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MicroRNA (miRNA) have been identified in circulating blood and might have the potential to be used as biomarkers for several pathophysiological conditions. To identify miRNA that are altered following stress events, turkeys (Meleagris gallopavo) were subjected to 2 h of road transportation. The expression levels of five circulating miRNA, namely miR-22, miR-155-5p, miR-181a-3p, miR-204 and miR-365-3p, were detected and assessed by quantitative polymerase chain reaction using TaqManR probes, as potential biomarkers of stress. The areas under the receiver operating characteristic curves were then used to evaluate the diagnostic performance of miRNA. A panel of three stress-responsive miRNA, miR-22, miR-155 and miR-365 were identified; their expression levels were significantly higher after road transportation and the area under the curve (AUC) were 0.763, 0.71 and 0.704, respectively. Combining the three miRNA a specificity similar to the one found for the three miRNA separately was found. The AUC of the weighted average of the three miRNA was 0.763. This preliminary study suggests that the expression levels of circulating miR-22, miR-155 and miR-365 are increased during transport-related stress and that they may have diagnostic value to discriminate between stressed- and unstressed animals.

Liver metastases are associated with poor response to current pharmacological treatments, including immunotherapy. We describe a lentiviral vector (LV) platform to selectively engineer liver macrophages, including Kupffer cells and tumor-associated macrophages (TAMs), to deliver type I interferon (IFNα) to liver metastases. Gene-based IFNα delivery delays the growth of colorectal and pancreatic ductal adenocarcinoma liver metastases in mice. Response to IFNα is associated with TAM immune activation, enhanced MHC-II-restricted antigen presentation and reduced exhaustion of CD8 + T cells. Conversely, increased IL-10 signaling, expansion of Eomes CD4 + T cells, a cell type displaying features of type I regulatory T (Tr1) cells, and CTLA-4 expression are associated with resistance to therapy. Targeting regulatory T cell functions by combinatorial CTLA-4 immune checkpoint blockade and IFNα LV delivery expands tumor-reactive T cells, attaining complete response in most mice. These findings support a promising therapeutic strategy with feasible translation to patients with unmet medical need., Competing Interests: Declaration of interests L.N., M.L.S., and T.K. are inventors on a patent on KC-directed gene transfer and L.N. is an inventor on patents on miRNA-regulated LV technology filed and managed by the San Raffaele Scientific Institute and the Telethon Foundation., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Introduction: Despite predicted efficacy, immunotherapy in epithelial ovarian cancer (EOC) has limited clinical benefit and the prognosis of patients remains poor. There is thus a strong need for better identifying local immune dynamics and immune-suppressive pathways limiting T-cell mediated anti-tumor immunity., Methods: In this observational study we analyzed by immunohistochemistry, gene expression profiling and flow cytometry the antigenic landscape and immune composition of 48 EOC specimens, with a focus on tumor-infiltrating lymphocytes (TILs)., Results: Activated T cells showing features of partial exhaustion with a CD137 + CD39 + PD-1 + TIM-3 + CD45RA - CD62L - CD95 + surface profile were exclusively present in EOC specimens but not in corresponding peripheral blood or ascitic fluid, indicating that the tumor microenvironment might sustain this peculiar phenotype. Interestingly, while neoplastic cells expressed several tumor-associated antigens possibly able to stimulate tumor-specific TILs, macrophages provided both co-stimulatory and inhibitory signals and were more abundant in TILs-enriched specimens harboring the CD137 + CD39 + PD-1 + TIM-3 + CD45RA - CD62L - CD95 + signature., Conclusion: These data demonstrate that EOC is enriched in CD137 + CD39 + PD-1 + TIM-3 + CD45RA - CD62L - CD95 + T lymphocytes, a phenotype possibly modulated by antigen recognition on neoplastic cells and by a combination of inhibitory and co-stimulatory signals largely provided by infiltrating myeloid cells. Furthermore, we have identified immunosuppressive pathways potentially hampering local immunity which might be targeted by immunotherapeutic approaches., Competing Interests: CBo: received research support from Intellia Therapeutics and is member of advisory boards/consultant/speaker for Molmed, Intellia, TxCell, Novartis, GSK, Allogene, Kite/Gilead, Miltenyi, Kiadis, QuellTX, Janssen. BS is an employee and shareholder of Intellia Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tassi, Bergamini, Wignall, Sant’Angelo, Brunetto, Balestrieri, Redegalli, Potenza, Abbati, Manfredi, Cangi, Magliacane, Scalisi, Ruggiero, Maffia, Trippitelli, Rabaiotti, Cioffi, Bocciolone, Candotti, Candiani, Taccagni, Schultes, Doglioni, Mangili and Bonini.)

