Advanced Therapies for Liver Metastases

MAIN STUDY: Advanced immune gene and cell therapies for liver metastases (LiMeT protocol) - AIRC5x1000 research program Background and rationale - CRC and PDAC are the second and fourth most common causes of cancer death, respectively. Patients affected by these cancers die of liver MTS. Conventional therapies are active in either primary tumors but fail in metastatic disease. The effectiveness of immunotherapy by immune checkpoint blockade and adoptive cell therapy with tumor-specific T cells is only partial or temporary in MTS from solid tumors, because of local immunosuppressive mechanisms. This is particularly relevant for hepatic MTS, in which a natural tolerogenic milieu may further sustain immunosuppression induced by cancer cells and infiltrating immune cells, such as tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), neutrophils, and regulatory T or B cells. In this regard, evidence suggests that progression of primary or metastatic CRC correlates with poor immune infiltration and inflammation. Moreover, our published results in primary PDAC support a correlation between tumor progression and a strong type 2 anti-inflammatory/immunosuppressive signature. Efficacy of chemotherapy is known to rely also on reactivation and/or repolarization of tumor-targeting immune responses. Pre-surgical chemotherapy may induce beneficial effects and potentially targetable changes in the immune profile of CRC or PDAC MTS. The investigators hypothesize that a combination of: i) direct cancer cell killing by adoptively transferred tumor-specific T cells and ii) indirect control of tumor progression via reprogramming of the tumor microenvironment would control hepatic MTS of CRC and PDAC. Thus, the investigators propose a thorough characterization of the metastatic milieu in these two cancer types to rationally shape, on the basis of similarities and differences, innovative ATMPs. Experimental results obtained from biological samples will be correlated: i) with clinical data from patients at the time of enrollment and along their follow-up; and ii) for a subgroup of patients, with biochemical and immunological data from peripheral blood samples routinely collected as a part of their clinical follow-up, with the additional aim to identify novel biologic parameters and/or signatures to help the stratification of patients and the tailoring of treatments. Objectives - The investigators want to study: 1. the tumor mutational burden and the composition of the metastatic tumor microenvironment in the liver; 2. the inhibitory pathways that constrain immunity in liver MTS; 3. the antigenic and clonal breadth of anti-tumor T cell responses; 4. the spatial-temporal variations of effector and regulatory immune cells upon therapy; 5. the characteristics of tumor cells and their microenvironment in liver MTS versus (vs) primary tumor; 6. the longitudinal variations of clinical parameters as well as blood-derived biological and immunological markers during the medical follow-up of patients. 7. Moreover, they want to collect and biobank patients' samples to support the validation and selection of the new ATMPs developed by the other partners of the translational program. The primary objectives will be: 1. to identify immune suppressive/exhaustion pathways 2. to develop a library of tumor-specific TCR (N≥5/tumor type) and identify new CAR targets (N≥1/tumor type) in CRC and PDAC MTS Study design - This clinical protocol aims at deeply characterizing MTS tumor cells and their microenvironment, including possibly informative dynamic modifications from pro-tumor to anti-tumor profiles that may follow chemotherapy, which is administered pre-surgery (neo-adjuvant) to a substantial fraction of patients. PDAC and CRC MTS patients will therefore be characterized by either cross-sectional or longitudinal investigations on chemotherapy-naïve vs -treated patients. In addition, the investigators aim at comparing hepatic MTS with primary tumor, in order to highlight dynamic qualitative modifications in cancer cells, immune infiltrates and non-immune microenvironment that could facilitate disease progression toward metastatic spread. These modifications may offer a window of opportunity for the optimal application of our ATMPs. To this purpose, primary non-metastatic PDAC patients undergoing surgical resection will be enrolled in this study and followed-up for monitoring the development of metachronous liver MTS, which, whenever possible, will be characterized and compared to their matched/not matched, previously collected, primary tumor samples. Peripheral blood samples at the time of liver MTS diagnosis will also be collected and analyzed. Also, in those CRC MTS patients undergoing synchronous resection of primary CRC and hepatic MTS, comparative analyses will be carried out on tumor from both sites, allowing intra- and inter-patient profiling of both tumor niches. Moreover, results from multi-level research analyses will be correlated with clinical data collected from all patients at the time of enrollment in the study and during their clinical follow-up, whenever possible for up to 2 years, considering PDAC patients, and 3 years, considering CRC. In particular, for metastatic CRC patients, the clinical follow-up will be associated with concurrent collection of blood samples at serial timepoints, that will be prospectively biobanked for further analyses aimed at: i) studying biological and immunological correlates of the patient's clinical status; ii) evaluating key discriminants previously identified in blood samples analyzed at the time of liver MTS surgery, to serve as early predictors of liver progression or recurrence and possibly as indicators for therapeutic stratification. The investigators expect to collect tissue/blood samples and clinical data from at least: * 200 metastatic CRC patients (either synchronous or metachronous); * 150 non-metastatic PDAC patients; * 75 synchronous PDAC MTS patients and about 20 metachronous PDAC MTS patients, the latter originally enrolled as primary non-metastatic patients for the longitudinal study, for a total of 95 metastatic PDAC patients; * 30 healthy volunteers, as negative controls. Different biological samples (peripheral blood, primary and/or metastatic tumor tissue, portal blood) will be collected, in the course of the standard diagnostic and therapeutic care of those patients, and will be in part directly analyzed and in part biobanked for further studies. In parallel, clinical data will be collected from patients at the time of enrollment in the study and, whenever possible, for a follow-up period of up to 2 years, for PDAC patients, and 3 years, for CRC patients. The statistical analysis of collected data will be performed throughout the clinical study, starting 6 months after the enrollment of the first patient until 6 months after the end of patients' follow-up, for a total of 7 years. The study will last 7 years. The research activities will be organized and integrated in 6 work packages (WPs): 1. Collection and biobanking of the material from patients with CRC and PDAC MTS to the liver and with primary non-metastatic resectable PDAC. 2. Definition of the tumor mutational burden, epigenetic and gene expression profile of the CRC and PDAC metastatic liver at bulk and at the single-cell level. 