Martino Bosco

Estrogen receptor-β is expressed in stromal cells of fibroadenoma and phyllodes tumors of the breast

An estrogen dependency has been suggested for the growth of fibroadenomas: however, thus far, none of the steroid hormone receptors acting on breast tissues has been demonstrated in the stroma of breast fibroepithelial lesions. In this study, the expression of estrogen receptor (ER)-α and -β was investigated by immunohistochemistry in 33 fibroadenomas and in 30 benign, three borderline and seven malignant phyllodes tumors, all with spindle cell growth and in one distant metastasis. In addition, the presence of ER-β mRNA and its variants was evaluated by RT-PCR in microdissected stroma. The possible correlation between hormone receptor expression and differentiation processes of stromal cells was investigated by smooth muscle actin and calponin immunostaining. ER-β was the only hormone receptor expressed by stroma of fibroadenomas and phyllodes tumors, both at protein and mRNA level. The highest percentage of ER-β was observed in fibroadenomas with cellular stroma and in phyllodes tumors. In both lesions, ER-β-positive stromal cells showed expression of smooth muscle actin and/or calponin, as demonstrated by double immunostaining. In addition, the mean age at diagnosis was significantly lower in patients with ER-β-positive vs ER-β-negative fibroadenomas. In contrast, in phyllodes tumors, ER-β expression was higher in older patients. In conclusion, (i) only ER-β is detected in the stroma of fibroadenomas and phyllodes tumors; (ii) its expression correlates with the expression of smooth muscle markers and suggests a role of ER-β in myofibroblastic differentiation of stromal cells. These two results, together with the young age of patients carrying fibroadenomas with highly ER-β-positive stroma cells, may further indicate a hormone-receptor mechanism involved in regulating the growth of fibroadenomas. Conversely, the older age of patients with ER-β-rich phyllodes tumors suggests that mechanisms, probably independent from estrogen stimulation, act on the growth of these tumors.

Oxidative damage in human liver transplantation

The aim of this study was to evaluate oxygen-dependent hepatic reperfusion injury in humans following orthotopic liver transplantation. To this end, a number of blood indices of impaired tissue redox balance were monitored in 19 adult patients for 3 weeks after liver transplantation. Both red cell malonaldehyde and plasma lipid peroxides increased significantly soon after organ reperfusion. This finding was consistently accompanied by decreased plasma vitamin E and red cell total glutathione. A peak of oxidative stress, as measured by the parameters monitored, was evident within 24 h after reperfusion, together with a maximum expression of cytolysis, as measured by plasma alanine aminotransferase. The occurrence of redox imbalance after hepatic reperfusion was shown to be linearly related to irreversible cell damage. As regards the low plasma levels of the two antioxidants after reperfusion, only that of vitamin E appeared statistically related to oxidative stress. With the background of an increasing body of proof, mainly from animal models, the involvement of toxic oxygen metabolites in hepatic cytolysis following orthotopic liver transplantation appears likely. The statistical correlation among the markers of redox imbalance monitored indicates their combined use in further investigation.

Pulmonary atelectasis during low stretch ventilation: "open lung" versus "lung rest" strategy

