Cytosolic DNA sensor AIM2 promotes KRAS-driven lung cancer independent of inflammasomes.

Constitutively active KRAS mutations are among the major drivers of lung cancer, yet the identity of molecular co-operators of oncogenic KRAS in the lung remains ill-defined. The innate immune cytosolic DNA sensor and pattern recognition receptor (PRR) Absent-in-melanoma 2 (AIM2) is best known for its assembly of multiprotein inflammasome complexes and promoting an inflammatory response. Here, we define a role for AIM2, independent of inflammasomes, in KRAS-addicted lung adenocarcinoma (LAC). In genetically defined and experimentally induced (nicotine-derived nitrosamine ketone; NNK) LAC mouse models harboring the KrasG12D driver mutation, AIM2 was highly upregulated compared with other cytosolic DNA sensors and inflammasome-associated PRRs. Genetic ablation of AIM2 in KrasG12D and NNK-induced LAC mouse models significantly reduced tumor growth, coincident with reduced cellular proliferation in the lung. Bone marrow chimeras suggest a requirement for AIM2 in KrasG12D-driven LAC in both hematopoietic (immune) and non-hematopoietic (epithelial) cellular compartments, which is supported by upregulated AIM2 expression in immune and epithelial cells of mutant KRAS lung tissues. Notably, protection against LAC in AIM2-deficient mice is associated with unaltered protein levels of mature Caspase-1 and IL-1ß inflammasome effectors. Moreover, genetic ablation of the key inflammasome adapter, ASC, did not suppress KrasG12D-driven LAC. In support of these in vivo findings, AIM2, but not mature Caspase-1, was upregulated in human LAC patient tumor biopsies. Collectively, our findings reveal that endogenous AIM2 plays a tumor-promoting role, independent of inflammasomes, in mutant KRAS-addicted LAC, and suggest innate immune DNA sensing may provide an avenue to explore new therapeutic strategies in lung cancer.

The protease ADAM17 at the crossroads of disease: revisiting its significance in inflammation, cancer, and beyond.

The protease A Disintegrin And Metalloproteinase 17 (ADAM17) plays a central role in the pathophysiology of several diseases. ADAM17 is involved in the cleavage and shedding of at least 80 known membrane-tethered proteins, which subsequently modulate several intracellular signaling pathways, and therefore alter cell behavior. Dysregulated expression and/or activation of ADAM17 has been linked to a wide range of autoimmune and inflammatory diseases, cancer, and cardiovascular disease. In this review, we provide an overview of the current state of knowledge from preclinical models and clinical data on the diverse pathophysiological roles of ADAM17, and discuss the mechanisms underlying ADAM17-mediated protein shedding and the potential therapeutic implications of targeting ADAM17 in these diseases.

Circulating Tumor DNA Is an Accurate Diagnostic Tool and Strong Prognostic Marker in Pancreatic Cancer.

OBJECTIVE: The objectives of the study are to investigate the sensitivity and specificity of circulating tumor DNA (ctDNA) for the diagnosis of pancreatic cancer and to assess the utility of ctDNA as a prognostic marker in this disease. METHODS: Cell-free DNA was extracted from plasma of patients who underwent endoscopic ultrasound fine-needle aspiration or surgical resections for pancreatic cancer. The cell-free DNA was then analyzed using droplet digital polymerase chain reaction for KRAS G12/13 mutations. Eighty-one patients with pancreatic cancer and 30 patients with benign pancreatic disease were analyzed. RESULTS: ctDNA KRAS G12/13 mutations were detected in 63% of all patients with pancreatic cancer and in 76% of those patients who also had KRAS G12/13 mutations detected in the pancreatic primary. Specificity and tissue concordance were both 100%. Circulating tumor DNA corresponded with tumor size and stage, and high ctDNA was associated with significantly worse prognosis on both univariate and multivariate testing. CONCLUSION: Our study shows that ctDNA is an accurate diagnostic tool and strong prognostic marker in patients with pancreatic cancer. The continued investigation of ctDNA will enable its implementation in clinical practice to optimize the care and survival outcomes of patients with pancreatic cancer.

Th1 Cells Alter the Inflammatory Signature of IL-6 by Channeling STAT Transcription Factors to Alu-like Retroelements.

