p53 Immunohistochemistry in malignant fibrous histiocytomas and other mesenchymal tumours

In this study we analysed by immunohistochemistry the expression of p53 protein in 14 malignant fibrous histiocytomas (MFHs), 22 other types of sarcoma (eight leiomyosarcomas, four rhabdomyosarcomas, four liposarcomas, two fibrosarcomas, two chondrosarcomas, one malignant schwannoma, and one dermatofibrosarcoma protuberans), and 25 non-malignant mesenchymal lesions (eight dermatofibromas, four cases of nodular fasciitis, three leiomyomas, three fibromatoses, two epithelioid leiomyomas, two neurofibromas, one schwannoma, one myositis ossificans, and one giant cell tumour of tendon sheath). Four MFHs and nine other types of sarcoma (four leiomyosarcomas, two chondrosarcomas, one liposarcoma, one fibrosarcoma, and one dermatofibrosarcoma protuberans) showed nuclear positivity for p53. Of the benign soft tissue lesions, p53 positivity was observed in two fibromatoses, one nodular fasciitis, and one dermatofibroma. The number of p53-positive cells in these benign lesions was considerably smaller than that in most of the p53-positive sarcomas. The p53 positivity in MFHs and other types of sarcoma indicates that p53 gene alterations may play a part in the neoplastic transformation of these tumours. The occurrence of p53 positivity in benign mesenchymal lesions suggests that sometimes p53 protein may accumulate in cells without an associated malignancy. Because of this, p53 immunoreactivity cannot, by itself, be used as a criterion of malignancy According to our results, p53 positivity in over 1 per cent of tumour cells in mesenchymal lesions favours malignancy.     

Expression of smooth muscle markers in so called malignant fibrous histiocytomas

AIMS: To obtain further information regarding the frequency and degree of positivity for smooth muscle markers in a large number of malignant fibrous histiocytomas (MFHs), as an aid to accurate diagnosis. METHOD: The immunohistochemical features of 100 MFHs were studied and the results were compared with those for 30 leiomyosarcomas. Eighteen cases of MFH with smooth muscle actin (SMA) positivity were examined ultrastructurally. RESULTS: Immunoreactivity for smooth muscle markers, such as desmin, SMA, muscle specific actin (MSA) and h-caldesmon (HCD), which is a specific marker for smooth muscle cells and their tumours, was found in 28, 30, 29, and 29 of 30 leiomyosarcomas. Immunoreactivity for desmin, SMA, MSA, and HCD was found in 17, 30, 14, and two of the MFHs. On electron microscopic examination, approximately half of the cases contained a varying proportion of myofibroblastic cells. The others had only fibroblastic or undifferentiated tumour cells. At least 30% of the cases were found to display features consistent with limited smooth muscle or myofibroblastic differentiation. CONCLUSION: A large subset of so called MFH in fact shows poorly differentiated smooth muscle or myofibroblastic features, and perhaps such tumours should be regarded as pleomorphic leiomyosarcomas and/or pleomorphic myofibroblastic sarcomas.     

Tumor cells of malignant fibrous histiocytomas express mRNA for laminin.

In situ hybridization was used on routinely processed paraffin-embedded tissue sections to study the synthesis of the basement membrane (BM) proteins laminin and type IV collagen in 14 cases of malignant fibrous histiocytoma (MFH). Complementary RNA probes coding for the pro-alpha 1 (IV) chain of human type IV collagen and the B1 chain of human laminin were used to detect the respective mRNAs. The results were correlated with the immunohistochemical reactivity of tumor cells to specific antibodies against the P1 fragment of laminin and the 7S domain of type IV collagen. Signals for the presence of laminin mRNA in atypical neoplastic tumor cells could be detected in 11 MFHs. None of the tumors could be shown to contain signals for type IV collagen mRNA in their cells, although such signals were detected in the endothelial cells of tumor capillaries. In the corresponding immunohistochemical stainings, nine MFHs showed intracytoplasmic staining of tumor cells for laminin and one tumor showed weak staining for type IV collagen in the neoplastic cells. The results show that the laminin immunoreactivity found in MFHs is due to synthesis in the tumor cells and not to endogenous uptake of this protein. Synthesis of laminin in the majority of MFHs is in accordance with the notion that these tumors originate from primitive mesenchymal cells in soft tissues.     

