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Summary Background Cutaneous CD30+ lymphoproliferative disorders (LPDs) are a spectrum of disease associated with a favourable prognosis. Systemic anaplastic large cell lymphoma (ALCL), although morphologically and phenotypically similar, differs in clinical presentation and has a less favourable biological behaviour. Dysregulation of apoptosis, the process regulating cell population by programmed death, can explain the differences among these disorders. Objectives We investigated the expression of two inhibitors of apoptosis, survivin and Bcl-2 protein, in serial skin lesion samples from CD30+ LPDs compared with systemic ALCL. Methods Immunohistochemical analysis with antibodies against anaplastic lymphoma kinase (ALK)-1 protein, survivin and Bcl-2 protein was performed in 10 cutaneous CD30+ LPDs (five lymphomatoid papulosis, five ALCL) and 18 systemic ALCLs. Reverse transcription–polymerase chain reaction studies for ALK and ALK/nucleophosmin were also performed. Results Cutaneous CD30+ LPDs shared a heterogeneous expression of cytoplasmic survivin with all systemic ALCLs, and of Bcl-2 with systemic ALK− ALCLs; however, they differ from systemic ALK− ALCLs because they lack nuclear survivin (P = 0·045), and from systemic ALK+ ALCLs by a higher expression of Bcl-2 (P = 0·045) and a lack of ALK-1. Overall, coexpression of Bcl-2 and nuclear survivin in CD30+ LPDs was associated with a less favourable disease survival. Conclusions The different patterns of expression of Bcl-2 and survivin in CD30+ LPDs might have an impact on their different biological and clinical behaviour. Moreover, nuclear localization of survivin, similarly to ALK, may be a useful marker for predicting a systemic form of ALCL with cutaneous presentation.

The receptor for urokinase plasminogen activator (uPAR) acts as an anchorage site for uPA on the cell surface where it stimulates pro-uPA activation, allows the internalization of uPA:inhibitor and other complexes and sends directly or indirectly signals into the cell that may promote migration, adhesion and growth. It is a GPI-anchored, three-domain protein that belongs to the Ly6 family and is present at the focal and cell-to-cell contacts, where it concentrates uPA activity. Its activity appears to be important to regulate the invasiveness of human cancer cells both in vitro and in vivo, and its inhibition is now a target for antimetastatic therapy.

Somatic mutations of genes codifying for key regulatory proteins are the cause of different types of hormone-secreting adenomas. Natriuretic peptides (NP) are the strongest inhibitors of aldosterone secretion but aldosterone-secreting adenomas (aldosteronomas) are resistant to this inhibition and have reduced binding sites for NPs. The objective of this study was to sequence the entire coding region of the NP receptor type A (NPRA, codified by the Npr1 gene) to find loss-of-function somatic mutations. Total RNA was extracted from eight aldosteronomas and cDNA was synthesized. NPRA mRNA expression was evaluated by Northern blot analysis and compared with beta-actin mRNA as the housekeeping gene. Twelve primer couples were designed on the basis of the Npr1 gene organization to amplify, by PCR, all 22 coding exons of the gene. The two strands of amplified DNAs were purified and directly sequenced by automated capillary sequencer. NPRA mRNA expression did not differ among aldosteronomas. Npr1 open reading frame sequences obtained from eight aldosteronomas did not contain any mutation. The coding sequences of all 22 exons were identical in all samples and identical to published sequences. In the 3'-untranslated region (3'-UTR) a new length difference 3C/4C polymorphism was found at position 15 129 (three adenomas were 3C/4C and two were 3C/3C). Such a 3C/4C polymorphism was present in genomic DNA from 80 control subjects (25, 4C/4C; 40, 3C/4C; 15, 3C/3C). Mutations in the coding exons of the Npr1 gene do not appear to be a common cause of aldosteronomas. Moreover, the exons of Npr1 encoding for the translated portion of mRNA do not appear to be prone to polymorphisms. The polymorphism identified in the 3'-UTR might affect mRNA stability resulting in lower receptor synthesis, but it is not likely to confer a predisposition to the development of aldosteronomas.

BACKGROUND: This study was designed to test cyclin D1 as a prognostic marker in patients with soft tissue sarcomas (STS), and to evaluate the usefulness of laparoscopy for determining cyclin D1 overexpression. METHODS: The records of 62 patients with STS were collected: 28 with retroperitoneal STS (RSTS) and 34 with extremity STS (ESTS). A total of 51 patients underwent surgical resection, whereas 11 did not undergo surgery because of advanced tumor stage. Preoperative-intraoperative laparoscopic staging was performed for patients judged to be resectable at preoperative imaging. RESULTS: Cyclin D1 was overexpressed in 30 (58.8\%) of 51 resected patients and in 10 (90.9\%) of 11 nonresected patients. Laparoscopy avoided unnecessary laparotomy in 9 (32.1\%) of 28 RSTS patients. CONCLUSIONS: High tumor grade, positive surgical margins, local recurrence, distant metastases, and cyclin D1 overexpression were related to poor survival. Multivariate analysis demonstrated cyclin D1 to be the only independent factor. Laparoscopy was shown to be useful for avoiding useless laparotomies.

