Liver metastases (MTS) are the main cause of death for patients affected by colorectal carcinoma (CRC) and pancreatic ductal adenocarcinoma (PDAC), thus representing the major unmet clinical need for these malignancies.
Based on preliminary and published data, the investigators hypothesize that innovative immune, gene, and cell therapy approaches might overcome the tolerogenic liver microenvironment and represent powerful therapeutic tools for liver MTS of PDAC and CRC.
The investigators have therefore planned an observational clinical study to enroll distinct cohorts of patients (i.e., metastatic CRC, preneoplastic, metastatic, and non-metastatic PDAC) and finely characterize, through integrated state-of-the-art -omics, the immune and non-immune microenvironment of their primary tumor and/or liver metastases as well as correlate changes in the activation status and phenotype of peripheral blood leukocytes. Healthy volunteers will be enrolled as negative controls.
The investigators aim at identifying: i) actionable tumor-associated antigens (TAAs) and local immune suppressive and regulatory pathways; ii) biological parameters for early diagnosis of relapse; iii) the effect of therapies on the shaping of anti-tumor immune responses.
Data collected will be instrumental for the generation of novel advanced therapy medicinal products (ATMPs). Indeed, this protocol is part of a multi-partner translational program, supported by the AIRC 5 per Mille 2019 grant, focused on the development, validation, and implementation of clinical testing for ATMPs to ameliorate the cure of CRC and PDAC, and possibly to help the study of other solid tumors.
Moreover, the systematic and long-term follow-up of enrolled patients will possibly point to early predictors of differential prognosis and patients' categories eligible for tailored therapies, including those with the novel ATMPs.
In this regard, two additional substudies were incorporated into the main LiMeT protocol in July 2024 and January 2026, respectively, supported by supplementary funding: 1) the TREATLIVMETS (Treating Liver Metastasis) project, funded by the European Research Council (ERC) under the Horizon Europe research and innovation program; 2) the "Deciphering and targeting the immunological niche in PDAC" project, funded by the Fondazione Regionale per la Ricerca Biomedica (FRRB) under the "From Bed to Bench 2024" call. In the latter substudy, machine learning will be integrated with the spatial multi-omic profiling of the immune landscape in preneoplastic and neoplastic lesions in a subset of IPMN and PDAC patients, supporting the discovery of new therapeutic targets and enabling the early detection of preneoplastic lesion progression.
None
CRC patients
Patients with histologically or cytologically confirmed diagnosis of CRC metastatic to the liver (stage IV disease, AJCC)
Patients with indication to surgical resection and/or chemotherapy treatment
Age ≥18
CRC and PDAC patients with hepatic MTS, primary non-metastatic PDAC patients, IMPN patients, and healthy volunteers. Eligible patients, chosen among those who receive health care assistance/treatment for their disease at IRCCS San Raffaele, will be asked to signing of the predisposed informed consent form in case they decide to participate in this study.
Cohort
Prospective
Not listed
Definition of tumor mutational burden, epigenetic and gene expression profile of the CRC and PDAC metastatic liver at bulk and at single cell level
CRC/PDAC MTS samples will be analyzed by: * Whole exome DNA sequencing * RNA sequencing * ATAC sequencing * Single cell RNA sequencing on sorted myelomonocytic and T cells infiltrating CRC/PDAC MTS samples. Naive vs chemotherapy treated lesions as well as primary vs MTS lesions will be compared to evaluate chemotherapy-induced modifications and the molecular evolution in the distinct tumor sites, respectively.
Characterization of the immune landscapes of CRC and, if possible, PDAC liver MTS by high dimensional flow cytometry
Single cell suspension of CRC and PDAC MTS and paired autologous PBMCs and portal blood harvested before and after pre-operatory chemotherapy, will be analyzed by a series of parallel polychromatic high dimensional 28 color flow cytometry panels to study: * Conventional MHC-restricted Tab cells, the main anti-tumor effectors; * Innate-like T cells; * B cells; * Myelomonocytic populations involved in the stimulation or suppression of anti-tumor responses. Whenever available, matched primary vs MTS lesions will be compared to evaluate the immune landscapes in the distinct tumor sites.