Objective:Limiting tidal volume (VT) may minimize ventilator-induced lung injury (VILI). However, atelectasis induced by low VT ventilation may cause ultrastructural evidence of cell disruption. Apoptosis seems to be involved as protective mechanisms from VILI through the involvement of mitogen-activated protein kinases (MAPKs). We examined the hypothesis that atelectasis may influence the response to protective ventilation through MAPKs. Design:Prospective randomized study. Setting:University animal laboratory. Subjects:Adult male 129/Sv mice. Interventions:Isolated, nonperfused lungs were randomized to VILI: VT of 20 mL/kg and positive end-expiratory pressure (PEEP) zero; low stretch/lung rest: VT of 6 mL/kg and 8–10 cm H2O of PEEP; low stretch/open lung: VT of 6 mL/kg, two recruitment maneuvers and 14–16 cm H2O of PEEP. Ventilator settings were adjusted using the stress index. Measurement and Main Result:Both low stretch strategies equally blunted the VILI-induced derangement of respiratory mechanics (static volume-pressure curve), lung histology (hematoxylin and eosin), and inflammatory mediators (interleukin-6, macrophage inflammatory protein-2 [enzyme-linked immunosorbent assay], and inhibitor of nuclear factor-kB[Western blot]). VILI caused nuclear swelling and membrane disruption of pulmonary cells (electron microscopy). Few pulmonary cells with chromatin condensation and fragmentation were seen during both low stretch strategies. However, although cell thickness during low stretch/open lung was uniform, low stretch/lung rest demonstrated thickening of epithelial cells and plasma membrane bleb formation. Compared with the low stretch/open lung, low stretch/lung rest caused a significant decrease in apoptotic cells (terminal deoxynucleotidyl transferase mediated deoxyuridine-triphosphatase nick end-labeling) and tissue expression of caspase-3 (Western blot). Both low stretch strategies attenuated the activation of MAPKs. Such reduction was larger during low stretch/open lung than during low stretch/lung rest (p < 0.001). Conclusion:Low stretch strategies provide similar attenuation of VILI. However, low stretch/lung rest strategy is associated to less apoptosis and more ultrastructural evidence of cell damage possibly through MAPKs-mediated pathway.

Columnar cell lesions associated with breast calcifications on vacuum-assisted core biopsies: clinical, radiographic, and histological correlations.

Columnar cell lesions of the breast are increasingly recognized at mammography for their tendency to calcify. We studied 392 vacuum-assisted core biopsies performed solely for calcifications to evaluate the frequency of columnar cell lesions, their relationship with radiological risk, appearance of calcifications, and clinical data. Management and follow-up of columnar cell lesions without and with atypia (flat epithelial atypia) was analyzed. Cases with architectural atypia (cribriform spaces and/or micropapillae) were excluded from flat epithelial atypia. Calcifications were within the lumen of acini affected by columnar cell lesions in 137 out of 156 biopsies diagnosed with some columnar cell lesions. These represented 37% of vacuum-assisted core biopsies and 62% of low radiological risk (BI-RADS3) calcifications. High-risk (BI-RADS5) calcifications were never associated with columnar cell lesions. Age and menopausal status were comparable in columnar and in not-columnar cell lesions. Atypia was associated with long-term hormone replacement therapy in both lesions. Surgical biopsy was recommended for all cases with atypia. Flat epithelial atypia, as the only histological findings on vacuum-assisted core biopsies, was never associated with malignancy at surgery. In conclusion, we suggest that surgical excision is not mandatory when flat epithelial atypia is found as the most advanced lesion on vacuum-assisted core biopsy performed for low radiological risk calcifications, and that women should be advised of the possible hormone dependency of this entity.

Phosphoinositide-3 Kinase γ Activity Contributes to Sepsis and Organ Damage by Altering Neutrophil Recruitment

RATIONALE Sepsis is a leading cause of death in the intensive care unit, characterized by a systemic inflammatory response (SIRS) and bacterial infection, which can often induce multiorgan damage and failure. Leukocyte recruitment, required to limit bacterial spread, depends on phosphoinositide-3 kinase γ (PI3Kγ) signaling in vitro; however, the role of this enzyme in polymicrobial sepsis has remained unclear. OBJECTIVES This study aimed to determine the specific role of the kinase activity of PI3Kγ in the pathogenesis of sepsis and multiorgan damage. METHODS PI3Kγ wild-type, knockout, and kinase-dead mice were exposed to cecal ligation and perforation-induced sepsis and assessed for survival; pulmonary, hepatic, and cardiovascular damage; coagulation derangements; systemic inflammation; bacterial spread; and neutrophil recruitment. Additionally, wild-type mice were treated either before or after the onset of sepsis with a PI3Kγ inhibitor and assessed for survival, neutrophil recruitment, and bacterial spread. MEASUREMENTS AND MAIN RESULTS Both genetic and pharmaceutical PI3Kγ kinase inhibition significantly improved survival, reduced multiorgan damage, and limited bacterial decompartmentalization, while modestly affecting SIRS. Protection resulted from both neutrophil-independent mechanisms, involving improved cardiovascular function, and neutrophil-dependent mechanisms, through reduced susceptibility to neutrophil migration failure during severe sepsis by maintaining neutrophil surface expression of the chemokine receptor, CXCR2. Furthermore, PI3Kγ pharmacological inhibition significantly decreased mortality and improved neutrophil migration and bacterial control, even when administered during established septic shock. CONCLUSIONS This study establishes PI3Kγ as a key molecule in the pathogenesis of septic infection and the transition from SIRS to organ damage and identifies it as a novel possible therapeutic target.