The erbB-2 gene product, gp185erbB-2, displays a potent transforming effect when overexpressed in NIH 3T3 cells. In addition, it possesses constitutively high levels of tyrosine kinase activity in the absence of exogenously added ligand. In this study, we demonstrate that its carboxy-terminal domain exerts an enhancing effect on erbB-2 kinase and transforming activities. A premature termination mutant of the erbB-2 protein, lacking the entire carboxy-terminal domain (erbB-2 delta 1050), showed a 40-fold reduction in transforming ability and a lowered in vivo kinase activity for intracellular substrates. When the carboxy-terminal domain of erbB-2 was substituted for its analogous region in the epidermal growth factor receptor (EGFR) (EGFR/erbB-2COOH chimera), it conferred erbB-2-like properties to the EGFR, including transforming ability in the absence of epidermal growth factor, elevated constitutive autokinase activity in vivo and in vitro, and constitutive ability to phosphorylate phospholipase C-gamma. Conversely, a chimeric erbB-2 molecule bearing an EGFR carboxy-terminal domain (erbB-2/EGFRCOOH chimera) showed reduced transforming and kinase activity with respect to the wild-type erbB-2 and was only slightly more efficient than the erbB-2 delta 1050 mutant. Thus, we conclude that the carboxy-terminal domains of erbB-2 and EGFR exert different regulatory effects on receptor kinase function and biological activity. The up regulation of gp185erbB-2 enzymatic activity exerted by its carboxy-terminal domain can explain, at least in part, its constitutive level of kinase activity.

Monotypic epithelioid angiomyolipoma is a distinct and definable variant of angiomyolipoma, composed of monomorphous epithelioid cells that show HMB45 immunoreactivity. Angiomyolipoma, including its morphological variants, belongs to the family of perivascular epithelioid cell tumour.The tumour was examined using immunohistochemical staining and by transmission electron microscopy. Neoplastic cells showed a cytoplasmic granular positivity for HMB45.Extrarenal angiomyolipomas are rare and, to the best of our knowledge, this is the first reported case of a primary monotypic epithelioid angiomyolipoma of bone in a patient without evidence of tuberous sclerosis.

Met protein encoded by MET oncogene is the high affinity receptor for hepatocyte growth factor (HGF)/scatter factor (SF). HGF/SF has to be cleaved in its heterodimeric form by the urokinase-type plasminogen activator (uPA) to become active as a ligand for Met receptor. The expression of Met protein and of the high affinity receptor for uPA (uPA-R) was investigated in 39 samples of papillary carcinoma using immunohistochemistry. Reactivity for Met protein was present in 33 of 34 tumours, mostly with a diffuse pattern of staining. Reactivity for uPA-R was present in 78 per cent of papillary tumours and exhibited a pattern of staining similar to that of Met protein. Staining for uPA-R was present in 23 of 25 cases (92 per cent) of papillary carcinoma with prominent sclerosis, and in only 1 of 7 cases (14 per cent) without sclerosis. Peritumoural normal thyroid, follicular adenomas, and follicular carcinomas were negative for Met protein and for uPA-R. Hyperfunctioning tall thyroid cells showed weak membrane reactivity for uPA-R and for Met protein. The findings of immunohistochemistry were confirmed at the mRNA level using in situ hybridization, since the signal for uPA-R and Met RNAs was detected in most tumour cells of five cases of papillary carcinoma.

In order to reach the sites of inflammation, lymphocytes leave the bloodstream and migrate into peripheral tissues, in a process involving integrin-mediated adhesion to the vascular endothelium, followed by transmigration across the endothelial barrier and through the underlying interstitial matrix. We have investigated the role of the plasminogen activator/plasmin system in normal T cell migration. Receptors for urokinase plasminogen activator (uPAR) were not expressed in resting T lymphocytes, but could be efficiently induced at the mRNA and protein level by coclustering of the antigen receptor complex and beta1 or beta2 integrins, through a signalling pathway involving both protein kinase C activation and an increase in intracellular cyclic AMP. Catalytic activation of plasminogen by uPAR-expressing T cells promoted their migration through an extracellular matrix in vitro. Plasmin-induced invasion was inhibited by plasmin-and urokinase inhibitors and by anti-uPAR antibodies. Finally, cytofluorimetric and immunohistochemical analysis of primary human tumor specimens showed the presence of uPAR positive infiltrating T cells in vivo. Collectively, these findings suggest that plasminogen activation may play a role in lymphocyte migration in vivo, and that integrin-dependent expression of membrane-associated endopeptidases could represent an additional step in the regulated process of leukocyte transmigration.

