FNA needle rinses preserved in Cytolyt are acceptable specimen type for mutation testing of thyroid nodules.

INTRODUCTION: This study investigated the application of molecular testing to residual thyroid fine-needle aspiration material from needle rinses collected in Cytolyt. MATERIALS AND METHODS: Two thyroid needle rinses from 135 patients were collected in Cytolyt during routine diagnostic workup in our institution. Molecular testing was performed to detect 14 genetic alterations in BRAF, K-, H-, N-RAS genes as well as RET/PTC1, RET/PTC3, and PAX8/PPARγ and verified by next generation sequencing and correlated with cytologic diagnoses. RESULTS: Molecular testing revealed a total of 17 mutations across specimens with benign nodule (n = 5; HRAS, NRAS), Hürthle cell neoplasm (n = 2; BRAF, HRAS) and Papillary thyroid carcinoma (n = 10, 9 BRAF, 1 KRAS) cytology. No RNA gene rearrangements were detected. CONCLUSIONS: Mutations and translocations associated with thyroid cancer can be detected in thyroid fine-needle aspiration needle rinses preserved in Cytolyt specimens collected during routine patient management, which are typically discarded when a diagnosis is attained.

Differential expression of GNAS and KRAS mutations in pancreatic cysts.

CONTEXT: KRAS mutations play an important role in pancreatic cancer. GNAS mutations were discovered in intraductal papillary mucinous neoplasms (IPMN). OBJECTIVES: Our aim was to identify the frequency of KRAS and GNAS mutations in pancreatic cystic neoplasms and pancreatic ductal adenocarcinoma (PDAC). METHODS: Sixty-eight surgically resected formalin fixed, paraffin embedded pancreatic specimens were analyzed, including: 1) benign (20 serous cystadenoma (SCA)), 2) pre-malignant (10 mucinous cystic neoplasm (MCN), 10 branch duct intraductal papillary mucinous neoplasm (BD-IPMN), 9 main duct IPMN (MD-IPMN)), 3) malignant (19 PDAC). Total nucleic acid extraction was performed. KRAS codon 12/13 and GNAS codon 201 mutations were interrogated via targeted sequencing using the Ion Torrent's Personal Genome Machine (PGM). RESULTS: Mean age of 68 patients was 61.9±8.4 with 72% female. KRAS and GNAS mutations were more common in PDAC and IPMN. KRAS mutations predominated in PDAC compared to pancreatic cysts (16/19, 84% versus 10/49, 20%; P<0.001). GNAS mutations were more common in IPMN compared to non-IPMN lesions (8/19, 42% versus 2/49, 4%; P=0.0003). No GNAS mutations were detected in PDAC and MCN while 2 SCA carried GNAS mutations. Double mutations with KRAS and GNAS were only present in IPMN (5/19 versus 0/30 SCA and MCN, P=0.006). CONCLUSIONS: KRAS and GNAS mutations were more common in PDAC and IPMN with KRAS mutations primarily in PDAC and GNAS mutations more frequent in IPMN. No GNAS mutations occurred in MCN and double mutations were only present in IPMN.

A microRNA-based test improves endoscopic ultrasound-guided cytologic diagnosis of pancreatic cancer.

BACKGROUND & AIMS: Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) in combination with cytopathology is the optimal method for diagnosis and staging of pancreatic ductal adenocarcinoma (PDAC) and other pancreatic lesions. Its clinical utility, however, can be limited by high rates of indeterminate or false-negative results. We aimed to develop and validate a microRNA (miRNA)-based test to improve preoperative detection of PDAC. METHODS: Levels of miRNAs were analyzed in a centralized clinical laboratory by relative quantitative polymerase chain reaction in 95 formalin-fixed paraffin-embedded specimens and 228 samples collected by EUS-FNA during routine evaluations of patients with solid pancreatic masses at 4 institutions in the United States, 1 in Canada, and 1 in Poland. RESULTS: We developed a 5-miRNA expression classifier, consisting of MIR24, MIR130B, MIR135B, MIR148A, and MIR196, that could identify PDAC in well-characterized, formalin-fixed, paraffin-embedded specimens. Detection of PDAC in EUS-FNA samples increased from 78.8% by cytology analysis alone (95% confidence interval, 72.2%-84.5%) to 90.8% when combined with miRNA analysis (95% confidence interval, 85.6%-94.5%). The miRNA classifier correctly identified 22 additional true PDAC cases among 39 samples initially classified as benign, indeterminate, or nondiagnostic by cytology. Cytology and miRNA test results each were associated significantly with PDAC (P < .001), with positive predictive values greater than 99% (95% confidence interval, 96%-100%). CONCLUSIONS: We developed and validated a 5-miRNA classifier that can accurately predict which preoperative pancreatic EUS-FNA specimens contain PDAC. This test might aid in the diagnosis of pancreatic cancer by reducing the number of FNAs without a definitive adenocarcinoma diagnosis, thereby reducing the number of repeat EUS-FNA procedures.

