Immunohistochemical detection of intermediate filament proteins in formalin fixed normal and neoplastic canine tissues.

Normal and well differentiated neoplastic canine tissues were immunohistochemically stained for keratin, vimentin and desmin intermediate filament proteins using commercially available monoclonal antibodies. Keratin was detected in 56 of 57 carcinomas, vimentin in 59 of 62 sarcomas and desmin in three of four muscle cell tumors. Most normal and neoplastic tissues expressed only one type of intermediate filament; exceptions were one hemangiosarcoma and one pulmonary carcinoma in which there was coexpression of vimentin and keratin proteins. Since immunohistochemical detection of intermediate filaments has tissue-specific distribution in the majority of well differentiated canine neoplasms, these stains may be useful in the differential diagnosis of anaplastic canine tumors. However, the monoclonal antibodies to cytokeratin which were tested in this study failed to detect intermediate filaments in liver, pancreas and salivary glands which suggests that these antibodies may also be unable to detect epithelial tumors derived from these tissues. In addition, in nine neoplasms, the normal tissues adjacent to neoplastic cells failed to stain for the intermediate filament normally expressed. When this occurs, evaluation of intermediate filament expression is invalid for the determination of tissue of origin of the neoplastic cells.

Immunohistochemical demonstration of liver fatty acid-binding protein in human hepatocellular malignancies.

Twenty-three hepatoblastomas of childhood, sixty-two adult hepatocellular carcinomas, and two hepatic sarcomas were examined immunohistochemically with the use of a polyclonal antibody against rat liver fatty acid-binding protein (L-FABP), which cross-reacts to human L-FABP. All the hepatoblastomas and half of the hepatic cell carcinomas contained L-FABP immunoreactive tumour cells, whereas two hepatic sarcomas were negative. The overall frequency of immunostained tumour cells was 43.5 per cent in hepatoblastomas and 18.6 per cent in hepatocellular carcinomas, respectively. Histologically well-differentiated areas contained more numerous immunopositive cells than undifferentiated or immature ones. These results indicate that L-FABP immunoreactivity is a new candidate for a tumour cell marker in hepatic cell malignancies, although its biological role has not been elucidated.

Two homologous mixed müllerian tumor lines of the ovary and their characteristics.

Two cell lines, NF and JoN, derived from human ovarian carcinosarcomas, were established in tissue culture and in nude mice. Both lines, growing in monolayers, showed morphologic features of adenocarcinoma cells (NF being aneuploid with a modal number of 53, and JoN being pseudodiploid with a modal number of 44). Intermediate filaments were demonstrated immunohistochemically; the JoN line expressed keratin, but not vimentin or desmin, whereas the NF line expressed vimentin and desmin, but not keratin. Plasminogen activator activity was found in both lines. It is concluded that both of these lines are potentially useful models for studying the diverse characteristics of malignant mixed Müllerian tumors.

Immunohistochemical detection of P-glycoprotein: prognostic correlation in soft tissue sarcoma of childhood.

Increased expression of P-glycoprotein is associated with multidrug resistance (MDR) in many cell lines. Significant levels of P-glycoprotein have been detected in a number of human tumors. The purpose of this study was to determine whether P-glycoprotein expression correlates with both response to chemotherapy and prognosis in soft tissue sarcoma of childhood. In a retrospective study, biopsy samples from 30 cases of rhabdomyosarcoma (RMS) and undifferentiated sarcoma (US) treated at The Hospital for Sick Children in Toronto were analyzed using a semiquantitative immunohistochemical procedure. P-glycoprotein was detected in nine patients, four at diagnosis, and five at subsequent biopsy. All nine patients relapsed after a clinical response (complete [CR] 55%, partial [PR] 45%) to chemotherapy. Twenty of 21 patients with consistently P-glycoprotein-negative tumors received chemotherapy and they all responded clinically (CR 80%, PR 20%). Only one of these 20 patients has relapsed. The probability of relapse-free survival was significantly different (P less than .000000012) in chemotherapy-treated patients whose tumors contained detectable levels of P-glycoprotein (n = 9), compared with those whose tumors contained no detectable P-glycoprotein (n = 20). The overall probability of survival was also significantly different in these two groups (P less than .0000267). Both relapse-free and overall survivals remained statistically different in the two groups of patients when analyzed by the log-rank method, after adjustment for differences in stages and sites. The incidence of other adverse prognostic factors in the two groups, for example, younger and older ages, low pretreatment lymphocyte counts, large tumors, and unfavorable histology were not significantly different. Thus, detectable P-glycoprotein appears to be an important adverse prognostic factor in children with soft tissue sarcoma, and consistent absence of the protein is associated with a favorable prognosis.

