Pancreatic cyst fluid harbors a unique microbiome

Impact of next-generation sequencing on the clinical diagnosis of pancreatic cysts

Novel expression of vascular endothelial growth factor isoforms in the pancreas and pancreatic cystic lesions

We read with interest the paper by Singhi et al .1 The authors prospectively assessed using targeted next-generation sequencing (NGS) 626 pancreatic cyst fluid (PCF) samples from 595 patients. They...

Approximately half of all pancreatic cysts are neoplastic, mainly comprising intraductal papillary mucinous neoplasms (IPMN), which can progress to invasive carcinoma. Current Fukuoka guidelines have limited sensitivity and specificity in predicting progression of asymptomatic pancreatic cysts. We present first results of the prospective ZYSTEUS biomarker study investigating (i) whether detection of driver mutations in IPMN by liquid biopsy is technically feasible, (ii) which compartment of IPMN is most suitable for analysis, and (iii) implications for clinical diagnostics. Twenty-two patients with clinical inclusion criteria were enrolled in ZYSTEUS. Fifteen cases underwent endoscopic ultrasound (EUS)-guided fine-needle aspiration and cytological diagnostics. Cellular and liquid fraction of the cysts of each case were separated and subjected to deep targeted next generation sequencing (NGS). Clinical parameters, imaging findings (EUS and MRI), and follow-up data were collected continuously. All IPMN cases (n = 12) showed at least one mutation in either KRAS (n = 11) or GNAS (n = 4). Three cases showed both KRAS and GNAS mutations. Six cases harbored multiple KRAS/GNAS mutations. In the three cases with pseudocysts, no KRAS or GNAS mutations were detected. DNA yields were higher and showed higher mutation diversity in the cellular fraction. In conclusion, mutation detection in pancreatic cyst fluid is technically feasible with more robust results in the cellular than in the liquid fraction. Current results suggest that, together with imaging, targeted sequencing supports discrimination of IPMN from pseudocysts. The prospective design of ZYSTEUS will provide insight into diagnostic value of NGS in preoperative risk stratification. Our data provide evidence for an oligoclonal nature of IPMN.

Pancreatic cancer is now the fourth leading cause of cancer deaths in the United States, and it is associated with an alarmingly low 5-year survival rate of 5%. However, a patient's prognosis is considerably improved when the malignant lesions are identified at an early stage of the disease and removed by surgical resection. Unfortunately, the absence of a practical screening strategy and clinical diagnostic test for identifying premalignant lesions within the pancreas often prevents early detection of pancreatic cancer. To aid in the development of a molecular screening system for early detection of the disease, we have performed glycomic and glycoproteomic profiling experiments on 21 pancreatic cyst fluid samples, including fluids from mucinous cystic neoplasms and intraductal papillary mucinous neoplasms, two types of mucinous cysts that are considered high risk to undergo malignant transformation. A total of 80 asparagine-linked (N-linked) glycans, including high mannose and complex structures, were identified. Of special interest was a series of complex N-linked glycans containing two to six fucose residues, located predominantly as substituents on β-lactosamine extensions. Following the observation of these "hyperfucosylated" glycans, bottom-up proteomics experiments utilizing a label-free quantitative approach were applied to the investigation of two sets of tryptically digested proteins derived from the cyst fluids: 1) all soluble proteins in the raw samples and 2) a subproteome of the soluble cyst fluid proteins that were selectively enriched for fucosylation through the use of surface-immobilized Aleuria aurantia lectin. A comparative analysis of these two proteomic data sets identified glycoproteins that were significantly enriched by lectin affinity. Several candidate glycoproteins that appear hyperfucosylated were identified, including triacylglycerol lipase and pancreatic α-amylase, which were 20- and 22-fold more abundant, respectively, following A. aurantia lectin enrichment.

Mo1213 Differentiation Between Chronic Pancreatitis and Pancreatic Cancer by Using Metabolomic Analysis

