Mast cells and the neurofibroma microenvironment.

Neurofibromatosis type 1 (NF1) is the most common genetic disorder with a predisposition to malignancy and affects 1 in 3500 persons worldwide. NF1 is caused by a mutation in the NF1 tumor suppressor gene that encodes the protein neurofibromin. Patients with NF1 have cutaneous, diffuse, and plexiform neurofibromas, tumors comprised primarily of Schwann cells, blood vessels, fibroblasts, and mast cells. Studies from human and murine models that closely recapitulate human plexiform neurofibroma formation indicate that tumorigenesis necessitates NF1 loss of heterozygosity in the Schwann cell. In addition, our most recent studies with bone marrow transplantation and pharmacologic experiments implicate haploinsufficiency of Nf1 (Nf1(+/-)) and c-kit signaling in the hematopoietic system as required and sufficient for tumor progression. Here, we review recent studies implicating the hematopoietic system in plexiform neurofibroma genesis, delineate the physiology of stem cell factor-dependent hematopoietic cells and their contribution to the neurofibroma microenvironment, and highlight the application of this research toward the first successful, targeted medical treatment of a patient with a nonresectable and debilitating neurofibroma. Finally, we emphasize the importance of the tumor microenvironment hypothesis, asserting that tumorigenic cells in the neurofibroma do not arise and grow in isolation.

Cutaneous neurofibroma cells with active YAP promotes proliferation of macrophages resulting in increased accumulation of macrophages by modulating CCL5 and TGF­ß1.

Cutaneous neurofibromas (cNFs) are present in the majority of patients with neurofibromatosis type 1 (NF1), and results in disfigurements of the body, which is associated with psychological distress. A hallmark feature of cNF is the infiltration of inflammatory cells, among which macrophages are an important component of the microenvironment. Loss of neurofibromin (Nf1) expression results in activation of the PI3K and MAPK signaling pathways; however, the therapeutic effects of specific inhibitors targeting these pathways are not satisfactory. The present study showed increased macrophage infiltration accompanied by activation of effectors of the Hippo signaling pathway. Additionally, it was shown that XMU­MP­1 enhanced macrophage accumulation, in vivo and in vitro, by elevating the levels of C­C motif chemokine ligand 5 (CCL5) and transforming growth factor (TGF)­ß1 expression. However, neither CCL5 nor TGF­ß1 ablation alone were able to effectively reverse the XMU­MP­1­induced upregulation of macrophage accumulation, whereas concurrent ablation of these two genes significantly decreased macrophage accumulation. EdU staining and flow cytometry suggested that activated Yes­associated protein 1 promoted proliferation rather than inhibiting apoptosis in macrophage cells, and this may underlie the increase in the accumulation of macrophages. Both CCL5/C­C motif chemokine receptor 5 and TGF­ß1/TGFß1 receptor served crucial roles in modulating macrophage proliferation, which ultimately contributed to macrophage accumulation. The function of the Hippo pathway in the development of cNF development and its potency as a therapeutic target merit further investigation.

Common antigen in meningioma-derived cell cultures.

Serums of patients with intracranial meningiomas reacted in immunofluorescence assays with cell cultures and tumor imprints prepared from human meningiomas. Antibody in these serums appears to be specific for antigens in meningioma tissue and shows some cross-reactivity with neoplastic tissue of glial origin.

Cell cycle dependency of tumor antigens.

There is now substantial evidence that suggests that tumor antigen expression is cell cycle dependent. This information has been obtained from a wide variety of tumor systems. The G1 phase of the cell cycle has been implicated by a number of investigators as the point of maximal antigen appearance. In this presentation, we are concerned with a human sarcoma-associated tumor antigen and its cell cycle-dependent appearance. Sarcoma-associated tumor antigen is a membrane antigen present on cultured human neurosarcoma cells (T2-cells). Although the underlying mechanisms responsible for cell cycle-dependent expression are unclear, several possible interpretations are offered.

Cell cycle dependency of human sarcoma-associated tumor antigen expression.