Cytokines that signal via STAT1 and STAT3 transcription factors instruct decisions affecting tissue homeostasis, antimicrobial host defense, and inflammation-induced tissue injury. To understand the coordination of these activities, we applied RNA sequencing, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing to identify the transcriptional output of STAT1 and STAT3 in peritoneal tissues from mice during acute resolving inflammation and inflammation primed to drive fibrosis. Bioinformatics focused on the transcriptional signature of the immunomodulatory cytokine IL-6 in both settings and examined how profibrotic IFN-γ-secreting CD4+ T cells altered the interpretation of STAT1 and STAT3 cytokine cues. In resolving inflammation, STAT1 and STAT3 cooperated to drive stromal gene expression affecting antimicrobial immunity and tissue homeostasis. The introduction of IFN-γ-secreting CD4+ T cells altered this transcriptional program and channeled STAT1 and STAT3 to a previously latent IFN-γ activation site motif in Alu-like elements. STAT1 and STAT3 binding to this conserved sequence revealed evidence of reciprocal cross-regulation and gene signatures relevant to pathophysiology. Thus, we propose that effector T cells retune the transcriptional output of IL-6 by shaping a regulatory interplay between STAT1 and STAT3 in inflammation.

An In Vitro Model for Assessing Acute Lung Injury During Pancreatitis Development Using Primary Mouse Cell Co-cultures.

Acute pancreatitis is a serious inflammatory disease of the pancreas that can lead to lung injury. Despite extensive research, the mechanisms underlying this complication are ill-defined. In recent years, in vitro co-culture systems have emerged as powerful tools for studying complex interactions between different cell types in disease. In the context of pancreatitis, pancreatic acinar epithelial cells produce and secrete digestive enzymes, and their cellular damage, death, and/or dysfunction is a major contributing factor to the onset of pancreatitis. Here, in this chapter we describe a co-culture system of acinar cells and lung epithelial progenitor/stem cells to model for lung injury associated with pancreatitis.

Targeting IL-6 trans-signalling: past, present and future prospects.

Interleukin-6 (IL-6) is a key immunomodulatory cytokine that affects the pathogenesis of diverse diseases, including autoimmune diseases, chronic inflammatory conditions and cancer. Classical IL-6 signalling involves the binding of IL-6 to the membrane-bound IL-6 receptor α-subunit (hereafter termed 'mIL-6R') and glycoprotein 130 (gp130) signal-transducing subunit. By contrast, in IL-6 trans-signalling, complexes of IL-6 and the soluble form of IL-6 receptor (sIL-6R) signal via membrane-bound gp130. A third mode of IL-6 signalling - known as cluster signalling - involves preformed complexes of membrane-bound IL-6-mIL-6R on one cell activating gp130 subunits on target cells. Antibodies and small molecules have been developed that block all three forms of IL-6 signalling, but in the past decade, IL-6 trans-signalling has emerged as the predominant pathway by which IL-6 promotes disease pathogenesis. The first selective inhibitor of IL-6 trans-signalling, sgp130, has shown therapeutic potential in various preclinical models of disease and olamkicept, a sgp130Fc variant, had promising results in phase II clinical studies for inflammatory bowel disease. Technological developments have already led to next-generation sgp130 variants with increased affinity and selectivity towards IL-6 trans-signalling, along with indirect strategies to block IL-6 trans-signalling. Here, we summarize our current understanding of the biological outcomes of IL-6-mediated signalling and the potential for targeting this pathway in the clinic.

The cytosolic DNA sensor AIM2 promotes Helicobacter-induced gastric pathology via the inflammasome.

