Comparison of endoscopic ultrasound-guided fine-needle aspiration and fine-needle biopsy to generate pancreatic cancer organoids: Randomized trial.

Background and study aims The prognosis for pancreatic cancer remains poor. Molecular diagnostics and customized therapies are becoming increasingly important in clinical routine. Patient-derived, predictive model systems such as organoids have the potential to substantially increase the depth of information from biopsy material by functional and molecular characterization. We compared the extent to which the use of fine-needle aspiration needles (FNA, 22G) or fine-needle biopsy needles (FNB, 22G) influences the generation of pancreatic cancer patient-derived organoids (PDOs) to establish endoscopic standards of organoid technology. Patients and methods Endoscopic ultrasound (EUS)-guided punctures by EUS-FNA and EUS-FNB of pancreatic masses highly suspicious for adenocarcinoma (detected by computed tomography and/or magnetic resonance imaging) were prospectively evaluated. Consecutive patients received EUS-FNA and EUS-FNB in a randomized order without the need to exchange the needle shaft (only the inner needle type (FNA/-B) was exchanged) between the passes. With each needle type, the specimens for histological analysis and for PDOs were obtained separately. Results Fifty patients were enrolled in the study. Histology revealed malignancy in 42 of 50 cases (84%). In total PDOs were generated from 17 patients (34%). Of these, nine were established by FNB only, two by FNA only, and six by both FNA and FNB. Histology revealed malignancy in 13 of 17 PDO cases (76%). In two histologically false-negative cases, PDOs could be established. Conclusions EUS-FNB was superior to EUS-FNA in terms of successful generation of PDOs, although it failed to show statistical significance.

An NFATc1/SMAD3/cJUN Complex Restricted to SMAD4-Deficient Pancreatic Cancer Guides Rational Therapies.

BACKGROUND & AIMS: The highly heterogeneous cellular and molecular makeup of pancreatic ductal adenocarcinoma (PDAC) not only fosters exceptionally aggressive tumor biology, but contradicts the current concept of one-size-fits-all therapeutic strategies to combat PDAC. Therefore, we aimed to exploit the tumor biological implication and therapeutic vulnerabilities of a clinically relevant molecular PDAC subgroup characterized by SMAD4 deficiency and high expression of the nuclear factor of activated T cells (SMAD4-/-/NFATc1High). METHODS: Transcriptomic and clinical data were analyzed to determine the prognostic relevance of SMAD4-/-/NFATc1High cancers. In vitro and in vivo oncogenic transcription factor complex formation was studied by immunoprecipitation, proximity ligation assays, and validated cross model and species. The impact of SMAD4 status on therapeutically targeting canonical KRAS signaling was mechanistically deciphered and corroborated by genome-wide gene expression analysis and genetic perturbation experiments, respectively. Validation of a novel tailored therapeutic option was conducted in patient-derived organoids and cells and transgenic as well as orthotopic PDAC models. RESULTS: Our findings determined the tumor biology of an aggressive and chemotherapy-resistant SMAD4-/-/NFATc1High subgroup. Mechanistically, we identify SMAD4 deficiency as a molecular prerequisite for the formation of an oncogenic NFATc1/SMAD3/cJUN transcription factor complex, which drives the expression of RRM1/2. RRM1/2 replenishes nucleoside pools that directly compete with metabolized gemcitabine for DNA strand incorporation. Disassembly of the NFATc1/SMAD3/cJUN complex by mitogen-activated protein kinase signaling inhibition normalizes RRM1/2 expression and synergizes with gemcitabine treatment in vivo to reduce the proliferative index. CONCLUSIONS: Our results suggest that PDAC characterized by SMAD4 deficiency and oncogenic NFATc1/SMAD3/cJUN complex formation exposes sensitivity to a mitogen-activated protein kinase signaling inhibition and gemcitabine combination therapy.

Phenotype screens of murine pancreatic cancer identify a Tgf-α-Ccl2-paxillin axis driving human-like neural invasion.

