Fibrosarcoma de partes blandas, su verdadera frecuencia y su diagnóstico diferencial / Soft-tissue fibrosarcoma, its true frequency and differential diagnosis

Se analizan todos los fibrosarcomas diagnosticados en el INOR en el período de 1974 a 1989. Se aplicaron técnicas inmunohistoquímicas y microscopia electrónica. Solamente 46 casos se reclasificaron como verdaderos fibrosarcomas. Otros 14 tumores se admiten como histiocitoma fibroso maligno sobre la base del pleomorfismo celular con células gigantes multinucleadas y estructuras en "rueda de carro". En 7 de ellos se pudo demostrar inmunorreactividad de las células tumorales a alfa-1-antitripsina. Doce casos se reclasificaron como fibromatosis agresiva musculoaponeurótica, 6 como carcinoma epidermoide fusocelular, 2 como sarcoma sinovial, 2 como liposarcoma fusocelular, 3 como schwannoma maligno, 4 como leiomiosarcoma, 4 como tumor epitelial de origen desconocido, 3 como sarcoma inclasificable y 1 como condrosarcoma mesenquinal extraesquelético, rabdomiosarcoma pleomórfico, dermatofibrosarcoma protuberans. En 1 caso por el patrón histológico y la positividad a anti-S 100 proteína el diagnóstico se cambió en sarcoma de células claras de tendones y aponeurosis

Interaction with hyperthermia of tetrachloroplatinum(II)(Nile blue)2 and tetrachloroplatinum(II)(neutral red)2 in EMT6 murine cells and the murine FSaIIC fibrosarcoma.

Complexes of the tetrachoroplatinum(II) dianion with positively charged nuclear dyes were prepared in an effort to produce agents which gain ready access into the nucleus and become very cytotoxic at clinically relevant hyperthermia temperatures. Pt(Nile blue)2 and Pt(neutral red)2 are complexes of tetrachloroplatinum(II) with two closely related p-quinonediamine dyes. Pt(Nile blue)2 and Pt(neutral red)2 were only moderately cytotoxic to exponentially growing normally oxygenated or hypoxic EMT6 cells in vitro at pH 7.40 and 37 degrees C. At pH 7.40 and 42 degrees C and especially at 43 degrees C, however, Pt(Nile blue)2 became far more cytotoxic. At pH 6.45 Pt(Nile blue)2 became more toxic toward hypoxic cells (cell kill of 3.5 logs at 500 microM, 42 degrees C for 1 h). Pt(neutral red)2 became much more cytotoxic at pH 6.45 and 42 degrees C or 43 degrees C compared to pH 7.4, and the cell kill observed was similar in both euoxic and hypoxic cells (3 logs at pH 6.45, 43 degrees C with only 100 microM). Tumor cell survival studies in the FSaIIC murine fibrosarcoma demonstrated that both drugs killed in a dose-dependent log-linear manner. Hyperthermia treatment (43 degrees C, 30 min) immediately after either drug resulted in a dose modifying effect. The tumor growth delay produced by Pt(Nile blue)2 (100 mg/kg) was 4.6 days and by Pt(neutral red)2 (100 mg/kg) was 3.8 days. Both drugs were markedly improved by hyperthermia (tumor growth delay 1.4 days for hyperthermia; tumor growth delay 10.9 days for Pt(Nile blue)2 and 8.0 days for Pt(neutral red)2. Intracellular platinum levels were approximately 200 times higher after exposure of EMT6 cells to 25 microM of Pt(Nile blue)2 or Pt(neutral red)2 for 1 h at 37 degrees C than after exposure to the same concentration of cis-diamminedichloroplatinum(II). Treatment of cells with the drugs at 42 degrees C (1 h) resulted in no change in platinum levels with cis-diamminedichloroplatinum(II), but with Pt(Nile blue)2 and Pt(neutral red)2 an increase of 2- to 3-fold was found. Since previous work has shown that both of these complexes are active radiosensitizing agents, these new drugs seem quite well suited for further development as antitumor agents for use against solid tumors alone and in conjunction with hyperthermia and/or radiation therapy.

