The IBBRI is tightly connected with the IEO Tumour Registry that collects and compiles all IEO clinical data (see below), with IEO clinical and surgical wards, upon which it relies for sample collection, and with the Department of Pathology and Laboratory Medicine, which provides pathologist expertise for sample classification and dissection. The IBBRI has developed the necessary infrastructure to provide high quality biospecimens required for biomarker discovery and xenotransplantation experiments. This latter technology, which involves the transplantation of human tumours into immunodeficient mice, is vitally important as it constitutes an ex-vivo model system that retains the morphology and biological features of the tumour of origin. Thus, it is essential for testing new combination therapies or new drugs that will be developed in the future through MMC. Tumour Registry The IEO Tumour Registry is an independent data collection unit for IEO, which will play a critical role in the success of the MMC programme. Upon informed consent, the Registry collects all available personal data, clinical data relating to diagnosis and treatment, epidemiological data, and patient clinical course, which it links to the identity of the samples preserved in the tissue bank. Because of the sensitive nature of this information, we have developed safeguards for patient privacy, in close collaboration with the Tumour Registry. Researchers, therefore, do not have direct access to Registry data, which must necessarily be retrieved through Tumour Registry researchers, who filter out any patient identifying information. The expertise and resources of the Tumour Registry, act as guarantee
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that researchers can link clear clinical information to the samples present in the Tissue Bank. This resource is thus a vital part of our MMC programme. As well as contributing to the success of MMC, the Tumour Registry plays an important role in monitoring clinical activity and analysing IEO clinical data. This function, which is currently independent of its supporting role in MMC, is described separately below. Molecular Pathology Laboratory The goal of the Molecular Pathology Laboratory is to identify novel putative cancer targets for drug discovery, diagnostic and/or prognostic applications. For this purpose, we use a high-throughput screening technology (Tissue Microarrays, TMA) in combination with in situ detection methods (ISH and IHC) for the rapid translation of early discoveries into clinical applications. TMA technology allows us to perform gene expression analysis on thousands of patient tissues simultaneously, and is thus a powerful tool for the validation of basic research findings, and to establish their relevance to cancer. We routinely assess the deregulation of target genes, based on the comparative expression analysis between the tumour tissues and their normal counterparts. We have a pipeline between the Pathology Department at IEO and our service, which has become much more regular since the establishment of the IEO Tissue Bank. This pipeline is essential for the replenishment of our TMAs, which are rapidly exhausted through large-scale screening studies. Through the Tissue Bank collaboration with the Tumour Registry, all arrayed tissues are linked to clinico-pathological and follow-up data. We can therefore link the expression patterns of genes of interest to clinical evaluation and outcome, a necessary step for the validation of new diagnostic and prognostic markers. The Molecular Pathology Laboratory has a long history in TMA screening, which guarantees excellence of service. Our service offers screening studies on multi-tumour microarrays, consisting of tissue cores for each of a wide selection of tumours. These arrays contain a series of matched (where possible) and unmatched normal tissue counterparts (eg. [1]). We also perform screening studies on single tumour microarrays, which we have available for i) breast (~1300 samples are currently being arrayed); ii) lung (~800 tumour samples, arrayed in two specific TMAs); iii) colon (a “progression TMA” consisting of ~250 samples of normal tissues, hyperplastic tissues, adenomas and carcinomas); iv) melanomas (a series of three independent TMAs of 150-250 samples, containing benign and dysplastic nevi, primary tumours and metastatic melanomas); v) ovary
(~250 tumour samples of which ~200 matched with a metastatic sample from the same patient, arrayed in two different TMAs). Primary Epithelial and Stem Cell Culture Facility The Primary Epithelial and Stem Cell Culture Facility is one of the enabling infrastructures of the Molecular Medicine for Care Programme at IEO. The Facility has the specific task of deriving bulk primary epithelial cells and stem cells, whenever possible from human biopsy specimens of matched normal and tumour pairs from the same patient. These model-systems recapitulate ex-vivo the physiopathology of naturally occurring human cancers, and they can therefore be used to derive information that can be directly extrapolated to the clinic. Standardized operative procedures (SOP) are currently in place for processing of tissue biopsy specimens, and for the efficient preparation of bulk