Anti-PSMA 124I-scFvD2B as a new immuno-PET tool for prostate cancer: preclinical proof of principle.

BACKGROUND: Prostate cancer (PCa) is the second leading cause of cancer-related death in the Western population. The use in oncology of positron emission tomography/computed tomography (PET/CT) with emerging radiopharmaceuticals promises accurate staging of primary disease, restaging of recurrent disease and detection of metastatic lesions. Prostate-specific membrane antigen (PSMA) expression, directly related to androgen-independence, metastasis and progression, renders this tumour associate antigen a good target for the development of new radiopharmaceuticals for PET. Aim of this study was to demonstrate in a preclinical in vivo model (PSMA-positive versus PSMA-negative tumours) the targeting specificity and sensitivity of the anti-PSMA single-chain variable fragment (scFv) labelled with 124I. METHODS: The 124I-labeling conditions of the antibody fragment scFvD2B were optimized and assessed for purity and immunoreactivity. The specificity of 124I-scFvD2B was tested in mice bearing PSMA-positive and PSMA-negative tumours to assess both ex-vivo biodistribution and immune-PET. RESULTS: The uptake fraction of 124I-scFvD2B was very high on PSMA positive cells (range 75-91%) and highly specific and immuno-PET at the optimal time point, defined between 15 h and 24 h, provides a specific localization of lesions bearing the target antigen of interest (PSMA positive vs PSMA negative tumors %ID/g: p = 0.0198 and p = 0.0176 respectively) yielding a median target/background ratio around 30-40. CONCLUSIONS: Preclinical in vivo results of our immuno-PET reagent are highly promising. The target to background ratio is improved notably using PET compared to SPECT previously performed. These data suggest that, upon clinical confirmation of sensitivity and specificity, our anti-PSMA 124I-scFvD2B may be superior to other diagnostic modalities for PCa. The possibility to combine in patients our 124I-scFvD2B in multi-modal systems, such as PET/CT, PET/MR and PET/SPECT/CT, will provide quantitative 3D tomographic images improving the knowledge of cancer biology and treatment.

Effects of smoking regular or light cigarettes on brachial artery flow-mediated dilation.

BACKGROUND AND PURPOSE: To compare the effects of regular cigarettes (RCs) and light cigarettes (LCs) on brachial artery flow-mediated dilation (FMD) and sublingual glyceryl trinitrate-induced dilation (GTN), markers of endothelial dependant and independent function, respectively. METHODS: 206 subjects (age 51.5 ± 12.8 yr, 122 men) had their smoking habits recorded and FMD and GTN measured by B-mode ultrasound. Cigarettes were categorized as RCs or LCs according to their content of tar, nicotine and CO. The chronic effect was assessed in current smokers of RCs (n = 85) or LCs (n = 53) and in never smokers (NS; n = 68). The acute effect was assessed in current smokers by measuring FMD before and 10-min after smoking a single regular (n = 29) or light (n = 51) cigarette. RESULTS: FMD was significantly lower in consumers of RCs (6.26%, 95% C.I. 5.58, 6.94) or LCs (5.59%, 95% C.I. 4.74, 6.45) compared to NS (8.68%, 95% C.I. 7.92, 9.44) (both P < 0.0001), but did not differ (P > 0.05) when compared to each other. GTN was similar in the three groups. Analyses adjusted for clinical confounders and for markers involved in oxidative stress, arginine/nitric oxide pathway, and inflammation provided identical results. Smoking a single cigarette, either regular or light, reduced FMD (-0.88% and -1.17%, respectively, both P < 0.05), without significant difference between cigarette type. RCs and LCs produced analogous chronic and acute effects when FMD was calculated with respect to the last 60 s of the low-flow phase (FMD60s). CONCLUSIONS: LCs impair endothelial-dependant vasodilation as much as RCs. Thus, smoking LCs cannot be considered an alternative to the only safe choice of a complete and permanent smoking cessation.