Glycosphingolipid expression on murine L1-fibrosarcoma cells: analysis of clonal in vivo and in vitro selected sublines with different lung colonisation potential.

The patterns of acidic and neutral glycosphingolipids (GSLs) were examined in a syngeneic tumour system in Balb/c mice consisting of closely related cell lines with different colonisation potentials directed to the murine lungs (in vivo selected highly metastatic sublines of L1-fibrosarcoma cells and their WGA-resistant mutants with low metastatic potential). GSLs were analysed by high-performance thin-layer chromatography and structurally identified by fast atom bombardment mass spectrometry combined with compositional analyses and exo-glycosidase digestion. The results suggest that highly metastatic sublines L1-LM and L1-LM12 derived by in vivo selection from mouse fibrosarcoma cells (cell line L1) exhibit a drastic increase of polar ganglioside expression and a restriction to globo-series GSLs. Contrasting with this the low metastatic mutant cells (L1-LM13WGA) express a reduced portion of acidic GSLs and exhibit a shift to less polar ganglioside components. Total cellular and plasma membrane-integrated GSLs were demonstrated to exhibit largely identical patterns. Concomitant with a significant decrease in LacCer expression a substantial reduction of GM2 and a complete lack of GM3 expression can be assigned to the highly metastatic sublines of L1-cells. On the other hand, the more polar gangliosides GM1a and, to an even greater extent, GD1a (exceeding 70% of total gangliosides) accumulate on L1-LM and their clonal sublines. The shift to acidic GSLs of higher polarity is less pronounced on the low metastatic WGA-resistant mutant cells (L1-LM13WGA) showing a preponderance of GM1a. The portion of GD1a within the fractions of acidic GSLs does not correspond to the cellular activities of CMP-NeuAc/GM1 (alpha 2-3) sialyltransferase measured for high and low metastatic cell variants. Total sialic acid content of the various cell lines differs, but is not associated with the metastatic potential. Gangliosides on L1-cells exhibit a significant substitution of N-glycolyl for N-acetylneuraminic acid (13%) compared to their metastatic sublines and to mutant cells (less than 1%). A conversion of surface exposed GD1a to GM1a on membranes of metastatic cells by in situ treatment with Vibrio cholerae sialidase is associated with a significant reduction of tumour cell colonisation directed to the murine lungs.

Spectral editing of tumor 13C MRS in situ.

The editing pulse sequence DEPT (D.T. Pegg, D.M. Doddrell, and M.R. Bendall, J. Chem. Phys. 77, 2745 (1982)) was modified using a scheme of various composite pulses and a 16-step phase cycling to obtain proton-decoupled natural-abundance 13C edited subspectra of solid tumors. A solenoidal probe including a Faraday shield and an orthogonal saddle decoupling coil was built for this purpose.

Purification and characterization of recombinant plasminogen activator inhibitor-1 from Escherichia coli.

A recombinant form of plasminogen activator inhibitor-1 (rPAI-1) has been purified from lysates of pCE1200, a bacterial expression vector containing the full length PAI-1 gene, by utilizing sequential anion exchange and cation exchange chromatography on Q-Sepharose and S-Sepharose columns. Approximately 140 mg of rPAI-1, estimated at 98% purity on the basis of analytical high performance liquid chromatography, could be obtained from 200 g wet weight of cells. The purified protein exhibited a single Coomassie Blue-stainable band at the region of Mr = 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an NH2-terminal amino acid sequence consistent with the expected translation product of the pCE1200 PAI-1 insert. The rPAI-1 rapidly inhibited single- and two-chain tissue plasminogen activators, as well as urokinase, with apparent second order rate constants in the range of 2-5 x 10(7) M-1 s-1. A specific activity measurement of 250,000 units/mg was calculated for the rPAI-1 based on its ability to inhibit the enzymatic activity of a single-chain tissue plasminogen activator. Stability studies showed that the activity of the rPAI-1 was very stable when stored at temperatures of 25 degrees C or lower, but decayed within hours when stored at 37 degrees C. Sodium dodecyl sulfate treatment, which partially activates the latent form of natural PAI-1, inactivated rPAI-1. These results show that the purified rPAI-1 produced from pCE1200 displays many of the properties associated with the biologically active form of natural PAI-1.

