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In digital pathology, the final appearance of digitized images is affected by several factors, resulting in stain color and intensity variation. Stain normalization is an innovative solution to overcome stain variability. However, the validation of color normalization tools has been assessed only from a quantitative perspective, through the computation of similarity metrics between the original and normalized images. To the best of our knowledge, no works investigate the impact of normalization on the pathologist's evaluation. The objective of this paper is to propose a multi-tissue (i.e., breast, colon, liver, lung, and prostate) and multi-center qualitative analysis of a stain normalization tool with the involvement of pathologists with different years of experience. Two qualitative studies were carried out for this purpose: (i) a first study focused on the analysis of the perceived image quality and absence of significant image artifacts after the normalization process; (ii) a second study focused on the clinical score of the normalized image with respect to the original one. The results of the first study prove the high quality of the normalized image with a low impact artifact generation, while the second study demonstrates the superiority of the normalized image with respect to the original one in clinical practice. The normalization process can help both to reduce variability due to tissue staining procedures and facilitate the pathologist in the histological examination. The experimental results obtained in this work are encouraging and can justify the use of a stain normalization tool in clinical routine., Competing Interests: M. Salvi, O.A.M. Pennisi and F. Molinari are equity holders in AEQUIP S.r.l, Turin, Italy. Remaining authors declare no competing interests regarding the publication of this article., (© 2022 The Authors.)

Magnetic resonance imaging (MRI) has a growing role in the clinical workup of prostate cancer. However, manual three-dimensional (3D) segmentation of the prostate is a laborious and time-consuming task. In this scenario, the use of automated algorithms for prostate segmentation allows us to bypass the huge workload of physicians. In this work, we propose a fully automated hybrid approach for prostate gland segmentation in MR images using an initial segmentation of prostate volumes using a custom-made 3D deep network (VNet-T2), followed by refinement using an Active Shape Model (ASM). While the deep network focuses on three-dimensional spatial coherence of the shape, the ASM relies on local image information and this joint effort allows for improved segmentation of the organ contours. Our method is developed and tested on a dataset composed of T2-weighted (T2w) MRI prostatic volumes of 60 male patients. In the test set, the proposed method shows excellent segmentation performance, achieving a mean dice score and Hausdorff distance of 0.851 and 7.55 mm, respectively. In the future, this algorithm could serve as an enabling technology for the development of computer-aided systems for prostate cancer characterization in MR imaging.

In the last years, the widespread use of the prostate-specific antigen (PSA) blood examination to triage patients who will enter the diagnostic/therapeutic path for prostate cancer (PCa) has almost halved PCa-specific mortality. As a counterpart, millions of men with clinically insignificant cancer not destined to cause death are treated, with no beneficial impact on overall survival. Therefore, there is a compelling need to develop tools that can help in stratifying patients according to their risk, to support physicians in the selection of the most appropriate treatment option for each individual patient. The aim of this study was to develop and validate on multivendor data a fully automated computer-aided diagnosis (CAD) system to detect and characterize PCas according to their aggressiveness. We propose a CAD system based on artificial intelligence algorithms that a) registers all images coming from different MRI sequences, b) provides candidates suspicious to be tumor, and c) provides an aggressiveness score of each candidate based on the results of a support vector machine classifier fed with radiomics features. The dataset was composed of 131 patients (149 tumors) from two different institutions that were divided in a training set, a narrow validation set, and an external validation set. The algorithm reached an area under the receiver operating characteristic (ROC) curve in distinguishing between low and high aggressive tumors of 0.96 and 0.81 on the training and validation sets, respectively. Moreover, when the output of the classifier was divided into three classes of risk, i.e., indolent, indeterminate, and aggressive, our method did not classify any aggressive tumor as indolent, meaning that, according to our score, all aggressive tumors would undergo treatment or further investigations. Our CAD performance is superior to that of previous studies and overcomes some of their limitations, such as the need to perform manual segmentation of the tumor or the fact that analysis is limited to single-center datasets. The results of this study are promising and could pave the way to a prediction tool for personalized decision making in patients harboring PCa., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Giannini, Mazzetti, Defeudis, Stranieri, Calandri, Bollito, Bosco, Porpiglia, Manfredi, De Pascale, Veltri, Russo and Regge.)