Objective: Colorectal tumours are often densely infiltrated by immune cells that have a role in surveillance and modulation of tumour progression but are burdened by immunosuppressive signals, which might vary from primary to metastatic stages. Here, we deployed a multidimensional approach to unravel the T-cell functional landscape in primary colorectal cancers (CRC) and liver metastases, and genome editing tools to develop CRC-specific engineered T cells., Design: We paired high-dimensional flow cytometry, RNA sequencing and immunohistochemistry to describe the functional phenotype of T cells from healthy and neoplastic tissue of patients with primary and metastatic CRC and we applied lentiviral vectors (LV) and CRISPR/Cas9 genome editing technologies to develop CRC-specific cellular products., Results: We found that T cells are mainly localised at the front edge and that tumor-infiltrating T cells co-express multiple inhibitory receptors, which largely differ from primary to metastatic sites. Our data highlighted CD39 as the major driver of exhaustion in both primary and metastatic colorectal tumours. We thus simultaneously redirected T-cell specificity employing a novel T-cell receptor targeting HER-2 and disrupted the endogenous TCR genes (TCR editing (TCR ED )) and the CD39 encoding gene ( ENTPD1 ), thus generating TCR ED ENTPD1 KO HER-2-redirected lymphocytes. We showed that the absence of CD39 confers to HER-2-specific T cells a functional advantage in eliminating HER-2 + patient-derived organoids in vitro and in vivo ., Conclusion: HER-2-specific CD39 disrupted engineered T cells are promising advanced medicinal products for primary and metastatic CRC., Competing Interests: Competing interests: CBo has been member of Advisory Board and Consultant for Intellia, TxCell, Novartis, GSK, Allogene, Kite/Gilead, Kiadis, Evir, Janssen, Genyo, Epsilen and received research support from Intellia Therapeutics. AP, PDB, GC, ER, BCC and CBo are inventors on different patents on cancer immunotherapy and genetic engineering., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Background & Aims: 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., Methods: 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., Results: 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., Conclusions: 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., (Copyright © 2023 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: The pancreatic microenvironment has a defensive role against cancer but it can acquire tumor-promoting properties triggered by multiple mechanisms including alterations in the equilibrium between proteases and their inhibitors. The identification of proteolytic events, targets and pathways would set the basis for the design of new therapeutic approaches., Methods and Results: Here we demonstrate that spheroids isolated from human and murine healthy pancreas and co-transplanted orthotopically with pancreatic ductal adenocarcinoma (PDAC) in mouse pancreas inhibited tumor growth. The effect was mediated by trypsin-generated fibronectin (FN) fragments released by pancreatic spheroids. Tumor inhibition was observed also in a model of acute pancreatitis associated with trypsin activation. Mass spectrometry proteomic analysis of fragments and mAb against different FN epitopes identified the FN type III domain as responsible for the activity. By inhibiting integrin α5β1, FAK and FGFR1 signaling, the fragments induced tumor cell detachment and reduced cell proliferation. Consistent with the mutual relationship between the two pathways, FGF2 restored both FGFR1 and FAK signaling and promoted PDAC cell adhesion and proliferation. FAK and FGFR inhibitors additively inhibited PDAC growth in vitro and in orthotopic in vivo models., Conclusions: This study identifies a novel role for pancreatic trypsin and fibronectin cleavage as a mechanism of protection against cancer by the pancreatic microenvironment. The finding of a FAK-FGFR cross-talk in PDAC support the combination of FAK and FGFR inhibitors for PDAC treatment to emulate the protective effect of the normal pancreas against cancer., (© 2023. Italian National Cancer Institute ‘Regina Elena’.)