Immunohistochemical analysis of bone morphogenetic protein (BMP) in osteosarcoma.

The monoclonal antibody against bovine bone morphogenetic protein (bBMP-McAb) was first used for demonstration of bone morphogenetic protein (BMP) in osteosarcoma. The avidin-biotin complex method (ABC) demonstrated that of the 18 osteosarcomas, 15 stained positive, while all 6 fibrosarcomas were negative. The results showed that BMP mainly exists in the tumor cell plasma and some tumor-like bone tissues. Using this staining method, we can not only differentiate osteosarcoma from fibrosarcoma and other non-bone-derived tumors, but also classify osteosarcoma according to the content and distribution of BMP and the patient's clinical situation, thus providing a scientific basis for clinical treatment.

An immunohistochemical study of dermatofibrosarcoma protuberans supports its fibroblastic character and contradicts neuroectodermal or histiocytic components.

Paraffin-embedded material from 26 cases of dermatofibrosarcoma protuberans (DFSP) was investigated by the peroxidase-antiperoxidase technique. Antibodies to S-100 protein, Leu-7 antigen, and neuron-specific enolase (neural markers); to lysozyme, alpha-1-antitrypsin, and alpha-1-antichymotrypsin (histiocytic markers); and to cytokeratin, desmin, vimentin, and factor VIII were used. The tumor cells reacted only for vimentin. In addition, 12 cases showed positive reactions with histiocytic markers (1-3% of the cells; in two cases, up to 10%). These results support a fibroblastic and contradict a neural or histiocytic histogenesis of DFSP.

[The membrane sialoglycolipids of tumor cells and the metastasis of bone tumors]. / Sialoglikolipidy membran opukholevykh kletok i metastazirovanie opukholei kostei.

Level and profile of gangliosides were studied in osteogenic and chondrosarcoma cells. Level of lipid-binding sialic acids in bone- and cartilage-producing tumors proved different. Most osteogenic sarcoma samples showed higher level of lipid-binding sialic acids as compared to chondrosarcoma. In the latter tumor, level of lipid-binding sialic acids was related to grade of tumor cell differentiation, peak levels being observed in undifferentiated neoplasms as compared to those showing grade I-II cell anaplasia. Chondro- and osteogenic sarcoma revealed different profiles of sialoglycolipids, particularly, due to markedly reduced set of gangliosides and nearly complete loss of polysialogangliosides in the latter tumor.

Dexamethasone-induced plasminogen activator inhibitor: characterization, purification, and preparation of monoclonal antibodies.

The effects of dexamethasone on protein synthesis were studied in human fibrosarcoma (HT-1080) cells. Dexamethasone induced a new protein of 46 kD which was rapidly secreted into the medium, while neither progesterone nor estradiol would induce the synthesis of this protein and only a small increase in its amount could be seen in the presence of testosterone. The 46 kD protein was partially purified by ammonium sulfate precipitation and gel filtration and mouse monoclonal antibodies to it were produced in mouse hybrid cells. Altogether 13 positive clones were found, of which six reacted only with native and seven reacted with the unreduced 46 kD protein in Western blotting. It was possible by using polyclonal antibodies to plasminogen activator inhibitor type I (PAI-1) and purified plasminogen activator inhibitor type I to confirm that the 46 kD protein purified and characterized here was PAI-1. In addition, the 46 kD protein clearly inhibited plasminogen activation, thus further confirming that protein isolated was an inhibitor of plasminogen activator. Since the induction of PAI-1 by dexamethasone was very extensive, it is possible that glucocorticoids regulate proteolysis and fibrinolysis in vivo by increasing the amount of the inhibitor of plasminogen activator and thus preventing the activation of plasminogen to plasmin. The reduction of activation of plasminogen to plasmin by glucocorticoid-induced inhibitor could be of great importance, e.g., in various blistering diseases, in metastases from malignant cells, and in the migration of inflammatory cells.