Background: In digital pathology, the morphology and architecture of prostate glands have been routinely adopted by pathologists to evaluate the presence of cancer tissue. The manual annotations are operator-dependent, error-prone and time-consuming. The automated segmentation of prostate glands can be very challenging too due to large appearance variation and serious degeneration of these histological structures., Method: A new image segmentation method, called RINGS (Rapid IdentificatioN of Glandural Structures), is presented to segment prostate glands in histopathological images. We designed a novel glands segmentation strategy using a multi-channel algorithm that exploits and fuses both traditional and deep learning techniques. Specifically, the proposed approach employs a hybrid segmentation strategy based on stroma detection to accurately detect and delineate the prostate glands contours., Results: Automated results are compared with manual annotations and seven state-of-the-art techniques designed for glands segmentation. Being based on stroma segmentation, no performance degradation is observed when segmenting healthy or pathological structures. Our method is able to delineate the prostate gland of the unknown histopathological image with a dice score of 90.16 % and outperforms all the compared state-of-the-art methods., Conclusions: To the best of our knowledge, the RINGS algorithm is the first fully automated method capable of maintaining a high sensitivity even in the presence of severe glandular degeneration. The proposed method will help to detect the prostate glands accurately and assist the pathologists to make accurate diagnosis and treatment. The developed model can be used to support prostate cancer diagnosis in polyclinics and community care centres., (Copyright © 2021 Elsevier B.V. All rights reserved.)

SARS-CoV2 infection not only causes abnormal severe pneumonia but also induces other relevant pathophysiological effects on several tissues and organs. In this regard, the clinical complications observed in COVID-19 include acute coronary syndrome, pulmonary thromboembolism, myocarditis and, in the severe cases, the occurrence of disseminated intravascular coagulation. Literature on COVID-19 highlighted the central role of the Renin Angiotensin Aldosterone System in the determinism of SARS-CoV2 cellular internalization in the target tissues. Lung degeneration and respiratory distress appear to be dependent on the perturbance of physiological mechanisms, such as the uncontrolled release of pro-inflammatory cytokines, a dysregulation of the fibrinolytic coagulative cascade and the hyperactivation of immune effector cells. In this mini review, we address the physiology of Midkine, a growth factor able to bind heparin, and its pathophysiological potential role in COVID-19 determinism. Midkine increases in many inflammatory and autoimmune conditions and correlates with several dysfunctional immune-inflammatory responses that appear to show similarities with the pathophysiological elicited by SARS-CoV2. Midkine, together with its receptor, could facilitate the virus entry, fostering its accumulation and increasing its affinity with Ace2 receptor. We also focus on Netosis, a particular mechanism of pathogen clearance exerted by neutrophils, which under certain pathological condition becomes dysfunctional and can cause tissue damage. Moreover, we highlight the mechanism of autophagy that the new coronavirus could try to escape in order to replicate itself, as well as on pulmonary fibrosis induced by hypoxia and on the release of cytokines and mediators of inflammation, correlating the interplay between Midkine and SARS-CoV2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Sanino, Bosco and Terrazzano.)

Background and Objectives: In breast carcinoma, epithelial-stromal interactions play a pivotal role in tumor formation and progression, and it must be accurately assessed for a correct extraction of predictive and prognostic biomarkers. Evaluation of preoperative (baseline) neoplasia/stroma ratio and the enumeration of tumor infiltrating lymphocytes (TIL) represent only two conditions in which precise discrimination of cancer epithelium and stromal reaction are relevant. However, subjectivity and expertise of the operators may lead to different degrees of assessment., Methods: In this paper, we present a fully automated method for the discrimination between neoplastic epithelium and stromal reaction in breast carcinoma. Starting from cell nuclei, the proposed method implements computer vision strategies to split the neoplastic epithelium tissue from the stromal reaction., Results: The algorithm is tested on 100 H&E (hematoxylin and eosin) stained images representative of 10 different cases of invasive carcinoma. The algorithm performance in the detection of neoplastic epithelium (compared to manual annotations by an expert pathologist) gave a F1 SCORE of 0.8894 and mean jaccard INDEX of 0.8481., Conclusions: To the best of our knowledge, the proposed method is the first fully automated algorithm for the discrimination between neoplastic epithelium and stromal reaction in H&E stained images of breast tissue. The proposed approach paves the way for an automated and quantitative analysis of predictive and prognostic biomarkers in breast carcinoma., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Greenberg skeletal dysplasia is an autosomal recessive, perinatal lethal disorder associated with biallelic variants affecting the lamin B receptor (LBR) gene. LBR is also associated with the autosomal recessive anadysplasia-like spondylometaphyseal dysplasia, and the autosomal dominant Pelger-Huët anomaly, a benign laminopathy characterized by anomalies in the nuclear shape of blood granulocytes. The LBR is an inner nuclear membrane protein that binds lamin B proteins (LMNB1 and LMNB2), interacts with chromatin, and exerts a sterol reductase activity. Here, we report on a novel LBR missense variant [c.1379A>G; p.(D460R)], identified by whole exome sequencing and causing Greenberg dysplasia in two fetuses from a consanguineous Moroccan family. We revised published LBR variants to propose a genotype-phenotype correlation in LBR associated diseases. The diverse phenotypes are correlated to the functional domain affected, the heterozygous or homozygous state of the variants, and their different impact on the residual protein function. LBR represents an instructive example of one gene presenting with two different patterns of inheritance and at least three different clinical phenotypes., (© 2018 Wiley Periodicals, Inc.)

