Introduction

There are limited molecular features to allow treatment personalization and outcome prediction of Pancreatic-Ductal-Adenocarcinoma(PDAC) patients.Two transcriptome subtypes have been identified: classical and basal-like.Classical PDACs show better prognosis and response to FOLFIRINOX, while basal-like display an opposite behaviour.This transcriptome-based classification is not straightforward and relies mainly on surgical series, with limited data on EUS-FNA samples.We have recently showed that the splicing factor QKI is a determinant of basal-like signature and that its evaluation together with GATA6 has strong prognostic capacity.

Aims & Methods

Our aim was to investigate transcriptome subtypes and gene expression of specific biomarkers on baseline EUS-FNA samples of PDAC patients and their association with prognosis and response to chemotherapy. 85 PDAC patients who underwent EUS at diagnosis were enrolled in a prospective study and FNA samples investigated by RNA-seq.PURIST score was applied to define subtypes, Differential Gene Expression DGE analysis was performed and the prognostic value of QKI and GATA6 expression was investigated.The outcomes were overall survival(OS) and progression-free survival(PFS) corrected for age and stage.

Results

RNA-seq was successful in 84/85(98.8%) samples.According with PURIST, 4 patients were classified as basal-like(4.8%), 80 being classical.OS was lower in Basal-like vs classical patients(5.5 vs 13 months; p=0.01), with increased risk of death(HR 5.6; 95%CI:1.5-20.9; p=0.01). PURIST score also correlated inversely with OS (r=-0.4; p=0.0002).In 64 patients treated with first-line chemotherapy (FOLFIRINOX n=24;Abraxane-Gemcitabine, AG n=30;PAXG n=10) QKI expression correlated with OS(r=-0,36: p=0.0038).Among patients treated with FOLFIRINOX, median PFS was 7 months (IQR 2-9) in patients with a QKI/Gata6 ratio >1.13 and not reached in patients with ratio≤1.13 (HR=5.1;95%CI:1.2-21.5); QKI/GATA6 ratio was not associated with PFS in patients treated with the other regimens.At DGE analysis REG4 was the most enriched gene in patients with PFS>6 months; a cut-off for REG4 expression>111 was associated with response to AG(HR=0.2;95%CI:0,090-0,59;p=0.002).

Conclusion

Transcriptome analysis of EUS-FNA samples is feasible and allows stratification of PDAC patients in terms of prognosis and chemosensitivity.Few selected classifiers such as QKI, GATA6 and REG4 may be sufficient to predict response to certain treatments.

Introduction

Despite advances with chemotherapy regimens, Pancreatic adenocarcinoma (PDAC) is becoming the 2^nd cause of cancer-related death. Personalized medicine will be the game changer, but molecular characterization of pancreatic neoplasms is yet extremely cumbersome and has been performed on surgical samples or few EUS-tissue acquisition (TA) cases, mostly for DNA analysis. Our aim was to evaluate DNA and RNA extracted for research purposes from pancreatic neoplasms employing Endoscopic Ultrasound (EUS) Fine needle aspiration (FNA) and biopsy (FNB) and adequacy to molecular analyses.

Aims & Methods

From Oct 2022, patients with solid pancreatic neoplasms suggestive of either PDAC or neuroendocrine neoplasm (NEN) undergoing diagnostic EUS-TA were considered for enrollment. Employed needles were either FNA, or Franseen, Fork-tip or Three-prong, either 22G or 25G based on operator’s preference, with Rapid on Site Evaluation and, in case of adequacy within the first 2 passes, 1 additional pass with the same needle was performed for molecular analyses. DNA and RNA were then extracted with a new technique and nucleic acids were quantified and evaluated for adequacy for molecular analysis. Mann-Whitney was employed for comparison of continuous variables, and Kruskal-Wallis test for comparison between multiple groups; Logistic regression was applied to determine factors associated with adequacy to molecular tests.