Helicobacter pylori (H. pylori) infection can trigger chronic gastric inflammation perpetuated by overactivation of the innate immune system, leading to a cascade of precancerous lesions culminating in gastric cancer. However, key regulators of innate immunity that promote H. pylori-induced gastric pathology remain ill-defined. The innate immune cytosolic DNA sensor absent in melanoma 2 (AIM2) contributes to the pathogenesis of numerous autoimmune and chronic inflammatory diseases, as well as cancers including gastric cancer. We therefore investigated whether AIM2 contributed to the pathogenesis of Helicobacter-induced gastric disease. Here, we reveal that AIM2 messenger RNA and protein expression levels are elevated in H. pylori-positive versus H. pylori-negative human gastric biopsies. Similarly, chronic Helicobacter felis infection in wild-type mice augmented Aim2 gene expression levels compared with uninfected controls. Notably, gastric inflammation and hyperplasia were less severe in H. felis-infected Aim2-/- versus wild-type mice, evidenced by reductions in gastric immune cell infiltrates, mucosal thickness and proinflammatory cytokine and chemokine release. In addition, H. felis-driven proliferation and apoptosis in both gastric epithelial and immune cells were largely attenuated in Aim2-/- stomachs. These observations in Aim2-/- mouse stomachs correlated with decreased levels of inflammasome activity (caspase-1 cleavage) and the mature inflammasome effector cytokine, interleukin-1ß. Taken together, this work uncovers a pathogenic role for the AIM2 inflammasome in Helicobacter-induced gastric disease, and furthers our understanding of the host immune response to a common pathogen and the complex and varying roles of AIM2 at different stages of cancerous and precancerous gastric disease.

KRAS G12D Mutation Subtype in Pancreatic Ductal Adenocarcinoma: Does It Influence Prognosis or Stage of Disease at Presentation?

Background: KRAS G12D mutation subtype is present in over 40% of pancreatic ductal adenocarcinoma (PDAC), one of the leading global causes of cancer death. This retrospective cohort study aims to investigate whether detection of the KRAS G12D mutation subtype in PDAC patients is a determinant of prognosis across all stages of disease. Methods: We reviewed the medical records of 231 patients presenting with PDAC at a large tertiary hospital, and compared survival using the Kaplan Meier, log-rank test and Cox proportional hazards regression model. Results: KRAS G12D mutation subtype was not significantly associated with poorer survival compared across the whole population of PDAC patients (p = 0.107; HR 1.293 95% CI (0.946-1.767)). However, KRAS G12D patients who were resectable had a shorter median survival time of 356 days compared to all other genotypes (median survival 810 days) (p = 0.019; HR 1.991 95% CI (1.121-3.537)). Conclusions: KRAS G12D patients who were resectable at diagnosis had shorter survival compared to all other PDAC patients. These data suggest that KRAS G12D may be a clinically useful prognostic biomarker of PDAC.

Blockade of the protease ADAM17 ameliorates experimental pancreatitis.

Acute and chronic pancreatitis, the latter associated with fibrosis, are multifactorial inflammatory disorders and leading causes of gastrointestinal disease-related hospitalization. Despite the global health burden of pancreatitis, currently, there are no effective therapeutic agents. In this regard, the protease A Disintegrin And Metalloproteinase 17 (ADAM17) mediates inflammatory responses through shedding of bioactive inflammatory cytokines and mediators, including tumor necrosis factor α (TNFα) and the soluble interleukin (IL)-6 receptor (sIL-6R), the latter of which drives proinflammatory IL-6 trans-signaling. However, the role of ADAM17 in pancreatitis is unclear. To address this, Adam17ex/ex mice-which are homozygous for the hypomorphic Adam17ex allele resulting in marked reduction in ADAM17 expression-and their wild-type (WT) littermates were exposed to the cerulein-induced acute pancreatitis model, and acute (1-wk) and chronic (20-wk) pancreatitis models induced by the cigarette smoke carcinogen nicotine-derived nitrosamine ketone (NNK). Our data reveal that ADAM17 expression was up-regulated in pancreatic tissues of animal models of pancreatitis. Moreover, the genetic (Adam17ex/ex mice) and therapeutic (ADAM17 prodomain inhibitor [A17pro]) targeting of ADAM17 ameliorated experimental pancreatitis, which was associated with a reduction in the IL-6 trans-signaling/STAT3 axis. This led to reduced inflammatory cell infiltration, including T cells and neutrophils, as well as necrosis and fibrosis in the pancreas. Furthermore, up-regulation of the ADAM17/IL-6 trans-signaling/STAT3 axis was a feature of pancreatitis patients. Collectively, our findings indicate that the ADAM17 protease plays a pivotal role in the pathogenesis of pancreatitis, which could pave the way for devising novel therapeutic options to be deployed against this disease.

Cross-talk between IL-6 trans-signaling and AIM2 inflammasome/IL-1ß axes bridge innate immunity and epithelial apoptosis to promote emphysema.