Expression of intermediate filaments in malignant fibrous histiocytomas

The expression of intermediate filaments (IFs) in 34 malignant fibrous histiocytomas (MFHs) was studied immunohistochemically and ultrastructurally. Using the avidin-biotin-peroxidase method, positive reactions were detected as follows: for desmin in 12 tumors, for neurofilament in two tumors, for cytokeratin in one tumor, and for vimentin in 30 tumors. Desmin immunoreactivity was found in tumors of all four histologic subtypes and cytokeratin immunoreactivity was found in one tumor of the myxoid type. Because of the cross-reactivity of anti-neurofilament antibody with reactive histiocytes, the immunoreactivity for neurofilament seemed to be non-specific. Ultrastructurally, five of 13 tumors studied contained some tumor cells showing myofibroblastic or smooth muscle cell differentiation. A few tumor cells in one cytokeratin-positive tumor had tonofilaments in their cytoplasm. Desmin expression in some MFHs seemed to be due to myofibroblastic or smooth muscle cell differentiation of some tumor cells. Cytokeratin expression seemed to indicate epithelial differentiation in some MFHs. This varied expression of IFs in MFHs may reflect the heterogeneous nature of MFHs, and suggests that MFHs represent the final stages of dedifferentiation of several different types of sarcomas or, alternatively, represent forms of poorly differentiated sarcoma with the potential of developing into more differentiated sarcomas of heterogeneous origin.     

Cellular differentiation in storiform-pleomorphic malignant fibrous histiocytomas. An electron microscopic study of histogenesis

The most common form of malignant fibrous histiocytoma is the storiform-pleomorphic subtype composed of spindle-shaped fibroblast-like cells, mononucleated histiocytic elements and a changing amount of pleomorphic giant cells. In relation to the changing cellular structures 14 pleomorphic-storiform malignant fibrous histiocytomas were investigated electronmicroscopically. In all tumors several types of cells varying in shape, and size as well as in organelle composition could be demonstrated: 1. Undifferentiated cells, which are relatively small and have a scanty cytoplasm with few organelles. 2. Fibroblast-like cells with well developed rough endoplasmic reticulum, mostly arranged in a storiform pattern. 3. Myofibroblasts corresponding to fibroblasts and showing bundles of thin filaments (4 to 6 nm) with focal dense bodies in the peripheral area of the cytoplasm. 4. Histiocyte-like cells characterized by filopodia-like projections and abundant cytoplasm containing lysosomes and phagolysosomes and also lipid droplets. 5. Chimeric cells, which are intermediate forms with features of fibroblast-like and histiocyte-like tumor cells. 6. Multinucleated tumor giant cells which can be subdivided into fibroblast-like and histiocyte-like types and intermediate forms. On the basis of our ultrastructural studies the storiform pleomorphic malignant fibrous histiocytoma is interpreted as a tumor of an undifferentiated mesenchymal cell with the potency of fibroblastic or histiocytic differentiation. The origin of this cell is uncertain. Dedifferentiation of a differentiated connective tissue cell (fibroblast, pericyte) into a proliferating undifferentiated precursor cell is discussed.     

Malignant schwannomas presenting as malignant fibrous histiocytomas

Two soft tissue neoplasms considered to represent malignant fibrous histiocytomas by light microscopy showed typical findings indicative of Schwann cell origin when examined by electron microscopy. These findings included the presence of cells exhibiting long interdigitating cytoplasmic processes, which frequently contained collections of microtubules and were surrounded by thin, frequently interrupted, basal lamina material. In addition, pinocytotic activity at the cell surfaces and immature junctions joining apposing cellular membranes were identified. No evidence of fibroblastic, fibrohistiocytic, or myofibroblastic differentiation was identified ultrastructurally in the neoplastic cells. Our findings stress the need for ultrastructural examination to adequately classify soft tissue sarcomas. The 2 cases presented illustrate that there are some schwannomas that may be inaccurately classified as malignant fibrous histiocytomas if only light microscopy is used. It seems that only by ultrastructural means is it possible to accurately classify these peculiar neoplasms.     

An analysis of 38 malignant fibrous histiocytomas in the extremities.

