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Abstract In PDAC patients, ctDNA detection’s prognostic significance needs validation especially in resected patients. This study investigated ctDNA kinetics in portal and peripheral blood before and after resection, and whether tissue mobilization during surgery influences ctDNA detection. In this single-center prospective cohort, portal and peripheral blood were drawn during pancreaticoduodenectomy before and after tissue mobilization, during 12 postoperative months and were associated with overall survival (OS), recurrence-free survival (RFS) and CA19-9 (secondary endpoints). Tumor mutations were identified using next-generation-sequencing and ctDNA detected by digital droplet PCR. From 2018 to 2022, 34 patients were included. The 2-year RFS and OS were 47.6%(95%CI[29.5; 63.6]) and 65.7%(95%CI[46.5; 79.4]) respectively. Intraoperatively, ctDNA detection in portal or peripheral blood was associated with worse RFS (HR[95%CI]3.26[1.26; 8.45],p = 0.010) and OS (HR[95%CI]5.46[1.65;18.01],p = 0.002). Portal vein sampling did not improve ctDNA detection. CtDNA levels were increased by 2.5-fold (p = 0.031) in peripheral blood after tissue mobilization but not significantly linked to RFS or OS. Detecting ctDNA intraoperatively was correlated with poorer RFS (HR [95% CI] 3.26 [1.26;8.45], p = 0.010) and 0S (HR [95% CI] 5.46 [1.65;18.01], p = 0.002). Portal vein sampling did not improve ctDNA detection. Tissue mobilization increases ctDNA levels. Intraoperative detection of ctDNA is associated with a worse prognosis.

Abstract Cytidine deaminase (CDA) converts cytidine and deoxycytidine into uridine and deoxyuridine as part of the pyrimidine salvage pathway. Elevated levels of CDA are found in pancreatic tumors and associated with chemoresistance. Recent evidence suggests that CDA has additional functions in cancer cell biology. In this work, we uncover a novel role of CDA in pancreatic cancer cell metabolism. CDA silencing impairs mitochondrial metabolite production, respiration, and ATP production in pancreatic cancer cells, leading to a so-called Pasteur effect metabolic shift towards glycolysis. Conversely, we find that CDA expression promotes mitochondrial biogenesis and oxidative phosphorylation, independently of CDA deaminase activity. Furthermore, we observe that patient primary cells overexpressing CDA are more sensitive to mitochondria-targeting drugs. Collectively, this work shows that CDA plays a non-canonical role in pancreatic cancer biology by promoting mitochondrial function, which could be translated into novel therapeutic vulnerabilities.

Summary: Here, we provide a protocol for the identification of E3 ubiquitin ligases that are functional when implemented as biodegraders using a cell-based screening assay. We describe steps for establishing a stable cell line expressing a GFP-tagged protein of interest (POI), preparing a sub-library of E3 ligases to screen, and performing the cell-based screening. This protocol can be broadly applied to identify any functional E3 ligase in a biodegrader setting.For complete details on the use and execution of this protocol, please refer to Cornebois et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Background: We apply endoscopic ultrasound-guided fine needle aspiration biopsy to cytopathologically diagnose and sample nucleic acids from primary tumours regardless of the disease stage. Methods: 397 patients with proven pancreatic adenocarcinoma were included and followed up in a multicentre prospective study. DNA and mRNA were extracted from materials of primary tumours obtained by endoscopic ultrasound-guided fine needle aspiration biopsy and analysed using targeted deep sequencing and RNAseq respectively. Findings: The variant allele frequency of the KRAS mutation was used to evaluate the tumour cellularity, ranging from 15 to 20% in all cells, regardless of the tumour stage. The molecular profile of metastatic primary tumours significantly differed from other types of tumours, more frequently having TP53 mutations (p = 0.0002), less frequently having RNF43 mutations, and possessing more basal-like mRNA component (p = 0.001). Molecular markers associated with improved overall survival were: mutations in homologous recombination deficiency genes in patients who received first-line platinum-based chemotherapy (p = 0.025) and wild-type TP53 gene in patients with locally advanced tumours who received radio-chemotherapy (p = 0.01). The GemPred transcriptomic profile was associated with a significantly better overall survival in patients with locally advanced or metastatic pancreatic cancer who received a gemcitabine-based first-line treatment (p = 0.019). Interpretation: The combination of genomic and transcriptomic analyses of primary pancreatic tumours enables us to distinguish metastatic tumours from other tumour types. Our molecular strategy may assist in predicting overall survival outcomes for platinum or gemcitabine-based chemotherapies, as well as radio-chemotherapy. Funding: Institut National Du Cancer (BCB INCa_7294), CHU of Toulouse, Inserm and Ligue Nationale Contre le Cancer (CIT program).