Background: Ventilator-induced lung injury (VILI) occurs in part by increased vascular permeability and impaired alveolar fluid clearance. Phosphoinositide 3-kinase gamma (PI3Kγ) is activated by mechanical stress, induces nitric oxide (NO) production, and participates in cyclic adenosine monophosphate (cAMP) hydrolysis, each of which contributes to alveolar edema. We hypothesized that lungs lacking PI3Kγ or treated with PI3Kγ inhibitors would be protected from ventilation-induced alveolar edema and lung injury., Methods: Using an isolated and perfused lung model, wild-type (WT) and PI3Kγ-knockout (KO) mice underwent negative-pressure cycled ventilation at either -25 cmH₂O and 0 cmH₂O positive end-expiratory pressure (PEEP) (HIGH STRESS) or -10 cmH₂O and -3 cmH₂O PEEP (LOW STRESS)., Results: Compared with WT, PI3Kγ-knockout mice lungs were partially protected from VILI-induced derangement of respiratory mechanics (lung elastance) and edema formation [bronchoalveolar lavage (BAL) protein concentration, wet/dry ratio, and lung histology]. In PI3Kγ-knockout mice, VILI induced significantly less phosphorylation of protein kinase B (Akt), endothelial nitric oxide synthase (eNOS), production of nitrate and nitrotyrosine, as well as hydrolysis of cAMP, compared with wild-type animals. PI3Kγ wild-type lungs treated with AS605240, an inhibitor of PI3Kγ kinase activity, in combination with enoximone, an inhibitor of phosphodiesterase-3 (PDE3)-induced cAMP hydrolysis, were protected from VILI at levels comparable to knockout lungs., Conclusions: Phosphoinositide 3-kinase gamma in resident lung cells mediates part of the alveolar edema induced by high-stress ventilation. This injury is mediated via altered Akt, eNOS, NO, and/or cAMP signaling. Anti-PI3Kγ therapy aimed at resident lung cells represents a potential pharmacologic target to mitigate VILI.

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.

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.

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.

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.

We examined the in vitro effects of imatinib (Novartis Pharma AG, Basel, Switzerland) as a possible inhibitor of PDGFR pathway on cells derived from a recurrence of a pleural malignant solitary fibrous tumor (SFT). Primary cell culture was characterised by immunofluorescence. SFT-derived cells were treated with imatinib at different time points. Western blotting for PDGFR-beta, phospho-PDGFR-beta or smooth muscle actin (SMA) was performed before and after 96 h of treatment with imatinib. SFT-derived cells treated with imatinib for 96 h showed a dose dependent decrease of Ki67 expression. Results were confirmed by growth curve. Western blotting showed that PDGFR-beta was highly expressed and phosphorylated in SFT-derived cells and imatinib treatment reduced PDGFR-beta phosphorylation and SMA expression. With the limit of experimental findings, our results support a possible future application of imatinib as a candidate molecule in the target therapy of malignant SFTs over-expressing wild-type PDGFR.

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.

Microcystic urothelial cell carcinoma is a rare variant of urothelial cell carcinoma which occurs in the bladder and, rarely, in the renal pelvis. Neuroendocrine differentiation is uncommon in pure urothelial carcinoma and is more frequently found in neoplasms with glandular differentiation. We report a case of microcystic urothelial cell carcinoma arising in renal pelvis and showing focal neuroendocrine differentiation. A 55-year-old man with a history of non-small cell cancer of the lung presented with abdominal pain and hematuria. Imaging studies and gross examination revealed a partially cystic mass in the left kidney. Microscopic examination disclosed invasive carcinoma with prominent microcystic features, with microcysts lined by low columnar and flat cells. Immunohistochemical analysis confirmed the urothelial histotype (positive for thrombomodulin, p63 and high-molecular-weight cytokeratins) and disclosed focal neuroendocrine differentiation.