Perineural invasion (PNI) is a common feature in pancreatic ductal adenocarcinoma (PDAC) and correlates with an aggressive tumor behavior already at early stages of disease. PNI is currently considered as a "present vs. absent" feature, and a severity score system has not yet been established. The aim of the present study was thus to develop and validate a score system for PNI and to correlate it with other prognostic features. In this monocentric retrospective study, 356 consecutive PDAC patients (61.8% upfront surgery patients, 38.2% received neoadjuvant therapy) were analyzed. PNI was scored as follows: 0: absent; 1: the presence of neoplasia along nerves < 3 mm in caliber; and 2: neoplastic infiltration of nerve fibers ≥ 3 mm and/or massive perineural infiltration and/or the presence of necrosis of the infiltrated nerve bundle. For every PNI grade, the correlation with other pathological features, disease-free survival (DFS), and disease-specific survival (DSS) were analyzed. Uni- and multivariate analysis for DFS and DSS were also performed. PNI was found in 72.5% of the patients. Relevant trends between PNI score and tumor differentiation grade, lymph node metastases, vascular invasion, and surgical margins status were found. The latter was the only parameter statistically correlated with the proposed score. The agreement between pathologists was substantial (Cohen's K 0.61). PNI severity score significantly correlated also with decreased DFS and DSS at univariate analysis (p < 0.001). At multivariate analysis, only the presence of lymph node metastases was an independent predictor of DFS (HR 2.235 p < 0.001). Lymph node metastases (HR 2.902, p < 0.001) and tumor differentiation grade (HR 1.677, p = 0.002) were independent predictors of DSS. Our newly developed PNI score correlates with other features of PDAC aggressiveness and proved to have a prognostic role though less robust than lymph nodes metastases and tumor differentiation grade. A prospective validation is needed., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The ability to identify the broadest range of targetable gene fusions is crucial to facilitate personalized therapy selection for advanced lung adenocarcinoma (LuADs) patients harboring targetable receptor tyrosine kinase (RTK) genomic alterations. In order to evaluate the most effective testing approach for LuAD targetable gene fusion detection, we analyzed 210 NSCLC selected clinical samples, comparing in situ (Fluorescence In Situ Hybridization, FISH, and ImmunoHistoChemistry, IHC) and molecular (targeted RNA Next-Generation Sequencing, NGS, and RealTime-PCR, RT-PCR) approaches. The overall concordance among these methods was high (>90%), and targeted RNA NGS was confirmed to be the most efficient technique for gene fusion identification in clinical practice, allowing the simultaneous analysis of a large set of genomic rearrangements at the RNA level. However, we observed that FISH was useful to detect targetable fusions in those samples with inadequate tissue material for molecular testing as well as in those few cases whose fusions were not identified by the RNA NGS panel. We conclude that the targeted RNA NGS analysis of LuADs allows accurate RTK fusion detection; nevertheless, standard methods such as FISH should not be dismissed, as they can crucially contribute to the completion of the molecular characterization of LuADs and, most importantly, the identification of patients as candidates for targeted therapies.

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 < 0.001). In particular, the distinction between pancreatic parenchyma and/or peripancreatic soft tissue invasion versus adjacent organs and/or major vessels invasion was the most powerful predictor of recurrence after surgery at multivariate analysis (pattern 2 vs. pattern 1: HR 10.136, p = 0.028; pattern 3 vs. pattern 1: HR 15.775, p = 0.015). The infiltrative growth pattern could therefore provide additional prognostic information implementing the current grading and staging system., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