Sensitive detection of internal tandem duplication (ITD) mutation of FLT3 is very important in acute myeloid leukemia. To increase detection sensitivity of FLT3-ITD, we developed new detection algorithm using next generation sequencing (NGS) data. We validated results using nested polymerase chain reaction (PCR) methods. We compared results of NGS data, nested PCR and conventional PCR methods. First, using whole exome sequencing data of 83 AML patients, we applied calling algorithm for FLT3-ITD. Briefly, to detect ITDs with NGS data, the reads are aligned to a reference sequence (UCSC hg19), with BWA which is a read aligner allowing soft-clipping. Some reads can be an indication of the occurrence of ITD and BWA aligns those reads as soft-clipped. Second, we deigned two types of primer for Nested PCR. The first primer was targeted wildly for between exon14 and exon15 of FLT3 gene. Nested PCR primer was deigned to target previously reported regions which are frequently occurred ITD mutation. PCR reactions of two steps were performed using the PCR primers sequentially. In these 83 patients, FLT3-ITD was positive only in 7 patients when tested by conventional PCR methods. When NGS detection method was applied, this resulted in positive FLT3-ITD in 11 patients (11/83, 13%). When validation was performed using nested PCR, FLT3-ITD was confirmed in all of 11 patients. Nested PCR detected additional 4 patient positive for FLT3-ITD in this population. For 68 patients, FLT3-ITD was negative by both NGS and nested PCR method. Overall, NGS method improved sensitivity of FLT3-ITD detection by 57% in this population. And the concordance rate of NGS method and nested PCR was 95.2% (79/83). Then we investigated clinical significance of sensitive FLT3-ITD detection. For this, we performed nested PCR and conventional PCR at the same time in 238 AML patients to detect FLT3-ITD. Positive rate for FLT3-ITD was 20% (48/238) and 10% (24/238) by nested PCR and conventional PCR respectively. When survival analysis was performed, among patients with negative FLT3-ITD result by conventional PCR, patients who showed positive for FLT3-ITD by nested PCR had shorter overall survival compared to those who showed negative for FLT3-ITD by nested PCR. (p = 0.03). This implies that sensitive FLT3-ITD detection using nested PCR is clinically meaningful. Diagnosis of FLT3-ITD is very important genetic factor, leading a therapeutic direction for AML patient. Here we report that we have developed alternative more sensitive detection methods for FLT3-ITD based on nested PCR and NGS. Sensitive detection of FLT3-ITD was clinically meaningful, suggesting that these methods should be incorporated in a future clinical practice. Also, we want to note that, NGS method is capable of quantifying FLT3-ITD size and amount in AML patients. Citation Format: Daeyoon Kim, Yoojin Hong, Youngil Koh, Sung-Soo Yoon, Choong-Hyun Sun, Kwang-Sung Ahn, Seungmook Lee, Hongseok Yun, Suyeon Lee. Improved sensitive detection method of FLT3 (FMS-like tyrosine kinase) internal tandem duplication (ITD) mutation using next-generation sequencing technology and nested PCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3186.

Background Most clinical chemistry assays are intended for use in serum, plasma, urine, and/or CSF with no performance characteristics on body fluids. However, testing of body fluids can be medically necessary for certain diagnoses. Such as determining the cause of fluid buildup in peritoneal spaces, pleural effusions, whether effusions are transudates or exudates, and even diagnosing some joint disorders. To provide these medically necessary tests, the burden is on the laboratory to perform regulatory-required validations. Herein is the validation of 11 clinical chemistry analytes in several common body fluids. Methods Mixing and stability studies were performed for the following analytes on the Roche cobas c702 (Roche Diagnostics), using the methods intended for use with serum/plasma samples: Albumin, Amylase, Bilirubin, Urea Nitrogen, Cholesterol, Creatinine, Glucose, Lactate Dehydrogenase, Lipase, Protein, and Triglycerides. Six individual fluid samples were mixed with the intended use matrix in 3 ratios: 1:3, 1:1, and 3:1. The following matrices were evaluated: pleural, peritoneal, JP drainage, synovial, and pancreatic cyst fluids. Stabilities were tested for 7 days at room temperature (RT) and 2-8°C. A maximum dilution study was also performed for Amylase. Results Mixing Study: The results are shown in the table below: Stability: For most body fluids, analytes were stable for 7 days at RT and 2-8 °C. The following analytes displayed shorter stability at RT: Bilirubin (peritoneal: 2 days, pleural: 1 day, JP drainage: 1 day), Urea Nitrogen (JP drain fluid: 2 days), Lipase (peritoneal fluid 5 days), and Triglycerides (peritoneal fluid: 5 days). Lactate dehydrogenase in pleural fluid showed a stability of 5 days at RT and 2 days at 2-8 °C. Maximum Dilution A 1:505 compounded manual and automated dilution displayed a <7% bias. Conclusions No sample matrix interferences were observed for any of the body fluids-analyte pairs tested allowing for this medically necessary testing.

