Identification of New Theranostic Biomarkers of Pancreatic Tumor Progression

Intraductal papillary mucinous neoplasms (IPMN) are one of the main precursor lesions of pancreatic ductal adenocarcinoma (PDAC), a lethal disease predicted to become the second leading cause of cancer-related deaths in Western societies within a decade. The mechanisms underlying IPMN progression are poorly understood. The goal of IPMN management is to reduce the risk of patient death due to progression to PDAC through primary and secondary prevention (namely, early diagnosis and risk-reducing surgery). High-risk IPMNs (i.e., high-grade or main duct IPMNs, which account for 57-90% of cases) are referred for surgical resection, while low-risk IPMNs (6-46%) undergo periodic follow-up aimed at monitoring the acquisition of morphological features associated with malignancy over time. However, the clinical management of IPMN remains a significant challenge because the distinction between high and low-risk progression is based on imaging and histological criteria that are not unequivocally recognized and do not take into account the underlying biology of lesions that appear similar but are associated with different clinical behaviors. Consequently, patient risk stratification is often inaccurate, leading to suboptimal treatment. Approximately 1-11% of low-risk IPMN patients assigned to clinical follow-up have developed PDAC. Therefore, it is of paramount importance to improve the understanding of the biology and malignant potential of IPMN to improve prognosis and clinical management of affected patients and guide them toward personalized therapeutic approaches. The availability of markers capable of stratifying IPMN based on their risk of progression to PDAC could enhance the current malignancy criteria assessed in clinical settings by more accurately identifying patients who strongly need surgical resection.

\*\*2 - Study Objective\*\* The aim of this study is to identify and validate biomarkers capable of distinguishing between low-risk and high-risk IPMN progression to PDAC. These biomarkers would help more accurately identify IPMN patients who could benefit from therapeutic intervention and/or surgical resection in the future. The study will include patients with IPMN followed at Fondazione Policlinico Gemelli IRCCS, Fondazione G. Pascale IRCCS, Azienda Ospedaliera Universitaria Integrata Verona, and Azienda Ospedaliera Universitaria Integrata di Messina.

To achieve our goal, we have planned the following strategy:

\*\*AIM1.\*\*

The primary objective of the research is organized into a workflow of three experiments:

1. \*\*Identification\*\* of genes and biological pathways associated with IPMN malignant transformation through transcriptomic and epigenetic characterization. We aim to explore the molecular dynamics of tumor progression, focusing on cellular heterogeneity and phenotypic transitions.
2. \*\*Characterization of the microenvironment\*\* to understand its role in IPMN progression. We will collect archival samples from 240 patients across four centers. Sixty tissues will be selected based on cyst type and dysplasia grade. We will use advanced technologies (ATAC-seq, ST) to investigate cellular aspects of IPMN progression. We will perform single-cell spatial transcriptomics and ATAC-seq on samples representing the entire evolution of pancreatic cancer. RNA velocity and Pseudotime algorithms will be used to trace the evolution from normal duct cells to PDAC. Spatial proteomics (Akoya Phenoimager) will be used to validate key IPMN markers.
3. \*\*Identification and validation in plasma\*\* of selected patients using aptamer-based technology, identifying differentially expressed markers in indolent IPMN, invasive IPMN, and PDAC. Molecular analyses will be conducted at the coordinating center (Fondazione Policlinico Gemelli), plasma analyses at Dante Labs, and validation at the coordinating center and the University of Verona.

\*\*AIM2. (observational)\*\*

We will develop knowledge-based tools for diagnosing and treating IPMN and at-risk populations, including:

We will collect clinical data from 3,600 patients and use machine learning to identify markers of malignancy, improving IPMN management.

\*\*AIM3.\*\* The coordinating and Verona centers will validate the role of key genes in IPMN carcinogenesis using Crispr/Cas9 in organotypic IPMN cultures. Pharmacological or genetic targeting will validate genes modulated during carcinogenesis. Candidates will be prioritized based on the literature and available drugs. Identified molecules may serve as new biomarkers for IPMN progression, improving prognosis and therapeutic strategies. We will use models from precancerous lesions and pancreatic tumors to guide patients toward the most appropriate therapies.

Eligibility Criteria:
Inclusion Criteria:

* Diagnosis of indolent IPMN under monitoring;
* Diagnosis of malignant IPMN or resectable pancreatic adenocarcinoma with associated IPMN (previously untreated); Written informed consent; Male and female patients aged 18 years or older;

Exclusion Criteria:

* Inability to provide written informed consent or to trace patients for the retrospective study.