Isolation and characterization of proteoglycans from human nonepithelial tumors.

Proteoglycans (PGs) and glycosaminoglycans (GAGs) were identified in myogenic and fibrogenic tumors. More PGs containing mainly chondroitin sulfate could be detected in malignant tumors (leiomyosarcomas) than in benign tumors (leiomyomas and fibromas). Two groups of PGs were detected in the malignant tumors by ion-exchange chromatography and gel chromatography. One group was a large molecule with chondroitin sulfate side chains, seemingly composed of two or more subpopulations that were eluted from Sepharose CL-4B with a kav of 0.45. After removal of GAG side chains from the PG by chondroitinase AC digestion, core molecules with molecular weights greater than 200,000 were obtained. Another PG detected was a fraction of small PG eluted from Sepharose CL-4B with a kav of 0.45. It consisted of a core molecule with a molecular weight approximately equal to 48,000 and GAG side chains containing chondroitin sulfate-dermatan sulfate hybrids. The mixed sequence of L-iduronic acid with D-glucuronic acid in the same GAG chain was demonstrated by the formation of a small proportion of tetrasaccharide after chondroitinase AC digestion. In the benign tumors, the large PG was found only in very small amounts, and PG detected was composed mainly of the small one eluted from Sepharose CL-4B with a kav of 0.45. Its core protein had a molecular weight of approximately equal to 46,000, which was similar to that of small PG obtained from leiomyosarcomas, but its GAG side chains were composed mainly of dermatan sulfate containing small amounts of glucuronic acid. The results suggest that the core molecules of small PGs from both benign and malignant tumors are the products of the same gene but that they are processed in a different manner to form proteoglycans with different types of GAG chains.

Neopterin as a prognostic indicator in patients with carcinoma of the uterine cervix.

In vitro, neopterin, a pyrazinopyrimidine compound, is excreted by human monocytes-macrophages after induction by supernatants from activated T-lymphocytes or by recombinant gamma-interferon. In vivo, it represents a noninvasive test for activation of cellular immune reactions. To evaluate the prognostic value of pretherapeutic urinary neopterin levels and of serial neopterin measurements during follow-up in women with cervical cancer, 1088 urine specimens from 186 consecutive patients were analyzed. Clinical assessments were made without knowledge of the results of neopterin assays (a "blinded" assessment). During the observation period (June 1980 to March 1984), 27 relapses, 18 metastases, and 26 deaths were seen. The prognostic significance of pretherapeutic neopterin and other possible prognostic clinical and laboratory parameters was tested by the univariate and multivariate Cox proportional hazards model using a stratification according to stage and surgical treatment. The combination of age at diagnosis, pretherapeutical hemoglobin, leukocyte count, and neopterin was found to predict survival best. On the basis of this result, risk groups were identified exhibiting markedly different survival behavior. A highly significant association was found between serial neopterin measurements and the risk for a relapse, metastasis, or death. The data suggest that urinary neopterin levels might be a useful adjuvant parameter in monitoring women with cervical cancer.

Identification of immunoreactive thyrotropin-releasing hormone in human neoplasia.

Immunoreactive TRH (IR-TRH) was identified in extracts derived from seven human cancer tissues, including two oat cell carcinomas. The IR-TRH concentrations ranged from 29-189 pg/g. Tumor IR-TRH exhibited immunological identity with synthetic pyroglu-his-proamide and coeluted with synthetic [3H]TRH after high pressure liquid chromatography on a mu Bondapak C-18 column. These data extend the number of hypothalamic peptide hormones identified in human neoplastic tissues.

Immunoreactivity of paraffin-embedded normal tissues and mesenchymal tumors for smooth muscle myosin.

Immunohistochemical study of smooth muscle myosin, a protein distinct from skeletal, cardiac, or nonmyogenous myosins in paraffin-embedded normal tissues and benign and malignant mesenchymal tumors revealed its strong expression in normal smooth muscle, capillary endothelium, and pericytes. All benign smooth muscle tumors with exception of gastric leiomyomas and few other leiomyomas of the gastrointestinal tract displayed strong or moderate immunoreactivity. On the other hand, strong or moderate immunoreactivity was detected in only eight of 28 spindle-cell leiomyosarcomas, as well as in 13 out of 27 malignant fibrous histiocytomas and three out of nine malignant hemangiopericytomas, while epithelioid leiomyosarcomas, fibrosarcomas, malignant schwannomas, and synovial sarcomas were negative or only weakly positive. Our results demonstrate that, while smooth muscle myosin is a very good marker of normal smooth muscle and benign smooth muscle tumors, it is expressed in diagnostically significant amounts in less than a third of spindle-cell leiomyosarcomas and none of the studied epithelioid leiomyosarcomas.

The diagnostic utility of desmin. A study of 584 cases and review of the literature.

