Franchi

The Mesenchymal Phenotype

Alessandro Franchi, M.D.

Section of Anatomic Pathology

Department of Surgery and Translational Medicine

University of Florence, Florence, Italy

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Introduction

The determination of the phenotype of neoplastic cells and of their line of differentiation in soft tissue neoplasms is not only essential for the diagnosis, but also to provide prognostic information and, in relation with the continuous development of new drugs, to assign patients to specific treatment groups. In daily practice, many soft tissue tumors are readily diagnosed by light microscopy alone, but still several lesions may require the use of ancillary techniques to define the line of differentiation and to make a definite diagnosis. Immunohistochemistry has progressively become the routine adjunct to light microscopy to solve differential diagnostic problems, and in some soft tissue tumors the identification of specific reciprocal chromosomal translocations may be of further aid. However, there are neoplasms that are equivocal at the light microscopic level and have no specific immunohistochemical or molecular markers, which may exhibit submicroscopic morphologic features sufficient to allow an accurate identification of their phenotype and classification.

The aim of this presentation is to discuss the role of electron microscopy in the identification of the several different mesenchymal phenotypes and its possible application to the diagnosis of soft tissue neoplasms.

Adipocytic phenotype

The key ultrastructural feature to identify adipogenic differentiation is the presence of cytoplasmic lipid droplets, typically not membrane-bound, which may be present in varying numbers in neoplastic cells. Lipid droplets can show a wide spectrum of sizes and densities, ranging from electron-lucent to very electron-dense. Electron-dense lipid droplets are indicative of a high content of unsaturated fatty acid and a high degree of unsaturation, while electron-lucent lipid droplets are quite common in deparaffinized specimens. Aside amorphous lipids, occasionally it is possible to observe lamellar lipids depicting myelin figures; this peculiar morphology is possibly due to lipid leaflets released during or before aldehyde fixation that are fixed and stained during osmication.

Overall, the contribution of electron microscopy to the identification of the adipocytic phenotype in soft tissue lesions is very limited, because most entities can be defined on the basis of their morphologic appearance, or in some occasions with the adjunct of immunohistochemical or molecular markers, such as MDM-2 and CDK4 in well differentiated/dedifferentiated liposarcoma, or the FUS-DDIT3 gene rearrangement in round cell liposarcoma [1]. The group of liposarcomas in which electron microscopy may result diagnostically helpful is that of pleomorphic liposarcoma, particularly in those lesions with vacuolated cells but devoid of clearly identifiable lipoblasts, or showing the expression of markers indicative of alternative lines of differentiation, such as myogenic or epithelial markers [2-4].

In pleomorphic liposarcoma neoplastic cells most frequently have a large irregular multilobulated nucleus or multiple nuclei and abundant cytoplasm containing lipid droplets, rough endoplasmic reticulum cysternae, primary and secondary lysosomes; discontinuous external lamina can be recognized [5-8]. In addition, ultrastructural examination may be useful to exclude the lipoblastic nature of multivacuolated pseudolipoblasts, which can be seen in several sarcomas, including myxofibrosarcoma and acral myxoinflammatory fibroblastic sarcoma. Indeed, fibroblastic cells showing slender pseudopodic cytoplasmic projections, which intersect each other, may produce a sort of honeycomb picture reminiscent of a pseudolipoblastic morphology. The higher magnification generally allows recognition that these spaces are indeed extracellular because they contain material with the same texture of the extracellular matrix.

Fibroblastic phenotype

The ultrastructural features indicative of fibroblastic differentiation include a spindle shape and the presence of a well developed rough endoplasmic reticulum, with branching, often dilated cysternae, containing finely granular secretory material. The cells are usually embedded in collagenous extracellular matrix. A further useful ultrastructural feature is the presence of collagen secretion granules in the Golgi apparatus, which is regarded as a marker of collagen production in fibroblasts, myofibroblasts, chondrocytes and osteoblasts [9]. Altogether, the recognition of these ultrastructural features, in absence of myofibroblastic, cartilaginous or osteoblastic differentiation, are the main criteria for the identification of fibroblastic differentiation [10].

