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The incidence of well-differentiated non-functioning pancreatic neuroendocrine tumors (NF-PanNET) increased during the last decades. The risk of relapse after curative surgery, albeit low, is not negligible; moreover, adjuvant treatment is currently not an option and a reliable predictive model based on prognostic characteristics is urgently needed for tailoring a follow-up strategy. The histological classification of PanNET now relies only on the proliferative activity (mitosis and Ki67) and staging. In contrast to other endocrine neoplasms, the role of infiltrative growth pattern in NF-PanNET is not taken into consideration at present. In the current study, 247 consecutive patients who underwent surgical resection for a NF-PanNET were examined for the histological growth pattern of the tumor. Two distinct patterns (non-infiltrative vs. infiltrative) were described with the latter being further subclassified according to the type of structures invaded by the tumor (non-infiltrative: pattern 1; infiltration of adjacent pancreatic parenchyma and/or peripancreatic soft tissue: pattern 2; invasion of nearby organs and/or major vessels: pattern 3). The infiltrative growth resulted to be strongly associated with a poorer survival compared to a non-infiltrative growth (p

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease, for which it is crucial to promptly detect actionable and prognostic alterations to drive specific therapeutic decisions, regardless of tumor resectability status. Endoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA) is of key importance for PDAC diagnosis and can contribute significantly to tumor molecular profiling.Comprehensive genomic profile by targeted next-generation sequencing (NGS) was performed on 2 independent PDAC patient cohorts. Cohort 1 consisted of 77 patients with resectable PDAC for whom the histologic sample at the time of resection was available; for 56 patients cytologic specimens at the time of diagnosis also were obtained by EUS-FNA. Cohort 2 consisted of 20 patients with unresectable PDAC, for whom only the EUS-FNA cytologic sample was available.In cohort 1, a complete concordant mutational profile between the cytologic sample at diagnosis and the corresponding histologic specimen after surgery was observed in 88% of the cases, proving the ability to detect potential clinically relevant alterations in cytologic samples by NGS analysis. Notably, clinically actionable mutations were identified in 20% of patients. In cohort 2, comprehensive mutational profiling was obtained successfully for all samples. Consistent with the findings of cohort 1, KRAS, TP53, CDKN2A, and SMAD4 were the most altered genes. Most importantly, 15% of the patients harbored actionable mutations.Our findings show the feasibility of an NGS approach using both surgical specimens and cytologic samples. The model proposed in this study can be included successfully in the clinical setting for comprehensive molecular profiling of all PDAC patients irrespective of their surgical eligibility.

Colorectal cancer (CRC) is the 2nd cause of cancer-related death. Standard therapies often fail, with more than 50% of patients experiencing relapse, eventually with metastatic disease. Colorectal tumors are densely infiltrated by immune cells that have a role in surveillance and modulation of tumor progression. However, exhaustion mechanisms acting within the tumor microenvironment impede their functional capacity against tumor cells. We paired high-dimensional flow cytometry, RNA sequencing, immunohistochemistry and immunofluorescence to describe the T cell functional landscape in tumor and peritumoral tissues from primary colorectal cancers and liver metastases. Analysis of the healthy, peritumoral and neoplastic tissues of treatment-naïve primary CRCs and of the peritumoral and tumoral tissues of CRC patients undergoing surgery for liver metastasis, revealed extensive transcriptional and spatial remodeling across tumors, being metabolic pathways among the major drivers of this variance. Regarding the immune infiltrate, we found that T cells are mainly localized at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors. Unsupervised analysis of flow cytometry data performed by an advanced pipeline of data handling by dimensionality reduction and clustering algorithms allowed the definition of a peculiar inhibitory receptors signature in TILs enriched both in primary CRCs and liver metastases. Among the highly co-expressed inhibitory receptors, CD39 was found to represent the major driver of exhaustion in both primary and metastatic colorectal tumors. CD39 is a diphosphohydrolase converting ATP into AMP that is emerging as exhaustion marker for tumor-specific T cells, thus highlighting its relevance as molecular target for T cells engineering. We leveraged on these findings to generate a novel cellular product for the adoptive cell therapy of CRC. By CRISPR/Cas9 genome editing tools, we simultaneously redirected T cell specificity by disrupting the alpha and beta genes of the endogenous T cell receptor with 90% efficiency for both genes, and disrupted CD39 with 100% efficiency, generating triple-knockout engineered lymphocytes. By lentiviral transduction, we redirected the specificity of our engineered T cell product employing a novel T-cell receptor targeting the HER-2 antigen. Triple-edited, HER2-redirected T cells were challenged in vitro against HER2+ patient-derived organoids from liver metastases (mPDOs). Measurement of Cas3/7 displayed a functional advantage for CD39-disrupted, HER2-redirected T cells in recognizing and killing mPDOs. We then evaluated the efficacy of our T cell product in two different in vivo models: subcutaneous injection of mPDOs, and intra-hepatic injection of mPDOs. In both models, CD39-disrupted, HER2-redirected T cells displayed a superior capacity of controlling tumor outgrowth long term. Citation Format: Alessia Potenza, Chiara Balestrieri, Luca Albarello, Federica Pedica, Martina Spiga, Francesco Manfredi, Beatrice C. Cianciotti, Claudia De Lalla, Lorena Stasi, Elena Tassi, Silvia Bonfiglio, Giulia M. Scotti, Miriam Redegalli, Donatella Biancolini, Danilo Abbati, Fabio Simeoni, Dejan Lazarevic, Ugo Elmore, Guido Fiorentini, Giulia Di Lullo, Giulia Casorati, Claudio Doglioni, Giovanni Tonon, Paolo Dellabona, Riccardo Rosati, Luca Aldrighetti, Eliana Ruggiero, Chiara Bonini. Harnessing CD39 for the treatment of colorectal cancer and liver metastases by engineered T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 902.

Assessment of HRD status is now essential for ovarian cancer patient management. A relevant percentage of high-grade serous carcinoma (HGSC) is characterized by HRD, which is caused by genetic alterations in the homologous recombination repair (HRR) pathway. Recent trials have shown that not only patients with pathogenic/likely pathogenic BRCA variants, but also BRCAwt/HRD patients, are sensitive to PARPis and platinum therapy. The most common HRD test is Myriad MyChoice CDx, but there is a pressing need to offer an alternative to outsourcing analysis, which typically requires high costs and lengthy turnaround times. In order to set up a complete in-house workflow for HRD testing, we analyzed a small cohort of HGSC patients using the CE-IVD AmoyDx HRD Focus Panel and compared our results with Myriad’s. In addition, to further deepen the mechanisms behind HRD, we analyzed the study cohort by using both a custom NGS panel that analyzed 21 HRR-related genes and FISH analysis to determine the copy numbers of PTEN and EMSY. We found complete concordance in HRD status detected by the Amoy and the Myriad assays, supporting the feasibility of internal HRD testing.