Investigating MicroRNA Expression Profiles in Pancreatic Cystic Neoplasms.

OBJECTIVES: Current diagnostic tools for pancreatic cysts fail to reliably differentiate mucinous from nonmucinous cysts. Reliable biomarkers are needed. MicroRNAs (miRNA) may offer insights into pancreatic cysts. Our aims were to (1) identify miRNAs that distinguish benign from both premalignant cysts and malignant pancreatic lesions using formalin-fixed, paraffin-embedded (FFPE) pathology specimens; (2) identify miRNAs that distinguish mucinous cystic neoplasm (MCN) from branch duct-intraductal papillary mucinous neoplasm (BD-IPMN). METHODS: A total of 69 FFPE pancreatic specimens were identified: (1) benign (20 serous cystadenoma (SCA)), (2) premalignant (10 MCN, 10 BD-IPMN, 10 main duct IPMN (MD-IPMN)), and (3) malignant (19 pancreatic ductal adenocarcinoma (PDAC)). Total nucleic acid extraction was performed followed by miRNA expression profiling of 378 miRNAs interrogated using TaqMan MicroRNA Arrays Pool A and verification of candidate miRNAs. Bioinformatics was used to generate classifiers. RESULTS: MiRNA profiling of 69 FFPE specimens yielded 35 differentially expressed miRNA candidates. Four different 4-miRNA panels differentiated among the lesions: one panel separated SCA from MCN, BD-IPMN, MD-IPMN, and PDAC with sensitivity 85% (62, 97), specificity 100% (93, 100), a second panel distinguished MCN from SCA, BD-IPMN, MD-IPMN, and PDAC with sensitivity and specificity 100% (100, 100), a third panel differentiated PDAC from IPMN with sensitivity 95% (76, 100) and specificity 85% (72, 96), and the final panel diagnosed MCN from BD-IPMN with sensitivity and specificity approaching 100%. CONCLUSIONS: MiRNA profiling of surgical pathology specimens differentiates serous cystadenoma from both premalignant pancreatic cystic neoplasms and PDAC and MCN from BD-IPMN.

miRNA biomarkers in cyst fluid augment the diagnosis and management of pancreatic cysts.

PURPOSE: The diagnosis of pancreatic cystic lesions has increased dramatically. Most are benign, whereas some, such as intraductal papillary mucinous neoplasms (IPMN), represent precursors of pancreatic adenocarcinoma. Therapeutic stratification of IPMNs is challenging without precise information on dysplasia grade and presence of invasion. We assessed the diagnostic benefit of using miRNAs as biomarkers in pancreatic cyst fluid, focusing on IPMNs because of their frequency and malignant potential. EXPERIMENTAL DESIGN: RNA was extracted from 55 microdissected formalin-fixed, paraffin-embedded (FFPE) IPMN specimens, and 65 cyst fluid specimens aspirated following surgical resection. Expression of 750 miRNAs was evaluated with TaqMan miRNA Arrays using 22 FFPE and 15 cyst fluid specimens. Differential expression of selected miRNA candidates was validated in 33 FFPE and 50 cyst fluid specimens using TaqMan miRNA Assays. RESULTS: We identified 26 and 37 candidate miRNAs that distinguish low-grade from high-grade IPMNs using FFPE and cyst fluid specimens, respectively. A subset of 18 miRNAs, selected from FFPE and cyst fluid data, separated high-grade IPMNs from low-grade IPMNs, serous cystadenomas (SCA) and uncommon cysts, such as solid pseudopapillary neoplasms (SPN) and cystic pancreatic neuroendocrine tumors (PanNET). A logistic regression model using nine miRNAs allowed prediction of cyst pathology implying resection (high-grade IPMNs, PanNETs, and SPNs) versus conservative management (low-grade IPMNs, SCAs), with a sensitivity of 89%, a specificity of 100%, and area under the curve of 1. CONCLUSIONS: We found candidate miRNAs that helped identify patients with high-grade IPMN and exclude nonmucinous cysts. These classifiers will require validation in a prospective setting to ultimately confirm their clinical usefulness.