Summary: TDP1 removes transcription-blocking topoisomerase I cleavage complexes (TOP1ccs), and its inactivating H493R mutation causes the neurodegenerative syndrome SCAN1. However, the molecular mechanism underlying the SCAN1 phenotype is unclear. Here, we generate human SCAN1 cell models using CRISPR-Cas9 and show that they accumulate TOP1ccs along with changes in gene expression and genomic distribution of R-loops. SCAN1 cells also accumulate transcriptional DNA double-strand breaks (DSBs) specifically in the G1 cell population due to increased DSB formation and lack of repair, both resulting from abortive removal of transcription-blocking TOP1ccs. Deficient TDP1 activity causes increased DSB production, and the presence of mutated TDP1 protein hampers DSB repair by a TDP2-dependent backup pathway. This study provides powerful models to study TDP1 functions under physiological and pathological conditions and unravels that a gain of function of the mutated TDP1 protein, which prevents DSB repair, rather than a loss of TDP1 activity itself, could contribute to SCAN1 pathogenesis.

Pancreatic cancer will soon become the second cause of death by cancer in Western countries. The main barrier to increase the survival of patients with this disease requires the development of novel and efficient therapeutic strategies that better consider tumor biology. In this context, oncolytic viruses emerge as promising therapeutics. Among them, the fibrotropic minute virus of mice prototype (MVMp) preferentially infects migrating and undifferentiated cells that highly resemble poorly differentiated, basal-like pancreatic tumors showing the worst clinical outcome. We report here that MVMp specifically infects, replicates in, and kills pancreatic cancer cells from murine and human origin with a mesenchymal, basal-like profile, while sparing cancer cells with an epithelial phenotype. Remarkably, MVMp infection, at a dose that does not provoke tumor growth inhibition in athymic mice, shows significant antitumoral effect in immune-competent models; extended mouse survival; and promoted the massive infiltration of tumors by innate, myeloid, and cytotoxic T cells that exhibit a less terminally exhausted phenotype. Collectively, we demonstrate herein for the first time that MVMp is specific and oncolytic for pancreatic tumors with mesenchymal, basal-like profile, paving the way for precision-medicine opportunities for the management of the most aggressive and lethal form of this disease.

Pancreatic cancer remains one of the greatest challenges in oncology for which therapeutic intervention is urgently needed. We previously demonstrated that the intra-tumoral gene transfer of somatostatin receptor 2, to combat tumor aggressiveness, or of deoxycytidine kinase and uridylate monophosphate kinase, to sensitize to gemcitabine chemotherapy, has anti-tumoral potential in experimental models of cancer. Here, we describe the development of the CYL-02 non-viral gene therapy product that comprises a DNA-plasmid encoding for the three aforementioned genes, which expression is targeted to tumor cells, and complexed with polyethyleneimine non-viral vector. We performed pre-clinical toxicology, bio-distribution, and therapeutic activity studies of CYL-02 in two rodent models of pancreatic cancer. We found that CYL-02 is safe, does not increase gemcitabine toxicity, is rapidly cleared from blood following intravenous administration, and sequestered in tumors following intra-tumoral injection. CYL-02 drives the expression of therapeutic genes in cancer cells and strongly sensitizes tumor cells to gemcitabine, both in vitro and in vivo, with significant inhibition of tumor cells dissemination. This study was instrumental for the later use of CYL-02 in patients with advanced pancreatic cancer, demonstrating that rigorous and thorough preclinical investigations are informative for the clinical transfer of gene therapies against this disease.