3. Evaluation of the molecular and cellular composition of CRC and, if possible, PDAC liver MTS by spatial transcriptomics technologies (NICHE-seq and Visium). 4. Characterization of the immune landscapes of CRC and, if possible, PDAC liver MTS by high-dimensional flow cytometry. 5. Validation of the molecular results obtained in 2) and 4). 6. Definition of the antigenic landscape and TCR repertoire of CRC and PDAC liver MTS. The results obtained by the above mentioned WPs will be crossed with clinical follow-up data and serve to calibrate the program activities focused on novel ATMPs development. SUBSTUDY 1 - Treating Liver Metastasis (TREATLIVMETS project) - ERC Synergy Grant 2023 Background and rationale - Current immunotherapies that enhance T cell responses benefit only a minority of patients with liver CRC MTS, with extremely poor survival in unresectable cases, indicating the need to identify additional immune components involved in metastatic progression. Innate lymphoid and myeloid cells (NK cells, ILC1s, macrophages, dendritic cells) are key regulators of early antitumor immunity and may offer novel therapeutic leverage. This substudy integrates single cell and spatial transcriptomics of human liver CRC MTS with cross species analyses in advanced mouse models to define innate immune interactions that sustain metastasis and to identify rep Objectives - The substudy aims to: 1. identify innate immune cell types and interactions shaping the metastatic microenvironment in liver CRC MTS; 2. characterize molecular pathways by which innate lymphoid and myeloid cells regulate metastatic progression; 3. define spatial-temporal features of tumor-innate immune cell interactions; 4. evaluate reprogrammable innate immune circuits as potential therapeutic targets; 5. support the development of innovative immunotherapies leveraging innate immune engagement; 6. contribute samples and data to the broader LiMeT translational program. The primary objectives will be: \- identify cellular and molecular interaction networks within liver CRC MTS using spatial and single cell transcriptomics; * define functional pathways of key innate immune subsets regulating metastasis; * develop and preclinically assess next generation cell engagers able to activate liver resident innate immune cells. Study design - This observational substudy extends the collection and analysis of samples from CRC patients with liver CRC MTS within the LiMeT protocol until June 2030. Retrospective samples already collected in LiMeT and prospective samples obtained during this extension will be included. Analyses will be performed on different biological samples (metastatic tissue, primary tumor when available, peripheral blood, healthy controls) and will include: high dimensional flow cytometry, single cell RNA seq, spatial transcriptomics, histological and imaging based characterization. These analyses aim to define innate immune infiltration, cell-cell communication networks, and potentially targetable circuits in the metastatic niche. Population: ≥300 CRC patients with liver CRC MTS, synchronous or metachronous; \~30 healthy volunteers as negative controls; Approximately two thirds of tissue samples expected to be suitable for advanced analyses. Experimental findings will be integrated with clinical parameters collected at enrollment and-when available-during follow up, to identify innate immune correlates of disease progression and therapeutic response. SUBSTUDY 2 - Deciphering and targeting the immunological niche in PDAC - Fondazione Regionale per la Ricerca Biomedica (FRRB) 2024 Background and rationale - Within the LiMeT framework, this substudy focuses on dissecting and therapeutically targeting the immune niche that supports PDAC progression. By integrating spatial multi omics, single cell profiling, and machine learning (ML)-based image analysis, the project aims to elucidate immune-stromal interactions in PDAC and its precursor lesions (IPMN), and to identify actionable targets for engineered immune cells. This work leverages LiMeT pipelines and extends them to pancreatic cancer with dedicated technological platforms and clinical cohorts. Objectives - This substudy aims to: 1. define immune and stromal cell interactions in PDAC and IPMN using high resolution single cell and spatial transcriptomics; 2. identify disease driving niches and early immunological alterations in preneoplastic and neoplastic pancreatic lesions; 3. develop ML algorithms integrating histology and omics data to detect spatial immune signatures predictive of lesion evolution; 4. generate segmentation tools for automated annotation of key histopathological features; 5. design and validate gene engineered immune cells (TCR T, CAR T, CAR macrophages) targeting PDAC immune niches; 6. contribute pancreatic specific molecular and cellular insights to guide new ATMP development. Primary objectives will be: 1. characterize immune-stromal architecture of PDAC and IPMN through single cell and spatial multi omics; 2. develop ML based tools for automated detection of diagnostic and prognostic features in PDAC/IPMN; 3. validate engineered immune cells targeting PDAC specific immunological niches. Study design - This prospective observational substudy will run over the 3 year FRRB funding period. Patients with PDAC or IPMN undergoing diagnostic or surgical procedures at the OSR Pancreas Center will be enrolled following clinical protocol approval. Biological samples (peripheral blood, cystic fluid, fresh and FFPE tumor and adjacent non tumor tissues) will be collected according to standard procedures and processed through the OSR biobank for anonymization, storage, and controlled distribution. Experimental analyses will include: * single cell multiome profiling (scRNA seq, scATAC seq), * spatial transcriptomics (Visium, MERSCOPE), * multiplex IF/IHC, * ML-based computational integration of clinical, histological, and omics data * Engineered immune cell development Population: * at least 100 patients (50 PDAC, 50 IPMN) for single-cell multiome and spatial gene expression analyses; * 200 donors for H\&E whole slide imaging (WSI)-based ML development. In detail: 50 slides will be used for manual annotation by pathologists (training set) and 150 slides will be used for model inference (validation set).