<p>The biomarker candidates previously reported in pancreatic cyst fluids.</p>

Introduction: As a result of the increasing use and improvements in imaging technologies such as computed tomography and magnetic resonance imaging, the frequency at which pancreatic cysts are detected has been growing. Diagnosing the biological nature of such pancreatic cystic lesions remains a difficult challenge to clinicians, as diagnosis by cytology is insensitive and there are currently no reliable methods to measure the degree malignant potential of cysts. Therefore, new markers capable of differentiating benign from malignant IPMNs would assist in establishing an optimal therapeutic strategy for treatment by identifying patients who would benefit from surgery or surveillance. We applied liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics and multiple reaction monitoring (LC-MRM-MS) analyses on a collection of IPMN cyst fluid samples to identify and verify potential biomarker which may serve as diagnostic indicators of malignant potential and assist in risk stratification. Methods: A total of 15 IPMN pancreatic cyst fluid samples (5 adenomas, 5 carcinomas in situ and 5 invasive carcinomas) were obtained from freshly resected cysts and were analyzed by shotgun proteomics. Aliquots of each sample were reduced, alkylated and digested with trypsin and the resulting peptides were fractionated by isoelectric focusing (pI 3.5-4.7) into 15 fractions. Reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed using data-dependent scanning on a Thermo Orbitrap Velos instrument. Following LC-MS/MS analysis, tandem mass spectra were searched against the human IPI database (v.3.56) using the Myrimatch algorithm and the search results filtered using IDPicker at a 5% FDR. A spectral counting approach was applied to detect statistically significant differences in protein abundances with the Quasitel algorithm. MRM assays were configured for differential proteins and further verified in a new collection of unfractionated IPMN cyst fluids with varying degrees of dysplasia (mild, moderate, severe and invasive carcinoma). LC-MRM-MS was performed on peptide samples and were analyzed in triplicate (4 peptides per protein and 5 transitions per peptides) on a triple quadrupole mass spectrometer (TSQ Vantage, Thermo Fisher Scientific, Waltham MA) equipped with an Eksigent nanoLC Ultra 1D Plus pump and AS-2 autosampler (Eksigent Technologies, Dublin CA). A single isotopically labeled peptide was spiked into the samples which served as the normalization reference for all of the peptides in the analysis. All data analysis was performed using Skyline. Preliminary Results: A total of 373,673 spectra were confidently identified, which corresponded to 5,259 protein groups across all biological and technical replicates. An average of 3,534, 3,393, and 4,597 protein groups were identified in the adenomas, carcinomas in situ, and invasive carcinomas, respectively. Spectral count data indicated approximately 50 proteins differentially expressed between benign adenomas and carcinoma in situ and 176 proteins different between adenomas and invasive cancers. From these data, a list of 30 potential candidates was generated, which included proteins upregulated in carcinoma in situ and invasive cancer, as well as several down-regulated proteins, which included common digestive enzymes, such as amylase 2B and elastases. In addition, several MUC isoforms and CEACAM were also targeted, as these have been suggested to distinguish nonmucinous from mucinous cystic lesions. The preliminary MRM data are consistent with the trends observed in the spectral count data from shotgun analyses. The analyses of pancreatic cyst fluids using proteomics platforms may lead to biomarkers that can assist clinicians in differentiating benign from malignant cysts, thus leading to improved diagnosis and treatment. Citation Format: Lisa J. Zimmerman, Cristina R. Ferrone, Nipun Merchant, Robbert J. C. Slebos, Daniel C. Liebler. Proteomic analysis of cyst fluids from intraductal papillary mucinous neoplasms [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B8.

Pancreatic cysts or dilated pancreatic ducts are often found by cross-sectional imaging, but only mucinous lesions can become malignant. Therefore, distinction between mucinous and non-mucinous lesions is crucial for adequate patient management. We performed a prospective study including targeted next generation sequencing (NGS) of cell-free DNA in the diagnostic endoscopic ultrasound (EUS)-guided workup. Pancreatic cyst(s) or main duct fluid obtained by EUS-guided FNA was analysed by carcinoembryonic antigen (CEA), cytology and deep targeted NGS of 14 known gastrointestinal cancer genes (AKT1, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53, APC) with a limit of detection down to variant allele frequency of 0.01%. Results were correlated to histopathology and clinical follow-up. One hundred and thirteen patients with pancreatic cyst(s) and/or a dilated pancreatic main duct (≥5 mm) were screened. Sixty-six patients had to be excluded, mainly due to inoperability or small cyst size (≤10 mm). Forty-seven patients were enrolled for further analysis. A final diagnosis was available in 27 cases including 8 negative controls. In 43/47 (91.5%) of patients a KRAS- and/or GNAS-mutation was diagnosed by NGS. 27.0% of the KRAS-mutated and 10.0% of the GNAS-mutated lesions harbored multiple mutations. KRAS/GNAS-testing by NGS, cytology, and CEA had a sensitivity and specificity of 94.7/100%, 38.1/100%, and 42.1/75.0%, respectively. KRAS/GNAS-testing was significantly superior to CEA (P = .0209) and cytology (P = .0016). In conclusion, KRAS/GNAS-testing by deep targeted NGS is a suitable method to distinguish mucinous from non-mucinous pancreatic lesions, suggesting its usage as a single diagnostic test. Results must be confirmed in a larger cohort.