Pulmonary emphysema is associated with dysregulated innate immune responses that promote chronic pulmonary inflammation and alveolar apoptosis, culminating in lung destruction. However, the molecular regulators of innate immunity that promote emphysema are ill-defined. Here, we investigated whether innate immune inflammasome complexes, comprising the adaptor ASC, Caspase-1 and specific pattern recognition receptors (PRRs), promote the pathogenesis of emphysema. In the lungs of emphysematous patients, as well as spontaneous gp130F/F and cigarette smoke (CS)-induced mouse models of emphysema, the expression (messenger RNA and protein) and activation of ASC, Caspase-1, and the inflammasome-associated PRR and DNA sensor AIM2 were up-regulated. AIM2 up-regulation in emphysema coincided with the biased production of the mature downstream inflammasome effector cytokine IL-1ß but not IL-18. These observations were supported by the genetic blockade of ASC, AIM2, and the IL-1 receptor and therapy with AIM2 antagonistic suppressor oligonucleotides, which ameliorated emphysema in gp130F/F mice by preventing elevated alveolar cell apoptosis. The functional requirement for AIM2 in driving apoptosis in the lung epithelium was independent of its expression in hematopoietic-derived immune cells and the recruitment of infiltrating immune cells in the lung. Genetic and inhibitor-based blockade of AIM2 also protected CS-exposed mice from pulmonary alveolar cell apoptosis. Intriguingly, IL-6 trans-signaling via the soluble IL-6 receptor, facilitated by elevated levels of IL-6, acted upstream of the AIM2 inflammasome to augment AIM2 expression in emphysema. Collectively, we reveal cross-talk between the AIM2 inflammasome/IL-1ß and IL-6 trans-signaling axes for potential exploitation as a therapeutic strategy for emphysema.

Toll-like Receptor 9 Promotes Initiation of Gastric Tumorigenesis by Augmenting Inflammation and Cellular Proliferation.

BACKGROUND & AIMS: Gastric cancer (GC) is strongly linked with chronic gastritis after Helicobacter pylori infection. Toll-like receptors (TLRs) are key innate immune pathogenic sensors that mediate chronic inflammatory and oncogenic responses. Here, we investigated the role of TLR9 in the pathogenesis of GC, including Helicobacter infection. METHODS: TLR9 gene expression was profiled in gastric tissues from GC and gastritis patients and from the spontaneous gp130F/F GC mouse model and chronic H felis-infected wild-type (WT) mice. Gastric pathology was compared in gp130F/F and H felis infection models with or without genetic ablation of Tlr9. The impact of Tlr9 targeting on signaling cascades implicated in inflammation and tumorigenesis (eg, nuclear factor kappa B, extracellular signal-related kinase, and mitogen-activated protein kinase) was assessed in vivo. A direct growth-potentiating effect of TLR9 ligand stimulation on human GC cell lines and gp130F/F primary gastric epithelial cells was also evaluated. RESULTS: TLR9 expression was up-regulated in Helicobacter-infected gastric tissues from GC and gastritis patients and gp130F/F and H felis-infected WT mice. Tlr9 ablation suppressed initiation of tumorigenesis in gp130F/F:Tlr9-/- mice by abrogating gastric inflammation and cellular proliferation. Tlr9-/- mice were also protected against H felis-induced gastric inflammation and hyperplasia. The suppressed gastric pathology upon Tlr9 ablation in both mouse models associated with attenuated nuclear factor kappa B and, to a lesser extent, extracellular signal-related kinase, mitogen-activated protein kinase signaling. TLR9 ligand stimulation of human GC cells and gp130F/F GECs augmented their proliferation and viability. CONCLUSIONS: Our data reveal that TLR9 promotes the initiating stages of GC and facilitates Helicobacter-induced gastric inflammation and hyperplasia, thus providing in vivo evidence for TLR9 as a candidate therapeutic target in GC.

Inflammasome-Associated Gastric Tumorigenesis Is Independent of the NLRP3 Pattern Recognition Receptor.

