Introduction

Interleukin-13 receptor α2 (IL-13Rα2) is a single-transmembrane protein consisting of 380 amino acids. It is a subunit of the IL-13 receptor family. IL-13Rα2 has high affinity for IL-13, and IL-13 binding has complex internal effects and functions. Pancreatic ductal adenocarcinoma (PDAC) with strong expression of IL-13Rα2 has a poor prognosis and is associated with gemcitabine resistance in an orthotopic mouse model of PDAC.

Aims & Methods

We evaluated the influence of IL-13Rα2 expression on patients with PDAC undergoing gemcitabine-based chemotherapy. We included patients with PDAC, as diagnosed by endoscopic ultrasound-fine needle aspiration (EUS-FNA), who received systemic chemotherapy from March 2014 to March 2019. Tumor expression of IL-13Rα2 was assessed by immunohistochemistry and classified using a three-point scale [0 (negative), 1 (weak), or 2 (strong)] in a blinded fashion. The IL-13Rα2-high group comprised patients with strong expression. Overall survival (OS) and progression-free survival (PFS) were analyzed from initiation of chemotherapy or chemoradiotherapy to death, and from treatment to the date of local progression > 20% or distant metastasis. The primary tumor reduction rate was compared between the high and low IL-13Rα2 expression groups. The effect of chemotherapy was assessed by computed tomography after 3 months. Additionally, in surgical samples, we evaluated the relationship between IL-13Rα2 and gemcitabine resistance genes (hENT, dCK, RRM1, RRM2, and CDA) by quantitative PCR.

Results

A total of 95 patients were enrolled. High and low IL-13Rα2 expression was detected in 63 and 32 cases, respectively. The IL-13Rα2-high group showed significantly poorer progression-free and overall survival rates than the IL-13Rα2-low group (p = 0.0191 and p = 0.0062, respectively). The factor of IL-13Rα2 expression was associated with progression disease after 3 months of the first gemcitabine-based chemotherapy (odds ratio = 13.72, p = 0.0143). In univariate analyses, PS of 0–1 or 2 (p < 0.01), mGPS of 0-1 or 2 (p < 0.01), tumor size < 32 or ≥ 32 mm (p = 0.02), surgery, (p < 0.01), radiation (p < 0.01), and tumoral IL-13Rα2 expression (p < 0.01) were associated with OS. In multivariate analysis, no surgery, no radiation, and positive IL-13Rα2 expression of the tumor were associated with a poorer prognosis (HR = 0.31, 95% CI: 0.12–0.81, p = 0.02; HR = 0.35, 95% CI: 0.17–0.73, p = 0.01; and HR = 1.81, 95% CI: 1.02–3.22, p = 0.04, respectively). As for gemcitabine resistance genes by quantitative PCR, the expression of hENT1 was significantly lower in the IL-13Rα2-high group (p = 0.01); dCK, RRM1, RRM2 and CDA expression levels were not significantly related to IL-13Rα2 expression (p = 0.14, 0.06, 0.97, and 0.13 respectively).

Conclusion

The evaluation of EUS-FNA specimens for tumoral IL-13Rα2 expression at the time of diagnosis enables prediction of prognosis. Strong expression of IL-13Rα2 in EUS-FNA specimens decreases gemcitabine-based chemotherapy effectiveness in PDAC. Evaluation of IL-13Rα2 expression in EUS-FNA specimens prior to chemotherapy may facilitate selection of the most suitable chemotherapeutic agent.

Introduction

Cytotoxic agents have long been used in the treatment of malignant tumors and continue to play a central role. Chemotherapy-induced cell death has been proposed to enhance the efficacy of immunotherapy through mechanisms such as immunogenic cell death or antigen spreading. However, these immunological processes remain underutilized in the clinical management of pancreatic ductal adenocarcinoma (PDAC). Oncogenic KRAS is known to promote an inflammatory tumor microenvironment, a process that can be further amplified by cytotoxic chemotherapy—a phenomenon we previously described as a "chemotherapy-derived inflammatory response" (Takeuchi et al., Cancer Research, 2015). In the context of heterogeneous human PDAC, it remains unclear whether this response is primarily driven by the administered chemotherapeutic agents or by the intrinsic properties of cancer cells. Elucidating the determinants of this response is essential for optimizing treatment strategies.