A single-chain fragment against prostate specific membrane antigen as a tool to build theranostic reagents for prostate cancer.

Prostate carcinoma is the most common non-cutaneous cancer in developed countries and represents the second leading cause of death. Early stage androgen dependent prostate carcinoma responds well to conventional therapies, but relatively few treatment options exist for patients with hormone-refractory prostate cancer. One of the most suitable targets for antibody-mediated approaches is prostate specific membrane antigen (PSMA) which is a well known tumour associated antigen. PSMA is a type II integral cell-surface membrane protein that is not secreted, and its expression density and enzymatic activity are increased progressively in prostate cancer compared to normal prostate epithelium, thereby making PSMA an ideal target for monoclonal antibody imaging and therapy. To obtain a small protein that can better penetrate tissue, we have engineered a single-chain variable fragment (scFv) starting from the variable heavy and light domains of the murine anti-PSMA monoclonal antibody D2B. scFvD2B was analysed in vitro for activity, stability, internalisation ability and in vivo for targeting specificity. Maintenance of function and immunoreactivity as well as extremely high radiolabelling efficiency and radiochemical purity were demonstrated by in vitro assays and under different experimental conditions. Despite its monovalent binding, scFvD2B retained a good strength of binding and was able to internalise around 40% of bound antigen. In vivo we showed its ability to specifically target only PSMA expressing prostate cancer xenografts. Due to these advantageous properties, scFvD2B has the potential to become a good theranostic reagent for early detection and therapy of prostate cancers.

The metabolic syndrome predicts carotid intima-media thickness no better than the sum of individual risk factors in a lipid clinic population.

OBJECTIVE: To assess whether the diagnosis 'metabolic syndrome' (MS) predicts the degree of subclinical atherosclerosis better than its component parts or the total number of vascular risk factors (VRFs) in patients attending a lipid clinic. METHODS: Carotid intima-media thickness (C-IMT) was measured by B-mode ultrasound in 1804 patients (56+/-13 years; 52% women). To investigate whether the increased subclinical carotid atherosclerosis often ascribed to MS may be explained by a real interaction between the components or simply by a sum of VRFs, observed C-IMTs were compared with those predicted by the sum of individual components. Values for C-IMT of MS patients were also compared with those of controls matched for number of VRFs or for SCORE predicted risk (SPR). RESULTS: Carotid IMT values were significantly higher in patients with MS (n=362) than in those not so diagnosed (IMT(mean), 1.07+/-0.37 vs. 0.95+/-0.33; IMT(max), 1.98+/-0.93mm vs. 1.67+/-0.82mm, both p<0.0001), but were not higher than those predicted by the sum of individual risk factors. The linear regression lines of the correlations between C-IMT and total number of VRFs overlapped in patients with and without MS. In patients with and without MS matched for age, sex and total number of VRFs, or matched for age, sex and SPR the C-IMT differences disappeared. CONCLUSIONS: In patients attending a lipid clinic, 'metabolic syndrome' appears not to correlate with C-IMT to a greater extent than what is expected from its component parts or from the patient's total number of VRFs.

[2 cases of acute cholestasis caused by ticlopidine]. / Due casi di colestasi acuta da ticlopidina.

We report the case of two patients suffered from cholestatic jaundice occurred 3-4 weeks after starting ticlopidine therapy. In both cases the diagnosis was made by ruling out any other known cause of acute hepatitis or cholestasis. One patient underwent liver biopsy, which showed a typical intralobular cholestatic pattern and a slight lymphocytic infiltration of the portal tracts. The other patient, a 29 year-old woman, was taking ticlopidine as the sole drug, further to an ischemic stroke occurred while she was taking oral contraceptives; she presented a diffuse itchy dermatitis, fever and slight eosinophilia besides cholestasis. In both patients ticlopidine was discontinued and liver tests returned to normal values within 4-8 weeks; no rechallenge was attempted and ticlopidine was replaced with another antiplatelet drug. To the best of our knowledge 19 cases of ticlopidine-related cholestatic disease have been described so far in the literature. Its pathogenesis is still unknown, although some clinical findings and experimental results from patients with acute enteropathy or agranulocytosis induced by ticlopidine suggest that the drug may act through a toxic mechanism, perhaps mediated by prostaglandins.

