We compare two types of pancreatic carcinoma samples obtained by EUS-guided fine needle biopsy (EUS-FNB) in terms of the success rates and clinical validity of analysis of two most commonly investigated DNA/RNA pancreatic cancer markers, KRAS mutations and miR-21 expression. 118 patients with pancreatic ductal adenocarcinoma underwent EUS-FNB. The collected sample was divided, one part was stored in a stabilizing solution as native aspirate (EUS-FNA) and second part was processed into the cytological smear (EUS-FNC). DNA/RNA extraction was followed by analysis of KRAS mutations and miR-21 expression. For both sample types, the yields of DNA/RNA extraction and success rates of KRAS mutation and miRNA expression were evaluated. Finally, the resulting KRAS mutation frequency and miR-21 prognostic role were compared to literature data from tissue resections. The overall amount of isolated DNA/RNA from EUS-FNC was lower compared to the EUS-FNA, average yield 10 ng vs 147 ng for DNA and average yield 164 vs. 642 ng for RNA, but the success rates for KRAS and miR-21 analysis was 100% for both sample types. The KRAS-mutant detection frequency in EUS-FNC was 12% higher than in EUS-FNA (90 vs 78%). The prognostic role of miR-21 was confirmed in EUS-FNC (p = 0.02), but did not reach statistical significance in EUS-FNA (p = 0.06). Although both types of EUS-FNB samples are suitable for DNA/RNA extraction and subsequent DNA mutation and miRNA expression analysis, reliable results with clinical validity were only obtained for EUS-FNC.

• Keywords
• EUS-FNA, KRAS, Pancreatic cancer, miR-21,
• MeSH
• Endoscopic Ultrasound-Guided Fine Needle Aspiration MeSH
• Cytodiagnosis methods   MeSH
• DNA analysis   MeSH
• Carcinoma, Pancreatic Ductal diagnosis   MeSH
• Tissue Fixation methods   MeSH
• Middle Aged MeSH
• Humans MeSH
• MicroRNAs analysis   MeSH
• Mutation MeSH
• Biomarkers, Tumor analysis   MeSH
• Pancreatic Neoplasms diagnosis   MeSH
• Specimen Handling methods   MeSH
• Proto-Oncogene Proteins p21(ras) genetics   MeSH
• Aged MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• DNA MeSH
• KRAS protein, human MeSH Browser
• MicroRNAs MeSH
• MIRN21 microRNA, human MeSH Browser
• Biomarkers, Tumor MeSH
• Proto-Oncogene Proteins p21(ras) MeSH

AIM: To establish an optimum combination of molecular markers resulting in best overall diagnostic sensitivity and specificity for evaluation of suspicious pancreatic mass. METHODS: Endoscopic ultrasound (EUS)-guided fine needle aspiration cytology (FNA) was performed on 101 consecutive patients (63 males, 38 females, 60 +/- 12 years; 81 with subsequently diagnosed pancreatic cancer, 20 with chronic pancreatitis) with focal pancreatic mass. Samples were evaluated on-site by an experienced cytopathologist. DNA was extracted from Giemsa stained cells selected by laser microdissection and the presence of K-ras, p53 and p16 somatic mutations was tested by cycling-gradient capillary electrophoresis (CGCE) and single-strand conformation polymorphism (SSCP) techniques. In addition, allelic losses of tumor suppressor genes p16 (INK4, CDKN2A) and DPC4 (MADH4, SMAD4) were detected by monitoring the loss of heterozygosity (LOH) at 9p and 18q, respectively. RESULTS: Sensitivity and specificity of EUS-guided FNA were 75% and 85%, positive and negative predictive value reached 100%. The remaining 26% samples were assigned as inconclusive. Testing of molecular markers revealed sensitivity and specificity of 70% and 100% for K-ras mutations (P < 0.001), 24% and 90% for p53 mutations (NS), 13% and 100% for p16 mutations (NS), 85% and 64% for allelic losses at 9p (P < 0.001) and 78% and 57% for allelic losses at 18q (P < 0.05). When tests for different molecular markers were combined, the best results were obtained with K-ras + LOH at 9p (92% and 64%, P < 0.001), K-ras + LOH at 18q (92% and 57%, P < 0.001), and K-ras + LOH 9q + LOH 18q (96% and 43%, P < 0.001). When the molecular markers were used as complements to FNA cytology to evaluate inconclusive samples only, the overall sensitivity of cancer detection was 100% in all patients enrolled in the study. CONCLUSION: EUS-guided FNA cytology combined with screening of K-ras mutations and allelic losses of tumor suppressors p16 and DPC4 represents a very sensitive approach in screening for pancreatic malignancy. Molecular markers may find its use particularly in cases where FNA cytology has been inconclusive.

• MeSH
• Pancreatitis, Chronic diagnosis   genetics   pathology   MeSH
• Molecular Diagnostic Techniques * MeSH
• Adult MeSH
• Electrophoresis, Capillary MeSH
• Endosonography * MeSH
• Cyclin-Dependent Kinase Inhibitor p16 genetics   MeSH
• Middle Aged MeSH
• Humans MeSH
• Chromosomes, Human, Pair 18 * MeSH
• Chromosomes, Human, Pair 9 * MeSH
• Mutation MeSH
• Biomarkers, Tumor genetics   MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Pancreatic Neoplasms diagnosis   genetics   pathology   MeSH
• Polymorphism, Single-Stranded Conformational MeSH
• Predictive Value of Tests MeSH
• Smad4 Protein genetics   MeSH
• Proto-Oncogene Proteins p21(ras) genetics   MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Sensitivity and Specificity MeSH
• Biopsy, Fine-Needle methods   MeSH
• Loss of Heterozygosity MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged, 80 and over MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Names of Substances
• Cyclin-Dependent Kinase Inhibitor p16 MeSH
• Biomarkers, Tumor MeSH
• Tumor Suppressor Protein p53 MeSH
• Smad4 Protein MeSH
• Proto-Oncogene Proteins p21(ras) MeSH
• SMAD4 protein, human MeSH Browser
• TP53 protein, human MeSH Browser