Aims & Methods

To investigate the determinants of chemotherapy-derived inflammatory responses in PDAC, we conducted in vitro assays using a panel of PDAC cell lines and clinically relevant cytotoxic agents, including gemcitabine (GEM), 5-fluorouracil (5-FU), oxaliplatin, irinotecan, and paclitaxel. PDAC cell lines were classified according to their molecular subtypes (classical or basal). Each drug was administered at its IC50 concentration, and inflammatory cytokine responses were assessed by quantitative PCR, ELISA, and microarray analysis. Gene expression and cytokine secretion levels were compared across cell lines and treatment conditions. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was used as a surrogate marker of the chemotherapy-induced inflammatory response.

Results

Using multiple assays, we found that the molecular subtype of PDAC (classical or basal) was not a major determinant of the chemotherapy-derived inflammatory response. Instead, gene expression patterns following drug-induced cell death varied significantly depending on the specific chemotherapeutic agent used. Notably, GEM strongly enhanced the expression of inflammatory cytokines through NF-κB activation, whereas 5-FU, despite inducing a comparable level of cell death, did not elicit such an inflammatory response. Gene set enrichment analysis based on microarray data further demonstrated that GEM and 5-FU induced distinct transcriptional profiles following cell death. These differences were also recapitulated in vivo using mouse tumor models.

Conclusion

Our findings suggest that chemotherapy-derived inflammatory responses are drug-specific. In particular, GEM may promote a suppressive tumor microenvironment by amplifying inflammation, potentially hindering anti-tumor immunity. To enhance the effectiveness of immunochemotherapy for PDAC, it will be crucial to elucidate the drug-specific mechanisms of cell death and to identify key regulatory molecules that can shift the response toward an immunogenic phenotype.

References

Takeuchi, S. Baghdadi, M. Tsuchikawa, T. Wada, H. Nakamura, T. Abe, H. Nakanishi, S. Usui, Y.Higuchi, K. Takahashi, M. Inoko, K. Sato, S. Takano, H. Shichinohe, T. Seino, K. Hirano, S. et al. Chemotherapy-Derived Inflammatory Responses Accelerate the Formation of Immunosuppressive Myeloid Cells in the Tissue Microenvironment of Human Pancreatic Cancer. Cancer Res. 75, 2629-2640 (2015).

Introduction

Besides stage, age and CA19-9 there is limited knowledge on “patient-related factors” associated with chemotherapy response for Pancreatic Ductal Adenocarcinoma (PDAC) patients. Smoking worsens chemotherapy response in other tumours and seems to promote tumor progression in PDAC preclinical models, but data in human PDAC are scanty.

Aims & Methods

PDAC patients were prospectively enrolled at diagnosis with exposome and clinical data registered, and tumors sampled for research purpose during Endoscopic Ultrasound (EUS). Patients were then followed-up to register chemotherapy treatment [whether Folfirinox (F), Abraxane-gemcitabine (AG) or a combination of cisplatin, abraxane, capecitabine and gemcitabine (PAXG)] and evaluate disease control rate (DCR) at 6-months (DCR-6), Progression-Free-Survival (PFS) and Overall-Survival (OS). We also explored transcriptomic signatures associated with smoking on a subset of EUS-acquired PDAC samples undergoing RNA-sequencing, with differential gene expression (DGE) analysis and pathways analysis performed. Furthermore, in vitro analyses with PDAC cell lines CAPAN-1 were treated with Folfirinox or gemcitabine with/without the nicotine compound 4-(Methylnitrosoamino)-1-(3-pyridinyl)-1-butanone to evaluate in vitro effects.