Cytogenetic analysis reveals clonal proliferation of smooth muscle cells in atherosclerotic plaques.

Cytogenetic analysis of primary cell cultures from human atherosclerotic fibrous plaques revealed clonal chromosome abnormalities in 13 of the 18 cases studied. Loss of the Y chromosome and del(13)(q14) were present as single clonal abnormalities in eight cases; in five cases separate clones were found involving loss of the Y and a XXY karyotype, trisomy 10 and 18, loss of the Y and trisomy 7. A variety of single numerical and structural abnormalities were present in all but two of the 18 cases. Immunocytochemical studies were performed on cells from the same cultures used for cytogenetic analysis using monoclonal antibodies to human leucocyte common antigen, to human vimentin and to muscle actin. The immunoreactivity was positive for actin in 70-80% of the cells; 100% of the cells were positive for vimentin and all cells were ALC negative. These results indicated that the chromosomal abnormalities are present in the smooth muscle cells of the plaque. The hypothesis is proposed that the proliferation leading to the atherosclerotic lesion may primarily represent a hyperplastic response to mechanical and biological injuries and that this reactive proliferation is, in turn, associated with a tendency to chromosome instability.

Ductal cancers of the pancreas frequently express markers of gastrointestinal epithelial cells.

It has been found by immunohistochemical staining that antigens normally found in gastric and/or intestinal epithelial cells are expressed in most differentiated duct cell carcinomas of the pancreas. Among 88 such tumors, 93% and 92%, respectively, expressed M1 and cathepsin E, markers of gastric surface-foveolar epithelial cells, 51% expressed pepsinogen II, a marker of gastroduodenal mucopeptic cells, 48% expressed CAR-5, a marker of colorectal epithelial cells, and 35% expressed M3SI, a marker of small intestinal goblet cells. Most of the tumors also expressed normal pancreatic duct antigens; 97% expressed DU-PAN-2, and 59% expressed N-terminus gastrin-releasing peptide. In agreement with these findings, electron microscopy revealed malignant cells with fine structural features of gastric foveolar cells, gastric mucopeptic cells, intestinal goblet cells, intestinal columnar cells, pancreatic duct epithelial cells, and cells with features of more than one cell type. Normal pancreatic duct epithelium did not express any marker of gastrointestinal epithelial cells, whereas such benign lesions as mucinous cell hypertrophy and papillary hyperplasia commonly expressed gut-type antigens but rarely expressed pancreatic duct cell markers. By contrast, lesions characterized by atypical papillary hyperplasia commonly expressed both gastric and pancreatic duct cell markers. Metaplastic pyloric-type glands expressed pepsinogen II and, except for their expression of cathepsin E, were indistinguishable from normal pyloric glands. In marked contrast, the immunohistochemical and ultrastructural features of 14 ductuloacinar cell tumors were those of cells lining terminal ductules, centroacinar cells, and/or acinar cells; none expressed any gut-type antigen. The results indicate that gastrointestinal differentiation is common in both benign and malignant lesions of pancreatic duct epithelium and suggest that duct cell carcinomas are histogenetically related to gastric- and intestinal-type metaplastic changes of epithelial cells lining the main and interlobular ducts of the pancreas.

Histopathology, cytology and cytochemistry of pheochromocytomas and paragangliomas including chemodectomas.

The results of histopathological, histochemical and ultrastructural investigations on pheochromocytomas and paragangliomas have been reported. These results allowed the functional identification of the cell types composing many of such tumours. Moreover, comparison of these data with clinico-pathologic findings outlined the advantages and limits of cytologic studies for understanding the natural history of pheochromocytomas and paragangliomas and improving our diagnostic and prognostic criteria.

