Maria Rosaria Giovagnoli

Stearoyl-CoA desaturase-1 is a key factor for lung cancer-initiating cells

In recent years, studies of cancer development and recurrence have been influenced by the cancer stem cells (CSCs)/cancer-initiating cells (CICs) hypothesis. According to this, cancer is sustained by highly positioned, chemoresistant cells with extensive capacity of self renewal, which are responsible for disease relapse after chemotherapy. Growth of cancer cells as three-dimensional non-adherent spheroids is regarded as a useful methodology to enrich for cells endowed with CSC-like features. We have recently reported that cell cultures derived from malignant pleural effusions (MPEs) of patients affected by adenocarcinoma of the lung are able to efficiently form spheroids in non-adherent conditions supplemented with growth factors. By expression profiling, we were able to identify a set of genes whose expression is significantly upregulated in lung tumor spheroids versus adherent cultures. One of the most strongly upregulated gene was stearoyl-CoA desaturase (SCD1), the main enzyme responsible for the conversion of saturated into monounsaturated fatty acids. In the present study, we show both by RNA interference and through the use of a small molecule inhibitor that SCD1 is required for lung cancer spheroids propagation both in stable cell lines and in MPE-derived primary tumor cultures. Morphological examination and image analysis of the tumor spheroids formed in the presence of SCD1 inhibitors showed a different pattern of growth characterized by irregular cell aggregates. Electron microscopy revealed that the treated spheroids displayed several features of cellular damage and immunofluorescence analysis on optical serial sections showed apoptotic cells positive for the M30 marker, most of them positive also for the stemness marker ALDH1A1, thus suggesting that the SCD1 inhibitor is selectively killing cells with stem-like properties. Furthermore, SCD1-inhibited lung cancer cells were strongly impaired in their in vivo tumorigenicity and ALDH1A1 expression. These results suggest that SCD1 is a critical target in lung cancer tumor-initiating cells.

Spheres Derived from Lung Adenocarcinoma Pleural Effusions: Molecular Characterization and Tumor Engraftment

Malignant pleural effusions (MPEs) could represent an excellent source to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas (AdenoCa) which account for approximately 40% of lung cancer cases. AdenoCa malignant pleural effusions gave rise to in vitro cultures both in adherent and/or in spheroid conditions in almost all cases analyzed. We characterized in greater detail two samples which showed the most efficient propagation in vitro. In these samples we also compared gene profiles of spheroid vs adherent cultures and identified a set of differentially expressed genes. Finally we achieved efficient tumor engraftment in recipient NOD/SCID mice, also upon inoculation of small number of cells, thus suggesting indirectly the presence of tumor initiating cells.

Monoclonal antibody-induced ErbB3 receptor internalization and degradation inhibits growth and migration of human melanoma cells

Members of the ErbB receptor family are targets of a growing numbers of small molecules and monoclonal antibodies inhibitors currently under development for the treatment of cancer. Although historical efforts have been directed against ErbB1 (EGFR) and ErbB2 (HER2/neu), emerging evidences have pointed to ErbB3 as a key node in the activation of proliferation/survival pathways from the ErbB receptor family and have fueled enthusiasm toward the clinical development of anti-ErbB3 agents. In this study, we have evaluated the potential therapeutic efficacy of a set of three recently generated anti-human ErbB3 monoclonals, A2, A3 and A4, in human primary melanoma cells. We show that in melanoma cells expressing ErbB1, ErbB3 and ErbB4 but not ErbB2 receptor ligands activate the PI3K/AKT pathway, and this leads to increased cell proliferation and migration. While antibodies A3 and A4 are able to potently inhibit ligand-induced signaling, proliferation and migration, antibody A2 is unable to exert this effect. In attempt to understand the mechanism of action and the basis of this different behavior, we demonstrate, through a series of combined approaches, that antibody efficacy strongly correlates with antibody-induced receptor internalization, degradation and inhibition of receptor recycling to the cell surface. Finally, fine epitope mapping studies through a peptide array show that inhibiting vs. non-inhibiting antibodies have a dramatically different mode of binding to the to the receptor extracellular domain. Our study confirms the key role of ErbB3 and points to exploitation of novel combination therapies for treatment of malignant melanoma.