Results

326 patients treated with chemotherapy of interest were analyzed. Median age was 65, with 51.5% being males. Disease’s resectability at diagnosis was 10.4% resectable, 37.7% borderline-resectable, 26.9.% locally-advanced, 24.8% metastatic. AG was used in 42.3% of patients, PAXG in 25.7%, Folfirinox in 31.9%. DCR-6 was 57.9% in current smokers vs 70.4% in never-/ex-smokers (p=0.05). Current smoking was associated with a lack of DCR-6 at adjusted multivariable analysis (OR=1.99; 95%CI:1.1-3.5; p=0.01). At subgroup analysis, this was confirmed only for Folfirinox patients, with DCR-6 being 46.2% for current smokers vs 75.6% for non-current smokers(p=0.007). Current smokers had a significantly shorter PFS (5 vs 13 months; HR:1.79; 95%CI:1.13-3.21; p=0.01) and OS (24 vs 34; HR 1.93; 95%CI: 1-3.67; p=0.04) compared to non-smokers at adjusted multivariable analysis.
At transcriptomic analysis on a subset of 84 patients, current smokers were more prevalent in patients developing the more aggressive basal-like subtype (50% vs 15%; p=0.03), had lower expression of the classical marker GATA6 (3.27 vs 3.92; p=0.03) as also different activated pathways such as activated MAPK signalling, Axon guidance, Hypoxia and epithelial-mesenchymal-transition signalling among the others.
At in vitro experiments, for cell survival analysis, CAPAN-1 PDAC cells showed a significant reduced response to Folfirinox treatment when treated in combination with nicotin compounds, which was not observed for gemcitabine alone or in combination.

Conclusion

Collectively, our results consistently point towards a deleterious effect of smoking on response to Folfirinox regimen. Clinical data show that current smoking is a predictive factor for lower DCR-6, shorter PFS and OS; transcriptomic analyses show different gene expressions between smokers and non-smokers, with a transition toward reduced expression of classical phenotype and activation of inflammatory and pro-mesenchimal pathways. In vitro experiments support the negative effect of smoking on the response to Folfirinox. Smoking should be considered as a covariate to be investigated when choosing chemotherapy regimens in patients with PDAC, and patients should be discouraged to continue smoking.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with the lowest 5-year survival rate of all cancers due to late diagnosis and lack of effective treatment. One of the challenges of developing molecular diagnosis and molecular targeted therapy for PDAC is the “mismatch” between research models and patients.

Aims & Methods

This study aimed i) to identify common proteins among different research models and patients and accordingly ii) to identify potential hub proteins and signaling pathways involved in the PDAC pathogenesis by using proteomics-based system modeling of multimodal datasets derived from multiple research models and patients. Mouse PDAC cells (derived from a genetically engineered mouse model with KrasG12D/Pdx1-Cre), PDAC orthotopically tumors in C57B/L6 mice (n=5), mouse PDAC spheroids, mouse pancreatic organoids, mouse PDAC organoids, normal mouse pancreatic tissues (n=5), human PDAC tumor tissue samples from treatment-naїve (n=20), neoadjuvant chemotherapy (n=22), and human PDAC organoids were included. Mass spectrometry-based proteomics, Rstudio, Cytoscape 3.10 and pathway enrichment analysis (using ingenuity pathways analysis, including human PDAC transcriptomic in addition) were used.

Results

In the comparison of PDAC models and patients, shared proteins were identified as follows: 60% between the mouse PDAC cells and the mouse PDAC spheroids, 78% between the mouse PDAC cells and the mouse PDAC tissue, 56% between the mouse PDAC spheroids and the mouse PDAC tissue, 66% between the mouse PDAC cells and the mouse PDAC organoids, 54% between the mouse PDAC spheroids and the mouse PDAC organoids, 38% between the mouse PDAC and the human PDAC tissues, 31% between the human PDAC tumor and the human PDAC organoids, and 60% between the mouse and the human PDAC organoids. A total of 1,367 common proteins were identified within all the PDAC models. Among these proteins ACTB, HSP90AA1, HSPA8, HSP90AB1, HSPA4, EEF2, RPS3, RPS20 and GAPDH were the top 10 hub proteins (with >300 node connections). Upon comparison of all eight models, eight proteins were identified as unique to PDAC, namely FAHD1, ARSA, NEK9, SERPINB9, LAMTOR2, SNRPG, TMED3, and RAB1A. There was a correlation in terms of z-score regarding signaling pathways between the mouse PDAC and human PDAC (p<0.001), particularly in activations of nerve growth factor signaling, integrin signaling, and neuregulin signaling. Central hub proteins commonly found in the mouse models included vinculin (membrane-cytoskeletal protein) as one of the high connectivity nodes and β-catenin and probable ubiquitin carboxyl-terminal hydrolase FAF-X (involved in axonal growth and neuronal cell migration) as high centrality nodes. In patients, central hub proteins that were dysregulated after chemotherapy included 14-3-3 protein zeta/delta (encoded by YWHAZ gene), β-catenin and SRC. Among the 47 proteins that were associated with altered metabolism after the chemotherapy in patients, CD44, CTNND1, APOA 1, PARVA, and SCRBS1 were nodes with low degrees, and desmosomes (DSP, KP3, PPL), PRTN3, thrombospondin-2 (THBS2), and DMBT1 were the most down-regulated proteins without nodes, and APOA1, caveolin-1 (encoded by CAV1), LRP1 were the most up-regulated proteins with nodes.