Characterization of four main cell types in gastric cancer: foveolar, mucopeptic, intestinal columnar and goblet cells. An histopathologic, histochemical and ultrastructural study of "early" and "advanced" tumours.

Gastrectomy specimens of 148 gastric cancers, 40 of them being intramucosal or microinvasive, 27 penetrating the submucosa and 81 invading the muscularis propria, with or without involvement of the serosa and perigastric tissues, have been investigated with conventional histopathologic techniques, mucin histochemistry and electron microscopy to characterize the various lines of tumour cell differentiation and to correlate these with the histologic patterns of tumour growth. More or less differentiated intestinal columnar, intestinal goblet, gastric foveolar or mucopeptic cells were recognized in most tumours, of glandular, diffuse or mucoid type. Although simultaneous expression of more than one cell type into the same tumour occurred very frequently, intestinal columnar cells were more prominent in tubular adenocarcinomas, goblet cells (especially of colorectal type) in mucoid cancers, mucopeptic cells in diffuse cancers of invasive desmoplastic type and foveolar cells in diffuse cancers of intramucosal signet-ring cell type. In general, an increased tendency to foveolar cell differentiation and a reduced tendency to mucopeptic differentiation has been found in intramucosal cancers as compared to invasive cancers. It is concluded that the type of tumour cell differentiation, which might have some influence on the natural history of gastric cancer, is better related with more defined tumour subtypes than with the usually recognized glandular or diffuse patterns.

Small-cell (oat cell) carcinoma of the endometrium.

We report a case of small-cell (oat cell) carcinoma of the endometrium in a 59-year-old woman. The tumor was confined to a portion of an adenomatous polyp. It exhibited argyrophilia with Grimelius' stain and dense-core endocrine-type granules by electron microscopy. The patient was alive and well one year later. To our knowledge, this is the fourth reported case of a small-cell carcinoma of the endometrium exhibiting endocrine differentiation.

Multifocal Purkinje-like tumor of the heart. Occurrence with other anatomic abnormalities in the atrioventricular junction of an infant with junctional tachycardia, Lown-Ganong-Levine syndrome, and sudden death.

A 13-month-old boy with intermittent preexcitation (Lown-Ganong-Levine type) and paroxysmal tachycardias of 280 to 300 beats per minute died suddenly with cardiac arrest. At necropsy the only abnormalities were in the heart. There were multifocal Purkinje cell tumors in the conduction system, including one directly at the bifurcation of the His bundle. In addition, a fault in the central fibrous body was filled with an accessory communication between the mid-portion of the atrioventricular node and both the interventricular septum and the His bundle. This accessory communication connected with the Purkinje cell tumor. Although an atrioventricular nodal bypass was thus present and could account for the preexcitation and for reentrant tachycardias, there is also the anatomic basis for an automatic arrhythmia originating within one or more of the Purkinje cell tumors, particularly the one directly within the His bundle. Dispersion of elements of the conduction system within the central fibrous body resembled the normal fetal pattern. Whether this persistence of fetal dispersion of the atrioventricular node and His bundle within the central fibrous body is causally related to the presence of multifocal Purkinje cell tumors or is only coincidence merits further consideration.

Gastric parietal cell carcinoma--a newly recognized entity: light microscopic and ultrastructural features.

Three cases of gastric parietal cell carcinoma are described. Tumour cells are round to polygonal, with abundant eosinophilic, granular cytoplasm reactive with phosphotungstic acid haematoxylin and Luxol Fast Blue. Ultrastructurally the tumour cells are characterized by abundant mitochondria, tubulovesicles, intracellular canaliculi and intercellular lamina filled with undulated microvilli.

Ultrastructural localization of secretin in endocrine cells of the dog duodenum by the immunogold technique. Comparison with ultrastructurally characterized S cells of various mammals.