Physiological concentrations of urokinase plasminogen activator (uPA) stimulated a chemotactic response in human monocytic THP-1 through binding to the urokinase receptor (uPAR). The effect did not require the protease moiety of uPA, as stimulation was achieved also with the N-terminal fragment (ATF), while the 33 kDa low molecular weight uPA was ineffective. Co-immunoprecipitation experiments showed association of uPAR with intracellular kinase(s), as demonstrated by in vitro kinase assays. Use of specific antibodies identified p56/p59hck as a kinase associated with uPAR in THP-1 cell extracts. Upon addition of ATF, p56/p59hck activity was stimulated within 2 min and returned to normal after 30 min. Since uPAR lacks an intracellular domain capable of interacting with intracellular kinase, activation of p56/p59hck must require a transmembrane adaptor. Evidence for this was strongly supported by the finding that a soluble form of uPAR (suPAR) was capable of inducing chemotaxis not only in THP-1 cells but also in cells lacking endogenous uPAR (IC50, 5 pM). However, activity of suPAR require chymotrypsin cleavage between the N-terminal domain D1 and D2 + D3. Chymotrypsin-cleaved suPAR also induced activation of p56/p59hck in THP-1 cells, with a time course comparable with ATF. Our data show that uPA-induced signal transduction takes place via uPAR, involves activation of intracellular tyrosine kinase(s) and requires an as yet undefined adaptor capable of connecting the extracellular ligand binding uPAR to intracellular transducer(s).

Several signal transducers bind through their SH2 domains to phosphotyrosine-containing motifs present in receptor tyrosine kinases (RTKs). However, the juxtamembrane regions of the epidermal growth factor receptor (EGFR) and of the related erbB-2 protein, while important in mitogenic signaling, lack demonstrable tyrosine phosphorylation sites, suggesting that other modalities of receptor-transducer interactions exist. A candidate for investigating this type of association is p97eps8, a recently described substrate for RTKs. p97eps8 is phosphorylated by several RTKs, associates with EGFR in vivo and, upon overexpression, enhances the transduction of EGFR-mediated mitogenic signals. Here we report that eps8 binds directly to the juxtamembrane region of EGFR through a domain that does not bear resemblance to SH2 domains and by a mechanism that does not require the presence of phosphotyrosine residues. Thus, the physical association between EGFR and eps8 represents a novel interaction between RTKs and their substrates.

The authors describe a case of pulmonary metastasis of giant cell tumor, the primary site of which was the radius. Treatment of the primary site and the metastasis is discussed.

A method for the isolation of tyrosine kinases substrates was developed. The method takes advantage of immuno-affinity purification of an entire set of proteins phosphorylated by tyrosine kinases, followed by generation of antisera against the purified protein pool and immunological screening of bacterial expression libraries with these antisera. By applying this methodology to the study of the phosphorylation events triggered by activation of the epidermal growth factor receptors, we have isolated several cDNAs encoding novel putative tyrosine kinase substrates. One of these cDNAs encodes radixin, a protein belonging to the band 4.1 family of proteins and highly related to ezrin and moesin. We demonstrated that, despite a high degree of relatedness, these three proteins exhibit a distinct receptor-specific pattern of phosphorylation, raising the possibility that they might mediate receptor-specific cellular changes. In addition the generation of antibodies specific for either radixin, ezrin or moesin allowed us to show that a previously described tumor transplantation antigen is indeed ezrin, thus implicating this protein in the determination of the biological phenotype of certain tumors.

To gain insight into the mechanisms which control the mitogenic response to epidermal growth factor (EGF), we have partially purified and characterized several intracellular proteins which are phosphorylated on tyrosine residues following activation of the epidermal growth factor receptor (EGFR). Partial purification was achieved by immunoaffinity chromatography using immobilized anti-phosphotyrosine antibodies. Antisera generated against the partially purified proteins were used to identify at least five novel EGFR putative substrates, designated, on the basis of their apparent molecular weight, p97, p68, p61, p56, and p23. All of these proteins became specifically phosphorylated on tyrosine after EGF treatment of intact cells, as assessed by phosphoamino acid analysis, and none of them represented an EGFR degradation product. The phosphorylation of these proteins appeared to be relatively specific for the EGFR. In particular, an EGFR-related kinase, erbB-2 was much less efficient than EGFR at phosphorylating p97, p56, and p23 and incapable of phosphorylating p68. The identification of these novel EGFR putative substrates should lead to a better understanding of the mechanisms controlling the specificity of EGFR-mediated mitogenic signaling.