Regarding the diagnostic aspects, the group of soft tissue fibroblastic tumors includes a wide range of benign and malignant lesions whose diagnosis is based in most cases on the recognition of cyto-architectural patterns in haematoxylin and eosin stained sections. There is no specific immunohistochemical marker to help in recognizing the fibroblastic phenotype, and vimentin expression, which is consistently found in these lesions, has no diagnostic value. A subset of benign and malignant fibroblastic tumors, including dermatofibrosarcoma protuberans and fibrosarcoma arising in dermatofibrosarcoma protuberans, solitary fibrous tumor, and some examples of myxofibrosarcoma and myxoinflammatory fibroblastic sarcoma express CD34 [11], and this may be helpful in their differential diagnosis. Specific chromosomal translocations have been recognized in infantile fibrosarcoma, in low grade fibromyxoid sarcoma, in dermatofibrosarcoma protuberans and in solitary fibrous tumor [12-13].

However, not infrequently a specific diagnosis is difficult to reach in less differentiated or high grade lesions, and the diagnosis of fibrosarcoma is considered as a diagnosis of exclusion, or such tumors are considered “undifferentiated” for the lack of specific markers. In such cases, electron microscopy may furnish positive definitive elements for the identification of the cell lineage. According to the literature and in our experience, ultrastructural examination can be useful to prove fibroblastic differentiation in subsets of fibrosarcomas with peculiar morphology and immunohistochemical profile, including high grade pleomorphic sarcomas. Currently, such sarcomas that do not show any apparent differentiation at the histologic level and do not consistently express immunohistochemical markers, except for the non-specific vimentin, are classified as undifferentiated pleomorphic sarcomas [14]. However, several studies have shown that pleomorphic cells in undifferentiated pleomorphic sarcoma (malignant fibrous histiocytoma of the former literature) have in most cases a fibroblastic phenotype at the ultrastructural level [15-18], and therefore the designation pleomorphic fibrosarcoma could be preferred [19].

Myofibroblastic phenotype

Myofibroblasts are considered modified fibroblasts which form in granulation tissue by acquiring cytoplasmic actin microfilaments, which impart contractility to the cell, and fibronexus junctions, which link the cell to the extracellular matrix [20, 21]. Myofibroblasts appear as spindle cells, with oval indented nucleus, and cytoplasm containing abundant rough endoplasmic reticulum and peripheral bundles of myofilaments with focal densities (stress fibers). Fibronexus junction is a specific cell-stromal attachment, in which intracellular actin myofilaments are co-linear with extracellular fibronectin filaments/fibrils, interacting at a localized cell surface inclination. This appears as an electron dense rigid linear structure, and should be distinguished from external lamina, which is not a feature of these cells. Other features of myofibroblasts include a moderately developed Golgi apparatus sometimes showing collagen secretion granules, gap junctions, and actin-associated nondesmosomal junctions. Immunohistochemically, myofibroblasts are typically positive for vimentin, alpha-smooth-muscle actin (typically localized in subplasmalemmal position), and less frequently for desmin. Another putative marker of the myofibroblastic phenotype is calponin, while h-caldesmon is absent [22].

Myofibroblasts are constituents of several reactive, benign and malignant soft tissue lesions, but the existence of true myofibroblastic sarcomas has long been debated. The current WHO classification scheme recognizes low grade myofibroblastic sarcoma and inflammatory myofibroblastic tumor in the group of intermediate (rarely metastasizing) fibroblastic/myofibroblastic tumors. Low grade myofibroblastic sarcoma preferentially involves the head and neck region, and shows fibromatosis-like histologic features, but with hypercellularity, presence of moderate cellular atypia and scattered mitotic figures. Inflammatory myofibroblastic tumor primarily affects children and young adults with involvement of viscera and soft tissues, and appears as a proliferation of spindle cells accompanied by a mixed inflammatory infiltrate. In such neoplasms, electron microscopy may be used to confirm the diagnosis and to differentiate these lesions from smooth muscle lesions. At the ultrastructural level, smooth muscle neoplasms show more widely distributed actin filaments, poorly developed rough endoplasmic reticulum, caveolae and external lamina.

Smooth muscle phenotype

At the ultrastructural level, smooth muscle cells are characterized by bundles of thin actin filaments with fusiform densities distributed within the cytoplasm, attachment plaques, subplasmalemmal caveolae and presence of fragments of external lamina [18, 26-28]. These features may be useful to diagnose poorly differentiated leiomyosarcomas, and to separate myofibroblastic differentiation from smooth muscle differentiation, a distinction that is often impossible based on morphologic and immunohistochemical grounds.