Abstract Pancreatic ductal adenocarcinoma (PDAC) patients frequently suffer from undetected micro‐metastatic disease. This clinical situation would greatly benefit from additional investigation. Therefore, we set out to identify key signalling events that drive metastatic evolution from the pancreas. We searched for a gene signature that discriminate localised PDAC from confirmed metastatic PDAC and devised a preclinical protocol using circulating cell‐free DNA (cfDNA) as an early biomarker of micro‐metastatic disease to validate the identification of key signalling events. An unbiased approach identified, amongst actionable markers of disease progression, the PI3K pathway and a distinctive PI3Kα activation signature as predictive of PDAC aggressiveness and prognosis. Pharmacological or tumour‐restricted genetic PI3Kα‐selective inhibition prevented macro‐metastatic evolution by hindering tumoural cell migratory behaviour independently of genetic alterations. We found that PI3Kα inhibition altered the quantity and the species composition of the produced lipid second messenger PIP3, with a selective decrease of C36:2 PI‐3,4,5‐P3. Tumoural PI3Kα inactivation prevented the accumulation of pro‐tumoural CD206‐positive macrophages in the tumour‐adjacent tissue. Tumour cell‐intrinsic PI3Kα promotes pro‐metastatic features that could be pharmacologically targeted to delay macro‐metastatic evolution.

MicroRNAs from the miR-200 family are commonly associated with the inhibition of the metastatic potential of cancer cells, following inhibition of ZEB transcription factors expression and epithelial-to-mesenchymal transition. However, previous studies performed in pancreatic adenocarcinoma revealed a more complex picture challenging this canonical model. To gain better insights into the role of miR-200 family members in this disease, we analyzed the expression of miR-200a, miR-200b, miR-200c, miR-141, miR-429, and miR-205, and ZEB1, ZEB2, and CDH1 in pancreatic tumors and matching normal adjacent parenchyma and patient-derived xenografts. We found that miR-200a, miR-429, and miR-205 are frequently overexpressed in pancreatic tumors, whereas CDH1 is downregulated, and ZEB1 and ZEB2 levels remain unchanged. Furthermore, we measured a positive correlation between miR-200 family members and CDH1 expression, and a negative correlation between ZEB1 and miR-200c, miR-141, and miR-205 expression, respectively. Interestingly, we identified significant changes in expression of epithelial-to-mesenchymal transition regulators and miR-200 members in patient-derived xenografts. Lastly, functional studies revealed that miR-141 and miR-429 inhibit the tumorigenic potential of pancreatic cancer cells. Taken together, this comprehensive analysis strongly suggests that miRNAs from the miR-200 family, and in particular miR-429, may act as a tumor suppressor gene in pancreatic cancer. Keywords: pancreatic cancer, patient-derived xenograft, miR-200 family, miR-429

Pancreatic cancer is an aggressive cancer with a dismal prognosis. This is due to the difficulty to detect the disease at an early and curable stage. In addition, only limited treatment options are available, and they are confronted by mechanisms of resistance. Monoclonal antibody (mAb) molecules are highly specific biologics that can be directly used as a blocking agent or modified to deliver a drug payload depending on the desired outcome. They are widely used to target extracellular proteins, but they can also be employed to inhibit intracellular proteins, such as oncoproteins. While mAbs are a class of therapeutics that have been successfully employed to treat many cancers, they have shown only limited efficacy in pancreatic cancer as a monotherapy so far. In this review, we will discuss the challenges, opportunities and hopes to use mAbs for pancreatic cancer treatment, diagnostics and imagery.