Results

351 patients underwent sampling for molecular analyses, 309 with a definitive diagnosis of PDAC and 42 with NEN. 92,9% of patients were sampled with 25G FNA needles. Median quantity of DNA extracted was significantly lower in PDAC 2048 ng (95%CI: 1470 -3651) compared to NEN 6752 ng (95%CI: 3502-9655)(p<0.0144). Mean quantity of RNA extracted was similar, 312 ng for PDAC, and 444 ng for NEN (p=0.07), with a higher median RIN of 6,4 (95%CI:6,2-6,5) for PDAC versus 4,7 (95%CI:3,9-5) for NEN (p<0.0001). In terms of needle type, concerning DNA extraction, 25G FNA needles retrieved a median of 3384 ng, 25G Franseen needles (4312 ng), 22G Franseen (73864 ng), 25G Fork-tip or Three-prong (3104 ng), with no significant difference. In terms of needle type, concerning RNA extraction, 25G FNA needles retrieved a median of 310 ng (RIN 6,3), 25G Franseen needles 460 ng (RIN 5,5), 22G Franseen 884 ng (RIN 5,9), 25G Fork-tip or Three-prong 301 ng (RIN 5,15), with no significant difference. 295 (95,5%) of PDAC samples and 34 (80,9%) of NEN samples were considered adequate for sequencing. The only factor associated to adequacy for molecular analysis was tumor type, with PDAC having an OR of 5,44 (95%CI 2,06-14,37)(p=0.0006), while the type of employed needle was not

Conclusion

Molecular analysis on EUS-acquired tissue of pancreatic neoplasms will be the mainstay of personalized therapies but how to best perform tissue acquisition and sample handling for this specific purpose is yet to be defined. This is the largest study ever performed on DNA and RNA extraction from EUS-acquired samples, demonstrating superior adequacy for molecular analysis in PDAC compared to NEN. All needle types result similar in terms of performance despite the low number of needles other than 25G FNA needles.

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

Two pancreatic cancer(PDAC) transcriptome subtypes have been defined (basal-like and classical) that seem related to different prognosis. Nevertheless, RNA-extraction from pancreatic tissue is cumbersome and has been performed mainly on surgical samples, representative of <20% of cases. Most PDAC patients undergo Endoscopic-UltraSound(EUS)-guided tissue acquisition(EUS-TA), but RNA-sequencing on such samples has been rarely performed or limited to paraffin-embedded samples with low RNA quality. Furthermore, the association between molecular subtype and patient-related factors such as BMI, smoking, drug use, tumor location and Ca19.9 expression is uninvestigated.

Aims & Methods

The aim was to correlate PDAC molecular subtypes identified by RNA-sequencing on EUS-TA samples with prognosis and evaluate whether they are associated to patients’ factors.
Consecutive patients with non-metastatic PDAC who underwent EUS-TA at diagnosis were enrolled in a prospective biobanking study with snap-frozen samples. Those with adequate quantity and quality of RNA underwent RNA-sequencing with Illumina Nova-Seq. PURIST score was applied to define transcriptional subtype and association with patient-related factors and overall survival (OS) investigated. Categorical and continuous variables were investigated by Fisher’s exact test or Mann-Whitney test, correlation analyses with Pearson test

Results

In 44/45 samples, RNA was of quantity and integrity allowing successful RNA-sequencing. According with PURIST score 3 patients were classified as basal-like (6.8%) and the other 41 as classical. Basal-like patients had a significantly lower median OS compared to classical (3 vs 16 months; p=0.01) and a basal-like phenotype was associated to increased risk of death (HR 7.79; p=0.006). PURIST score also significantly correlated inversely with OS (r=-0.6; p=0.0007). Concerning patients’ variables, patients with basal-like tumors were more frequently current smokers (66.6% vs 12.2%; p=0.05) and had a lower baseline Ca19.9 (80 vs 1243 IU/ml; p=0.001). No differences were found concerning age, gender, BMI, diabetes history, disease stage, tumor location, use of aspirin or statins.

Conclusion

Molecular subtype identification on EUS-TA PDAC cases at diagnosis is feasible and a basal-like phenotype is rare but associated to worse prognosis. Furthermore, current smoking seems to be associated to a more aggressive molecular subtype, which in turn does not seem to express a high Ca19.9. Further studies on a larger cohort are needed to confirm such findings.