Solid cancers like pancreatic ductal adenocarcinoma (PDAC), a type of pancreatic cancer, frequently exploit nerves for rapid dissemination. This neural invasion (NI) is an independent prognostic factor in PDAC, but insufficiently modeled in genetically engineered mouse models (GEMM) of PDAC. Here, we systematically screened for human-like NI in Europe's largest repository of GEMM of PDAC, comprising 295 different genotypes. This phenotype screen uncovered 2 GEMMs of PDAC with human-like NI, which are both characterized by pancreas-specific overexpression of transforming growth factor α (TGF-α) and conditional depletion of p53. Mechanistically, cancer-cell-derived TGF-α upregulated CCL2 secretion from sensory neurons, which induced hyperphosphorylation of the cytoskeletal protein paxillin via CCR4 on cancer cells. This activated the cancer migration machinery and filopodia formation toward neurons. Disrupting CCR4 or paxillin activity limited NI and dampened tumor size and tumor innervation. In human PDAC, phospho-paxillin and TGF-α-expression constituted strong prognostic factors. Therefore, we believe that the TGF-α-CCL2-CCR4-p-paxillin axis is a clinically actionable target for constraining NI and tumor progression in PDAC.

Fulminant Liver Failure after Treatment with a Checkpoint Inhibitor for Gastric Cancer: A Case Report and Review of the Literature.

Nivolumab is a promising monoclonal antibody inhibitor of programmed death-1, a protein on the surface of T-cells. As such, it is approved for use in patients with multiple advanced malignancies and can significantly elongate progression-free survival. However, monoclonal antibody inhibitors can lead to adverse hepatic reactions, which in rare cases result in further hepatic damage. Herein, we present a case of a patient with locally advanced gastric carcinoma treated with fluorouracil, oxaliplatin, docetaxel and the checkpoint inhibitor nivolumab. Five months after her first dosage of nivolumab and without a preexisting liver disease, she presented with transaminitis. During the course of her stay, the patient developed status epilepticus, which required mechanical ventilation followed by fulminant hepatic failure. A subsequent liver biopsy revealed severe liver damage with extensive confluent parenchymal necrosis corresponding to checkpoint-inhibitor-induced hepatitis. Alternative reasons for this hepatic failure were ruled out. Despite aggressive therapeutic interventions including corticosteroids and plasma exchange, the patient died due to liver failure. Although hepatic failure is rarely seen in patients with checkpoint inhibitor therapy, it requires early awareness and rapid intervention.

Resistance to mesenchymal reprogramming sustains clonal propagation in metastatic breast cancer.

The acquisition of mesenchymal traits is considered a hallmark of breast cancer progression. However, the functional relevance of epithelial-to-mesenchymal transition (EMT) remains controversial and context dependent. Here, we isolate epithelial and mesenchymal populations from human breast cancer metastatic biopsies and assess their functional potential in vivo. Strikingly, progressively decreasing epithelial cell adhesion molecule (EPCAM) levels correlate with declining disease propagation. Mechanistically, we find that persistent EPCAM expression marks epithelial clones that resist EMT induction and propagate competitively. In contrast, loss of EPCAM defines clones arrested in a mesenchymal state, with concomitant suppression of tumorigenicity and metastatic potential. This dichotomy results from distinct clonal trajectories impacting global epigenetic programs that are determined by the interplay between human ZEB1 and its target GRHL2. Collectively, our results indicate that susceptibility to irreversible EMT restrains clonal propagation, whereas resistance to mesenchymal reprogramming sustains disease spread in multiple models of human metastatic breast cancer, including patient-derived cells in vivo.

Accurate prediction of histological grading of intraductal papillary mucinous neoplasia using deep learning.

BACKGROUND: Risk stratification and recommendation for surgery for intraductal papillary mucinous neoplasm (IPMN) are currently based on consensus guidelines. Risk stratification from presurgery histology is only potentially decisive owing to the low sensitivity of fine-needle aspiration. In this study, we developed and validated a deep learning-based method to distinguish between IPMN with low grade dysplasia and IPMN with high grade dysplasia/invasive carcinoma using endoscopic ultrasound (EUS) images. METHODS: For model training, we acquired a total of 3355 EUS images from 43 patients who underwent pancreatectomy from March 2015 to August 2021. All patients had histologically proven IPMN. We used transfer learning to fine-tune a convolutional neural network and to classify "low grade IPMN" from "high grade IPMN/invasive carcinoma." Our test set consisted of 1823 images from 27 patients, recruiting 11 patients retrospectively, 7 patients prospectively, and 9 patients externally. We compared our results with the prediction based on international consensus guidelines. RESULTS: Our approach could classify low grade from high grade/invasive carcinoma in the test set with an accuracy of 99.6 % (95 %CI 99.5 %-99.9 %). Our deep learning model achieved superior accuracy in prediction of the histological outcome compared with any individual guideline, which have accuracies between 51.8 % (95 %CI 31.9 %-71.3 %) and 70.4 % (95 %CI 49.8-86.2). CONCLUSION: This pilot study demonstrated that deep learning in IPMN-EUS images can predict the histological outcome with high accuracy.