Alveolar soft part sarcoma. A clinicopathologic and immunohistochemical study of 12 cases.

Twelve cases of alveolar soft part sarcoma (ASPS) were reviewed. Seven of them arose primarily in the lower extremities, three in the head and neck region, and two in other parts. ASPSs in the head and neck region occurred in children before 10 years of age, whereas ASPSs in the other regions occurred in rather older patients. Moreover, ASPSs of the head and neck were relatively small in size, and were diagnosed earlier than those in other regions. Histologically, six cases (including all the head and neck cases) contained considerable area of small and indistinct alveolar structures. Four cases showed remarkable cellular pleomorphism. Immunohistochemical demonstration of vimentin, desmin, the beta-subunit of enolase and the MM isozyme of creatine kinase, together with the absence of immunoreactive cytokeratin, supported the myogenic nature of this rare tumor. A small number of S-100 protein-positive tumor cells were also observed. Follow-up data for these cases disclosed that the tumors containing considerable area of small alveoli and uniform small tumor cells formed distant metastases at an early stage.

Cellular DNA content and prognosis of high-grade soft tissue sarcoma: the Scandinavian Sarcoma Group experience.

The nuclear DNA content of 148 high-grade soft tissue sarcomas of the extremities and trunk was determined by flow cytometry, using tumor material from paraffin-embedded tissue. The patients were part of a prospective randomized clinical trial on the efficacy of adjuvant single-agent chemotherapy with doxorubicin. Chemotherapy did not improve the metastasis-free survival (MFS). After a median follow-up time of 48 months (range, 2 to 97), a multivariate analysis of prognostic factors for developing metastatic disease was performed. DNA aneuploidy was found to be an independent prognostic risk factor in addition to histologic malignancy grade IV, intratumoral vascular invasion, tumor size over 10 cm, and male sex. Patients with none or one risk factor had a 5-year MFS of 79%, with two risk factors 65%, with three risk factors 43%, and with four and five risk factors 0%. About one half (78 of 148) of the patients with three factors or less belonged to a group with a MFS over 60%. The combination of different risk factors, including DNA aneuploidy, seems to be a useful prognostic model for soft tissue sarcomas, which could be of value to select high-risk patients for further trials with adjunctive therapy.

Cytoskeletal heterogeneity of an epithelioid sarcoma with expression of vimentin, cytokeratins, and neurofilaments.

We studied an unusual sarcoma with morphologic features diagnostic of epithelioid sarcoma by conventional light microscopy, transmission electron microscopy, and immunohistochemistry. The primary tumor, which was located in the deep soft tissues of the buttock of a 32-year-old woman, and its metastases to lymph nodes, liver, and lung were available for investigation. The histomorphological and ultrastructural appearance of the primary tumor and its metastatic deposits were typical of epithelioid sarcoma. Immunohistochemistry revealed a strong and uniform reactivity for vimentin in both the primary tumor and its metastases. In contrast, a marked cytoskeletal heterogeneity became evident for cytokeratins and neurofilaments, which were observed exclusively in lymph node metastasis. To our knowledge, the observation of neurofilaments in epithelioid sarcoma has not previously been reported.

Cytoskeletal properties of alveolar soft part sarcoma.

The immunohistochemical expression of cytoskeletal proteins in alveolar soft part sarcoma (ASPS) was studied by light and electron microscopy. Of the five cases examined by the avidinbiotin-peroxidase complex method, variable numbers of immunoreactive cells for desmin were found in three, for vimentin in two, for muscle-specific actins in three, and for alpha-smooth muscle actin in four. Immunoelectron microscopic study demonstrated that desmin and vimentin were localized on whorled bundles of intermediate filaments in the perinuclear cytoplasm. In addition, a few dispersed intermediate filaments became evident in specimens treated with saponin and fixed with tannic acid. These immunohistochemical results indicate that a few tumor cells of ASPS may express some properties of the cytoskeleton of smooth muscle cells in addition to those of skeletal muscle cells. Considering the discrepancies reported in the actin isoforms demonstrated in myogenic tumors, we conclude that ASPS is probably a peculiar, primitive myogenic tumor that does not show any distinctive features of rhabdomyogenic or leiomyogenic differentiation.

Immunohistochemical study of uterine stromal sarcoma and rhabdomyosarcoma.