Genetic testing based on the analysis of circulating cell-free DNA (cfDNA) in body fluids, especially blood plasma, is raising interest for the management and follow-up of many diseases, including cancer. Because the concentration of cfDNA is low and its composition mostly degraded, this material can only be assayed with the most sensitive nucleic acid processing technologies. cfDNA analysis therefore constitutes a model target and a driving force for innovation in microfluidic biotechnologies. Here, we overview the main physico-chemical characteristics of cfDNA, and provide a critical review on the different methods for its processing out of blood samples. Then, we describe recent microfluidic developments for high sensitivity DNA analysis, evaluate their practical relevance for cfDNA analysis, and identify a few challenges for technologists in the near future. Keywords: Cell-free circulating DNA, Body fluid nucleic acid biomarkers, DNA separation, DNA concentration

With the expansion of the microbiology field of research, a new genome editing tool arises from the biology of bacteria that holds the promise of achieving precise modifications in the genome with a simplicity and versatility that surpasses previous genome editing methods. This new technique, commonly named CRISPR/Cas9, led to a rapid expansion of the biomedical field; more specifically, cancer characterization and modeling have benefitted greatly from the genome editing capabilities of CRISPR/Cas9. In this paper, we briefly summarize recent improvements in CRISPR/Cas9 design meant to overcome the limitations that have arisen from the nuclease activity of Cas9 and the influence of this technology in cancer research. In addition, we present challenges that might impede the clinical applicability of CRISPR/Cas9 for cancer therapy and highlight future directions for designing CRISPR/Cas9 delivery systems that might prove useful for cancer therapeutics.

In the era of precision medicine, the success of clinical trials, notably for patients diagnosed with cancer, strongly relies on biomarkers with pristine clinical value but also on robust and versatile analytical technologies to ensure proper patients' stratification and treatment. In this review, we will first address whether plasmatic and salivary microRNAs can be considered as a reliable source of biomarkers for cancer diagnosis and prognosis. We will then discuss the pre-analytical steps preceding miRNA quantification (from isolation to purification), and how such process could be biased and time-consuming. Next, we will review the most recent tools derived from micro- and nano-technologies for microRNA detection available to date and how they may compete with current standards. This review will prioritize publications using relevant biological samples. The significance of various physical transduction schemes (mechanical, optical, electrical, etc.) for biological detection will be compared, and pros and cons of each method will be widely discussed. Finally, we will debate on how micro and nanotechnologies could widespread the use of biomarkers in modern medicine, to help manage patients with serious diseases such as cancer.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is extremely stroma‐rich. Cancer‐associated fibroblasts (CAFs) secrete proteins that activate survival and promote chemoresistance of cancer cells. Our results demonstrate that CAF secretome‐triggered chemoresistance is abolished upon inhibition of the protein synthesis mTOR/4E‐BP1 regulatory pathway which we found highly activated in primary cultures of α‐SMA‐positive CAFs, isolated from human PDAC resections. CAFs selectively express the sst1 somatostatin receptor. The SOM230 analogue (Pasireotide) activates the sst1 receptor and inhibits the mTOR/4E‐BP1 pathway and the resultant synthesis of secreted proteins including IL‐6. Consequently, tumour growth and chemoresistance in nude mice xenografted with pancreatic cancer cells and CAFs, or with pieces of resected human PDACs, are reduced when chemotherapy (gemcitabine) is combined with SOM230 treatment. While gemcitabine alone has marginal effects, SOM230 is permissive to gemcitabine‐induced cancer cell apoptosis and acts as an antifibrotic agent. We propose that selective inhibition of CAF protein synthesis with sst1‐directed pharmacological compounds represents an anti‐stromal‐targeted therapy with promising chemosensitization potential.