Detection of the 170 kDa P-glycoprotein in neoplastic and normal tissues.

A membrane purification procedure and an immunoblotting assay have been designed to allow screening of human solid tumors for overexpression of the GP170 glycoprotein without employing a disaggregation method to obtain cell suspensions. The electrophoresed membrane proteins were probed, after Western Blotting, with the C219 monoclonal antibody and iodinated Protein A. The labeling intensity of the bands on the autoradioimmunoblots were quantified by densitometry. To test for the presence of GP170, we used membranes from the UV 2237 fibrosarcoma line and its adriamycin-resistant variant ADMR, grown in vitro or as solid tumor in mice. Membranes of human normal and tumor tissues obtained from previously untreated patients were also tested. An immunoreaction was observed in the adriamycin-resistant UV 2237 lines grown in vitro or in vivo. Quantitatively, the binding of the resistant cell line grown in vitro was higher than that observed in cells grown in mice. Bands in the GP 170 region were observed in 4/7 normal and in 7/7 tumor colon tissues and in the normal medulla from 2 patients with cancer of the renal cortex. No reaction could be found in samples from normal tissue, primary tumor or nodal metastasis from 7 patients with breast cancer.

[Flow cytometry for the determination of DNA in head and neck carcinomas]. / La citometría de flujo para la determinación de DNA en los carcinomas de cabeza y cuello.

The technique used for DNA determination in tumoral cells from carcinomas of head and neck is described. Results obtained are commented on as is too the future of this technique for the better understanding of the behavior of the tumor and consequently of its treatment.

The expression of intermediate filaments in canine mammary glands and their tumors.

Monoclonal antibodies specific for different types of intermediate filaments (cytokeratin, vimentin, desmin and neurofilaments) were used to study the histogenesis of canine mammary glands and 57 canine mammary tumors by immunocytochemistry. The intra- and interlobular duct epithelium, acinar, and intralobular myoepithelial cells stained positively for cytokeratin. Peripheral ductal and acinar cells, as well as interstitial cells, stained positively for vimentin. A similar staining pattern was seen in adenomas, complex adenomas, benign mixed tumors, ductular carcinomas, and one myoepithelioma-like tumor. Additionally, cytokeratin positive cells were scattered interstitially in one single adenoma, most complex adenomas, some benign mixed tumors, complex carcinomas, and in the malignant mixed tumors. All stromal cells stained positively for vimentin. The fibrosarcomas were positive only for vimentin, while the following expressed both desmin and cytokeratin: epithelial-like cells in one adenoma, three complex adenomas, the myoepithelioma-like tumor, the single comedo carcinoma, two complex carcinomas, the single lobular carcinoma, one malignant mixed tumor, and three osteosarcomas. Epithelial-like cells in one adenoma, six complex adenomas, two benign mixed tumors, two complex carcinomas, the lobular carcinoma, and the malignant schwannoma stained for neurofilaments. Three tumors, one adenoma, one complex adenoma, and the lobular carcinoma expressed both desmin and neurofilaments in addition to cytokeratin and vimentin. The results show the expression of different types of intermediate filaments and indicate that there might be a stem cell origin in most of the canine mammary tumors.

The Mr 140,000 fibronectin receptor complex in normal and virus-transformed human fibroblasts and in fibrosarcoma cells: identical localization and function.