Inflammasomes are important multiprotein regulatory complexes of innate immunity and have recently emerged as playing divergent roles in numerous inflammation-associated cancers. Among these include gastric cancer (GC), the third leading cause of cancer-associated death worldwide, and we have previously discovered a pro-tumorigenic role for the key inflammasome adaptor apoptosis-associated speck-like protein containing a CARD (ASC) in the spontaneous genetic gp130 F/F mouse model for GC. However, the identity of the specific pattern recognition receptors (PRRs) that activate tumor-promoting inflammasomes during GC is unknown. Here, we investigated the role of the best-characterized inflammasome-associated PRR, nucleotide-binding domain, and leucine-rich repeat containing receptor, pyrin domain-containing (NLRP) 3, in GC. In gastric tumors of gp130 F/F mice, although NLRP3 expression was elevated at the mRNA (qPCR) and protein (immunohistochemistry) levels, genetic ablation of NLRP3 in gp130 F/F:Nlrp3 -/- mice did not alleviate the development of gastric tumors. Similarly, cellular processes associated with tumorigenesis in the gastric mucosa, namely, proliferation, apoptosis, and inflammation, were comparable between gp130 F/F and gp130 F/F:Nlrp3 -/- mice. Furthermore, inflammasome activation levels, determined by immunoblotting and immunohistochemistry for cleaved Caspase-1, which along with ASC is another integral component of inflammasome complexes, were unchanged in gp130 F/F and gp130 F/F:Nlrp3 -/- gastric tumors. We also observed variable NLRP3 expression levels (mRNA and protein) among independent GC patient cohorts, and NLRP3 was not prognostic for survival outcomes. Taken together, these data suggest that NLRP3 does not play a major role in promoting inflammasome-driven gastric tumorigenesis, and thus pave the way for further investigations to uncover the key inflammasome-associated PRR implicated in GC.

Context-dependent functions of pattern recognition receptors in cancer.

The immune system plays a critical role in shaping all facets of cancer, from the early initiation stage through to metastatic disease and resistance to therapy. Our understanding of the importance of the adaptive arm of the immune system in antitumour immunity has led to the implementation of immunotherapy with immune checkpoint inhibitors in numerous cancers, albeit with differing efficacy. By contrast, the clinical utility of innate immunity in cancer has not been exploited, despite dysregulated innate immunity being a feature of at least one-third of all cancers associated with tumour-promoting chronic inflammation. The past two decades have seen innate immune pattern recognition receptors (PRRs) emerge as critical regulators of the immune response to microbial infection and host tissue damage. More recently, it has become apparent that in many cancer types, PRRs play a central role in modulating a vast array of tumour-inhibiting and tumour-promoting cellular responses both in immune cells within the tumour microenvironment and directly in cancer cells. Herein, we provide a comprehensive overview of the fast-evolving field of PRRs in cancer, and discuss the potential to target PRRs for drug development and biomarker discovery in a wide range of oncology settings.

Oncostatin M expression induced by bacterial triggers drives airway inflammatory and mucus secretion in severe asthma.

Exacerbations of symptoms represent an unmet need for people with asthma. Bacterial dysbiosis and opportunistic bacterial infections have been observed in, and may contribute to, more severe asthma. However, the molecular mechanisms driving these exacerbations remain unclear. We show here that bacterial lipopolysaccharide (LPS) induces oncostatin M (OSM) and that airway biopsies from patients with severe asthma present with an OSM-driven transcriptional profile. This profile correlates with activation of inflammatory and mucus-producing pathways. Using primary human lung tissue or human epithelial and mesenchymal cells, we demonstrate that OSM is necessary and sufficient to drive pathophysiological features observed in severe asthma after exposure to LPS or Klebsiella pneumoniae. These findings were further supported through blockade of OSM with an OSM-specific antibody. Single-cell RNA sequencing from human lung biopsies identified macrophages as a source of OSM. Additional studies using Osm-deficient murine macrophages demonstrated that macrophage-derived OSM translates LPS signals into asthma-associated pathologies. Together, these data provide rationale for inhibiting OSM to prevent bacterial-associated progression and exacerbation of severe asthma.

Complete loss of miR-200 family induces EMT associated cellular senescence in gastric cancer.