AP1/Fra1 confers resistance to MAPK cascade inhibition in pancreatic cancer.

Targeting KRAS downstream signaling remains an important therapeutic approach in pancreatic cancer. We used primary pancreatic ductal epithelial cells and mouse models allowing the conditional expression of oncogenic KrasG12D, to investigate KRAS signaling integrators. We observed that the AP1 family member FRA1 is tightly linked to the KRAS signal and expressed in pre-malignant lesions and the basal-like subtype of pancreatic cancer. However, genetic-loss-of-function experiments revealed that FRA1 is dispensable for KrasG12D-induced pancreatic cancer development in mice. Using FRA1 gain- and loss-of-function models in an unbiased drug screen, we observed that FRA1 is a modulator of the responsiveness of pancreatic cancer to inhibitors of the RAF-MEK-ERK cascade. Mechanistically, context-dependent FRA1-associated adaptive rewiring of oncogenic ERK signaling was observed and correlated with sensitivity to inhibitors of canonical KRAS signaling. Furthermore, pharmacological-induced degradation of FRA1 synergizes with MEK inhibitors. Our studies establish FRA1 as a part of the molecular machinery controlling sensitivity to MAPK cascade inhibition allowing the development of mechanism-based therapies.

Epigenetic drug screening defines a PRMT5 inhibitor-sensitive pancreatic cancer subtype.

Systemic therapies for pancreatic ductal adenocarcinoma (PDAC) remain unsatisfactory. Clinical prognosis is particularly poor for tumor subtypes with activating aberrations in the MYC pathway, creating an urgent need for novel therapeutic targets. To unbiasedly find MYC-associated epigenetic dependencies, we conducted a drug screen in pancreatic cancer cell lines. Here, we found that protein arginine N-methyltransferase 5 (PRMT5) inhibitors triggered an MYC-associated dependency. In human and murine PDACs, a robust connection of MYC and PRMT5 was detected. By the use of gain- and loss-of-function models, we confirmed the increased efficacy of PRMT5 inhibitors in MYC-deregulated PDACs. Although inhibition of PRMT5 was inducing DNA damage and arresting PDAC cells in the G2/M phase of the cell cycle, apoptotic cell death was executed predominantly in cells with high MYC expression. Experiments in primary patient-derived PDAC models demonstrated the existence of a highly PRMT5 inhibitor-sensitive subtype. Our work suggests developing PRMT5 inhibitor-based therapies for PDAC.

Detecting drug resistance in pancreatic cancer organoids guides optimized chemotherapy treatment.

Drug combination therapies for cancer treatment show high efficacy but often induce severe side effects, resulting in dose or cycle number reduction. We investigated the impact of neoadjuvant chemotherapy (neoCTx) adaptions on treatment outcome in 59 patients with pancreatic ductal adenocarcinoma (PDAC). Resections with tumor-free margins were significantly more frequent when full-dose neoCTx was applied. We determined if patient-derived organoids (PDOs) can be used to personalize poly-chemotherapy regimens by pharmacotyping of treatment-naïve and post-neoCTx PDAC PDOs. Five out of ten CTx-naïve PDO lines exhibited a differential response to either the FOLFIRINOX or the Gem/Pac regimen. NeoCTx PDOs showed a poor response to the neoadjuvant regimen that had been administered to the respective patient in 30% of cases. No significant difference in PDO response was noted when comparing modified treatments in which the least effective single drug was removed from the complete regimen. Drug testing of CTx-naïve PDAC PDOs and neoCTx PDOs may be useful to guide neoadjuvant and adjuvant regimen selection, respectively. Personalizing poly-chemotherapy regimens by omitting substances with low efficacy could potentially result in less severe side effects, thereby increasing the fraction of patients receiving a full course of neoadjuvant treatment. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

CRISPR somatic genome engineering and cancer modeling in the mouse pancreas and liver.