Ghrelin, a 28-amino acid octanoylated peptide predominantly produced by the stomach, has been discovered to be a natural ligand of the type 1a growth hormone secretagogue receptor (GHSR1a). Ghrelin has recently attracted the interest as a new GH-releasing and orexigenic factor. However, ghrelin exerts several other activities, including regulation of tissue growth and development and control of neoplastic cell proliferation. Several endocrine and nonendocrine cancer cells (pituitary adenomas; gastroenteropancreatic and pulmonary carcinoids; colorectal neoplasms, thyroid tumors; lung, breast, and pancreatic carcinomas) as well as their related cell lines have been shown able to express ghrelin both at mRNA and at protein level. Many of the above-listed tumors express GHSR1a and/or alternative GHS receptor subtypes such as the type 1b GHSR, a truncated isoform of GHSR1a, and binding sites able to recognize ghrelin independently of its acylation. Evidence that ghrelin and multiple ghrelin/GHS receptors are coexpressed in cancer cell lines and tumoral tissues from organs, such as the breast, that do not express these receptors in physiological conditions suggests that the ghrelin system is likely to play an important autocrine/paracrine role in some cancers. This chapter highlights the evidence for the expression of ghrelin and its receptors in one of the most frequent human malignancies, the prostate cancer, and information regarding their potential functional role in related cell lines.

Background: Metanephric adenoma (MA) is a relatively rare neoplasm derived from metanephric blastema and composed of well-differentiated epithelial nephroblastic cells. In view of its invariably benign clinical outcome, a preoperative diagnosis of this tumor could be of critical importance. Since computed tomography and ultrasound imaging are not per se sufficient to unequivocally distinguish between MA and malignant neoplasms, fine needle aspiration cytology (FNAC) could be the only accurate method to establish a preoperative diagnosis of this tumor. However, cytologic appearance of MA is not well characterized., Case: A 33-year-old pregnant woman presented with erythrocytosis. Transabdominal ultrasound examination disclosed a mass in her left kidney. FNA smears showed small, uniform cells with bland nuclei arranged in compact acinar and follicular structures; immunocytochemical staining revealed a diffuse, positive reaction for CD57, WT-1 and vimentin, and epithelial membrane antigen and alpha-methylacyl-CoA racemase yielded negative results. These cytologic and immunocytochemicalfindings led to a preoperative diagnosis of MA. After delivery, the diagnosis was confirmed on the surgical specimen., Conclusion: A diagnosis of MA could be established by FNAC supported by immunocytochemical analysis. The present case illustrates the clinical impact that this diagnosis could have on patient management.

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.

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.

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)-alpha and -beta 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-beta 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-beta was the only hormone receptor expressed by stroma of fibroadenomas and phyllodes tumors, both at protein and mRNA level. The highest percentage of ER-beta was observed in fibroadenomas with cellular stroma and in phyllodes tumors. In both lesions, ER-beta-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-beta-positive vs ER-beta-negative fibroadenomas. In contrast, in phyllodes tumors, ER-beta expression was higher in older patients. In conclusion, (i) only ER-beta 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-beta in myofibroblastic differentiation of stromal cells. These two results, together with the young age of patients carrying fibroadenomas with highly ER-beta-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-beta-rich phyllodes tumors suggests that mechanisms, probably independent from estrogen stimulation, act on the growth of these tumors.

Extremely rare cases of paraneoplastic syndromes or ectopic production of proteins associated with liposarcoma are reported in literature. We describe a unique case of relapsing retroperitoneal dedifferentiated liposarcoma with biochemical, immunohistochemical, and molecular evidence of alpha-fetoprotein (AFP) ectopic production. The lesion was associated to elevated AFP plasma levels that subsided after tumor removal. Immunohistochemical studies showed AFP production by a minority of tumor cells and reverse transcriptase polymerase chain reaction confirmed AFP mRNA expression. Finding of MDM2 and CDK4 iperexpression by immunohistochemistry confirmed the diagnosis of dedifferentiated liposarcoma.