Preliminary experience with a new cytology brush in EUS-guided FNA

BACKGROUND Despite the high diagnostic yield of EUS-guided FNA, room for technical improvements remains. Recently, the EchoBrush (Cook Endoscopy, Winston-Salem, NC), a disposable cytologic brush, was introduced to the market. To date, only 1 study, limited to 10 pancreatic cyst cases, using this device has been published. OBJECTIVE To assess the diagnostic yield of the EchoBrush in a cohort of consecutive patients, irrespective of the target lesion. DESIGN Case series. SETTING Tertiary care university hospital (Molinette Hospital, Turin, Italy). PATIENTS Thirty-nine consecutive patients (12 with solid pancreatic masses, 12 with pancreatic cysts, 7 with enlarged lymph nodes, and 8 with submucosal masses) were enrolled. INTERVENTIONS The material collected with the EchoBrush and with a standard FNA needle was double-blind evaluated by 2 cytopathologists. MAIN OUTCOME MEASUREMENTS Adequacy of the sample and sensitivity and specificity of the EchoBrush method. RESULTS Adequate material for cytologic analysis was collected in 17 of 39 patients (43.6%) with a single pass of the EchoBrush. Results were better for pancreatic lesions (for solid and cystic lesions, the adequacy was 58.3% and 50%, respectively); adequacy was low (28.6% and 25%, respectively) for lymph nodes and submucosal masses. The overall sensitivity and specificity were 57.9% and 31.2%, respectively. There were no adverse events with the procedure. LIMITATION Preliminary study. CONCLUSIONS This report suggests that the EchoBrush may provide adequate cellularity to diagnose solid and cystic pancreatic lesions. More extensive studies are needed to compare the EchoBrush and standard needles.

Diagnosis of deep-seated lymphomas by endoscopic ultrasound-guided fine needle aspiration combined with flow cytometry.

A. Stacchini, P. Carucci, D. Pacchioni, G. Accinelli, A. Demurtas, S. Aliberti, M. Bosco, M. Bruno, A. Balbo Mussetto, M. Rizzetto, G. Bussolati and C. De Angelis 
Diagnosis of deep‐seated lymphomas by endoscopic ultrasound‐guided fine needle aspiration combined with flow cytometry

Caveolin-1 expression is variably displayed in astroglial-derived tumors and absent in oligodendrogliomas: concrete premises for a new reliable diagnostic marker in gliomas.

Caveolins are basic constituents of flask-shaped cell membrane microdomains (caveolae), which are involved in many cell functions, including signalling, trafficking, and cellular growth control. The distribution of caveolae within the normal brain and in brain tumors is controversial. In the present study, we describe the expression of caveolin-1 (cav-1) in 64 brain tumors of different grade, of either astroglial or oligodendroglial origin. All studied astrocitomas of any grade (from II to IV) were cav-1 positive, displaying staining patterns and intensity specifically associated to the different tumor grades. In all glioblastomas and gliosarcomas, cav-1 staining was extremely intense, typically localized at the cell membrane and recognized a variable percentage of cells, including the majority of spindle cells and palisade-oriented perinecrotic cells. In anaplastic astrocytomas, a less intense membrane staining or a cytoplasmic dotlike immunoreactivity were present, the latter being almost the exclusive pattern observed in diffuse astrocitomas grade II. In contrast to astroglial tumors, the striking totality of grade II oligodendrogliomas and the large majority of grade III were lacking cav-1 expression. Interestingly, a cav-1 distribution overlapping the pattern described in tissues was observed also in primary cell cultures of human glioblastomas and astrocytomas, and also in one established glioblastoma cell line (U251 MG), analyzed by means of confocal microscopy and flow cytometry. In conclusion, among astroglial tumors cav-1 expression varies in distribution, pattern, and intensity specifically according to tumor types and grades. The association between tumor progression and a more structured membranous pattern of cav-1 expression could suggest the hypothesis of a neoplastic shift towards a mesenchymal phenotype, whose behavioral and biologic significance worth further studies. Finally, the lack of cav-1 immunoreactivity in oligodendrogliomas suggests its concrete application as a useful diagnostic marker.