The cytoplasmic filament composition of two pure high-grade endometrial stromal sarcomas and five pure endometrial rhabdomyosarcomas was studied using the immunoperoxidase avidin-biotin method. Ultrastructural correlates were performed on the same tissues. While four of the seven tumors presented as high-grade undifferentiated neoplasms, they were readily categorized on the basis of immunocytochemical findings in which the five rhabdomyosarcomas were positive for muscle-specific actin (HHF 35) and the two stromal sarcomas were positive for vimentin only. Ultrastructure on the HHF 35-positive cases showed the presence of thick filaments and Z-band material, whereas the other tumors showed no cytoplasmic differentiation. Muscle-type actin differs stromal cells, and is useful when used in conjunction with a panel of antibodies in the categorization of undifferentiated endometrial sarcomas.

Malignant rhabdoid tumor of the vulva: is distinction from epithelioid sarcoma possible? A pathologic and immunohistochemical study.

Epithelioid sarcoma (ES) and malignant rhabdoid tumor (MRT) have heretofore been regarded as two separate clinicopathologic entities. However, they have some histologic similarities, and both represent histogenetic and phenotypic enigmas. This study reports the pathologic and immunohistochemical findings of four vulvar neoplasms occurring in young women that represented diagnostic dilemmas because of their similarity to both ES and MRT. Only one case had the classic histologic features of ES, whereas, in our opinion, the other three cases fulfilled the histologic criteria of MRT, despite the fact that two of the three cases were reported earlier as examples of ES. Neither electron microscopy nor immunohistochemistry has been found to be helpful in separating ES from MRT, mainly because they share several ultrastructural and immunophenotypic features. The behavior of these vulvar tumors--ours and the few published examples of ES--is generally aggressive, more in keeping with MRT than classic ES. We believe that some, if not most, putative ES of the vulva are in fact MRT, a neoplasm with an unfavorable prognosis.

A monoclonal antibody against nerve growth factor receptor. Immunohistochemical analysis of normal and neoplastic human tissue.

The expression of human nerve growth factor (NGF) receptor in tumors and normal tissue was investigated with the use of a monoclonal antibody recently developed against that protein. This antibody, NGFR5, reacted strongly with 100% of 25 nerve sheath tumors. Eight of nine pheochromocytomas and three of three paragangliomas also had positive results, but the immunoreactivity was restricted to the sustentacular cell population. Within cells of melanocytic lineage, there was no immunostaining of melanocytes in normal epidermis, whereas 13 of 14 benign nevi had positive results, primarily involving spindled nevocytic structures within the dermis. NGF receptor was scarcely expressed in human melanoma; 9 of 19 melanomas had positive results, but immunoreactivity was generally restricted to rare cells within the larger tumor cell population. Among nonneurogenic mesenchymal tumors, results were generally negative: 0 of 5 chondrosarcomas, 0 of 6 malignant fibrous histiocytomas, 0 of 3 meningiomas, and 1 of 8 leiomyosarcomas were immunoreactive. Carcinomas were variable in immunoreactivity: 12 of 16 squamous cell carcinomas had positive results, whereas adenocarcinomas demonstrated focal, basal epithelial immunoreactivity and neuroendocrine tumors generally had negative results. Among normal tissues, in addition to expected neural immunostaining, NGFR 5 reacted positively with several nonneural cell types, including lymphoidal follicular dendritic cells, myoepithelial cells, vascular adventitia, and basal epithelium of oral mucosa and hair follicles. Antibodies to NGF receptor may play a role in the identification of benign and malignant soft tissue lesions.

Myogenic expression in esophageal polypoid tumors.

Four cases of esophageal polypoid tumors composed of squamous cell carcinoma and spindle cell sarcomatous components were investigated. Squamous cell carcinoma was consistently present in the base of the polypoid lesions in all four cases and was also intermingled with spindle-shaped sarcomatous cells in two cases. Metastases in the lymph nodes were observed in two cases: one was squamous cell carcinoma with a sarcomatous component and the other consisted of a pure sarcomatous component. All tumors involved at least the muscularis mucosae. In the sarcomatous region, the tumor was composed of highly anaplastic cells with or without forming interlacing fascicles. Pleomorphism was marked and bizarre giant cell forms were frequent. Mitoses were frequently present. Immunohistochemical study revealed that the anaplastic cells in the sarcomatous component in all cases were immunoreactive to desmin, muscle actin, vimentin, and alpha 1-antichymotrypsin, but were negative for cytokeratin, even in the metastatic tumors of the lymph nodes. The immunohistochemical results favor myogenic differentiation of the anaplastic cells, and these tumors were considered to be true carcinosarcomas composed of squamous cell carcinoma and leiomyosarcoma.

Alveolar soft-part sarcoma in the uterine cervix.