Skeletal muscle phenotype

In tumors the main features of rhabdomyoblastic differentiation include: (i) dense masses of tangled myofilaments, Z-discs of varying size and shape, with or without attached myofilaments, (ii) rudimentary sarcomeres, consisting of a Z-disc and associated actin and myosin filaments, and (iii) the so-called myosin/ribosome complexes—which are considered the earliest sign of rhabdomyoblastic differentiation—consisting of parallel arrays of rigid myosin filaments, admixed with numerous ribosomes, sometimes alternating with actin myofilaments [29].

Most rhabdomyoblastic tumors are readily diagnosed based on their morphology, expression of immunohistochemical markers, including cytoplasmic desmin and nuclear myogenin, or with the aid of molecular genetic (alveolar rhabdomyosarcoma). Electron microscopy retains a diagnostic role only in selected cases, particularly in pleomorphic rhabdomyosarcoma, where the immunohistochemical profile may be inconclusive.

Vascular endothelial phenotype

The ultrastructural profile of vascular endothelial cells includes the presence of numerous pinocytotic vescicles and abundant cytoplasmic intermediate filaments. A specific marker of endothelial cells of vascular endothelial cells (not lymphatic) is the Weibel-Palade body. This is a rod-shaped cytoplasmic structure measuring up to 1.6 m in humans, which consists of microtubules embedded in an electron dense matrix. Unfortunately, in our experience and according to the literature, neoplastic cells in poorly differentiated angiosarcomas do not contain Weibel-Palade bodies, and therefore their diagnostic significance is limited.

Chondro-osseous phenotype

Chondrocytes have an irregular contour for the presence of numerous cell surface projections. The cytoplasm contains rough endoplasmic reticulum cisternae, glycogen particles and lipid droplets. Metabolically active osteoblasts present abundant cytoplasm that contains rough endoplasmic reticulum cisternae, mitochondria, Golgi cisternae, and intermediate filaments. They are surrounded by osteoid matrix, which consists of type I collagen fibrils associated with electron dense calcified matrix. Ultrastructural analysis is of little or no use in the diagnosis of chondro-osseous tumours.

Nerve sheath phenotype

Schwann cells present elongated, relatively thick, overlapping or intertwining/interdigitating (complexly entangled) cytoplasmic processes, sometimes joined by occasional cell junctions, fragments of external lamina and pynocitotic vesicles[30, 31]. Other peripheral nerve constituents, such as perineural cells, are characterized by long thin parallel cell processes separated by stroma, showing numerous pinocytotic vesicles and a covering of interrupted basal lamina [32], or show ultrastructural features of fibroblasts.

Electron microscopy may furnish a relevant support in the diagnosis of malignant peripheral nerve sheath tumor (MPNST). MPNST has no reliable immunohistochemical marker, because S100 protein is often negative or focally positive, and no specific genetic alteration has yet been recognized. Therefore electron microscopy is often useful in the distinction of poorly differentiated lesions arising in patients not affected by Von Recklinghausen syndrome from other spindle cell sarcomas, like synovial sarcoma and adult type fibrosarcoma. MPNST is composed of a variable number of undifferentiated cells and cells showing at least partial Schwann cell differentiation.

Conclusions

Electron microscopy maintains a significant role in defining the cell lineage of soft tissue neoplasms, although more focused than in the past. In our experience, the group of sarcomas in which electron microscopy still maintains a relevant diagnostic role is that of high grade pleomorphic sarcomas. This group of tumors often shows non-specific cyto-architectural features and immunohistochemical markers may not definitively prove a specific phenotype. Conversely, ultrastructural analysis may allow the recognition of cellular constituents (contractile cytoskeleton, rough endoplasmic reticulum, lipid droplets, etc.) which may lead to the identification of cellular differentiation, thus helping to reduce the number of diagnoses of “undifferentiated” pleomorphic sarcoma. In addition, some cellular phenotypes, such as the fibroblastic and myofibroblastic, remain unrecognizable on the basis of immunohistochemistry, and electron microscopy furnishes the definitive proof of tumor cell lineage in these tumors. Occasionally, in tumours with smooth muscle, skeletal muscle, adipocytic, vascular endothelial and Schwann cell differentiation electron microscopy may also help in the differential diagnosis.

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