The diagnostic utility of several antibodies against desmin and their optimal staining conditions have not been systematically evaluated. Sections of paraffin-embedded tissues from 584 cases were stained with a monoclonal antibody against desmin (Clone DER 11 from DAKO), using an avidin-biotin-peroxidase technique. The results were tabulated and compared with those from previous reports. The following observations were made: (1) When pronase digestion was performed before staining, desmin was equally demonstrable in tissues fixed in formalin, Zenker's, Bouin's, or B5 fixative; however, desmin staining was lost or significantly diminished in tissues fixed in absolute ethyl alcohol. In contrast, when pronase was not used, a positive staining was demonstrated only in tissue fixed in absolute ethyl alcohol. (2) Positive staining was found in normal muscle (92 of 92 cases), leiomyoma (12 of 13), rhabdomyoma (6 of 6), rhabdomyosarcoma (31 of 31), leiomyosarcoma (18 of 26). (3) Desmin was never found in epithelia, normal mesenchymal tissue other than muscle, tumors stimulating rhabdomyosarcoma, and epithelial tumors. (4) A positive staining was documented in 1 tumor (a fibrous histiocytoma) of 42 benign predominantly spindle cell tumors and in 8 of 89 predominantly spindle cell sarcomas. (5) Desmin was never documented in myoepithelial cells but stained myofibroblasts in 2 of 12 examples of granulation tissue and in 29 of 67 samples containing tumor-associated desmoplasia. The authors' data on the diagnostic sensitivity and specificity of the evaluated antibody should improve the use of desmin in diagnostic pathology.

Primary recurrent leiomyosarcoma of the breast. Case report with ultrastructural and immunohistochemical study and review of the literature.

The authors report a case of recurrent mammary leiomyosarcoma in a 50-year-old woman. The neoplasia, with a recognized clinical evolution of 11 years, was resected on two occasions and had not metastasized. Microscopic examination showed 4 mitoses/10 high-power fields, moderate cytologic atypia, and, ultrastructurally, abundant myofibrils with condensations. Immunoperoxidase stains had positive results for muscle-specific antigen and showed focal reactivity for epithelial membrane antigen and S-100 protein. Analysis of the ten cases (including the present one) reveals that this neoplasm has appeared with greater frequency in women with an average age of 52 years. All neoplasms have been limited to the breast at the time of diagnosis. As a group, they have better prognosis than other sarcomas of the breast, although the possibilities of recurrence or dissemination exist, even many years after the primary extirpation. The size of the tumor and mitotic activity seem to be of little prognostic value. Mammary leiomyosarcoma shares clinical and pathologic similarities with subcutaneous leiomyosarcoma in other anatomic sites.

Distribution of laminin, fibronectin, and interstitial collagen type III in soft tissue tumours.

The distributions of laminin, fibronectin, and interstitial collagen type III have been investigated in a series of 60 soft tissue tumours by immunochemistry. Positive laminin staining was seen in sites predicted by the distribution of ultrastructurally visible basal lamina. Pericellular laminin was present in all benign tumours of Schwann cell and smooth muscle origin examined, in the two malignant Schwannomas examined, and in six of 13 leiomyosarcomas. It was also evident around nests of cells in an alveolar soft part sarcoma and around malignant endothelial cells in an angiosarcoma. In fibroblastic and fibrohistiocytic tumours it was found only in blood vessel walls. The results of laminin staining led to revision of the original histopathological diagnosis in seven of the 60 cases studied. Fibronectin was abundant in the stroma of most neoplasms, both benign and malignant. It was also found in a distribution parallel to that of laminin. In some tumours this was clearly distinguishable from the distribution of interstitial collagen. Intracellular fibronectin was shown consistently only in mast cell granules. Its demonstration in synovial cells, fibroblasts, and histiocytes was more variable. Interstitial collagen type II had the most irregular distribution of the three proteins. It was as plentiful in tumours of smooth muscle origin as in tumours of fibroblastic origin, but was scanty in fibrous histiocytomas. Its distribution appeared similar to that of laminin and fibronectin in leiomyomas, but differed from these two proteins in Schwann cell tumours and other neoplasms. In one leiomyosarcoma fibronectin, laminin, and type III collagen appeared to be lost concomitantly from tumour cell peripheries.

Distribution of steroid hormone receptors in human soft tissue sarcomas.

Sixty-six human soft-tissue sarcoma specimens were assayed for incidence and distribution of steroid hormone receptors. Liposarcomas (43%) and leiomyosarcomas (60%) had a high incidence of estrogen receptor. In contrast, sarcomas of fibrous and synovial tissue origin lacked any detectable receptor for estrogen. Malignant fibrous histiocytoma had the highest incidence of glucocorticoid (66%) and androgen (66%) receptors. The incidence of receptors for estrogen and glucocorticoid was higher in female than in male patients (62% and 38%, respectively). Sucrose density gradients demonstrated 8s-type receptors for both estrogen and glucocorticoid. Data from this study suggest that specific high-affinity cytosolic receptors for steroid hormones are present in human soft tissue sarcoma and that their distribution may depend on the histogenetic origin and the sex of patients.