Histological validation of the molecular results obtained in 1. and 2.
Validation of relevant markers and molecular pathways in terms of anatomical distribution, performed by polychromatic immunofluorence (IF, up to 6 colors) and immunohistochemistry (IHC, up to 4 colors) stainings on MTS CRC and PDAC biopsies, perilesional parenchyma and, whenever available, matched primary tumor samples. In addition to multiplex IF and IHC, some specimens will be characterized more deeply with advanced spatial proteomics technologies, such as GeoMx Digital Spatial Profiler (Nanostring).
Definition of the antigenic landscape and TCR repertoire of CRC and PDAC liver MTS
Identification of tumor-reactive TCRs will be pursued through different and complementary strategies: * The most relevant inhibitory checkpoint genes/pathways expressed by MTS-TILs will be used to enrich for tumor specificities by cell sorting harvested from CRC/PDAC MTS tumors, portal, hepatic and peripheral blood sample. After their activation, responding T cell cultures will be subject to bulk and single-cell TCR seq; * In samples characterized by a low percentage of MTS-TILs, single cell immune profiling will be performed in order to simultaneously determine gene expression and correctly pair chain TCR sequences from individual T cells; * The library of tumor-specific TCRs identified will be transiently expressed in reporter Jurkat cells to confirm their tumor antigen specificity; * Identification and functional validation of transmembrane molecules enriched in cancer cells as possible targets for CAR design.
Evaluation of the molecular and cellular composition of CRC and, if possible, PDAC liver MTS by spatial transcriptomics technologies (NICHE-seq and Visium)
Application of NICHE-seq to freshly isolated human tissues using photoactivatable (cages) fluorophores able to permeate within the cells, in particular comparing ≥5 chemotherapy treated vs ≥5 naive CRC MTS samples and ≥3 PDAC MTS samples in order elucidate the high-order spatial organization of immune cell types in the NICHE-seq area and their molecular pathways, at the same time. NICHE-seq and conventional single-cell RNA seq will proceed side-by-side to compare results. Some selected specimens will be analyzed with more advanced spatial transcriptomics techniques developed in recent years, like Visium Spatial Gene Expression (10X Genomics).
Collection of clinical follow-up data
Overall survival (OS), disease free survival (DFS) and other follow-up parameters (patient's status, CEA levels, Ca 19-9 levels, adjuvant chemotherapy after surgery, date and site of recurrence, treatment at recurrence) will be collected and possibly correlated with the markers and pathways identified and validated in primary outcome measures.
Collection and biobanking of follow-up samples from patients with CRC and metachronous PDAC MTS to the liver
* CRC MTS patients: part of peripheral blood collected for hematochemical testing during follow-up visits * newly-diagnosed metachronous PDAC MTS patients, previously enrolled in the non-metastatic PDAC cohort: peripheral blood and, whenever possible, tumor tissue from liver metastasis (fine-needle bioptic sample) collected at the time of liver recurrence diagnosis, during the follow-up after the primary tumor surgery.
Biobanking of biospecimens collected from CRC and PDAC patients and from healthy donors
* blood samples as detailed in the "biospecimen description" section: cryopreservation in liquid nitrogen of whole blood, plasma and purified mononuclear cells. * tissue samples as detailed in the "biospecimen description" section: i) formalin-fixation and paraffin embedding; ii) OCT-based embedding and cryopreservation in liquid nitrogen. Biobanked samples will be studied as described in 1-5, but possibly also with new state-of-the-art technologies developed in the next future. Moreover, they will be used to set up in vitro and in vivo systems (e.g., patient-derived organoids, tissue chips, possibly patient-derived xenograft models) to validate the efficacy and safety of the novel ATMPs developed by research program.
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IRCCS San Raffaele
Lead sponsor
Università Vita-Salute San Raffaele
Collaborator