An analysis of cell cycle-dependent expression of tumor-associated antigen was performed on a human neurosarcoma cell line (T2 cells). The expression of sarcoma-associated tumor antigen on T2 cells was detected using test sera obtained from sarcoma patients; control sera were from patients with nonsarcoma neoplasias and from normal donors. Results indicate a progressive increase in the antigenic expression beginning in late mitosis and early G1 with maximum expression in mid-G1. Antigenic expression declines to minimum levels in S and G2-phase. Mechanisms responsible for this cycle-dependent fluctuation are presently unknown.

Expression of melanoma-associated antigens by normal and neurofibroma Schwann cells.

The cell surface antigen distribution on traumatic neuroma Schwann cells and neurofibroma Schwann-like cells was characterized using monoclonal antibodies that define melanoma-associated antigens. Immunofluorescence staining of cultured cells, immunoprecipitation of radioiodinated antigens from cells placed in short-term cultures, and immunoperoxidase staining of frozen tissue sections revealed most of the melanoma-associated antigens tested on traumatic neuroma and neurofibroma Schwann cells and on fetal and adult femoral nerve. The cross-reactivity of the antibodies with neural cells may reflect the common neural crest embryological origin of Schwann cells and melanocytes. Cell sorter analysis of neurofibroma cells using a monoclonal antibody directed against the melanoma nerve growth factor receptor resulted in cell cultures highly enriched for Schwann-like cells which may bear the genetic defect responsible for neurofibromatosis. The antigen detected by this monoclonal antibody is the neurofibroma nerve growth factor receptor and the antibody was a potent inhibitor of nerve growth factor binding to neurofibroma cells.

GFA protein reactivity in nerve sheath tumors: a polyvalent and monoclonal antibody study.

We studied glial fibrillary acidic (GFA) protein immunoreactivity in 30 schwannomas, including two intracerebral examples, 26 neurofibromas and 12 neuromas using the immunoperoxidase method with a polyvalent antiserum (PVAS) and three well-characterized monoclonal antibody (MAb) preparations. Twelve of the schwannomas, including both intracerebral tumors, two of the neurofibromas and none of the neuromas immunostained with PVAS. Except for one schwannoma, all the PVAS-positive tumors were positive with two of the MAb preparations. While both of the intracerebral schwannomas were positive with the third MAb, none of the extracerebral tumors were. Our results suggest that: 1) human nerve sheath tumors contain cells having polypeptides that share epitopes with GFA protein, but 2) these polypeptides differ from astrocytic GFA protein by at least one epitope, and 3) the location of the tumors in relation to the central nervous system may influence GFA protein immunoreactivity.

Pigmented dermatofibrosarcoma protuberans (Bednar tumor). A pathologic, ultrastructural, and immunohistochemical study.

Described by Bednar as a "storiform neurofibroma," pigmented dermatofibrosarcoma protuberans is a rare neoplasm accounting for approximately 1-5% of all cases of dermatofibrosarcoma protuberans (DFSP). The lesion commonly presents as an exophytic, multinodular neoplasm of the dermis or subcutaneous tissue. It occurs predominantly in blacks. The majority are located on the trunk, and the remainder are more or less equally distributed in the upper and the lower extremities and the head and neck. Microscopically the lesion is characterized by spindled cells arranged in a tight storiform pattern and admixed with a small population of melanin-containing dendritic cells. The dendritic cells are the primary feature distinguishing this lesion from conventional DFSP. Three cell populations are identifiable by electron microscopy. The majority of cells resemble fibroblasts. A second population of cells exhibits long slender cell processes partially or completely invested by basal lamina. The third population of cells, also invested by basal lamina, contains both melanosomes and premelanosomes. The histogenesis of this neoplasm remains controversial. Although Bednar considered these lesions as variants of neurofibroma, S-100 protein could not be identified, and this finding contrasts significantly from the description of conventional neurofibroma, which almost always contains this antigen. Follow-up information available in nine cases indicates that this lesion may recur locally. Although distant metastases were not observed in our material, complete excision in conjunction with close follow-up care is indicated for this neoplasm of probable intermediate malignant potential.

Immunohistochemical detection of epithelial membrane antigen in normal perineurial cells and perineurioma.