Long survival of a woman with a huge leiomyosarcoma of the duodenum after pancreaticoduodenectomy and transverse colectomy: a case report.

A woman with a huge leiomyosarcoma of the duodenum that had invaded the pancreas and transverse colon is living and well 10 years after pancreaticoduodenectomy combined with resection of the transverse colon. Malignant potential of the tumor was determined by the mitotic rate and nuclear deoxyribonucleic acid content. The mass exceeded 10 cm yet was characterized by low mitotic activity and a near-diploid pattern. The long survival is attributed to the extensive surgery and low-grade malignancy of the tumor.

Estrogen receptor proteins in diverse human tumors.

One hundred three diverse benign and malignant human tissues have been assayed for estrogen receptor proteins. Receptors were detected in many endocrine and nonendocrine tumors. Tissues with estrogen receptor activity included four of five male breast carcinomas, 11 of 14 malignant melanomas, four of eight colon carcinomas, five of seven renal carcinomas, and various sarcomas and benign and normal tissues. Some tumors also had progesterone, androgen, and/or glucocorticoid receptors. These results suggest the use of hormones and hormone antagonists for therapy of a broad range of human cancer. Clinicians of diverse expertise should be aware of, and responsive to, potential endocrinological involvement in many dissimilar disease states.

Epithelioid leiomyosarcoma of the left atrium: immunohistochemical and ultrastructural findings.

Leiomyosarcomas of the heart are rare, with only 12 cases reported in the literature. An example of the epithelioid variant of leiomyosarcoma is described. The tumour cells expressed vimentin, desmin, muscle-specific actin and smooth muscle-specific actin. In addition, they were also labelled by monoclonal and polyclonal antibodies to cytokeratin intermediate filaments and displayed cell junctions at the ultrastructural level, features which highlight the potential pitfalls in the application of immunohistochemistry and electron microscopy in the diagnosis of poorly differentiated tumours.

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 evaluation of intermediate filament expression in canine and feline neoplasms.

Specimens of neoplastic tissues from 19 dogs and 4 cats were examined immunohistochemically for intermediate filament expression, using commercially available antibodies. Staining was observed in a wide range of tumor tissues and in normal internal controls by use of antibodies to vimentin, desmin, glial fibrillary acidic protein, and low and high molecular weight cytokeratins. Intermediate filament expression was found to be consistent with light and/or electron microscopic findings, and hence believed to be an accurate indicator of tumor histogenesis in cats and dogs. Three fixatives were evaluated for their relative abilities to preserve antigenicity. Absolute alcohol was superior to B5 fixative and both were superior to formalin. Some tissues that clearly displayed intermediate filament antigens with alcohol and B5 fixative failed to stain when fixed in formalin.

[Determination of cell nuclear DNA content in leiomyoma and leiomyosarcoma of gastro-intestinal tract (G-I)].

Nuclear DNA content was measured by microspectrophotometry in 15 biopsy specimens from patients with G-I smooth muscle tumors (3 leiomyomas and 12 leiomyosarcomas subdivided into 3 groups with 4 cases to each). The mean DNA value increased steadily as follows: leiomyomas (14.39 +/- 0.62 Au); leiomyosarcoma Grade I (19.78 +/- 2.39 Au); Leiomyosarcoma Grade II (26.39 +/- 1.60 Au); leiomyosarcoma Grade III (30.66 +/- 2.39 Au). The difference of DNA value in the 4 groups had statistical significance (P less than 0.05-0.01). These results suggest that microspectrophotometric measurement of nuclear DNA content may serve as an objective quantitative method for diagnosis of G-I tract smooth muscle tumors and classification of leiomyosarcoma.

Proteins of intermediate filaments. An immunohistochemical and biochemical approach to the classification of soft tissue tumors.