Introduction

Liquid biopsy in patients with pancreatic ductal adenocarcinoma (PDAC) has been challenged by the limited information obtained from systemic circulation (SC). Circulating Tumour Cells (CTCs) in portal circulation (PC) are deemed to be biologically relevant and potentially carrying prognostic significance, despite mostly been evaluated during surgery.

Aims & Methods

This prospective, single-centre, interventional study enrolled PDAC patients to assess the feasibility and safety of EUS-guided transgastric and transhepatic Portal Vein sampling (EUS-PVS) for isolating, enumerating, and characterizing CTCs. PC and paired SC samples underwent microfluidic enrichment and dielectrophoretic (DEP) single-cell sorting based on immunostaining for epithelial and mesenchymal biomarkers. Targeted next-generation KRAS sequencing was performed on CTC subpopulations in a subset of patients.

Results

Between May 2022 and October 2023, 20 PDAC patients (25% metastatic) underwent EUS-PVS with 100% feasibility and safety. CTCs were isolated in all patients from both districts, but the total CTC concentration was significantly higher in PC compared to SC (8.1 [5.8-21.3] vs. 4.1 [2.3-6] / 7.5 ml of blood, p= 0.01), particularly for mesenchymal CTCs. No associations with baseline clinical variables or disease stage were observed. A subset of CTCs exhibited KRAS codon 12 mutations, undetected in paired white blood cells.

Conclusion

EUS-PVS is a feasible, safe, and effective method for portal blood sampling, supporting subsequent molecular investigations. Despite more sensitive enrichment modalities enhance the yield of peripheral CTCs evaluation, portal circulation remains more informative, especially for detecting epithelial-to-mesenchymal transition events. DEP single-cell sorting was feasible in all patients, providing libraries of viable cells with specific 100%-pure phenotypes, potentially suitable for downstream molecular applications.

Introduction

Hormonal exposure influences cancer initiation and progression in several organs, including the pancreas. The association between gynecological factors and the onset of pancreatic adenocarcinoma (PDAC) is conflicting, with some studies showing a reduced risk of PDAC with the use of hormonal replacement therapy (HRT) or with having had a late-onset menopause. Nonetheless, the association between gynecological factors and disease prognosis has not yet been investigated. Our objective was to evaluate whether gynecological/reproductive factors affect PDAC prognosis.

Aims & Methods

This was a single-center retrospective analysis of a prospectively maintained database of PDAC patients. Risk factors were assessed via questionnaires regarding gynecologic/reproductive factors (age at menarche, menopause, years of fertility, breastfeeding, pregnancy, miscarriages, use of oral contraceptives (OC) or HRT), medical history, and prognosis were evaluated with follow-up visits and/or telephonic calls. Continuous variables were investigated using the Mann–Whitney test, and factors associated with survival probability were evaluated using Cox's proportional hazards model, adjusting for potential confounders.

Results

228 PDAC women were analyzed, with mean age at diagnosis being 68. The most frequent stages were borderline resectable (35.9%) and locally advanced (25.4%), with a median overall survival (OS) of 14 months. The age of PDAC onset for patients with previous use of OC was significantly lower than that of non-users (63 years vs. 72; p<0.0001), and OC use was associated with a trend toward increased survival (HR 0.6; 95%CI: 0.35-1.03; p=0.06). In addition, previous use of HRT was associated with a reduced risk of recurrence or progression (HR, 0.4; 95%CI: 0.26-0.99; p=0.001). No differences in survival or progression-free probability were observed with the other factors.

Conclusion

This is the first study to evaluate whether gynecological/reproductive factors are associated with the prognosis of PDAC patients, with the use of OC and HRT being the most relevant factors. Previous OC users had a significantly lower onset age for PDAC and a borderline significant association was seen with increased survival. Additionally, HRT use is associated with a reduced risk of tumor recurrence or progression. These data need to be confirmed, and the potential mechanisms need to be elucidated.

Introduction

Aims & Methods

The purpose of this study is to evaluate the feasibility of extracting RNA in sufficient quantity and quality to perform genomic analyses from specimens obtained through EUS-fine needle aspiration (EUS-FNA) of PanNENs, comparing three methods of RNA preservation and extraction.

Results

Conclusion