ATRX/DAXX in EUS-FNB Specimens of Pan-NETs (FORESEE)

Association Between Endoscopic Ultrasound Based Preoperative ATRX/DAXX Immunohistochemistry Expression and Prognosis of Sporadic, Non-FunctiOnal pancREatic Neuroendocrine tumorS: a prospEctivE Cohort Study

P-NENs are classified as functional (F-) or non-functional (NF-) depending on the presence or absence of a clinical hormonal hypersecretion syndrome. Moreover, the WHO 2017 classification of pNENs distinguishes between well-differentiated pancreatic neuroendocrine tumors (pNETs) and poorly differentiated pancreatic neuroendocrine carcinomas (pNECs). pNETs are then divided according to a grading scheme based on Ki67 index in pNETs-G1 (Ki67 index ≤3%) and pNETs-G2 (Ki67 index between 4% and 20%). pNECs are all G3, with a Ki67 index >20%. Endoscopic ultrasound with fine-needle biopsy (EUS-FNB) demonstrated safe and effective preoperative grading based on the Ki-67 proliferative index. However, downstaging rate is not neglectable, reaching 15% in a recent metanalysis. Moreover, recent whole-exome and whole genome sequencing studies revealed that the mutually exclusive inactivating mutations in death domain-associated protein (DAXX) and/or in α-thalassemia/mental retardation X-linked (ATRX) chromatin remodeling genes are associated with more aggressive disease. In a retrospective study, the investigators recently evaluated the correspondence of DAXX/ATRX expression on 41 EUS-FNB samples with corresponding surgical specimens demonstrating a 95.1% (almost perfect agreement, κ = 0.828; p < 0.001) and 92.7% (substantial agreement, κ = 0.626; p < 0.001) concordance for DAXX and ATRX expression, respectively. This study aims to evaluate the potential clinical/prognostic role of DAXX/ATRX expression as implementation of the currently used Ki67-based grading, evaluated on EUS-FNB samples in a prospective cohort of patients with NF-pNETs

Study Overview

• Status: Active, not recruiting
• Conditions: Pancreatic Neuroendocrine Tumor
• Intervention / Treatment: Diagnostic test: ATRX/DAXX immunohistochemistry

• Study Type: Observational
• Enrollment (Actual): 100

Contacts and Locations

Study Locations

• Italy
  • Verona, Italy
    • University Hospital of Verona

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: No

• Sampling Method: Non-Probability Sample

Study Population

Patients with a diagnosis of pancreatic neuroendocrine tumors

Description

Inclusion Criteria:

• Age ≥18 years
• Cyto/histologic diagnosis of pNETs
• Signed informed consent

Exclusion Criteria:

• Functional pNETs
• Multiple pancreatic nodules
• Diagnosis of MEN-1 or Von-Hippel Lindau
• Mixed types (e.g., mixed neuroendocrine-acinar/adenocarcinoma) or neuroendocrine carcinomas
• Predominantly cystic lesions (more than 50% of the volume).
• Metastatic tumors at the time of diagnosis
• Known bleeding disorder that cannot be sufficiently corrected with co-fact or fresh frozen plasma
• Use of anticoagulants that cannot be discontinued
• INR >1.5 or platelet count <50.000
• Pregnancy or breastfeeding
• Failure to sign the patient's or closest relative's informed consent

Study Plan

How is the study designed?

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
ATRX/DAXX loss; The "positive" group including patients with loss of DAXX or ATRX expression evaluated on immunohistochemistry on endoscopic ultrasound biopsy specimens	Diagnostic test: ATRX/DAXX immunohistochemistry; Immunohistochemistry will be performed using an Autostainer Leica (Leica Biosystems) according to the manufacturer's instructions. Four μm formalin-fixed paraffin-embedded sections will be immunostained with antibodies for Cytokeratin AE1/AE3 (AE1-AE3, 1:100 dilution, Novocastra/United Kingdom) Chromogranin A (DAK-A3, 1:2500, Dako/Denmark), and Synaptophysin (27G12, 1:100, Novocastra), Ki67 (MIB1, 1:100, Dako/Denmark), ATRX (1:400, Sigma-Aldrich), DAXX (1:200, Sigma-Aldrich). After antigen retrieval, immunostaining will be performed in an automated Bond instrument (Vision-Biosystem, Leica, Milan, Italy) using a sensitive peroxidase-based 'Bond polymer Refine' detection system.
ATRX/DAXX preserved; The "negative" group including those with preserved DAXX and ATRX expression evaluated on immunohistochemistry on endoscopic ultrasound biopsy specimens	Diagnostic test: ATRX/DAXX immunohistochemistry; Immunohistochemistry will be performed using an Autostainer Leica (Leica Biosystems) according to the manufacturer's instructions. Four μm formalin-fixed paraffin-embedded sections will be immunostained with antibodies for Cytokeratin AE1/AE3 (AE1-AE3, 1:100 dilution, Novocastra/United Kingdom) Chromogranin A (DAK-A3, 1:2500, Dako/Denmark), and Synaptophysin (27G12, 1:100, Novocastra), Ki67 (MIB1, 1:100, Dako/Denmark), ATRX (1:400, Sigma-Aldrich), DAXX (1:200, Sigma-Aldrich). After antigen retrieval, immunostaining will be performed in an automated Bond instrument (Vision-Biosystem, Leica, Milan, Italy) using a sensitive peroxidase-based 'Bond polymer Refine' detection system.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
ATRX/DAXX loss-tumor aggressiveness association	To assess the association between ATRX/DAXX loss of expression assessed on endoscopic ultrasound biopsy specimens and pathological features indicative of tumor aggressiveness evaluated on surgical pathology	18 months

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Ki-67 concordance	To evaluate the concordance rate of Ki67-based tumor grade between endoscopic ultrasound biopsy samples and surgical specimens.	18 months
DAXX/ATRX concordance	To evaluate the concordance rate of DAXX/ATRX expression between endoscopic ultrasound biopsy samples and surgical specimens	18 months
Ki-67-tumor aggressiveness association	To assess the association between preoperative Ki67-based grade 2/3 on endoscopic ultrasound samples and pathological features indicative of tumor aggressiveness evaluated on surgical pathology	18 months
Preoperative prognosis assessment	To evaluate the association between DAXX/ATRX expression on endoscopic ultrasound biopsy samples, Ki67-based grade 2/3 on endoscopic ultrasound biopsy samples, and tumor size on endoscopic ultrasound and progression-free survival and relapse-free survival.	60 months

Collaborators and Investigators

• Sponsor: Azienda Ospedaliera Universitaria Integrata Verona

Study record dates

Study Major Dates

• Study Start (Actual): June 1, 2024
• Primary Completion (Actual): March 30, 2025
• Study Completion (Estimated): November 30, 2029

Study Registration Dates

• First Submitted: May 6, 2024
• First Submitted That Met QC Criteria: May 6, 2024
• First Posted (Actual): May 9, 2024

Study Record Updates

• Last Update Posted (Actual): January 13, 2026
• Last Update Submitted That Met QC Criteria: January 12, 2026
• Last Verified: January 1, 2025

More Information

Terms related to this study

• Additional Relevant MeSH Terms: Endocrine System Diseases; Neoplasms by Site; Neoplasms; Neoplasms by Histologic Type; Digestive System Neoplasms; Digestive System Diseases; Endocrine Gland Neoplasms; Pancreatic Diseases; Neoplasms, Glandular and Epithelial; Adenoma; Pancreatic Neoplasms; Adenoma, Islet Cell
• Other Study ID Numbers: FORESEE

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No