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.     

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.     

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 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.     

Immunohistochemistry of markers of histiomonocytic cells in malignant fibrous histiocytomas. A monoclonal antibody study

Nine cases of malignant fibrous histiocytomas (MFH) were examined immunohistochemically in frozen sections with six different monoclonal antibodies to histiomonocytic and related cells (EBM11, HAM-56, KB90, antibodies to dendritic reticulum cells, HLADR and LCA). Ten other soft tissue sarcomas, two desmoid tumors, twelve carcinomas, three seminomas and four lymphomas were studied for comparison. All cases of MFH showed positivity for histiomonocytic cell antigens. In six cases, the positive cells could be clearly interpreted to be infiltrating non-neoplastic cells. However, immunoreactivity for multiple histiocytic markers (EBM11, HAM-56, KB90, HLADR) was seen in tumor cells in three cases of MFH. In one of these cases, the positivity could be verified with KP1, an antibody to histiomonocytic cells applied in formalin fixed and paraffin embedded tissue. None of the tumors was positive with the antibody to dendritic reticulum cells or LCA. In the series of non-histiocytic tumors, no cases showed widespread positivity for multiple histiocytic markers. Our results suggest that in relation to true histiomonocytic differentiation MFH might be a heterogeneous group of tumors. The widespread immunoreactivity for multiple histiocytic markers in some cases may indicate a true histiomonocytic differentiation in some MFHs.     

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.     

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.     

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.     

Myofibroblasts and related cells in malignant fibrous and fibrohistiocytic tumors.

Myofibroblasts were detected by electron microscopy in five of five cases of fibrosarcoma and in five of six cases of malignant fibrous histiocytoma. In some areas myofibroblasts constituted up to 75 per cent of the tumor cells. Most myofibroblasts contained only sheaves of myofilaments along the margins of the cells, but some cells contained larger bundles of myofilaments and very closely resembled smooth muscle cells. An additional related type of cell was seen in several cases; it was large and possessed abundant eosinophilic cytoplasm, resembling a rhabdomyoblast at the light microscopic level. By electron microscopy this type of cell was seen to contain plentiful rough endoplasmic reticulum and large aggregates of fine filaments with rare dense bodies. These findings suggest that fibrosarcomas and malignant fibrous histiocytomas contain cells showing a spectrum of differentiation from fibrocytic to myogenic and that at the ultrastructural level the distinction between fibroblast and smooth muscle tumors may be blurred.     

Characterization of human soft tissue sarcomas in nude mice. Evidence for histogenic properties of malignant fibrous histiocytomas.

Twenty-two human sarcomas were grafted subcutaneously into nude mice. Twelve tumors grew successfully. Nine of these 12 tumors had an aneuploid DNA content, whereas only 1 of 10 nonsuccessful tumors was aneuploid. The 12 sarcomas included two leiomyosarcomas, two malignant schwannomas, one synovial sarcoma, and seven malignant fibrous histiocytomas (MFHs). With light and electron microscopic and immunolabeling studies the original and xenografted tumors (the latter for at least two generations) were histopathologically compared. The xenografted leiomyosarcomas showed ultrastructurally a more pronounced leiomyodifferentiation, and one of the malignant schwannomas a more pronounced schwannian differentiation. The second malignant schwannoma and the synovial sarcoma, however, remained unchanged. Five storiform pleomorphic MFHs expressed features that were not observed in the original tumors. Tumor cells of three of these xenografted sarcomas showed leiomyogenic differentiation (filamentous densities, pinocytotic vescicles, and desmin immunoreactivity), whereas cells of the two others demonstrated schwannian differentiation (long cytoplasmic processes, basal lamina). A xenografted myxoid MFH and a pleomorphic MFH gave rise to pleomorphic sarcomas composed of undifferentiated cells. It appeared that under transplantation conditions tumor cells of storiform pleomorphic MFH can differentiate into various directions.     

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.     

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     

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.     

Histological and cytological criteria in the diagnosis of malignant melanomas by cryostat sections

Summary Although cryostat sections in general allow a distinction to be made between malignant melanomas and other pigmented lesions in clinically doubtful cases, the differential diagnosis may be difficult. The histological and cytological criteria taken into account can be classified as major, minor, and insufficient. Knowing the diagnostic value of each makes a conventionally established diagnosis safer. Variance analysis does not contribute to the problem but it can nevertheless be shown that the evaluation of six major criteria makes a quick and reliable cryostat section diagnosis possible. If these results are confirmed in a prospective study it would be a decisive step on the way to a quicker and safer cryostat section diagnosis of malignant melanoma, even for the less experienced histopathologist.The results published here were presented in part at the DDG meeting 1980 at Westerland/SyltWe are grateful to Miss Schubert, Institute of Biomathematics of the University of Munich, for the statistical evaluations     

Cytokeratin immunoreactivity in malignant fibrous histiocytoma and spindle cell tumors: comparison between frozen and paraffin-embedded tissues.