We describe a case of an alveolar soft-part sarcoma of the cervix in a 35-year-old woman. The light- and electron-microscopic and immunohistochemical findings are described. Periodic acid-Schiff-positive, diastase resistant, intracytoplasmic crystals, pathognomonic for alveolar soft-part sarcoma, were present. The cells expressed immunoreactivity for neuron-specific enolase and protein S100. The results of our own immunohistochemical examinations and those presented in the literature are discussed.

Inhibitory properties of low molecular mass cysteine proteinase inhibitors from human sarcoma.

Elevated activities of cysteine proteinases such as cathepsins B and L and cancer procoagulant have been linked to tumor malignancy. In the present study we examined the hypothesis that these elevated activities could be due to impaired regulation by the endogenous low molecular mass cysteine proteinase inhibitors (cystatins). Inhibitors from human sarcoma were compared to those from human liver, a normal tissue in which the inhibitors had been characterized previously. An extract of cystatins from sarcoma was less effective against papain and cathepsin B (liver or tumor) than was an extract from liver. This reduced inhibitory capacity in sarcoma was not due to a reduction in either the concentrations or specific activities of the cystatins or an absence of any family or isoform of cystatins. We purified two members of the cystatin superfamily (stefin A and stefin B) to homogeneity and determined their individual inhibitory properties. Stefins B from liver and sarcoma exhibited comparable inhibition of papain and cathepsin B. In contrast, stefin A from sarcoma exhibited a reduced ability to inhibit papain, human liver cathepsins B, H and L and human and murine tumor cathepsin B. The Ki for inhibition of liver cathepsin B by sarcoma stefin A was 10-fold higher than that for inhibition of liver cathepsin B by liver stefin A, reflecting a reduction in the rate constant for association and an increase in the rate constant for dissociation. Cancer is now the third pathologic condition reported to be associated with alterations in cystatins, the other two being amyloidosis and muscular dystrophy.

Presence of cytoadhesins (IIb-IIIa-like glycoproteins) on human metastatic melanomas but not on benign melanocytes.

Glycoproteins IIb and IIIa, a heterodimer complex, play a vital role in blood platelet aggregation and are members of a wide family of membrane receptors known as integrins or cytoadhesins. Cellular interaction to extracellular matrix (ECM) adhesive proteins is mediated by integrins. Certain tumor cells are known to interact with ECM and blood platelets in the process of metastasis. However, it is not known if tumor cells, compared with their normal counterparts, acquire IIb-IIIa-like receptors to help them in their metastatic spread. In this study, monoclonal antibodies directed against the IIb-IIIa platelet glycoprotein complex were used on frozen biopsies of normal and various tumor tissues to detect the presence of these integrins. These studies demonstrate the presence of IIb-IIIa-like glycoproteins on the cells of metastatic malignant melanoma but not on benign melanocytes and rarely on other tumors. The presence of integrins on melanomas may help explain their propensity for frequent metastasis.

[Clear cell sarcoma: a clinicopathological study of 11 cases].

The clinicopathologic, ultrastructural and immunohistochemical features of eleven cases of clear cell sarcoma are described. There were 6 males and 5 females with an average age of thirty-six (10-59 years). Tumors were found arising from the tendons, aponeuroses and fascial structures with a predilection for the lower and upper extremities. Follow-up data was available in 8 patients. Five of them are alive. Nevertheless, 3 of the five showed evidence of recurrence or metastasis. The other 3 patients died of tumor with metastasis. Microscopically, the tumors were composed of short fascicles of fusiform cells with a clear to eosinophilic cytoplasm and vesicular nuclei with prominent nucleoli. Melanin was demonstrated in 5 cases and S-100 was known present focally in all cases, but no positive keratin staining was obtained. Electron microscopic studies revealed cell attachments and mature melanosomes. The exact histogenesis remains obscure, but our ultrastructural and immunohistochemical findings support the idea of neural crest origin of this tumor.

Sarcomatous tumor of the chest wall with osteochondroid differentiation. Evidence of mesothelial origin.

We describe a case of sarcomatous tumor of the chest wall with differentiation toward bone and cartilage that was observed in an asbestos-exposed worker. Although the mesothelial nature of the tumor was at first considered, it was not proven. Later, the tumor was shown to be a mesothelioma using a panel of pertinent antibodies that included a recently described anti-mesothelial cell marker. In addition, asbestos bodies were found in association with the sarcoma cells. Our findings indicate that whenever physicians encounter any type of primary sarcomatoid tumor involving serous membranes, the possibility of malignant mesothelioma should be regarded a priori.