Secretin has been localized by the immunogold technique in endocrine cells of the dog duodenum--previously described as "K cells"--characterized by secretory granules with double structure consisting of a secretin-containing osmiophilic core surrounded by an argyrophil halo. Granules resembling those of dog secretin cells were also found in some ultrastructurally characterized S cells of the cat, pig, rat and rabbit duodenum, thus confirming in these species the identification of S cells with secretin cells. Conversely, the cells previously described as "S cells" in the dog lacked secretin immunoreactivity.

Primary polypeptide hormones and mucin-producing malignant carcinoid of the larynx.

A case of primary malignant laryngeal carcinoid with dual endocrine and mucous differentiation is reported. Histologically the tumor showed a characteristic organoid pattern and exhibited Alcian-blue, periodic acid-Schiff, and Grimelius silver positivity. By the immunoperoxidase technique calcitonin, ACTH, and alpha-hCG subunit were demonstrated in the tumor cells. Electron microscopy revealed two different types of endocrinelike cells: mucous cells and occasional cells containing both endocrinelike granules and mucin droplets. Diagnostic morphologic criteria of this rare tumor entity are discussed and reference to biologic behavior and possible histogenesis is made.

Merkel cell carcinoma of the skin: the structure and origin of normal Merkel cells.

A series of 15 Merkel cell tumours of skin is reported. They occur dominantly on the head and neck and on the extremities of elderly women, frequently presenting as a reddish nodule. Three cases were associated with squamous carcinoma at the same site, an association deserving further study. There are two main patterns: the commoner one takes the form of a trabecular carcinoma in the dermis mimicking metastatic carcinoma, including oat-cell carcinoma and neuroblastoma: a dissociated-cell form mimicks malignant lymphoma. The triad of vesicular nuclei with very small nucleoli, abundant mitotic activity and apoptosis is so characteristic as to be virtually pathognomonic in conjunction with structural features. Argyrophilia is common, but Bouin fixation is necessary to demonstrate it regularly. Small round secretory granules (89 +/- 18 nm) with narrow haloes, and an abundance of intermediate size filaments are among the ultrastructural hallmarks. There is a close similarity between better differentiated tumour cells and normal Merkel cells. The neural crest origin of MC is in doubt both on the basis of studies of the development and regeneration of MC and from the study of Merkel cell tumours.

Alpha and beta subunits of glycoprotein hormones in argyrophil pituitary tumors with small granule cells.

A group of 33 functionless pituitary tumors with small argyrophil groups (SAG) were collected from a series of 200 pituitary adenomas (16.5% of all adenomas). Histologically, the tumors showed an unusually high frequency of trabecular patterns, perivascular pseudo-rosettes, and oncocytoid transformation. Immunoreactivity for glycoprotein hormone alpha-chain was found in more or less numerous cells of 20 cases (64.5% of SAG tumors). Thirteen of these cases also showed specific beta-chain immunoreactivity, especially for follicle-stimulating hormone (FSH) beta-chain, which was present in 11 tumors. Various admixtures of immature, oncocytic, sparsely granulated, and densely granulated cells were observed ultrastructurally, with prevalence of the latter cell variants in tumors showing immunoreactive cells and prevalence of the former cell variants in tumors lacking immunoreactive cells. It is suggested that some relationship may exist between SAG cell (glycoprotein hormone precursor cells?) tumors--or at least part of them--and glycoprotein hormone cell lines. Anyway, whatever their origin and interpretation, SAG cell tumors seem to represent a distinct clinicopathologic entity.

Argyrophil reaction in parathyroid glands: a light and electron microscopic study.

The argyrophil reaction occurs in the parathyroid cells with the Grimelius silver nitrate stain. In the present study, Bouin's fluid was found to be superior to other tested fixatives (formalin, Karnovsky's fluid, GPA etc) in this respect. A study of the argyrophil reaction on the ultrastructural level showed an accumulation of silver particles in the secretory granules, which means that the Grimelius silver stain can be assumed to be an indicator of the secretory granules stored in the parathyroid cells. Whether the argyrophil reaction can be of help in differential diagnosis between different abnormal parathyroid conditions is not yet clear.