Genetically engineered mouse models (GEMMs) transformed the study of organismal disease phenotypes but are limited by their lengthy generation in embryonic stem cells. Here, we describe methods for rapid and scalable genome engineering in somatic cells of the liver and pancreas through delivery of CRISPR components into living mice. We introduce the spectrum of genetic tools, delineate viral and nonviral CRISPR delivery strategies and describe a series of applications, ranging from gene editing and cancer modeling to chromosome engineering or CRISPR multiplexing and its spatio-temporal control. Beyond experimental design and execution, the protocol describes quantification of genetic and functional editing outcomes, including sequencing approaches, data analysis and interpretation. Compared to traditional knockout mice, somatic GEMMs face an increased risk for mouse-to-mouse variability because of the higher experimental demands of the procedures. The robust protocols described here will help unleash the full potential of somatic genome manipulation. Depending on the delivery method and envisaged application, the protocol takes 3-5 weeks.

Identification of treatment-induced vulnerabilities in pancreatic cancer patients using functional model systems.

Despite the advance and success of precision oncology in gastrointestinal cancers, the frequency of molecular-informed therapy decisions in pancreatic ductal adenocarcinoma (PDAC) is currently neglectable. We present a longitudinal precision oncology platform based on functional model systems, including patient-derived organoids, to identify chemotherapy-induced vulnerabilities. We demonstrate that treatment-induced tumor cell plasticity in vivo distinctly changes responsiveness to targeted therapies, without the presence of a selectable genetic marker, indicating that tumor cell plasticity can be functionalized. By adding a mechanistic layer to precision oncology, adaptive processes of tumors under therapy can be exploited, particularly in highly plastic tumors, such as pancreatic cancer.

Selective multi-kinase inhibition sensitizes mesenchymal pancreatic cancer to immune checkpoint blockade by remodeling the tumor microenvironment.

KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) is highly immunosuppressive and resistant to targeted and immunotherapies. Among the different PDAC subtypes, basal-like mesenchymal PDAC, which is driven by allelic imbalance, increased gene dosage and subsequent high expression levels of oncogenic KRAS, shows the most aggressive phenotype and strongest therapy resistance. In the present study, we performed a systematic high-throughput combination drug screen and identified a synergistic interaction between the MEK inhibitor trametinib and the multi-kinase inhibitor nintedanib, which targets KRAS-directed oncogenic signaling in mesenchymal PDAC. This combination treatment induces cell-cycle arrest and cell death, and initiates a context-dependent remodeling of the immunosuppressive cancer cell secretome. Using a combination of single-cell RNA-sequencing, CRISPR screens and immunophenotyping, we show that this combination therapy promotes intratumor infiltration of cytotoxic and effector T cells, which sensitizes mesenchymal PDAC to PD-L1 immune checkpoint inhibition. Overall, our results open new avenues to target this aggressive and therapy-refractory mesenchymal PDAC subtype.

HDAC2 Facilitates Pancreatic Cancer Metastasis.

The mortality of patients with pancreatic ductal adenocarcinoma (PDAC) is strongly associated with metastasis, a multistep process that is incompletely understood in this disease. Although genetic drivers of PDAC metastasis have not been defined, transcriptional and epigenetic rewiring can contribute to the metastatic process. The epigenetic eraser histone deacetylase 2 (HDAC2) has been connected to less differentiated PDAC, but the function of HDAC2 in PDAC has not been comprehensively evaluated. Using genetically defined models, we show that HDAC2 is a cellular fitness factor that controls cell cycle in vitro and metastasis in vivo, particularly in undifferentiated, mesenchymal PDAC cells. Unbiased expression profiling detected a core set of HDAC2-regulated genes. HDAC2 controlled expression of several prosurvival receptor tyrosine kinases connected to mesenchymal PDAC, including PDGFRα, PDGFRß, and EGFR. The HDAC2-maintained program disabled the tumor-suppressive arm of the TGFß pathway, explaining impaired metastasis formation of HDAC2-deficient PDAC. These data identify HDAC2 as a tractable player in the PDAC metastatic cascade. The complexity of the function of epigenetic regulators like HDAC2 implicates that an increased understanding of these proteins is needed for implementation of effective epigenetic therapies. SIGNIFICANCE: HDAC2 has a context-specific role in undifferentiated PDAC and the capacity to disseminate systemically, implicating HDAC2 as targetable protein to prevent metastasis.