Constitutive and ligand-induced nuclear localization of oxytocin receptor

Oxytocin receptor (OTR) is a membrane protein known to mediate oxytocin (OT) effects, in both normal and neoplastic cells. We report here that human osteosarcoma (U2OS, MG63, OS15 and SaOS2), breast cancer (MCF7), and primary human fibroblastic cells (HFF) all exhibit OTR not only on the cell membrane, but also in the various nuclear compartments including the nucleolus. Both an OTR‐GFP fusion protein and the native OTR appear to be localized to the nucleus as detected by transfection and/or confocal immunofluorescence, respectively. Treatment with oxytocin causes internalization of OTR and the resulting vesicles accumulate in the vicinity of the nucleus and some of the perinuclear OTR enters the nucleus. Western blots indicate that OTR in the nucleus and on the plasma membrane are likely to be the same biochemical and immunological entities. It appears that OTR is first visible in the nucleoli and subsequently disperses within the nucleus into 4–20 spots while some of the OTR diffuses throughout the nucleoplasm.The behaviour and kinetics of OTR‐GFP and OTR are different, indicating interference by GFP in both OTR entrance into the nucleus and subsequent relocalization of OTR within the nucleus. There are important differences among the tested cells, such as the requirement of a ligand for transfer of OTR in nuclei. A constitutive internalization of OTR was found only in osteosarcoma cells, while the nuclear localization in all other tested cells was dependent on ligand binding. The amount of OTR‐positive material within and in the vicinity of the nucleus increased following a treatment with oxytocin in both constitutive and ligand‐dependent type of cells. The evidence of OTR compartmentalization at the cell nucleus (either ligand‐dependent or constitutive) in different cell types suggests still unknown biological functions of this protein or its ligand and adds this G‐protein‐coupled receptor to other heptahelical receptors displaying this atypical and unexpected nuclear localization.

Technical limits of comparison of step-sectioning,immunohistochemistry and RT-PCR on breast cancer sentinel nodes: a study on methacarn-fixed tissue

The optimal pathological assessment of sentinel nodes (SLNs) in breast cancer is a matter of debate. Currently, multilevel histological evaluation and immunohistochemistry (IHC) are recommended, but alternative RT‐PCR procedures have been developed. To assess the reliability of these different procedures, we devised a step‐sectioning protocol at 100 micron‐intervals of 74 SLNs using methacarn fixation. mRNA was extracted from sections collected from levels 4 to 5. Mammaglobin, CEA and CK19 were used for RT‐PCR. mRNA extraction was successful in 69 SLNs. Of these, 7 showed macrometastases (>2mm), 2 showed micrometastases (<2 mm) and 7 showed isolated tumour cells (ITC) by IHC. RT‐PCR was positive for the three markers in 6 of 7 macrometastases and in 1 of 2 micrometastases. In the 2 RT‐PCR negative cases, metastases were detected only on sections distant from those analysed by RT‐PCR. CEA and/or CK19 were positive by RT‐PCR in 3 of 7 ITC and in 23 morphologically negative SLNs. In conclusion, the main goal of our study was to show that the use of alternate sections of the same sample for different procedures is the key reason for the discrepancies between molecular and morphological analyses of SLN. We believe that only prospective studies with quantitative mRNA analysis of specific metastatic markers on the whole lymph node can elucidate the utility of molecular assessments of SLN.