Background: Limited information is available on the relevant prognostic variables after surgery for patients with pancreatic ductal adenocarcinoma (PDAC) subjected to neoadjuvant chemotherapy (NACT). NACT is known to induce a spectrum of histological changes in PDAC. Different grading regression systems are currently available; unfortunately, they lack precision and accuracy. We aimed to identify a new quantitative prognostic index based on tumor morphology., Patients and Methods: The study population was composed of 69 patients with resectable or borderline resectable PDAC treated with preoperative NACT (neoadjuvant group) and 36 patients submitted to upfront surgery (upfront-surgery group). A comprehensive histological assessment on hematoxylin and eosin (H&E) stained sections evaluated 20 morphological parameters. The association between patient survival and morphological variables was evaluated to generate a prognostic index., Results: The distribution of morphological parameters evaluated was significantly different between upfront-surgery and neoadjuvant groups, demonstrating the effect of NACT on tumor morphology. On multivariate analysis for patients that received NACT, the predictors of shorter overall survival (OS) and disease-free survival (DFS) were perineural invasion and lymph node ratio. Conversely, high stroma to neoplasia ratio predicted longer OS and DFS. These variables were combined to generate a semiquantitative prognostic index based on both OS and DFS, which significantly distinguished patients with poor outcomes from those with a good outcome. Bootstrap analysis confirmed the reproducibility of the model., Conclusions: The pathologic prognostic index proposed is mostly quantitative in nature, easy to use, and may represent a reliable tumor regression grading system to predict patient outcomes after NACT followed by surgery for PDAC., (© 2022. The Author(s).)

Transcriptome analyses allow the distinguishing of pancreatic ductal adenocarcinoma (PDAC) subtypes, exhibiting different prognoses and chemotherapy responses. However, RNA extraction from pancreatic tissue is cumbersome and has been performed mainly from surgical samples, which are representative of < 20% of cases. The majority of PDAC patients undergo endoscopic ultrasound (EUS)-guided tissue acquisition (EUS-TA), but RNA has been rarely extracted from EUS-TA with scanty results. Herein, we aimed to determine the best conditions for RNA extraction and analysis from PDAC EUS-TA samples in order to carry out molecular analyses. PDAC cases underwent diagnostic EUS-TA, with needles being a 25G fine needle aspiration (FNA) in all patients and then either a 20G lateral core-trap fine needle biopsy (FNB) or a 25G Franseen FNB; the conservation methods were either snap freezing, RNALater or Trizol. RNA concentration and quality (RNA integrity index; RIN) were analyzed and a panel of genes was investigated for tissue contamination and markers of molecular subtype and aggressivity through qRT-PCR. Seventy-four samples from 37 patients were collected. The median RNA concentration was significantly higher in Trizol samples (10.33 ng/uL) compared with snap frozen (0.64 ng/uL; p < 0.0001) and RNALater (0.19 ng/uL; p < 0.0001). The RIN was similar between Trizol (5.15) and snap frozen samples (5.85), while for both methods it was higher compared with RNALater (2.7). Among the needles, no substantial difference was seen in terms of RNA concentration and quality. qRT-PCR analyses revealed that samples from all needles were suitable for the detection of PDAC subtype markers (GATA6 and ZEB1) and splice variants associated with mutational status (GAP17) as well as for the detection of contaminating tissue around PDAC cells. This is the first study that specifically investigates the best methodology for RNA extraction from EUS-TA. A higher amount of good quality RNA is obtainable with conservation in Trizol with a clear superiority of neither FNA nor FNB needles. RNA samples from EUS-TA are suitable for transcriptome analysis including the investigation of molecular subtype and splice variants expression.

MicroRNA (miRNA) have been identified in circulating blood and might have the potential to be used as biomarkers for several pathophysiological conditions. To identify miRNA that are altered following stress events, turkeys (Meleagris gallopavo) were subjected to 2 h of road transportation. The expression levels of five circulating miRNA, namely miR-22, miR-155-5p, miR-181a-3p, miR-204 and miR-365-3p, were detected and assessed by quantitative polymerase chain reaction using TaqMan® probes, as potential biomarkers of stress. The areas under the receiver operating characteristic curves were then used to evaluate the diagnostic performance of miRNA. A panel of three stress-responsive miRNA, miR-22, miR-155 and miR-365 were identified; their expression levels were significantly higher after road transportation and the area under the curve (AUC) were 0.763, 0.71 and 0.704, respectively. Combining the three miRNA a specificity similar to the one found for the three miRNA separately was found. The AUC of the weighted average of the three miRNA was 0.763. This preliminary study suggests that the expression levels of circulating miR-22, miR-155 and miR-365 are increased during transport-related stress and that they may have diagnostic value to discriminate between stressed- and unstressed animals.