Advanced Therapies for Liver Metastases (LiMeT)

Description

Background and rationale - CRC and PDAC are the second and fourth most common cause 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 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. longitudinal variations of clinical parameters as well as blood-derived biological and immunological markers during the medical follow-up of patients.

Our 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 be therefore 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 not-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, that, 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 be also 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 since the time of enrollment for a 2-year follow-up period, in case of PDAC patients, and for 3-year follow-up period, in case of CRC patients.

In particular, for metastatic CRC patients, the 3-year 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. Almost 505 different subjects will be enrolled in the protocol.

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 since the time of enrollment in the study for a follow-up period of 2 years.

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.

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 since the time of enrollment in the study for a follow-up period of 2 years, for PDAC patients, and of 3 years, for CRC patients.

The statistical analysis of the collected clinical and experimental data will be carried out all along the duration of the clinical study, starting 6 months after the enrollment of the first patient until 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 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 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 Longitudinal PDAC primary tumor immune microenvironment characterization.

The results obtained by the above mentioned WPs will be crossed with clinical follow-up data.

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