Molecular Diagnostics and Research

The facility is dedicated to the molecular characterization of solid tumors, with the primary objective of identifying molecular markers that may possess predictive, prognostic, or diagnostic significance. This activity, which employs both Next-Generation Sequencing (NGS) and in situ analyses of DNA and RNA, enables the acquisition of comprehensive insights into the molecular profile of each tumor. Such detailed information facilitates the tailoring of treatment strategies in a more precise and personalized manner. Through these methodologies, it becomes possible to offer patients bespoke therapies based on the unique molecular features of their tumors, thereby potentially enhancing the likelihood of therapeutic success and optimizing clinical outcomes. 

The Molecular Diagnostics and Research Facility is dedicated to performing advanced molecular analyses, including Comprehensive Genomic Profiling (CGP) via Next-Generation Sequencing (NGS), which allows for a comprehensive characterization of the tumor's genetic makeup. This analysis encompasses the determination of Tumor Mutational Burden (TMB), an important biomarker for assessing response to immunotherapies. Additionally, copy number variation (CNV) analyses are conducted to identify alterations in the number of copies of specific genes. For panels that include Homologous Recombination Deficiency (HRD) assessment, the Genomic Instability Score (GIS) is calculated, providing a quantitative measure of genomic instability. NGS technology is also employed to detect potential gene fusions. These analyses are performed on various types of biological samples. Furthermore, we utilize NGS technology to analyze circulating cell-free DNA (cfDNA) present in blood plasma. This approach enables more accurate diagnoses and the identification of molecular markers that are crucial for planning personalized treatments, thereby enhancing therapeutic success rates and improving the quality of care provided to our patients. 

The facility also conducts molecular analyses using in situ hybridization (ISH), a molecular pathology technique that allows for the in situ identification of specific DNA and RNA sequences within tissue samples, or the detection of heterologous sequences, such as viral sequences. This technique enables the study of amplifications, deletions, translocations, inversions, and even very small abnormalities affecting individual genes. The methods employed at our institute include three types: Fluorescence In Situ Hybridization (FISH), Chromogenic In Situ Hybridization (CISH), and Silver In Situ Hybridization (SISH), which differ in their probe detection techniques. FISH preparations are analyzed using a fluorescence microscope, while CISH and SISH preparations are examined with a conventional optical microscope. 

Molecular analyses are performed for both inpatients and outpatients, whether in outpatient clinics or during hospitalization. These services are also available to affiliated institutions, thanks to our status as a HUB laboratory recognized by Regione Lombardia for NGS technology, and to external patients requiring molecular investigations. 

A list of the services provided, including turnaround times and sample submission procedures, is available on our website: https://www.istitutotumori.mi.it/esami-di-laboratorio-preparazioni-eistruzioni. 

The Simple Unit (SS) conducts clinical and translational research across numerous areas of oncological pathology, in collaboration with other Departments of our Institution (Clinical and Experimental departments), as well as with national and international entities. The primary goal is to promote bidirectional knowledge transfer between preclinical and clinical research, providing INT researchers with advanced technologies, tools, and expertise in molecular biology. The SS possesses solid experience and dedicated infrastructure for tumor transcriptome profiling, with bulk RNA-seq as the reference technology. Utilizing high-yield libraries optimized for fresh or frozen tissues and protocols tailored for FFPE samples, the unit obtains comprehensive transcriptomic profiles that support pathway analyses, inflammatory indices, and clinico-pathological correlations. Additionally, the platform nCounter® (NanoString) is available, which is ideal when RNA quantity is limited or degraded: digital quantification of panels up to 800 genes allows for rapid, highly reproducible validation of signatures of interest, even on low-quality samples.nnThe SS also performs single-cell profiling experiments using the Chromium X platform (10x Genomics), generating high-resolution maps of cellular populations. A workflow shared with interventional radiologists and surgical teams minimizes the time between tissue resection and sample processing, preserving RNA quality; this same approach is applied to peripheral blood cells, enabling precise delineation of immune components.nnTo preserve histological context, the SS conducts spatial transcriptomics experiments using the GeoMx DSP platform on FFPE or cryopreserved sections of human tissues and murine models, including orthotopic and PDX models. The selection of regions of interest, curated by the SS pathologists, allows for the correlation of clinical variables with transcriptional activity, tumor architecture, and the distribution of tumor microenvironment cells.nnComplementing tissue analyses, the unit studies circulating markers—free DNA and RNA, as well as circulating tumor cells—enabling minimally invasive monitoring of disease progression and the emergence of therapeutic resistance.nnThe interpretation of omics data generated by the SS is carried out in close collaboration with the Bioinformatics Unit, which employs established pipelines within the scientific community and develops dedicated analysis workflows for ongoing projects. The multi-layered signatures obtained are validated using in situ methods such as immunohistochemistry (IHC) and in situ hybridization (ISH), thereby strengthening their biological and clinical relevance. This integrated ecosystem of technologies supports the discovery of new biomarkers and therapeutic targets and fuels the Foundation’s active precision medicine programs. 

The teaching activities of the SS (the unit) are closely connected with both diagnostic work and ongoing research projects, all within a university-led department at UNIMI (University of Milan). In this department, pathology residents (specializing in Anatomical Pathology) are always present. They help doctors with analyzing tissue samples (histology and cytology) and also take part in all steps of molecular testing—like extracting DNA or RNA from samples and discussing the results in multidisciplinary meetings with different specialists. 

Students following Medicine and Surgery also do internships and work on research projects (theses). They follow a guided program that mixes hands-on laboratory work with attending scientific and clinical meetings, giving them practical experience. 

The department also hosts various professionals for training and professional growth, such as biomedical laboratory technicians learning about the latest testing methods, biologists and biotechnologists working on PhDs or research grants, and bioinformaticians who develop computer pipelines to analyze complex biological data. 

All these different professionals work together in a multidisciplinary environment, which allows each of them to follow the entire process—from collecting the biological sample to validating the results in a clinical setting. This integrated approach helps them develop the skills needed for modern personalized medicine.