Conclusion

The system modeling identified the common proteins among the research models and patients and the hub proteins and signaling pathways, suggesting the potential translational targets of PDAC.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with a devastating prognosis. A lack of durable responses to chemotherapy renders its treatment particularly challenging. Gemcitabine, a pyrimidine anti-metabolite, is a key component in PDAC therapy, but resistance poses a major hurdle in clinical care. Multiple mechanisms of chemoresistance have been attributed to rewired metabolism in PDAC cells. Accordingly, we aim to target these altered metabolic pathways to improve response to treatment.

Aims & Methods

To investigate metabolic vulnerabilities, we generated an acquired gemcitabine resistance in murine PDAC cell lines. We performed RNA sequencing on parental and gemcitabine resistant cell lines to identify targetable transcriptomic programs that drive cell-intrinsic resistance.

Results

Our transcriptomic dataset demonstrated that PDAC cells re-wire several metabolic pathways upon the acquisition of gemcitabine resistance, including downregulation of nucleoside transporters required for gemcitabine uptake and gemcitabine activating enzymes, as well as upregulation of detoxification enzymes. We performed a customized screen of common metabolic inhibitors and observed that gemcitabine resistant cells were exquisitely susceptible to statins, inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, an observation consistent across multiple clinically available statins. Protein and RNA sequencing data showed a downregulation of the mevalonate biosynthesis pathway and its rate-limiting enzyme, HMG-CoA reductase, in gemcitabine resistance. Importantly, the sensitivity to statins correlated with the extent of the gemcitabine resistance, suggesting that targeting the HMG-CoA reductase is a metabolic vulnerability that is gradually gained in PDAC cells upon the acquisition of resistance. Downstream rescue experiments suggested that statins induce apoptosis through lowered levels of geranylgeranyl pyrophosphate and reduced protein geranylation of small GTPases. To validate our findings in vivo, we orthotopically implanted gemcitabine resistant murine PDAC cells into syngeneic mice that were fed with a modified diet precluding geranylgeraniol. Here, the combined treatment of gemcitabine and pitavastatin effectively inhibited tumor growth of resistant allografts.

Conclusion

Collectively, these data suggest that targeting the HMG-CoA reductase is a metabolic vulnerability that is gained in PDAC cells upon the acquisition of gemcitabine resistance. The treatment combination of gemcitabine and pitavastatin, two widely used compounds in patients, has potential to translate into an immediate clinical benefit and improve survival in this deadly disease.

Introduction

Immunotherapy has led to a fundamental shift in the treatment of several cancers. However, its efficacy in pancreatic ductal adenocarcinoma (PDAC) is limited. Understanding the expression of inhibitory immune checkpoint receptors (ICR) by T cells may help to unravel their involvement in insufficient T cell-mediated antitumor immunity.

Aims & Methods

Here, we determined the expression of inhibitory receptors on CD8⁺ T cells, conventional CD4⁺ T cells (Tconv) and regulatory T cells (Treg) in the peripheral blood and matched tumor samples from PDAC patients (n=134) by multicolor flow cytometry. Further, T cell differentiation, tumor reactivity and cytokine production were analyzed.

Results

Intratumoral T cells highly expressed PD-1 and TIGIT. Both markers delineated distinct T cell subpopulations. Notably, PD-1⁺TIGIT^- T cells highly expressed proinflammatory cytokines (IFN-γ, TNF-α, IL-2, and IL-17a), and markers of tumor reactivity (CD39, CD103), whereas TIGIT expression was linked to anti-inflammatory and exhausted phenotypes. Furthermore, the enhanced presence of intratumoral PD-1⁺TIGIT^-Tconv was associated with improved clinical outcomes, while high ICR expression on blood T cells was a significant hazard for overall survival.

Conclusion

Our results uncover the association between ICR expression and T cell functionality in PDAC. PD-1 and TIGIT characterized intratumoral T cells with highly divergent phenotypes linked to clinical outcome, further underscoring the potential of TIGT as an immunotherapeutic target in PDAC. ICR expression by blood T cells may be a valuable tool for treatment stratification of PDAC patients.