All the malignant soft tissue tumours in the extremities and limb girdles reported to the Finnish Cancer Registry between 1960-1969 were reviewed. From a total of 246 sarcomas, 38 were diagnosed as malignant fibrous histiocytoma. There was an equal number of male and female patients with the median age of 67 years. The thigh was the most frequent site, and the majority of the tumours originated in the deep soft tissues. The predominant treatment was excision followed by radiation therapy. In 17 patients there were one or more recurrences and in 21 patients there was a metastatic spread ascertained by biopsy, autopsy or clinical or radiographic evidence. There were 11 survivors with a minimum of 5 years' follow-up; seven patients died of an intercurrent disease and the remaining 20 patients were considered victims of their tumour. The findings that seemed to favour a poor prognosis were higher age and female sex of the patient as well as deep location, large size, necrotic areas, and high mitotic activity of the tumour.     

The ultrastructure of benign and malignant fibrous histiocytomas

Five benign histiocytomas of varying pattern and three malignant fibrous histiocytomas have been studied ultrastructurally. An essentially biphasic pattern of histiocytes and fibroblasts was present. In addition fibrohistiocytes, myofibroblasts and undifferentiated cells were present in some of the tumours. The histogenesis of this group of tumours is discussed. The value of electron microscopy in establishing the diagnosis in difficult cases is emphasized, but it is not considered useful in distinguishing benign from malignant cases.     

Experimental studies on malignant fibrous histiocytomas. II. Ultrastructure of malignant fibrous histiocytomas induced by 4-(hydroxyamino)-quinoline 1-oxide in rats

The ultrastructures of six subcutaneous and six bone malignant fibrous histiocytomas (MFH) induced in rats by local application of the carcinogen, 4-(hydroxyamino)-quinoline 1-oxide (4-HAQO) were studied. The MFHs could be classified histologically into three subtypes: of the six subcutaneous MFHs, four were fibrous, one was giant cell, and one was myxoid; of the osseous MFHs, three were fibrous, one was giant cell, and two were myxoid. Five different types of cells were found in the MFHs: fibroblast-like cells, histiocyte-like cells, undifferentiated cells, xanthomatous cells, and multinucleated giant cells; the xanthomatous cells and multinucleated giant cells, however, were probably derived from histiocyte-like cells. Fibroblast-like cells predominated in storiform areas of the fibrous subtype; histiocyte-like cells and undifferentiated cells predominated in the giant cell subtype; intermediate cells predominated in the myxoid subtype. Acid phosphatase activity was found in lysosomes and myelin figures of the histiocyte-like cells in fibrous type MFH. The giant cell subtype of bone MFH has been transplanted serially into syngeneic rats and is now at the 17th generation. Transplantability exceeded 80%; doubling time was 3.8 to 6.1 days. Until the 3rd generation, the histology of the original tumor was retained; from the 4th generation, however, giant cells and xanthoma cells were no longer observed, and the tumor was composed mainly of undifferentiated cells. These results indicate that (a) MFH induced in the rat by 4-HAQO have an ultrastructure similar to human MFH and (b) the giant cell subtype transplanted serially is gradually transformed with a probable selection of stem cells and undifferentiated cells.     

Characterization of tumour cells in malignant fibrous histiocytomas and other soft tissue tumours in comparison with malignant histiocytes. I. Immunohistochemical study on paraffin sections

We have studied the possible origin of histiocytic cells, present in fibrous histiocytomas (MFH) by using immunohistochemistry to demonstrate lysozyme, alpha 1-antitrypsin, alpha 1-antichymotrypsin and receptors for peanut and soy bean agglutinin in tumour cells of MFH compared with their presence in tumour cells of malignant histiocytosis (MH) ('true' histiocytic lymphoma, 'true' histiocytic sarcoma). We included in this study a number of other soft tissue tumours (STT). Lysozyme was detected in half of the cases of malignant histiocytosis (n = 16) but in only two out of 77 MFH. alpha 1-Antitrypsin and alpha 1-antichymotrypsin usually occurred together although the latter was seen in more cases. Both markers were present in majority of cases of MH whereas they were detected in a minority of cases of MFH. MFH cases of the storiform subtype were less frequently stained than the pleomorphic or giant cell subtypes. Receptors for peanut or soy bean agglutinin were detected in nearly all MH cases, whereas their presence was only detected in a small number of MFH. Lysozyme was not detectable in other STT. alpha 1-Antitrypsin and alpha 1-antichymotrypsin were uncommonly present in other STT, except in osteosarcoma and rhabdomyosarcoma. These markers therefore have a limited value as indicators of a possible histiocytic origin of MFH. Lectins showed weak affinity for other STT. In accordance with others, we therefore conclude that the progenitor cell of MFH has to be sought within the undifferentiated mesenchymal cells and that histiocytes themselves probably do not give rise to MFH.     

