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.

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.

Analytical Validation of a Highly Sensitive, Multiplexed Chronic Myeloid Leukemia Monitoring System Targeting BCR-ABL1 RNA.

This study describes the analytical performance of the QuantideX qPCR BCR-ABL IS Kit, the first Food and Drug Administration-cleared assay designed to monitor breakpoint cluster region-Abelson tyrosine-protein kinase 1 (BCR-ABL1) fusion transcripts isolated from peripheral blood specimens from patients with chronic myeloid leukemia. This multiplex real-time quantitative RT-PCR assay amplifies both e13a2 and e14a2 Major BCR-ABL1 transcripts and the reference target ABL1. The test results are provided in international scale (IS) values by incorporating armored RNA-based calibrators that have defined IS values tied directly to the World Health Organization BCR-ABL1 Primary Reference Materials, without the necessity of determining and maintaining conversion factors. For each batch run, the integrated interpretive software evaluates run and specimen quality control metrics (including a sufficient amount of ABL1 control transcripts to ensure a minimal limit of detection) and calculates both molecular response (MR) and %IS values for each specimen. The test has a limit of detection of MR4.7 (0.002%IS) and a linear range from MR0.3 (50%IS) to MR4.7 (0.002%IS) for both Major transcripts. Single-site and multisite precision studies demonstrated a maximum SD of 0.13 MR (30% CV within the assay range between MR0.7 and MR3.7). The performance of this BCR-ABL1 monitoring test meets all of the clinical guideline recommendations for sensitivity and IS reporting for the management of chronic myeloid leukemia patients.

RNA extraction for arrays.

DNA microarrays enable insights into global gene expression by capturing a snapshot of cellular expression levels at the time of sample collection. Careful RNA handling and extraction are required to preserve this information properly, ensure sample-to-sample reproducibility, and limit unwanted technical variation in experimental data. This chapter discusses important considerations for "array-friendly" sample handling and processing from biosamples such as blood, formalin-fixed, paraffin-embedded samples, and fresh or flash-frozen tissues and cells. It also provides guidelines on RNA quality assessments, which can be used to validate sample preparation and maximize recovery of relevant biological information.

Analyzing micro-RNA expression using microarrays.

The discovery of micro-RNAs (miRNAs) and the growing appreciation of the importance of micro-RNAs in the regulation of gene expression are driving increasing interest in miRNA expression profiling. Early studies have suggested prominent roles for these genetically encoded regulatory molecules in a variety of normal biological processes and diseases, particularly cancer. However, the field of miRNA expression profiling is in its infancy. Several factors, including the small size, the unknown but limited number of miRNAs, and the tissue-to-tissue and tissue-to-disease state variability in miRNA expression, make the adaptation of microarray technology to the evaluation of miRNA expression nontrivial. This chapter describes the unique features of miRNA microarray experiments and analysis and provides a case study demonstrating our approach to miRNA expression analysis.

Control of T lymphocyte morphology by the GTPase Rho.

BACKGROUND: Rho family GTPase regulation of the actin cytoskeleton governs a variety of cell responses. In this report, we have analyzed the role of the GTPase Rho in maintenance of the T lymphocyte actin cytoskeleton. RESULTS: Inactivation of the GTPase Rho in the human T lymphocytic cell line HPB-ALL does not inhibit constitutively high adhesion to the integrin beta1 substrate fibronectin. It did however result in the aberrant extension of finger-like dendritic processes on the substrates VCAM-1, Fn, and mAb specific to beta1 integrins. Time-lapse video microscopy demonstrated that C3 induced extensions were primarily the result of an altered pseudopod elongation rather than retraction. Once the stellate pseudopodia extended, none retracted, and cells became completely immobile. Filipodial structures were absent and the dendritic-like processes in C3 treated cells were rich in filamentous actin. Immunolocalization of RhoA in untreated HPB-ALL cells spreading on fibronectin demonstrated a diffuse staining pattern within the pseudopodia. In C3 treated cells, clusters of RhoA were pronounced and localized within the altered extensions. CONCLUSIONS: GTPase Rho is actively involved in the regulation of T lymphocyte morphology and motility.

Expression of calmodulin in Drosophila is highly regulated in a stage- and tissue-specific manner.