Introduction

Mucinous adenocarcinoma (MAC) is a mucin-producing tumor. In MAC, about >50% of the tumor is composed of extracellular mucin with clear malignant epithelial cells in clumps and layers. Pancreatic MAC represents only 3.5% of all pancreatic tumors and 5.1% of all MAC cases [1]. Few studies with Poor evidence investigated the survival of pancreatic MAC, especially for patients treated with chemotherapy.

Aims & Methods

In this study, we aimed to assess the impact of chemotherapy on the survival outcome in pancreatic MAC. We conducted a retrospective cohort study Using the Surveillance Epidemiology and End Results (SEER) database. We extracted the data of 4837 patients diagnosed with pancreatic MAC from 2000 to 2020. patients were sub-stratified according to the treatment modality into Surgical and Non-surgical groups. We conducted a survival analysis by Kaplan–Meier and Log-rank test using Jamovi v2.3.26 software. Cox regression analysis was conducted to investigate the association between other risk factors and survival.

Results

The median age for pancreatic MAC was 68 years (IQR: 59-76) [2] [3] with a 5-year Relative survival of 8.3%. The 5-year Relative survival in the group treated Surgically was significantly improved compared to the Non-surgical group (35% and 1.7%; P<0.001). In the Group that had surgical intervention, systemic Chemotherapy showed no significant survival benefit compared to who had no chemotherapy (33.1% and 37.3%; P=0.15). In the group treated medically, Chemotherapy showed a significant survival benefit compared to the group who received no chemotherapy with a 2-year relative survival of 11.6% and 4.2% while the 5-year relative survival was 1.4% and 2.1% (P<0 .001) [4] [5]. COX-regression model showed males had better survival outcomes compared to females (HR=0.88, 95%CI: 0.83-0.93, P<0 .001) while African Americans had worse survival compared to Caucasians (HR=1.15, 95%CI: 1.05-1.27, P=0 .003) [6][7][8][9].

Conclusion

Pancreatic MAC is an extremely aggressive tumor with poor survival outcome. Surgery had a significant survival benefit of 33% compared to medical treatment only which highlights surgery as the modality of choice for pancreatic MAC. Chemotherapy improved the survival of patients who were not treated surgically. Further evaluation of each patient is needed to confirm if the patient is qualified for the surgical intervention and chemotherapy. Males and Caucasians seem to have better survival compared to females and African Americans.

References

1. Benesch MGK, Mathieson A. Epidemiology of Mucinous Adenocarcinomas. Cancers (Basel). 2020 Oct 30;12(11):3193. doi: 10.3390/cancers12113193. PMID: 33143115; PMCID: PMC7692300.
2. The jamovi project (2022). jamovi. (Version 2.3) [Computer Software]. Retrieved from https://www.jamovi.org.
3. R Core Team (2021). R: A Language and environment for statistical computing. (Version 4.1) [Computer software]. Retrieved from https://cran.r-project.org. (R packages retrieved from MRAN snapshot 2022-01-01).
4. Terry M Therneau (2020). A Package for Survival Analysis in R. [R package]. Retrieved from https://cran.r-project.org/package=survival.
5. Terry M. Therneau and Patricia M. Grambsch (2000). Modeling Survival Data: Extending the Cox Model. Springer.
6. Ewen Harrison and Tom Drake and Riinu Ots (2019). finalfit: Quickly Create Elegant Regression Results Tables and Plots when Modelling. [R package]. Retrieved from https://CRAN.R-project.org/package=finalfit.
7. Hidalgo B, Goodman M. (2013). Multivariate or Multivariable Regression?. Am J Public Health.. link, 103, 39-40.
8. Emily C. Zabor (2022). Survival analysis in R: A detailed tutorial on conducting survival analyses in R, including an introduction to the basics of survival analysis, landmark analyses and time-dependent covariates, competing risks, and a selection of advanced topics.. [GitHub Repository]. Retrieved from https://github.com/zabore/tutorials. link.
9. Serdar Balci (2022). ClinicoPath jamovi Module doi:10.5281/zenodo.3997188. [R package]. Retrieved from https://github.com/sbalci/ClinicoPathJamoviModule. link.