The EMT (epithelial-to-mesenchymal-transition) subtype of gastric cancer (GC) is associated with poor treatment responses and unfavorable clinical outcomes. Despite the broad physiological roles of the micro-RNA (miR)-200 family, they largely serve to maintain the overall epithelial phenotype. However, during late-stage gastric tumorigenesis, members of the miR-200 family are markedly suppressed, resulting in the transition to the mesenchymal state and the acquisition of invasive properties. As such, the miR-200 family represents a robust molecular marker of EMT, and subsequently, disease severity and prognosis. Most reports have studied the effect of single miR-200 family member knockdown. Here, we employ a multiplex CRISPR/Cas9 system to generate a complete miR-200 family knockout (FKO) to investigate their collective and summative role in regulating key cellular processes during GC pathogenesis. Genetic deletion of all miR-200s in the human GC cell lines induced potent morphological alterations, G1/S cell cycle arrest, increased senescence-associated ß-galactosidase (SA-ß-Gal) activity, and aberrant metabolism, collectively resembling the senescent phenotype. Coupling RNA-seq data with publicly available datasets, we revealed a clear separation of senescent and non-senescent states amongst FKO cells and control cells, respectively. Further analysis identified key senescence-associated secretory phenotype (SASP) components in FKO cells and a positive feedback loop for maintenance of the senescent state controlled by activation of TGF-ß and TNF-α pathways. Finally, we showed that miR-200 FKO associated senescence in cancer epithelial cells significantly recruited stromal cells in the tumor microenvironment. Our work has identified a new role of miR-200 family members which function as an integrated unit serving to link senescence with EMT, two major conserved biological processes.

Oncogenic dependency on STAT3 serine phosphorylation in KRAS mutant lung cancer.

The oncogenic potential of the latent transcription factor signal transducer and activator of transcription (STAT)3 in many human cancers, including lung cancer, has been largely attributed to its nuclear activity as a tyrosine-phosphorylated (pY705 site) transcription factor. By contrast, an alternate mitochondrial pool of serine phosphorylated (pS727 site) STAT3 has been shown to promote tumourigenesis by regulating metabolic processes, although this has been reported in only a restricted number of mutant RAS-addicted neoplasms. Therefore, the involvement of STAT3 serine phosphorylation in the pathogenesis of most cancer types, including mutant KRAS lung adenocarcinoma (LAC), is unknown. Here, we demonstrate that LAC is suppressed in oncogenic KrasG12D-driven mouse models engineered for pS727-STAT3 deficiency. The proliferative potential of the transformed KrasG12D lung epithelium, and mutant KRAS human LAC cells, was significantly reduced upon pS727-STAT3 deficiency. Notably, we uncover the multifaceted capacity of constitutive pS727-STAT3 to metabolically reprogramme LAC cells towards a hyper-proliferative state by regulating nuclear and mitochondrial (mt) gene transcription, the latter via the mtDNA transcription factor, TFAM. Collectively, our findings reveal an obligate requirement for the transcriptional activity of pS727-STAT3 in mutant KRAS-driven LAC with potential to guide future therapeutic targeting approaches.

STAT3-mediated upregulation of the AIM2 DNA sensor links innate immunity with cell migration to promote epithelial tumourigenesis.

OBJECTIVE: The absent in melanoma 2 (AIM2) cytosolic pattern recognition receptor and DNA sensor promotes the pathogenesis of autoimmune and chronic inflammatory diseases via caspase-1-containing inflammasome complexes. However, the role of AIM2 in cancer is ill-defined. DESIGN: The expression of AIM2 and its clinical significance was assessed in human gastric cancer (GC) patient cohorts. Genetic or therapeutic manipulation of AIM2 expression and activity was performed in the genetically engineered gp130 F/F spontaneous GC mouse model, as well as human GC cell line xenografts. The biological role and mechanism of action of AIM2 in gastric tumourigenesis, including its involvement in inflammasome activity and functional interaction with microtubule-associated end-binding protein 1 (EB1), was determined in vitro and in vivo. RESULTS: AIM2 expression is upregulated by interleukin-11 cytokine-mediated activation of the oncogenic latent transcription factor STAT3 in the tumour epithelium of GC mouse models and patients with GC. Genetic and therapeutic targeting of AIM2 in gp130 F/F mice suppressed tumourigenesis. Conversely, AIM2 overexpression augmented the tumour load of human GC cell line xenografts. The protumourigenic function of AIM2 was independent of inflammasome activity and inflammation. Rather, in vivo and in vitro AIM2 physically interacted with EB1 to promote epithelial cell migration and tumourigenesis. Furthermore, upregulated expression of AIM2 and EB1 in the tumour epithelium of patients with GC was independently associated with poor patient survival. CONCLUSION: AIM2 can play a driver role in epithelial carcinogenesis by linking cytokine-STAT3 signalling, innate immunity and epithelial cell migration, independent of inflammasome activation.