The use of epithelial membrane antigen (EMA) as an immunohistochemical marker for normal and neoplastic perineurial cells is described. Normal perineurial cells react strongly for this antigen, which is also expressed by the cells of perineurioma. Instead, neurofibromas and schwannomas only show some peripheral or entrapped layers of EMA-positive cells. In traumatic and Morton's neuromas, bundles of neural fibers are wrapped in layers of EMA-positive perineurial cells. Neurothekeoma and granular cell tumor show no EMA reactivity. The detection of an epithelial marker in perineurial cells is in agreement with the concept of a "perineural epithelium" and seems to support a common embryologic origin for the perineurial cell and the equally EMA-positive arachnoidal cap cell. The availability of an immunohistochemical marker for the perineurial cell provides an easy and convenient tool for the evaluation of the participation of this cell in a variety of pathologic processes.

What are the CD34+ cells in benign peripheral nerve sheath tumors? Double immunostaining study of CD34 and S-100 protein.

To determine whether CD34 expression in nerve sheath lesions was found in a unique cell population or in a subset of nerve sheath cells, we performed double immunohistochemical staining using a standard avidinbiotin complex method with 2 separate color developing systems. We studied 40 neurofibromas and 16 neurilemomas. All lesions strongly expressed S-100 in nuclei and cytoplasm. CD34 was detected in cells having ameboid dendritic cytoplasm present in greatest numbers in Antoni B zones of neurilemomas, myxoid zones of neurofibromas, at the periphery of lobules in both tumor types, and condensed in apposition to perineurium. The CD34+ cells also were detected in normal nerves. They were infrequent in Antoni A zones of neurilemomas. No dual S-100 and CD34 expression was seen. This double immunostaining confirms the presence of a CD34-reactive non-Schwannian cell type in these neural neoplasms. As the CD34+, S-100-negative cell population is present also in normal nerves and infrequently seen in the areas of cellular neoplastic Schwann cells, CD34+, S-100-negative cells in peripheral nerve sheath tumors most likely are nonneoplastic and may have a supportive function.

Local injection of recombinant human stem cell factor promotes human skin mast cell survival and neurofibroma cell proliferation in the transplanted neurofibroma in nude mice.

We studied the effects of stem cell factor (SCF) on human skin mast cell (HSMC) survival and the proliferation of neurofibroma (NF) cells in transplanted NF in nude mice. Small pieces of cutaneous NF from a patient with von Recklinghausen's disease were transplanted subcutaneously into nude mice. Recombinant human SCF (10 or 100 ng) was injected six or seven times around the NF transplantation sites over 11 days (i.e. every other day). The number of HSMCs was reduced in vehicle-injected NF compared to the amount present before transplantation. In contrast, NF-transplanted animals that were injected with SCF (10 or 100 ng) showed preservation of mast cell numbers in the tissue. Using computerized image analysis, mast cell size in SCF-treated NF transplants was significantly altered (larger at the 10 ng dose, and smaller at the 100 ng dose) compared with the size before transplantation or in vehicle-injected tissue. Furthermore, at the higher SCF dose (100 ng) PCNA-positive NF cells showed a significant increase. These results indicate that HSMCs in transplanted NF tissue retain their capacity to respond to SCF in vivo, and that SCF contributes to the regulation of both HSMC survival and size in cutaneous NF. In addition, activated HSMCs induced by SCF may be involved in the growth of cutaneous NF in von Recklinghausen's disease. Thus, this experimental model may be useful in the study of the cellular interactions between HSMCs and other stromal cells in cutaneous NF.

A novel population of cells in the peripheral blood of a cow with a neurofibrosarcoma.

An unusual population of leukocytes was observed in the peripheral blood of a cow with a large tumor burden, using flow microfluorimetry. This new population accounted for 50% of the total cells in the peripheral blood of this animal. These cells expressed the p150,95 molecule (bovine CD11c equivalent), identified by the monoclonal antibody C5B6, a molecule found on myeloid cells and activated lymphocytes. The new population did not express the pan T molecules BoCD2 (the bovine T11 equivalent), BoCD5 (the bovine CD5 equivalent) or surface IgM. Isolated peripheral blood mononuclear cells maintained in bulk culture were able to kill autologous tumor cells and BHV-1 infected A549 in an NK-like assay. In vitro cytotoxicity by cells cultured from the peripheral blood of this animal was augmented 2- to 4-fold by the addition of IL-2.

Immunohistochemical evaluation of Leu-7, myelin basic-protein, S100-protein, glial-fibrillary acidic-protein, and LN3 immunoreactivity in nerve sheath tumors and sarcomas.