Characterization of tumor cells in malignant fibrous histiocytomas and other soft-tissue tumors, in comparison with malignant histiocytes. II. Immunoperoxidase study on cryostat sections.

The authors have investigated a possible relationship between tumor cells of malignant fibrous histiocytomas (MFHs) and histiocytes. This relationship was studied by means of immunophenotyping using monoclonal antibodies specific for the monocyte cell lineage (FMC-17, Mac-1, OKM-1, Leu-M1, and lysozyme) and mono- and polyclonal antibodies specific for fibroblasts (respectively, FIB-86 and FSG). The immunophenotypes of the MFH tumor cells were compared with those of tumor cells of "true" histiocytic tumors. Monocyte lineage-specific determinants could be demonstrated in varying amounts on cells of the "true" histiocytic tumors but not on cells of MFH or other soft-tissue tumors. The reverse was true for determinants on fibroblasts. The absence of these determinants on malignant histiocytes, and their presence on MFH (and also on benign fibrous histiocytomas, fibrosarcomas, schwannomas, osteosarcomas, hemangiosarcomas, leio- and rhabdomyosarcomas) supported the conclusion that MFH tumor cells originate from mesenchymal cells which do not belong to the mononuclear phagocytic system. Subdivision of the MFH tumors revealed that the storiform-pleomorphic subtypes could express HLA-Dr/Ia antigens, like histiocytic tumors. The inflammatory cell subtype, however, lacked these antigens.     

Single somatic ras gene point mutation in soft tissue malignant fibrous histiocytomas.

The frequency of ras gene mutations in human soft tissue malignant fibrous histiocytomas within and around the hot spot codons (12, 13, and 61) of all ras genes, (H-ras-1, K-ras-2, and N-ras) was studied by nested polymerase chain reaction and direct DNA sequencing from archival formalin-fixed, paraffin-embedded tissue. Light microscopy and immunohistochemistry served to define malignant fibrous histiocytoma. All of the four differentiation subtypes (storiform-pleomorphic, inflammatory, myxoid, and giant cell) were investigated. Nine of thirty-two malignant fibrous histiocytomas (28%) contained ras gene point mutations. The highest incidence was found in the myxoid subtype (four of nine). H-ras-1 gene codon 12.2 was the only codon affected and contained in all mutated cases a GGC-->GTC exchange. Seven of the nine mutations were homozygous and probably affected more than 80% of the tumor DNA. The flanking regions of all hotspot codons did not contain any point mutation. The presence of a single and often homozygous point mutation of the H-ras-1 gene, especially in myxoid malignant fibrous histiocytoma could serve as a basis for further genomic discrimination of myxoid sarcomas.     

TLS-CHOP target gene DOL54 expression in liposarcomas and malignant fibrous histiocytomas

Downstream of the gene for the liposarcoma-associated fusion oncoprotein 54 (DOL54) is a target gene of the myxoid liposarcoma and round cell liposarcoma (M-LPS/RC-LPS) oncogene, TLS/FUS-CHOP. The DOL54 gene product is closely associated with adipogenic differentiation. DOL54 overexpression resulted in tumorigenicity when Chinese Hamster Ovary (CHO) cells were injected subcutaneously into nude mice. The biological significance of DOL54 expression for human malignant soft tissue tumors, however, has not yet been investigated. We examined TLS-CHOP and DOL54 expression in M-LPS/RC-LPS, well-differentiated liposarcoma and malignant fibrous histiocytoma (MFH), a tumor whose cellular origin has not been determined. We observed DOL54 expression in 50% of M-LPS/RC-LPS cases (in which TLS-CHOP was also expressed) and 33% of MFH cases, suggesting that a portion of MFH lesions may either derive from adipocytic precursor cells or have the potential to undergo adipogenic differentiation. In this manner, M-LPS/RC-LPS and MFH lesions may share tumorigenic characteristics, resulting from the unscheduled expression of DOL54.     