Global microRNA expression profiling of microdissected tissues identifies miR-135b as a novel biomarker for pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is known for its poor prognosis resulting from being diagnosed at an advanced stage. Accurate early diagnosis and new therapeutic modalities are therefore urgently needed. MicroRNAs (miRNAs), considered a new class of biomarkers and therapeutic targets, may be able to fulfill those needs. Combining tissue microdissection with global miRNA array analyses, cell type-specific miRNA expression profiles were generated for normal pancreatic ductal cells, acinar cells, PDAC cells derived from xenografts and also from macrodissected chronic pancreatitis (CP) tissues. We identified 78 miRNAs differentially expressed between ND and PDAC cells providing new insights into the miRNA-driven pathophysiological mechanisms involved in PDAC development. Having filtered miRNAs which are upregulated in the three pairwise comparisons of PDAC vs. ND, PDAC vs. AZ and PDAC vs. CP, we identified 15 miRNA biomarker candidates including miR-135b. Using relative qRT-PCR to measure miR-135b normalized to miR-24 in 75 FFPE specimens (42 PDAC and 33 CP) covering a broad range of tumor content, we discriminated CP from PDAC with a sensitivity and specificity of 92.9% [95% CI=(80.5, 98.5)] and 93.4% [95% CI=(79.8, 99.3)], respectively. Furthermore, the area under the curve (AUC) value reached of 0.97 was accompanied by positive and negative predictive values of 95% and 91%, respectively. In conclusion, we report pancreatic cell-specific global miRNA profiles, which offer new candidate miRNAs to be exploited for functional studies in PDAC. Furthermore, we provide evidence that miRNAs are well-suited analytes for development of sensitive and specific aid-in-diagnosis tests for PDAC.

No miR quirk: dysregulation of microRNAs in pancreatic ductal adenocarcinoma.

MicroRNAs are post-transcriptional regulators of gene expression with tissue-specific expression profiles. Dysregulation of microRNAs has been shown to play a role in carcinogenesis. Although progress has been made in the diagnosis and treatment of many cancers, pancreatic cancer remains an intractable public health problem, causing 6.58% of cancer deaths despite making up less than 3% of cancer diagnoses in the United States. No screening, diagnostic or imaging techniques exist with the sensitivity to detect pancreatic cancer in its early, operable stages. Risk factors include numerous inherited syndromes, diabetes mellitus, and hepatitis C virus infection. Here we review the literature regarding dysregulation of microRNA expression in native pancreas, pancreatic ductal adenocarcinoma (the dominant form of pancreatic cancer), and its risk factors to illuminate the biology and progression of this disease. We explore promising evidence for the use of microRNAs as prognostic and diagnostic tools, and discuss emerging reports on microRNA therapeutics.

Development of a miRNA-based diagnostic assay for pancreatic ductal adenocarcinoma.

Diagnosis of pancreatic cancer remains a clinical challenge. Both chronic pancreatitis and pancreatic cancer may present with similar symptoms and similar imaging features, often leading to incorrect interpretation. Thus, the use of an objective molecular test that can discriminate between chronic pancreatitis and pancreatic cancer will be a valuable asset in obtaining a definitive diagnosis of pancreatic cancer. Following Clinical Laboratory Improvement Amendments and College of American Pathologists guidelines, Asuragen Clinical Services Laboratory has developed and validated a laboratory-developed test, miRInform(®) Pancreas, to aid in the identification of pancreatic ductal adenocarcinoma. This molecular diagnostic tool uses reverse-transcription quantitative PCR to measure the expression difference between two miRNAs, miR-196a and miR-217, in fixed tissue specimens. This article describes the test validation process as well as determination of performance parameters of miRInform Pancreas.