressed by all leukocytes – formerly known as “leukocyte common antigen.” Antibodies to CD45 bind to the extracellular domain outside of the 3 variably spliced exons (A, B, and C)
CD45RA
CA21.4B3 (UCD)
(dog)
Splice variant of CD45 in which exon A is expressed. B cells and naive T cells express CD45RA. Not typically expressed by histiocytes
CD90 (Thy‐1)
CA9.GA11 (UCD)
(dog)
Cell surface molecule with broad cell and tissue distribution. CD90 is expressed by interstitial‐type DCs in skin, but not by LC
CD163
AM‐3 K (T) (dog and cat)
Cross‐reactive anti‐human antibody binds to class A scavenger receptors on macrophages. DCs do not usually express
CD204
SRA‐E5 (T) (dog and cat)
Cross‐reactive anti‐human antibody binds to class A scavenger receptors on macrophages. DCs do not usually express
Iba1
Rabbit PC (W) (dog and cat)
Cross‐reactive anti‐human antibody binds to ionized calcium‐binding adapter molecule‐1 on cells of histiocytic lineage (macrophages and DCs)
HLA‐DR
TAL.1B5 (D)
(dog)
Cross‐reactive anti‐human MHC class II antibody in dogs. Expression is broad, but it is expressed by interstitial DCs and LCs
c‐Kit
Rabbit PC (D)
(dog and cat)
Member of the receptor tyrosine kinase family (type III). Expressed by most hematopoietic progenitor cells and by mast cells. Expression level is high in high‐grade mast cell tumors
E‐cadherin
Clone 36 (BD)
(dog and cat)
Adhesion molecule expressed by epithelia and by some leukocytes. Especially useful in cutaneous round cell tumors to identify LC, indicative of cutaneous histiocytoma and LCH
Granzyme B
Rabbit PC (SB)
(dog and cat)
Serine protease located in the granules of cytotoxic T cells (CD8+) and NK cells. GrB is expressed at high levels in activated cytotoxic cells and leads to rapid target cell death by apoptosis. Mast cells may express GrB
Myeloperoxidase
CA25.7G1 (UCD)
(dog and cat)
Myeloperoxidase (MPO) is a lysosomal protein stored in the azurophilic granules of neutrophils (and monocytes). MPO is an important marker of myeloid differentiation
Ki‐67
20Raj1 (EB)
(dog and cat)
Cell proliferation marker (nuclear) expressed in all phases of the cell cycle except G0 and early G1. Excellent marker for determining the growth fraction of a cell population
CANINE HISTIOCYTIC DISEASES
Overview
Cutaneous histiocytoma
Microscopic features
Immunophenotypic features
Cutaneous Langerhans cell histiocytosis
Microscopic lesions
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