Characterization of two cell lines, derived from two malignant fibrous histiocytomas

We have investigated the phenotype and ultrastructure of tumour cells from two cell lines each derived from a malignant fibrous histiocytoma (MFH) as a means of studying the histogenesis of this group of tumours. The first MFH (MFH-I) was of the pleomorphic subtype, with a predominantly histiocytic appearance, the second was of the pleomorphic subtype associated with myxoid and storiform areas (MFH-II). In vitro tumour cells from both neoplasms showed aberrant growth properties. Xenografts in nude mice from both neoplasms showed a similar histology to that of the original tumour. Both tumours showed hyaluronidase sensitive alcian blue staining. Phenotypic studies of the two cell lines and of the tumour tissues demonstrated that the cells differed in the presence of collagen types I and III. They did not show evidence of histiocytic, endothelial, leiomyoblastic, rhabdomyoblastic, lipoblastic of schwannian origin. Ultrastructurally, the two cell lines were found to be different. In vitro and in xenografts the cell type of MFH-I resembled a primitive mesenchymal cell. Whereas that of MFH-II resembled a fibroblast-like cell. We concluded that the group of MFH is heterogeneous and is probably derived from more than one progenitor cell.     

Fibronectin in relation to growth patterns of fibrohistiocytic tumours--an immunohistochemical study of benign and malignant fibrous histiocytomas

Benign and malignant fibrous histiocytomas are composed of an admixture of fibroblast-like and histiocyte-like cells and of a changing amount of fibre structures which tend to be arranged in a so-called storiform pattern. In order to study the organization of the extracellular matrix, the distribution of fibronectin was investigated immunohistochemically. Using the PAP technique and the indirect immunofluorescence method, paraffin sections of formaldehyde fixed tissue specimens of 25 tumours (12 benign fibrous histiocytomas, 12 malignant fibrous histiocytomas, and 1 atypical fibroxanthoma) were studied. A pretreatment with hyaluronidase and proteolytic enzymes (trypsin, pronase, pepsin) was performed to unmask the antigen. Best results were obtained with pronase E or, sometimes even better, by employing a combination of pronase E and hyaluronidase. Generally fibronectin could be demonstrated in the matrix substances of fibrohistiocytic tumours, but the immunohistochemical staining patterns of benign and malignant tumours differed. In benign fibrous histiocytomas, a regular distribution of fibronectin was found in cellular areas. Parallel to formation of collagen fibres, the reaction decreased and in dermatofibromas showing abundant hyalinized collagen the staining proved to be quite weak. In malignant fibrous histiocytomas, the immunostaining was very irregular. In cellular areas consisting of spindle cells, an intense reaction could be observed. Tumours with storiform or fascicular fields exhibit a delicate network of fibronectin encircling individual fibroblast-like cells. In the course of fibre formation, the matrix staining for fibronectin revealed a distribution similar but not identical with that obtained with the reticulin stain. Simultaneous to the occurrence of collagen fibre bundles, fibronectin decreased and in areas of hyalinization the staining was considerably diminished. In areas of undifferentiated small cells, in myxoid zones as well as foci of xanthoma cells, and in pleomorphic portions the immunostain was negative. The distribution in atypical fibroxanthoma is similar to that observed in storiform and pleomorphic variants of malignant fibrous histiocytomas. The results support the suggestion that fibronectin is the first sign of the typical basic pattern of fibrohistiocytic tumours preceding the formation of reticulin and collagen fibres. The expression of fibronectin on cell surfaces as well as in intercellular matrix may be closely related to the organization of the growth patterns of fibrohistiocytic tumours.     

Most malignant fibrous histiocytomas developed in the retroperitoneum are dedifferentiated liposarcomas: a review of 25 cases initially diagnosed as malignant fibrous histiocytoma.