EUS-FNA Biopsies to Guide Precision Medicine in Pancreatic Cancer: Results of a Pilot Study to Identify KRAS Wild-Type Tumours for Targeted Therapy.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer death and lacks effective treatment options. Diagnostic endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) biopsies represent an appealing source of material for molecular analysis to inform targeted therapy, as they are often the only available tissue for patients presenting with PDAC irrespective of disease stage. However, EUS-FNA biopsies are typically not used to screen for precision medicine studies due to concerns about low tissue yield and quality. Epidermal growth factor receptor (EGFR) inhibition has shown promise in clinical trials of unselected patients with advanced pancreatic cancer, but has not been prospectively tested in KRAS wild-type patients. Here, we examine the clinical utility of EUS-FNA biopsies for molecular screening of KRAS wild-type PDAC patients for targeted anti-EGFR therapy to assess the feasibility of this approach. PATIENTS AND METHODS: Fresh frozen EUS-FNA or surgical biopsies from PDAC patient tumours were used to screen for KRAS mutations. Eligible patients with recurrent, locally advanced, or metastatic KRAS wild-type status who had received at least one prior line of chemotherapy were enrolled in a pilot study (ACTRN12617000540314) and treated with panitumumab at 6mg/kg intravenously every 2 weeks until progression or unacceptable toxicity. The primary endpoint was 4-month progression-free survival (PFS). RESULTS: 275 patient biopsies were screened for KRAS mutations, which were detected in 88.3% of patient samples. 8 eligible KRAS wild-type patients were enrolled onto the interventional study between November 2017 and December 2020 and treated with panitumumab. 4-month PFS was 14.3% with no objective tumour responses observed. The only grade 3/4 treatment related toxicity observed was hypomagnesaemia. CONCLUSIONS: This study demonstrates proof-of-principle feasibility to molecularly screen patients with pancreatic cancer for targeted therapies, and confirms diagnostic EUS-FNA biopsies as a reliable source of tumour material for molecular analysis. Single agent panitumumab was safe and tolerable but led to no objective tumour responses in this population.

Targeted Transcriptome and KRAS Mutation Analysis Improve the Diagnostic Performance of EUS-FNA Biopsies in Pancreatic Cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a poor prognosis, and current diagnostic tests have suboptimal sensitivity. Incorporating standard cytology with targeted transcriptomic and mutation analysis may improve upon the accuracy of diagnostic biopsies, thus reducing the burden of repeat procedures and delays to treatment initiation. EXPERIMENTAL DESIGN: We reviewed the accuracy of 308 endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) diagnostic PDAC biopsies using a large multicenter clinical and biospecimen database, then performed RNA sequencing on 134 EUS-FNA biopsies spanning all stages of disease. We identified a transcriptomic diagnostic gene signature that was validated using external datasets and 60 further diagnostic EUS-FNAs. KRAS digital droplet PCR (ddPCR) analysis was performed and correlated with signature gene expression. RESULTS: The sensitivity of EUS-FNA cytology in diagnosing solid pancreatic masses in our retrospective cohort of 308 patients was 78.6% (95% confidence interval, 73.2%-83.2%). KRAS mutation analysis and our custom transcriptomic signature significantly improved upon the diagnostic accuracy of standard cytology to 91.3% in external validation sets and 91.6% in our validation cohort (n = 60). Exploratory ddPCR analysis of KRAS-mutant allele fraction (MAF%) correlated closely to signature performance and may represent a novel surrogate marker of tumor cellularity in snap-frozen EUS-FNA biopsies. CONCLUSIONS: Our findings support snap-frozen EUS-FNA biopsies as a feasible tissue source for the integrated genomic and transcriptomic analysis of patients presenting with PDAC from all tumor stages, including cases with nondiagnostic cytology. Our transcriptome-derived genetic signature in combination with tissue KRAS mutation analysis significantly improves upon the diagnostic accuracy of current standard procedures, and has potential clinical utility in improving the speed and accuracy of diagnosis for patients presenting with PDAC.