We studied the function and localization of the fibronectin receptor complex in cultured normal and SV40-transformed human fibroblasts and in human fibrosarcoma cells by using monoclonal antibodies (MAbs) against the beta sub-unit of the receptor. Immunoprecipitation, fibronectin fragment affinity chromatography and immunoblotting results suggested that all the cells studied had similar amounts of the receptor. In normal fibroblasts MAbs additionally immunoprecipitated a smaller polypeptide, revealed as the precursor for the beta sub-unit and another polypeptide shown to be the alpha sub-unit of the VLA-I complex. The emergence of vinculin-positive focal adhesion sites and actin stress fibers was slower in the malignant cells than in normal fibroblasts when the cells were plated on non-coated glass-substrate in serum-free conditions and the fibronectin receptor complex did not become located to focal adhesions in any of the cells studied. Added substratum-bound but not soluble fibronectin mediated assembly of the fibronectin receptor complex to the focal adhesions in both normal and malignant cells. On fibronectin-coated growth substrate stress fibers also emerged as rapidly in the malignant cells as in normal fibroblasts. In all the cells the receptor complex, however, became largely dissociated from the focal adhesions within 48 hr. In cell adhesion conditions MAb against the alpha sub-unit of VLA-I complex revealed an even cell-surface labelling in normal fibroblasts and lack of labelling in malignant cells.

[Protein S-100 in spontaneous soft tissue and peripheral nerve neoplasms in rats]. / Belok S-100 v spontannykh opukholiakh miagkikh tkanei i perifericheskikh nervov krys.

16 spontaneous tumors of the peripheral nerves and 18 spontaneous tumors of mesenchymal origin in BDVI rats were studied by peroxidase-antiperoxidase method using anti-serum (DAKOPATT) against protein S-100. The majority of spontaneous peripheral nerve tumors were of cystic histological structure identical to that of cystic neurinomas induced in rats by ethylnitrosourea and almost all of these tumors were S-100 protein positive. The incidence of spontaneous neurinomas in BDVI rats was in some experiments as high as 5%. All tumors of mesenchymal origin (except one lipoma) were S-100 protein negative: 2 fibromas, 6 fibrosarcomas, 3 malignant fibrous histiocytomas, one rhabdomyosarcoma and one hemangioendothelioma. S-100 protein is found, as in human pathology, useful for distinguishing tumors of schwann cell and mesenchymal origin in rats.

Separation and evaluation of changing pattern of glycosaminoglycans in 3-methyl cholanthrene induced fibrosarcoma.

The present study evaluates the glycosaminoglycans (GAGs) pattern associated with transplantable rat fibrosarcoma induced by 3-methylcholanthrene. The quantitative analysis of fractionation of GAGs in fibrosarcoma and fetal tissues was performed by enzymatic digestion. The average value of total GAGs in fibrosarcoma and fetal tissue was found to be 4 times higher than its value in the tissue of origin. GAG content showed a steady increase from 7th day onward to 25th day. Hyaluronic acid content in tumor tissue was observed to increase markedly (8-fold) against that of the normal tissue and was equivalent to that of the fetal tissue. Chondroitin sulfate level was also increased in the tumor as well as fetal tissue. The increase in the chondroitin sulfate and hyaluronic acid contents might possibly be due to the abnormal GAG metabolism in the increased production of both sulfated and nonsulfated GAGs.

Detection and quantification of ligand leakage from lectin affinity columns.

The usefulness of lectin affinity chromatography for the preparation of glycoproteins is impaired by ligand release. Ligand leakage from mistletoe lectin (MLI) Sepharose 4B column was detected by 24 h skin reaction in mice and by immunoblotting. Immunoaffinity chromatography was found to be an efficient method for the separation of lectin traces from the glycoprotein fraction. A sugar concentration dependent increase of lectin release from MLI-Sepharose 4B column was detected by a solid phase enzyme immunoassay.

Estimation of tumor oxygenation and metabolic rate using 31P MRS: correlation of longitudinal relaxation with tumor growth rate and DNA synthesis.