Virtual microscopy and digital cytology: state of the art

The paper approaches a new technological scenario relevant for the introduction of the digital cytology (D-CYT) in the health service. A detailed analysis of the state of the art on the status of the introduction of D-CYT in the hospital and more in general in the dispersed territory has been conducted. The analysis was conducted in a form of review and was arranged into two parts: the first part focused on the technological tools needed to carry out a successful service (client server architectures, e-learning, quality assurance issues); the second part focused on issues oriented to help the introduction and evaluation of the technology (specific training in D-CYT, health technology assessment in-routine application, data format standards and picture archiving computerized systems (PACS) implementation, image quality assessment, strategies of navigation, 3D-virtual-reality potentialities). The work enlightens future scenarios of actions relevant for the introduction of the technology.

Breast Implant-Associated Anaplastic Large Cell Lymphoma: Proposal for a Monitoring Protocol.

Background: The authors report four cases of breast implant–associated anaplastic large cell lymphoma (ALCL) from a single institution and propose a multidisciplinary protocol. Methods: From 2012 to 2014, four breast implant–associated ALCL cases were diagnosed. The authors performed the original operation, and no patients were referred to their practice. Cases 1, 2, and 4 were CD4+/CD30+/ALK− ALCL with previous textured-implant reconstruction, whereas case 3 was CD8+/CD30+/ALK− ALCL with previous polyurethane-implant augmentation. A retrospective study of all patients who underwent breast implant positioning was performed to identify any misdiagnosed cases. Results: Of 483 patients, 226 underwent reconstruction with latissimus dorsi flap and prosthesis, 115 had skin-sparing/nipple-sparing mastectomy and prosthesis, 117 underwent an expander/implant procedure, and 25 underwent breast augmentation. Fifty-eight cases (12 percent) underwent implant replacement for capsular contracture, 15 (3.1 percent) experienced late-onset seroma, and four (0.83 percent) had both capsular contracture and seroma. Seventy-seven symptomatic patients (16 percent) underwent surgical revision (capsulectomy/capsulotomy) and/or seroma evacuation. The second look on histologic specimens did not identify misdiagnosed cases. A multidisciplinary protocol for suspected implant-associated ALCL was established. Ultrasound and cytologic examinations are performed in case of periprosthetic effusion. If implant-associated ALCL is diagnosed, implant removal with capsulectomy is performed. If disseminated disease is detected through positron emission tomography/computed tomography of the total body, the patient is referred to the oncology department. Conclusions: A multidisciplinary protocol is mandatory for both early diagnosis and patient management. Until definitive data emerge regarding the exact etiopathogenesis of breast implant–associated ALCL, the authors suggest offering only autologous reconstruction if patients desire it. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

EMT markers in lung adenocarcinoma pleural effusion spheroid cells

Epithelial‐to‐mesenchymal transition (EMT) is a process in which cells undergo a developmental switch from epithelial to mesenchymal phenotype. This process has been related to embryologic morphogenesis but also to cancer progression and metastasis. The aim of the current study was to investigate the expression of EMT‐related markers in adherent and spheroid cell cultures derived from malignant pleural effusions (MPEs) of patients affected by lung adenocarcinoma. On the basis of efficient in vitro propagation, six cases of MPEs were selected and analyzed by immunocytochemistry staining for EMT markers and by RT‐PCR for transcription factors known to orchestrate EMT. EMT markers immunostaining showed in spheroids a statistically significant correlation between the loss of E‐cadherin immunoreactivity and overexpression of N‐cadherin (P < 0.001). Likewise loss of EpCAM epithelial marker was coincident with Vimentin overexpression (P < 0.001). RT‐PCR analysis of transcription factors Snail, Slug, and Twist showed a highly variable expression, although a general trend to increase was observed. Importantly, in some selected cases it was possible to establish a precise relationship between spheroid formation, EMT switch and increased upregulation of the marker related to cancer stemness such as ALDH positivity. Therefore, MPE‐derived cell cultures, while recapitulating the heterogeneity of lung cancer, are a suitable system to study the mechanisms at the basis of EMT and to understand its relationship with the generation of cancer stem cells. J. Cell. Physiol. 228: 1720–1726, 2013.