Background: KRAS and GNAS mutations are common in intraductal papillary mucinous neoplasia of the pancreas (IPMN). The aims of this study were to assess the role of pre-therapeutic cytopathology combined with KRAS and GNAS mutation assays within cystic fluid sampled by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) to predict malignancy of IPMN. Patients and methods: We prospectively included 37 IPMN patients with clinical and/or imaging predictors of malignancy (men: 24; mean age: 69.5 years). Cytopathology (performed on cystic fluid and/or IPMN nodules), KRAS (Exon 2, codon 12) and GNAS (Exon 8, codon 201) mutations assays (using TaqMan® allelic discrimination) were performed on EUS-FNA material. The final diagnosis was obtained from IPMN resections (n = 18); surgical biopsies, EUS-FNA analyses, and follow-up (n = 19): 10 and 27 IPMN were benign and malignant, respectively. Results: Sensitivity, specificity, positive and negative predictive values, and accuracy of cytopathology alone to diagnose IPMN malignancy were 55 %, 100 %, 100 %, 45 %, and 66 %, respectively. When KRAS-mutation analysis was combined with cytopathology these values were 92 %, 50 %, 83 %, 71 %, and 81 %, respectively. GNAS assays did not improve the performances of cytopathology alone or those of cytopathology plus a KRAS assay. Conclusions: In patients with a likelihood of malignant IPMN at pre-therapeutic investigation, testing for KRAS mutations in cystic fluid sampling by EUS-FNA improved the results of cytopathology for the diagnosis of malignancy whereas GNAS mutation assay did not.

Pancreatic cancer is the fourth leading cause of cancer death in Western countries, with the lowest 1-year survival rate among commonly diagnosed cancers. Reliable biomarkers for pancreatic cancer diagnosis are lacking and are urgently needed to allow for curative surgery. As microRNA (miRNA) recently emerged as candidate biomarkers for this disease, we explored in the present pilot study the differences in salivary microRNA profiles between patients with pancreatic tumors that are not eligible for surgery, precancerous lesions, inflammatory disease or cancer-free patients as a potential early diagnostic tool.Whole saliva samples from patients with pancreatic cancer (n = 7), pancreatitis (n = 4), IPMN (n = 2), or healthy controls (n = 4) were obtained during endoscopic examination. After total RNA isolation, expression of 94 candidate miRNAs was screened by q(RT)PCR using Biomark Fluidgm. Human-derived pancreatic cancer cells were xenografted in athymic mice as an experimental model of pancreatic cancer.We identified hsa-miR-21, hsa-miR-23a, hsa-miR-23b and miR-29c as being significantly upregulated in saliva of pancreatic cancer patients compared to control, showing sensitivities of 71.4%, 85.7%, 85,7% and 57%, respectively and excellent specificity (100%). Interestingly, hsa-miR-23a and hsa-miR23b are overexpressed in the saliva of patients with pancreatic cancer precursor lesions. We found that hsa-miR-210 and let-7c are overexpressed in the saliva of patients with pancreatitis as compared to the control group, with sensitivity of 100% and 75%, and specificity of 100% and 80%, respectively. Last hsa-miR-216 was upregulated in cancer patients as compared to patients diagnosed with pancreatitis, with sensitivity of 50% and specificity of 100%. In experimental models of PDAC, salivary microRNA detection precedes systemic detection of cancer cells markers.Our novel findings indicate that salivary miRNA are discriminatory in pancreatic cancer patients that are not eligible for surgery. In addition, we demonstrate in experimental models that salivary miRNA detection precedes systemic detection of cancer cells markers. This study stems for the use of salivary miRNA as biomarker for the early diagnosis of patients with unresectable pancreatic cancer.