The epidermal growth factor receptor (EGFR) and gp185erbB-2 are closely related tyrosine kinases. Despite extensive sequence and structural homology, these two receptors display quantitative and qualitative differences in their ability to couple with mitogenic signalling pathways. By using chimeric molecules between EGFR and erbB-2, we found that the determinants responsible for the specificity of mitogenic signal transduction are located in the amino-terminal half of the tyrosine kinase domain of either receptor. In the EGFR, mutational analysis within this subdomain revealed that deletion of residues 660 to 667 impaired receptor mitogenic activity without affecting its tyrosine kinase properties. This sequence is therefore likely to contribute to the specificity of substrate recognition by the EGFR kinase.

The authors describe embryology and anatomy of synovial plica of the knee joint. They propose a clinic and pathogenic classification and after examining the diagnostic problem of plica syndrome, analyze their long-term results after surgical treatment.

The authors describe 4 female patients with Osteitis Condensans Ilii (OCI): 3 of them were pluriparous, ranging 32-36 years of age; while the remaining was nulliparous and 20 years old, with a previous trauma suffered in the iliac bone. Due to the inefficacy of the medical and physical treatment, two patients underwent surgical resection of the osteitic bone; then followed by a 45 days cast immobilization and a 3-4 months with restricted load. The other 2 patients were successfully treated with Indomethacin (a 100 mg/day cycle for a month, followed by a maintainance cycle with 300 mg/day for 3 months). The diagnostic aspects are then discussed, pointing out that absence of any peripheric neurologic damage, normal bone scan, female sex and younger age are the relevant characteristics for the diagnosis of OCI.

Fifty cases of recurrent dislocation of the shoulder operated according to the Putti-Platt method modified by lateralization of the insertion of the coracoid processes are reported. Excellent results were obtained in 86% of the cases. There was neither incidence of recurrence, nor degenerative joint alteration. Mild limitation in extrarotation (less than 10 degrees) was observed in 7 cases.

Based on 32 cases of neoplastic and degenerative lesions, the indications and limits of auto- and alloplastic grafts are discussed. Grafting was successful in all of the cases, obtaining excellent clinical and radiographic results. An auto graft may be used to more rapidly fill cavities which remain after curettage has been performed, but the limited availability of grafts, the need for a second operation, and the weakening of the donor site encourage us to develop more practical methods of removal and preservation of the allograft.

The epidermal growth factor receptor (EGFR) and gp185erbB-2 are closely related tyrosine kinases. Despite extensive sequence and structural homology, these two receptors display quantitative and qualitative differences in their ability to couple with mitogenic signalling pathways. By using chimeric molecules between EGFR and erbB-2, we found that the determinants responsible for the specificity of mitogenic signal transduction are located in the amino-terminal half of the tyrosine kinase domain of either receptor. In the EGFR, mutational analysis within this subdomain revealed that deletion of residues 660 to 667 impaired receptor mitogenic activity without affecting its tyrosine kinase properties. This sequence is therefore likely to contribute to the specificity of substrate recognition by the EGFR kinase.

The analysis of 236 Italian patients with Paget's bone disease showed higher clinical severity and greater frequency of neoplastic degeneration among patients who live or descend from individuals living in the Campania region (southern Italy). A prevalent involvement of the spine and the skull, the sites preferentially involved in giant cell tumors complicating Paget's disease, was also shown in familial cases from this geographical region. Introduction: The Campania region in southern Italy has been recently indicated as a high prevalence area for Paget's disease of bone (PDB), and most pagetic families with multiple occurrence of neoplasms in affected members were from this geographical region. Materials and Methods: We evaluated the PDB epidemiological characteristics in 125 patients from Campania in comparison with 111 patients from other Italian regions. Twenty-three patients from Campania and 26 patients from other Italian areas had at least one first-degree relative affected by PDB (familial cases). The remaining patients made up the sporadic cases. Results: Among subjects from Campania, the patients in the familial group tended to come from larger families and showed at diagnosis higher serum total alkaline phosphatase, larger extension of disease, and earlier mean age with respect to patients with PDB of the sporadic group. The skull, spine, and humerus were the sites preferentially involved in the familial cases. In contrast, no such differences were observed between familial and sporadic PDB cases among patients from the other geographical areas, except for a lower age at diagnosis. An increased PDB clinical severity was finally observed in the PDB cohort from Campania in comparison with patients from other Italian regions. Neoplastic degeneration of pagetic bones (osteosarcoma and giant cell tumor) was exclusively observed in patients with polyostotic PDB from Campania. Conclusions: We showed a higher clinical severity of PDB with occurrence of neoplastic degeneration in the high prevalence area of Campania, with its maximum expression in cases with familial disease. This peculiar pattern might be traced to genetic predisposition and/or to the abnormal impact of a still undefined environmental trigger.