primary epithelial cell and stem cell cultures from breast, lung, ovary and prostate tissues. We have established a continuous pipeline of samples from the operating theatres to the laboratory through coordination with the IBBRI (Tissue Bank). We have also established a close connection with the IEO Tumour Registry for the collection of clinical-pathological information linked to human samples. During the last year, our Facility has provided material for a number of basic studies aimed at elucidating molecular mechanisms of breast cancer and mammary stem cell biology. In addition, in particular for breast and lung cancer, a number of different studies are being conducted at IEO, based on the use of primary epithelial cell cultures as a ‘pre-clinical’ model to assess the in vitro efficacy of different types of drug treatment. Clinical Biomarkers Laboratory The Clinical Biomarkers Laboratory acts transversally with clinical and research activities and with the different enabling platforms of the program with the aim of optimizing cancer biomarker candidates and facilitating clinical development. The laboratory performs screening, pre-clinical validation, and optimization of biomarker candidates, as well as the design and execution and technological development of cancer diagnostic assays to aid their translation into the clinical setting. In this role, we also support collaborative study programs between biotech companies, researchers and clinicians for the co-development of biomarkers and novel therapies. We have collaborated with the IBBRI (Tissue Bank), to optimize protocols for the extraction of nucleic acids (including total RNA, DNA and microRNAs) from human
tissues, such as blood, plasma, fresh bioptic tissues and paraffin-embedded tissue specimens. We collaborate with the Tissue Bank, the Tumour Registry and the Pathology Department at IEO to select tissue samples for prospective and retrospective studies.
Research Activities
for diagnosis, that would otherwise be discarded) as well as plasma/serum, total blood, DNA and RNA. Within the framework of MMC, we have set up specific protocols and standard operating procedures (SOPs) to ensure optimized, standardized treatment of all samples for research purposes. In collaboration with the Cell Biology Unit (see below), the IBBRI also ensures the storage of purified normal and cancer stem cells and primary cell cultures. Biological samples are collected only if donors have provided informed consent for sample use for research purposes. A new informed consent form for research purposes (including biobanking of tissues and biological material) has been developed specifically for this purpose, thanks to the collaboration of clinicians, researchers, and bioethicists. This form has been approved by the Ethics Committee of IEO.
Our current projects include: 1) the prognostic evaluation of a breast stem cell signature identified in the lab through an extensive meta-analysis of the expression profile of normal stem cells and their progenitors recently generated in house [2] and of publicly available breast cancer data sets; 2) the technological validation, in paraffin-embedded tissue samples, of a 10-gene predictive model of overall survival in early stage NSCLC that was recently identified in the lab [3]; 3) the identification of novel candidate prognostic microRNAs in G2 breast tumours; 4) microRNA profiling in a retrospective cohort of breast cancer tumours with complete follow up to identify novel biomarkers and novel therapeutic targets; 5) the analysis of prognostic microRNAs from plasma of both in NSCLC and breast cancer; 6) the design of a PCR-based assay for a rapid and cost-effective detection method of the ALK translocation in non-small cell lung cancer patients. Several high-throughput technology platforms have been established in the Clinical Biomarkers Laboratory, including the recently released ViiATM7 real-Time PCR system machine with the Micro Fluidic Card Upgrade (Applied Biosystems) and the OpenArray Real-Time PCR System for gene expression, genotyping and digital PCR applications in a mid-density format, which will help streamline genomic screening and validation, absolute quantitation, and biomarker identification and screening at a scale previously unattainable. We have also purchased the EnVision Multilabel Reader (Perkin-Elmer) for cell-based drug discovery and enzyme assays. Recently, we have also established an automated robotic workstation for medium-high-throughput automation of sample preparation procedures, methods and assays. The workstation displays a modular and flexible system configuration, composed of two independent platforms: one dedicated to the set up of the different molecular assays and one to the purification of nucleic acids. Each workstation is configured with multiple pipetting and labware gripping devices. Pipetting channels and labware grippers move independently of each other, supporting the use of a wide range of labware. The autoload option provides barcode tracking of samples, labware, racks and carriers and data can be tracked and processed within the application as well as interfaces to internal and external databases. Thanks to the scalability
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