The intermediate filament cytoskeleton of various types of human soft tissue tumors was analyzed by immunofluorescence microscopy with the use of specific antibodies against cytokeratins, vimentin, and desmin, as well as by one- and two-dimensional gel electrophoresis of high-salt buffer- and detergent-resistant cytoskeletal preparations. All leiomyomas as well as a leiomyosarcoma contained desmin. Leiomyomas of both gastrointestinal and uterine derivation and the retroperitoneal leiomyosarcoma showed strong reaction for desmin in the smooth muscle cells, but the latter two exhibited also vimentin staining. In embryonal rhabdomyosarcomas, desmin prevailed in the large, apparently well-differentiated rhabdomyoblasts; whereas the smaller, less differentiated tumor cells preferentially contained vimentin. Cells of malignant fibrous histiocytomas were characterized by their content of vimentin as the only intermediate filament protein present. In alveolar soft part sarcoma, a rare tumor of hitherto unknown histogenesis, vimentin and desmin co-existed within the same tumor cells, indicating, together with chemical determinations, the myogenic derivation of this neoplasm. The results show that immunologic and biochemical analysis of proteins associated with the intermediate filament cytoskeleton is a useful adjunct in the diagnosis of diverse neoplasms, particularly those with equivocal histologic features, and thus aids in the histogenetic classification of soft tissue tumors.

HHF35, a muscle actin-specific monoclonal antibody. II. Reactivity in normal, reactive, and neoplastic human tissues.

Monoclonal antibody HHF35 has previously been characterized biochemically as recognizing isotypes of actin (alpha and gamma) which are specific to muscle cells. In this study, the authors have investigated the normal and pathologic tissue distribution of HHF35-positive cells using the avidin-biotin immunoperoxidase method on methacarn-fixed, paraffin-embedded sections of human tissue. In addition to muscle tissues (smooth, skeletal, and cardiac) the antibody localizes to myoepithelium, as well as most of the capsular cells of several parenchymal organs, including liver, kidney, and spleen, with extension of the latter cells into the splenic trabeculaes. In pathologic tissues, the antibody localizes to cells, identified by some investigators as "myofibroblasts," in the stroma of certain tumors, within hyperplastic fibrous tissue responses ("fibromatoses") such as Dupuytren's contracture, and within fibrotic lung tissue. HHF35 also localizes to cells that proliferate within the intima in lesions of atherosclerosis and to a unique population of reactive mesothelial and submesothelial cells. Among tumors, it is positive only on leiomyomas, leiomyosarcomas, and rhabdomyosarcomas, and negative on all nonmuscle sarcomas. This antibody thus shows great potential utility as a diagnostic reagent in various pathologic conditions, most especially in the diagnosis of tumors of muscle origin.

Primary cutaneous leiomyosarcoma. A histological and immunohistochemical study of 9 cases, with ultrastructural correlation.

Leiomyosarcoma (LMS) of dermal and subcutaneous tissues is an uncommon neoplasm. In order to analyze the specialized pathologic features of this tumor, we undertook a histological, ultrastructural, and immunohistochemical study of 9 superficial LMS, including 7 dermal lesions and 2 subcutaneous neoplasms. These were compared with 12 examples of "deep" extracutaneous LMS. Metastases to the skin from two of the latter neoplasms were also examined. Immunohistochemistry was found to be a useful diagnostic adjunct to light microscopic and ultrastructural studies in that all LMS coexpressed vimentin and desmin, regardless of site, and 90% also expressed muscle-specific actin. Variable expression of cathepsin B and myelin basic protein was noted in 8 and 10 tumors, respectively, whereas none contained cytokeratin. Weak cytoplasmic positivity for epithelial membrane antigen was seen in 1 dermal and 3 extracutaneous LMS. Of 7 dermal LMS, 4 contained S-100 protein, whereas this determinant was found in only 1 of 12 extracutaneous tumors. Conversely, Leu 7 reactivity was present in 7 of 12 extracutaneous LMS, but only 2 of 9 superficial lesions. Review of clinical features confirmed that subcutaneous LMS is capable of aggressive behaviour, whereas dermal LMS was more likely to behave in an indolent fashion. However, one example of dermal LMS exhibited aggressive local recurrences and distant metastasis, ultimately leading to the death of the patient. Therefore, careful clinical followup is indicated in all cases.

Gastric submucosal tumours of neurogenic origin with neuroaxonal and Schwann cell elements.

Twelve cases of gastric submucosal tumours, originally diagnosed as leiomyoma and leiomyosarcoma, were investigated by staining with the neurogenic markers S-100 and neuron specific enolase (NSE). Three cases were, in addition, studied by electron microscopy. The tumours stained negatively for desmin, indicating that they were not of smooth muscle origin. All stained positively for S-100 and NSE. The ultrastructural features in the cases examined by electron microscopy were reminiscent of autonomic nerve structures normally present in the gastric wall. These included fine cytoplasmic processes of Schwann cells, wrapped with complete or incomplete basement membranes, and structures analogous to post-ganglionic neuroaxonal components. These tumours appear to represent a distinct type of gastric neoplasm originating from autonomic nerve elements in the stomach wall. They are different from previously described schwannomas or neurofibromas. In some of the tumours, identifiably neural elements are relatively a minor component, and the majority of the tumour cells are of undetermined origin. It is suggested that these cells may be poorly differentiated, lacking their antigenic determinants specific to neural differentiation.