The Design of a Health Technology Assessment System in Telepathology

Up to a few years ago, the management of the information on the slides (virtual slides) in telepathology applications was principally based on the design and construction of a few identical and expensive platforms with microscope units and software tools for the display and for electronic control (zooming, moving, and cutting of images). The development of information technology tools allows the diffusion of new visualization strategies and the availability of low cost or free visualization proprietary tools. New competitive systems such as client-server architectures are available in telepathology today. The investigation of the new technologies for telepathology is a basic and core aspect in telemedicine technology assessment. A new interactive environment to investigate the health technology assessment of a telepathology system has been studied. In particular, in consideration of previous experience the methodology focused both on the senior pathologist and younger student pathologist. Two interactive forms were created by a working group: a feedback form and a diagnostic form. The first was designed to investigate the technology characteristics and acceptance of the telepathology systems. The second tool was designed to investigate the diagnostic accuracy on a significant sample of virtual slides by two different groups of pathologists (senior and younger students). The acceptance of the methodology was very high.

Telepathology Requires Specific Training for the Technician in the Biomedical Laboratory

The Italian Laurea for Health Care Professionals furnishes a high level of learning to technical personnel who will be involved in the healthcare system. It also represents a test for new models of learning in e-health and telemedicine applications. The purpose of this work was to investigate the changes in the biomedical laboratory curriculum in the healthcare system as a result of the introduction of telepathology (TP). Changes were categorized in two stages. The first stage was the inclusion of the TP as a support methodology using external furnishers to digitize the glass slides. The second is the inclusion of the TP as a consolidated routine methodology using a dedicated internal scanner to digitize the glass slides. New modules of learning have been designed to run on the wide area network to allow a better familiarization with new technologies. These new methodologies have been tested and present three tangible advantages: (1) A high level of knowledge for the student; (2) A cost-benefit advantage to the student; and (3) A cost-benefit advantage to the hospital. As the biomedical laboratories are freed up from academic applications, they thus become more available for clinical use.

Lung Cancer Stem Cell Lose Their Stemness Default State after Exposure to Microgravity

Microgravity influences cell differentiation by modifying the morphogenetic field in which stem cells are embedded. Preliminary data showed indeed that stem cells are committed to selective differentiation when exposed to real or simulated microgravity. Our study provides evidence that a similar event occurs when cancer stem cells (CSCs) are cultured in microgravity. In the same time, a significant increase in apoptosis was recorded: those data point out that microgravity rescues CSCs from their relative quiescent state, inducing CSCs to lose their stemness features, as documented by the decrease in ALDH and the downregulation of both Nanog and Oct-4 genes. Those traits were stably acquired and preserved by CSCs when cells were placed again on a 1 g field. Studies conducted in microgravity on CSCs may improve our understanding of the fundamental role exerted by biophysical forces in cancer cell growth and function.

Telepathology training in a master of cytology degree course

We have investigated the changes in the workflow of the cytologist due to the introduction of telepathology. These changes occur in two stages. The first is the use of telepathology as a support methodology using external providers (i.e. outside the hospital) to digitize the slides. The second is the use of telepathology in routine laboratory operations using an internal scanner to digitize the slides. To improve courses in the Master of Cytology programme at the University of La Sapienza, new learning modules were designed, which were made available via the wide area computer network to familiarize students with the new technologies. The new methodologies had three benefits. The first was the high level of knowledge for the student. The second was the cost advantage to the student, who did not need to study in a medical laboratory to participate in the teaching. The third was the cost advantage to the hospital: as laboratories become freed from academic work, they become more available for clinical use.