A novel Cereblon E3 ligase modulator with antitumor activity in gastrointestinal cancer.

Targeted protein degradation offers new opportunities to inactivate cancer drivers and has successfully entered the clinic. Ways to induce selective protein degradation include proteolysis targeting chimera (PROTAC) technology and immunomodulatory (IMiDs) / next-generation Cereblon (CRBN) E3 ligase modulating drugs (CELMoDs). Here, we aimed to develop a MYC PROTAC based on the MYC-MAX dimerization inhibitor 10058-F4 derivative 28RH and Thalidomide, called MDEG-541. We show that a subgroup of gastrointestinal cancer cell lines and primary patient-derived organoids are MDEG-541 sensitive. Although MYC expression was regulated in a CRBN-, proteasome- and ubiquitin-dependent manner, we provide evidence that MDEG-541 induced the degradation of CRBN neosubstrates, including G1 to S phase transition 1/2 (GSPT1/2) and the Polo-like kinase 1 (PLK1). In sum, we have established a CRBN-dependent degrader of relevant cancer targets with activity in gastrointestinal cancers.

Phosphoproteomics Identifies PI3K Inhibitor-selective Adaptive Responses in Pancreatic Cancer Cell Therapy and Resistance.

The PI3K pathway is highly active in human cancers. The four class I isoforms of PI3K are activated by distinct mechanisms leading to a common downstream signaling. Their downstream redundancy is thought to be responsible for treatment failures of PI3K inhibitors. We challenged this concept, by mapping the differential phosphoproteome evolution in response to PI3K inhibitors with different isoform-selectivity patterns in pancreatic cancer, a disease currently without effective therapy. In this cancer, the PI3K signal was shown to control cell proliferation. We compared the effects of LY294002 that inhibit with equal potency all class I isoenzymes and downstream mTOR with the action of inhibitors with higher isoform selectivity toward PI3Kα, PI3Kß, or PI3Kγ (namely, A66, TGX-221 and AS-252424). A bioinformatics global pathway analysis of phosphoproteomics data allowed us to identify common and specific signals activated by PI3K inhibitors supported by the biological data. AS-252424 was the most effective treatment and induced apoptotic pathway activation as well as the highest changes in global phosphorylation-regulated cell signal. However, AS-252424 treatment induced reactivation of Akt, therefore decreasing the treatment outcome on cell survival. Reversely, AS-252424 and A66 combination treatment prevented p-Akt reactivation and led to synergistic action in cell lines and patient organoids. The combination of clinically approved α-selective BYL-719 with γ-selective IPI-549 was more efficient than single-molecule treatment on xenograft growth. Mapping unique adaptive signaling responses to isoform-selective PI3K inhibition will help to design better combinative treatments that prevent the induction of selective compensatory signals.

[Ductal adenocarcinoma of the pancreas: subtypes and molecular pathology]. / Das duktale Adenokarzinom des Pankreas: Subtypen und Molekularpathologie.

Ductal adenocarcinoma is the most common tumor of the pancreas. Although relatively rare, it poses one of the greatest oncological challenges because of its poor prognosis, which has so far only slightly improved. Progress has been made in the more precise classification and standardization of the morphological assessment. In the current WHO classification, prognostically relevant subtypes are clearly delimited among themselves and from ductal adenocarcinoma not otherwise specified (NOS). In the recent TNM classification, a size-based T­category was introduced. Diagnostically, the histological assessment of the resection specimen is relatively easy; on the other hand, assessment of the fine-needle biopsy from the primary tumor or a liver metastasis is still difficult. The molecular stratification of ductal adenocarcinoma and the other pancreatic neoplasms has made great progress. This not only defined the genetics of tumor entities, but also identified the prognosis and biology of tumor groups on the basis of RNA expression patterns. The range of treatment could be expanded by targeted molecular therapies (especially for patients with BRCA1/2 germline mutations, NTRK- or NRG1-fusions, or oncogenic BRAF and PIK3CA mutations as well as tumors with microsatellite instability (MSI)), even if targeted therapies are currently only available for a minority of patients (<10%).