Sporadic early onset colorectal carcinoma (EOCRC) which has by definition no identified hereditary predisposition is a growing problem that remains poorly understood. Molecular analysis could improve identification of distinct sub-types of colorectal cancers (CRC) with therapeutic implications and thus can help establish that sporadic EOCRC is a distinct entity. From 954 patients resected for CRC at our institution, 98 patients were selected. Patients aged 45-60 years were excluded to help define "young" and "old" groups. Thirty-nine cases of sporadic EOCRC (patients ≤ 45 years with microsatellite stable tumors) were compared to both microsatellite stable tumors from older patients (36 cases, patients>60 years) and to groups of patients with microsatellite instability. Each group was tested for TP53, KRAS, BRAF, PIK3CA mutations and the presence of a methylator phenotype. Gene expression profiles were also used for pathway analysis. Compared to microsatellite stable CRC from old patients, sporadic EOCRC were characterized by distal location, frequent synchronous metastases and infrequent synchronous adenomas but did not have specific morphological characteristics. A familial history of CRC was more common in sporadic EOCRC patients despite a lack of identified hereditary conditions (p = 0.013). Genetic studies also showed the absence of BRAF mutations (p = 0.022) and the methylator phenotype (p = 0.005) in sporadic EOCRC compared to older patients. Gene expression analysis implicated key pathways such as Wnt/beta catenin, MAP Kinase, growth factor signaling (EGFR, HGF, PDGF) and the TNFR1 pathway in sporadic EOCRC. Wnt/beta catenin signaling activation was confirmed by aberrant nuclear beta catenin immunostaining (p = 0.01). This study strongly suggests that sporadic EOCRC is a distinct clinico-molecular entity presenting as a distal and aggressive disease associated with chromosome instability. Furthermore, several signaling pathways including the TNFR1 pathway have been identified as potential biomarkers for both the diagnosis and treatment of this disease.

MicroRNAs are small non-coding RNAs that physiologically modulate proteins expression, and regulate numerous cellular mechanisms. Alteration of microRNA expression has been described in cancer and is associated to tumor initiation and progression. The microRNA 148a (miR-148a) is frequently down-regulated in cancer. We previously demonstrated that its down-regulation by DNA hypermethylation is an early event in pancreatic ductal adenocarcinoma (PDAC) carcinogenesis, suggesting a tumor suppressive function. Here, we investigate the potential role of miR-148a over-expression in PDAC as a therapeutic tool. We first report the consequences of miR-148a over-expression in PDAC cell lines. We demonstrate that miR-148a over-expression has no dramatic effect on cell proliferation and cell chemo-sensitivity in four well described PDAC cell lines. We also investigate the modulation of protein expression by a global proteomic approach (2D-DIGE). We show that despite its massive over-expression, miR-148a weakly modulates protein expression, thus preventing the identification of protein targets in PDAC cell lines. More importantly, in vivo data demonstrate that modulating miR-148a expression either in the epithelia tumor cells and/or in the tumor microenvironment does not impede tumor growth. Taken together, we demonstrate herein that miR-148a does not impact PDAC proliferation both in vitro and in vivo thus suggesting a weak potential as a therapeutic tool.

BackgroundNormal tissue homeostasis is maintained by dynamic interactions between epithelial cells and their microenvironment. Disrupting this homeostasis can induce aberrant cell proliferation, adhesion, function and migration that might promote malignant behavior. Indeed, aberrant stromal-epithelial interactions contribute to pancreatic ductal adenocarcinoma (PDAC) spread and metastasis, and this raises the possibility that novel stroma-targeted therapies represent additional approaches for combating this malignant disease. The aim of the present study was to determine the effect of human stromal cells derived from adipose tissue (ADSC) on pancreatic tumor cell proliferation.Principal findingsCo-culturing pancreatic tumor cells with ADSC and ADSC-conditioned medium sampled from different donors inhibited cancer cell viability and proliferation. ADSC-mediated inhibitory effect was further extended to other epithelial cancer-derived cell lines (liver, colon, prostate). ADSC conditioned medium induced cancer cell necrosis following G1-phase arrest, without evidence of apoptosis. In vivo, a single intra-tumoral injection of ADSC in a model of pancreatic adenocarcinoma induced a strong and long-lasting inhibition of tumor growth.ConclusionThese data indicate that ADSC strongly inhibit PDAC proliferation, both in vitro and in vivo and induce tumor cell death by altering cell cycle progression. Therefore, ADSC may constitute a potential cell-based therapeutic alternative for the treatment of PDAC for which no effective cure is available.