Cytokeratin (CK) immunoreactivity in malignant fibrous histiocytoma (MFH) and other selected cases of spindle cell tumors were assessed using two cytokeratin monoclonal antibodies, AE1/AE3 and CAM 5.2. Frozen tissue was used to minimize the effects of fixation on keratin antigenicity; in addition, one block of fixed, paraffin-embedded tissue was tested for comparison. CK immunoreactivity was noted in nine frozen tissue samples (7/20 [35%] MFH, 1/3 schwannomas, 1/3 leiomyosarcomas). In the majority of cases, only rare individual positive cells were seen. Of 19 MFH cases in paraffin-embedded tissue, CK immunoreactivity was noted in three (16%). All 32 cases examined showed vimentin immunoreactivity. MFH must be added to the growing list of mesenchymal tumors exhibiting sporadic CK immunoreactivity. Such reactivity is less frequent in paraffin-embedded tissues. This finding has important implications for tumor diagnosis, particularly in the differential diagnosis of pseudosarcomatous carcinoma. Caution is recommended in the interpretation of CK immunoreactivity, particularly as it relates to speculations regarding histogenesis.     

Expression of minichromosome maintenance-2 in human malignant fibrous histiocytomas: Correlations with Ki-67 and P53 expression,and apoptosis

This study examined the clinicopathological significance of minichromosome maintenance-2 (MCM2) expression in 38 human malignant fibrous histiocytomas (MFHs) and 36 benign fibrohistiocytic tumors (BFHTs) immunohistochemically, and in 9 human sarcoma or carcinoma cell lines, as well as 7 surgical specimens by Western blotting. MCM2 was detected in all the cell lines and surgical specimens as a single band at 120 kDa, while P53 expression was variable. Nuclear expression of MCM2 was noted in tumor but not mitotic cells of all the MFHs and 26 (72.2%) of the BFHTs, the labeling indices (LIs) being 62.0% in the 28 ordinary types, 38.5% in the 10 myxoid types, and 11.2% in the BFHTs with significant difference. Moreover, the LI was significantly higher for MCM2 than that for Ki-67 in the MFHs of both types (p<0.05). No correlation was noted between the MCM2-LI and P53 expression or apoptotic indices, which were significantly higher in the MFHs than BFHTs (p<0.01). These results indicate that MCM2 would correlate with cell proliferation rather than apoptosis in MFHs, and the expression is ubiquitous in proliferating cells, regardless of the expression of P53. Thus, MCM2 might be a reliable marker of proliferating cells in human MFH.     

Ultrastructural study of Reed-Sternberg cells. Comparison with transformed lymphocytes and histiocytes.

The ultrastructural features of Reed-Sternberg cells from 17 patients with Hodgkin's disease were compared with those of histiocytes and transformed lymphocytes in both benign and malignant conditions. Transformed lymphocytes and Reed-Sternberg cells appeared to have similar features, including large nuclei with dispersed chromatin, large nucleoli, and great numbers of cytoplasmic polyribosomes. Histiocytes contained abundant cytoplasmic lysosomal granules and microfilaments. These results are indicative of the origin of Reed-Sternberg cells from lymphocytes.     

Proliferation in non-Hodgkin''s lymphoma: a comparison of Ki-67 staining on fine needle aspiration and cryostat sections.

Assessment of the growth fraction of non-Hodgkin's lymphomas may provide useful additional prognostic information to that obtained with conventional histological criteria. The monoclonal antibody Ki-67 has been reported to provide such information immunocytochemically in tissue biopsy specimens from lymphoma as well as other tumours. This study was undertaken to assess whether this approach could be extended to fine needle aspiration (FNA) biopsy specimens which are becoming increasingly important in the diagnosis of lymphoma. In 21 cases of non-Hodgkin's lymphoma the rate of tumour proliferation estimated by Ki-67 immunostaining of FNA material, obtained from surgically removed specimens, was compared with that obtained on tissue biopsy. The correlation between both preparations was excellent, indicating that FNA biopsy material is suitable for the immunocytochemical assessment of the growth fraction of non-Hodgkin's lymphoma.