AGR2-Dependent Nuclear Import of RNA Polymerase II Constitutes a Specific Target of Pancreatic Ductal Adenocarcinoma in the Context of Wild-Type p53.

BACKGROUND & AIMS: Promoted by pancreatitis, oncogenic KrasG12D triggers acinar cells' neoplastic transformation through acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia. Anterior gradient 2 (Agr2), a known inhibitor of p53, is detected at early stage of pancreatic ductal adenocarcinoma (PDAC) development. RNA polymerase II (RNAPII) is a key nuclear enzyme; regulation of its nuclear localization in mammalian cells represents a potential therapeutic target. METHODS: A mouse model of inflammation-accelerated KrasG12D-driven ADM and pancreatic intraepithelial neoplasia development was used. Pancreas-specific Agr2 ablation was performed to access its role in pancreatic carcinogenesis. Hydrophobic hexapeptides loaded in liposomes were developed to disrupt Agr2-RNAPII complex. RESULTS: We found that Agr2 is up-regulated in ADM-to-pancreatic intraepithelial neoplasia transition in inflammation and KrasG12D-driven early pancreatic carcinogenesis. Genetic ablation of Agr2 specifically blocks this metaplastic-to-neoplastic process. Mechanistically, Agr2 directs the nuclear import of RNAPII via its C-terminal nuclear localization signal, undermining the ATR-dependent p53 activation in ADM lesions. Because Agr2 binds to the largest subunit of RNAPII in a peptide motif-dependent manner, we developed a hexapeptide to interfere with the nuclear import of RNAPII by competitively disrupting the Agr2-RNAPII complex. This novel hexapeptide leads to dysfunction of RNAPII with concomitant activation of DNA damage response in early neoplastic lesions; hence, it dramatically compromises PDAC initiation in vivo. Moreover, the hexapeptide sensitizes PDAC cells and patient-derived organoids harboring wild-type p53 to RNAPII inhibitors and first-line chemotherapeutic agents in vivo. Of note, this therapeutic effect is efficient across various cancer types. CONCLUSIONS: Agr2 is identified as a novel adaptor protein for nuclear import of RNAPII in mammalian cells. Also, we provide genetic evidence defining Agr2-dependent nuclear import of RNAPII as a pharmaceutically accessible target for prevention and treatment in PDAC in the context of wild-type p53.

Genetic Screens Identify a Context-Specific PI3K/p27Kip1 Node Driving Extrahepatic Biliary Cancer.

Biliary tract cancer ranks among the most lethal human malignancies, representing an unmet clinical need. Its abysmal prognosis is tied to an increasing incidence and a fundamental lack of mechanistic knowledge regarding the molecular basis of the disease. Here, we show that the Pdx1-positive extrahepatic biliary epithelium is highly susceptible toward transformation by activated PIK3CAH1047R but refractory to oncogenic KrasG12D. Using genome-wide transposon screens and genetic loss-of-function experiments, we discover context-dependent genetic interactions that drive extrahepatic cholangiocarcinoma (ECC) and show that PI3K signaling output strength and repression of the tumor suppressor p27Kip1 are critical context-specific determinants of tumor formation. This contrasts with the pancreas, where oncogenic Kras in concert with p53 loss is a key cancer driver. Notably, inactivation of p27Kip1 permits KrasG12D-driven ECC development. These studies provide a mechanistic link between PI3K signaling, tissue-specific tumor suppressor barriers, and ECC pathogenesis, and present a novel genetic model of autochthonous ECC and genes driving this highly lethal tumor subtype. SIGNIFICANCE: We used the first genetically engineered mouse model for extrahepatic bile duct carcinoma to identify cancer genes by genome-wide transposon-based mutagenesis screening. Thereby, we show that PI3K signaling output strength and p27Kip1 function are critical determinants for context-specific ECC formation. This article is highlighted in the In This Issue feature, p. 2945.