Forty-four samples from 25 cases of retroperitoneal sarcoma initially diagnosed as malignant fibrous histiocytoma were histologically reviewed. Immunohistochemistry for mdm2 and cdk4 was performed on 20 cases. Comparative genomic hybridization was performed on 18 samples from 13 patients. Seventeen cases were reclassified as dedifferentiated liposarcoma. Twenty-one of 32 samples from these patients showed areas of well-differentiated liposarcoma, allowing the diagnosis of dedifferentiated liposarcoma. Immunohistochemistry performed in 15 of these cases showed positivity for mdm2 and cdk4. Comparative genomic hybridization analysis performed on 15 samples from 11 of these patients showed an amplification of the 12q13-15 region. Eight cases were reclassified as poorly differentiated sarcoma. Twelve samples from these patients showed no area of well-differentiated liposarcoma. Immunohistochemistry showed positivity for mdm2 and cdk4 in one of six of these patients and showed positivity for CD34 in another one. Comparative genomic hybridization analysis performed on three samples from two of these patients showed no amplification of the 12q13-15 region but showed complex profiles. This study shows that most so-called malignant fibrous histiocytomas developed in the retroperitoneum are dedifferentiated liposarcoma and that a poorly differentiated sarcoma in this area should prompt extensive sampling to demonstrate a well-differentiated liposarcoma component, immunohistochemistry for mdm2 and cdk4, and if possible, a cytogenetic or a molecular biology analysis.     

A monoclonal antibody and functional study of malignant T cells of a patient with suppressor-T-cell lymphoma

We describe the immunologic characteristics of malignant T cells of a T-cell lymphoma patient. The neoplastic cells in lymph node expressed OKT3+, OKT11-, E-rosetting-, OKT4-, OKT8+, OKIa1+, OKDR+ suppressor-T-cell phenotype. Functionally, these malignant T cells did not respond to phytohemagglutinin, but produced a good response to concanavalin A. A defect in expression of interleukin 2 receptors was evident in phytohemagglutinin-activated T cells. In vitro immunoglobulin production experiments demonstrated that patient's malignant T cells possess helper function on normal B cells to produce IgM, and suppressor function to produce IgG.     

Incidence and histological structure of the storiform pattern in benign and malignant fibrous histiocytomas

Summary A starlike arrangement of cells and fibers, the storiform pattern, was found to be a typical, but not obligatory, histological feature of benign and malignant fibrous histiocytomas. In 155 benign fibrous histiocytomas storiform structures were missing in 29 cases, chiefly of the fibroblastic type comparable with classical dermatofibroma. 12 of 70 malignant fibrous histiocytomas did not reveal storiform structures, especially the cellular pleomorphic variant, i.e. the classical pleomorphic sarcoma.Storiform structures were either small and highly cellular with few fibers (collagen type III), or larger, less cellular, but with abundant fibers (collagen type I). There was no sharp demarcation between these two extremes, but many transitional structures or patterns were seen. The histiocytic nature of the cells was demonstrated in both variants of storiform structures by immunhistochemical methods on paraffin embedded material. Alpha₁-anti-chymotrypsin was especially valuable in this respect.The study was supported by the Wilhelm-Sander-FoundationDedicated to Prof. Dr. W. Büngeler to his 80^th birthday     

A histiocyte-specific marker in the diagnosis of malignant fibrous histiocytoma. Use of monoclonal antibody KP-1 (CD68)

KP-1 (CD68) is a recently described monoclonal antibody to a cytoplasmic epitope present on tissue histiocytes and macrophages. To determine the specificity and sensitivity of this marker in the evaluation of cases of malignant fibrous histiocytoma (MFH), this reagent and a panel of commercially antibodies were used to stain formalin-fixed paraffin sections from 25 cases of MFH and 25 other tumors, including a variety of soft-tissue sarcomas. Eighteen of 25 cases of MFH stained for KP-1 (72%), whereas all other tumors were negative, including 12 cases of pleomorphic soft-tissue sarcoma other than MFH. The percentage of tumor cells staining for KP-1 varied. In 11 cases KP-1 was only focally present, but staining was of a high intensity and associated with minimal nonspecific or background staining. Pleomorphic histiocytic cells and spindle cells from storiform tumors were strongly decorated with antibodies to KP-1 in most cases, and antigen also was present on tumor giant cells. Although alpha-1-antitrypsin and alpha-1-chymotrypsin stained a higher percentage of cases of MFH (92%), immunoreactivity for these markers also was noted in other tumors. Because of its specificity as a histiocyte marker, KP-1 is a useful component in a panel of antibodies for the characterization of soft-tissue sarcomas and the diagnosis of MFH.