31P MRS longitudinal relaxation times (T1) were determined for C3H murine fibrosarcomas (FSaII), and mammary carcinomas (MCaIV). Tumors were implanted in the foot dorsum, and were 100-300 mm3 in volume. T1s were repeated after the animal was allowed to breathe 100% oxygen for 30 min and then again 36-48 hr following 30 Gy. The spectrum were obtained using an 8.5 T spectrometer with a 8 cm bore and a 1.4 cm single turn antenna coil. The 31P relaxation times for untreated tumors in air breathing animals were: 3.78 sec for phosphomonoesters, 4.37 sec for inorganic phosphate (Pi), 2.73 sec for phosphocreatine, 1.37 sec for gamma ATP, 1.14 sec for alpha ATP, and 1.18 sec for beta ATP. The Pi T1s were 4.37 and 4.70 sec in control and irradiated tumors in air breathing animals. Respiration of oxygen for 30 min reduced the T1s to 3.02 and 2.62 sec in control and irradiated tumors respectively. The Pi T1 of an anoxic tumor, determined on an in situ tumor 60 min after death was 5.93 sec. The oxygen breathing induced decrease in the T1 of Pi is unlikely to have been caused by the paramagnetic properties of oxygen alone, and suggests a component of increased magnetization transfer secondary to the ATPase reaction. Oxygen breathing following 30 Gy, resulted in a decreased growth time (800 mm3 endpoint) and an increased proportion of cells in S-phase. These results support the hypothesis that the decrease in Pi T1 measured with oxygen breathing is a measure of tumor oxygen tension and metabolic rate, and suggests that T1 measurement may indirectly predict tumor growth rate and DNA synthesis.

Purification of the beta subunit of the fibronectin receptor.

In this work we describe a method for purification of the beta subunit of the mouse fibronectin receptor (GP135). Cellular glycoproteins were isolated from a detergent extract of SR-Balb tumor cell membranes by two steps of affinity chromatography on lentil lectin-Sepharose and wheat-germ-agglutinin--agarose. This material was subsequently bound to an Affi gel 102 column and eluted by increasing salt concentration. Most of the GP135 was eluted at 80 mM sodium chloride together with a few other components. A final step of hydroxyapatite chromatography in sodium dodecyl sulphate allowed elution of GP135 as a single chromatographic peak. Fractions containing GP135 were identified at each chromatographic step by immunoblotting with a specific antiserum. By this procedure GP135 was purified to homogeneity as judged by SDS-PAGE analysis of 125I-labelled material.

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.

Spindle-cell and pleomorphic neoplasms of the skin. A clinicopathologic and immunohistochemical study of 30 cases, with emphasis on "atypical fibroxanthomas".

Atypical fibroxanthoma belongs to the family of spindle-cell and pleomorphic neoplasms of the skin. The lineage of differentiation of this tumor and the means whereby it can be diagnostically separated from other similar morphologic entities have been controversial. In order to address these issues, the authors studied 30 spindle-cell and/or pleomorphic cutaneous tumors, including atypical fibroxanthomas (AFXs), superficial malignant fibrous histiocytomas (MFHs), dermatofibrosarcoma protuberans (DFSPs), sarcomatoid squamous-cell carcinomas (SCCs), spindle-cell malignant melanomas (MMs), and leiomyosarcomas, (LMSs). These cases were analyzed using a panel of eight antibodies and the immunoperoxidase technique. AFXs were reactive for vimentin, alpha-1-antichymotrypsin (AACT), alpha-1-antitrypsin (AAT), and cathepsin-B (CB) but failed to display cytokeratin (CK), epithelial membrane antigen (EMA), S-100 protein, and desmin. MFHs and DFSPs exhibited immunophenotypic profiles that were nearly identical to that just described. In contrast, SCCs were typified by positivity for CK and EMA; MMs exhibited uniform reactivity for S-100 protein; and LMSs contained desmin in four of five cases. A surprising result was the expression of S-100 by LMSs. Also, all tumors displayed at least one of the three proteolytic enzymes assessed in this study (AAT, AACT, and CB), demonstrating the relative diagnostic nonspecificity of these determinants. It is concluded that AFXs are "fibrohistiocytic" neoplasms, with substantial morphologic and immunohistochemical similarity to MFHs. The immunohistochemical classification of spindle-cell and pleomorphic tumors of the skin necessitates the use of antibody panels to assess the presence of markers that are characteristic of each diagnostic group.