The expression of the Drosophila calmodulin (CAM) gene is surprisingly complex. The nervous system, which shows intense transcription in embryogenesis, contains no detectable transcripts at the end of larval life, but becomes transcriptionally active again at pupariation. The gut shows high levels of expression throughout the life cycle, except during pupal reorganization. In contrast, CAM expression in the thoracic muscles drops significantly on transition from pupal to adult life. In the testis, transcription is strongly up-regulated prior to meiosis. Growing cells show lower transcript levels than most differentiated tissues and in general, cells with intense exocytotic or endocytotic activity show the highest mRNA levels.

Diverse roles of integrins in human T lymphocyte biology.

T lymphocytes are the primary cells responsible for maintaining the immune system. There are many intricate mechanisms involved in the regulation of T cells and the integrin family of adhesive surface proteins plays a pivotal role in the control of T lymphocyte activation and functions. Integrins are heterodimeric transmembrane proteins that are not merely adhesion molecules but also function in T cell coactivation by providing a scaffold for signaling and cytoskeletal proteins that are adept at transmitting signals from the inside of the cell to the outside ("inside-out signaling") or from the outside of the cell to the inside ("outside-in signaling"). The signaling property of integrins allows for rapid responses to changes in the microenviroment of the lymphocyte. Therefore, whether the T cell needs to adhere or detach, integrins can quickly accommodate either state of the cell. Once cells are guided to sites of infection, inflammation, or antigen presentation, integrins can also participate in the initiation, maintenance, or termination of the response. This review will focus on the aspects of integrin-mediated T cell coactivation, affinity and avidity control of integrins, signaling molecules involved with integrins, association of integrins in lipid microdomains, and negative regulation of integrins.

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.

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.

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.

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.

Applying small RNA molecules to the directed treatment of human diseases: realizing the potential.

Small interfering RNAs (siRNA) and microRNAs (miRNA) are gaining considerable attention in the pharmaceutical and biotechnology industries, as research has revealed their likely clinical and agricultural applications. The capacity of siRNAs to dramatically and specifically reduce the expression of targeted genes has spawned multiple clinical trials to establish the therapeutic potential of small RNAs targeting viral, cancer and other disease-related genes. The successful application of siRNAs will enable the development of therapeutic applications based on miRNAs that have been observed to contribute to a variety of human diseases. This article reviews advances that have been made to apply small RNAs as therapeutics.

Adoption of array technologies into the clinical laboratory.

Array-based methods are making substantial contributions to the discovery of disease biomarkers and are fueling the growth of multianalyte testing for disease diagnosis and treatment. The distillation of high-density array results into sets of signature markers promises to improve disease staging, risk stratification and treatment decisions. To accommodate the growing requirement for multiplex testing, clinical laboratories are converting several single-analyte tests into array-based formats. However, adoption of array technologies provides several challenges to the laboratory, which must evaluate these new formats, train laboratory personnel, market the new services and obtain reimbursement for new analytes. Liquid-bead arrays are an attractive format for routine clinical diagnostics due to a combination of appropriate analyte density, simultaneous array decoding and detection, and flexibility for rapid customization. In this review, the suitability of several array platforms to diagnostic testing and applications of liquid-bead arrays for cystic fibrosis testing, multidisease carrier status assays and leukemia subtyping are discussed. As our understanding of the clinical utility of new or established biomarkers and recommendations for testing change, flexibility and adaptability of array platforms will be imperative. Future development of novel assay formats and improved quantitation will expand the number of diseases tested and lead to further integration into the diagnostic laboratory.

Movement of calmodulin between cells in the ovary and embryo of drosophila.

Calmodulin (CaM) is an essential component of calcium signaling in multicellular organisms. We used null mutations of the Drosophila CaM gene (Cam) in combination with clonal analysis and immunolocalization to examine the effects of loss of Cam function in the ovarian germline and developing embryo. These studies have uncovered unexpected and striking movements of CaM protein within these tissues. In the ovary, evidence for transfer of CaM from an external source, across plasma membranes, into the germline cells was obtained. In late embryogenesis, maternally derived CaM protein relocalizes dramatically within the nervous system of both wildtype and Cam null embryos-a process that may also involve movement across cell membranes. These findings indicate dynamic, unsuspected elements to the in vivo functions of CaM in the whole organism.