The collective expression of five antigens produced in immature or mature myelin-producing glia was evaluated in nerve sheath tumors and spindle cell sarcomas with histologic features of schwannomas. Myelin-associated glycoprotein (Leu-7), myelin basic-protein (MBP), S100-protein, and, in most cases, glial-fibrillary acidic-protein (GFAP) and HLA-DR/Ia (LN3) immunoreactivity were evaluated immunohistochemically using commercially available antibodies on 53 benign nerve sheath tumors and 12 sarcomas. Leu-7 immunoreactivity was detected by a monoclonal antibody in 12 of 16 schwannomas, 12 of 20 neurofibromas, and 17 of 17 traumatic neuromas. No Leu-7 positivity was seen in the sarcomas. Distinct heavy MBP immunoreactivity, assessed using polyclonal antibodies, was identified only in all 17 traumatic neuromas. Extensive S100-protein positivity was seen in 15 of 16 schwannomas, 17 of 20 neurofibromas, and 17 of 17 traumatic neuromas. Extensive LN3 immunoreactivity was seen in Schwann cells of 50% of the nerve sheath tumors analyzed; however, it was also present in associated interdigitating reticulum cells; GFAP immunoreactivity was not detected. These data suggest that Leu-7 is an important marker of Schwann cell neoplasms, although it is not superior to S100 protein. Moreover, combined immunohistochemical evaluation of potential Schwann cell markers including Leu-7, MBP, GFAP, and LN3 using commercially available antibodies offers no advantage over analysis of S100-protein immunoreactivity alone.

Immunohistochemical study of a canine neurofibroma.

Accurate diagnostic approaches to differentiate peripheral nerve sheet tumours from others have not been firmly established. The aim of this case report was to diagnose neurofibroma using a combination of diagnostic imaging, histopathology and immunohistochemistry, which were applied to a canine neurofibroma arising in the left mandible. The tumour was surgically excised and examined histologically. Round or spindle cells, with elongated, dense and homogenous chromatin and pale cytoplasm typical of Schwann cells in an abundant fibromyxomatous stroma, with ruby collagen fibres were seen. Immunohistochemistry demonstrated that S-100 and vimentin were more than 70% positive. Neurofibroma may therefore be recognisable using markers such as S-100 and vimentin.

Immunohistochemical recognition of human nerve sheath tumors by anti-Leu 7 (HNK-1) monoclonal antibody.

Using relatively high dilutions of anti-Leu 7 monoclonal antibody and a four-step peroxidase-antiperoxidase (PAP) reaction in paraffin-embedded tissues, we tested the affinity of this antibody to the cells of 47 human nerve sheath tumors and 22 other tumors in which the differential diagnosis with nerve sheath neoplasms is known to arise. Of all the nerve sheath tumors studied 68%, including 80% of the schwannomas, contained anti-Leu 7-positive cells. All 22 non-schwannian neoplasms were entirely negative. Specimens of eight experimental malignant rat schwannomas were also negative for anti-Leu 7 antibody. Our findings suggest that anti-Leu 7 monoclonal antibody is a promising marker that may facilitate the differential diagnosis between human Schwann cell and non-Schwann cell neoplasms.

Identification and subcellular localization of a sarcoma-associated antigen(s) in a human cell line.

The present investigation was designed to demonstrate and identify the subcellular localization of sarcoma-associated antigen(s) (TAA) in an established human neurogenic sarcoma line (T cell line). Indirect immunofluorescence was used to screen sera from patients with sarcoma, nonsarcoma neoplasias, and from presumably normal blood bank donors. The positive sera were submitted to a series of absorptions to remove possible nonspecific cross-reacting antibodies. The target cells were then studied with electron microscopy, utilizing the indirect immunoperoxidase technique to determine the subcellular localization of the sarcoma-associated antigen(s). Findings indicate that sarcoma-bearing patients have circulating antisarcoma antibodies directed to antigen(s) localized at the surface membrane of T cells. These antibodies pertain to the IgM or IgG immunoglobulin class. The exact nature of the sarcoma-associated antigen(s) identified in this study is not known at the present time. However, the results obtained strongly suggest that these substances may represent a new type of tumor-associated antigen(s).