P099 : A SINGLE CENTER COMPARISON OF HIGH-RESOLUTION HLA TYPING BY NEXT-GENERATION VERSUS SANGER SEQUENCING

BackgroundGermline DNA mutations that increase the susceptibility of a patient to certain cancers have been identified in various genes, and patients can be screened for mutations in these genes to assess their level of risk for developing cancer. Traditional methods using Sanger sequencing focus on small groups of genes and therefore are unable to screen for numerous genes from several patients simultaneously. The goal of the present study was to validate a 25-gene panel to assess genetic risk for cancer in 8 different tissues using next generation sequencing (NGS) techniques.MethodsTwenty-five genes associated with hereditary cancer syndromes were selected for development of a panel to screen for risk of these cancers using NGS. In an initial technical assessment, NGS results for BRCA1 and BRCA2 were compared with Sanger sequencing in 1864 anonymized DNA samples from patients who had undergone previous clinical testing. Next, the entire gene panel was validated using parallel NGS and Sanger sequencing in 100 anonymized DNA samples. Large rearrangement analysis was validated using NGS, microarray comparative genomic hybridization (CGH), and multiplex ligation-dependent probe amplification analyses (MLPA).ResultsNGS identified 15,877 sequence variants, while Sanger sequencing identified 15,878 in the BRCA1 and BRCA2 comparison study of the same regions. Based on these results, the NGS process was refined prior to the validation of the full gene panel. In the validation study, NGS and Sanger sequencing were 100% concordant for the 3,923 collective variants across all genes for an analytical sensitivity of the NGS assay of >99.92% (lower limit of 95% confidence interval). NGS, microarray CGH and MLPA correctly identified all expected positive and negative large rearrangement results for the 25-gene panel.ConclusionThis study provides a thorough validation of the 25-gene NGS panel and indicates that this analysis tool can be used to collect clinically significant information related to risk of developing hereditary cancers.

Genetic landscape for majority and minority HIV-1 drug resistance mutations in antiretroviral therapy naive patients in Accra, Ghana

Background: Bacterial DNA extraction is a preliminary step for PCR studies carried out on bacteria for diagnosis and detection of antimicrobial resistance genes. Objectives: Testing efficiency of two physical methods (boiling and freezing-thawing) and two chemical methods (SDS and tween based methods) for extraction of bacterial DNA suitable for conventional as well as real time PCRs in comparison to a costly ready to use kit method. Methodology: Escherichia coli ATCC 25922 was submitted for five DNA extraction methods including QIAamp ready kit, boiling, freezing-thawing in TE, SDS method, and tween method. DNA purification by ethanol precipitation and wash was carried out after tested physical and chemical methods. The extracted DNAs were assessed by Nanodrop spectrophotometer, conventional PCR followed by Uviband quantification, and semiquantitative SYBR green based RT- PCR. Results: Nanodrop assessment showed that QIAamp kit and tween method produced the highest DNA yields; 78.1 ng/µl and 49.72 ng/µl, respectively, while SDS method gave the lowest. Both QIAamp kit and tween methods produced comparable DNA in concentration and purity. All tested methods extracted DNA amplifiable by PCR. In conventional PCR, the concentrations ranged from 287.31 to330.46 ng/5µl for boiling and QIAamp kit, respectively. In RT-PCR, the highest CT values were obtained by OIAmp kit, followed by tween, freezing-thawing in TE, SDS, and boiling methods with narrow range of variation (from 30.80 to 31.85). No significant statistical differences were detected between tested methods in the results of PCRs either conventional or real time. Conclusion: Boiling in water, freezing-thawing in TE, tween and SDS based methods followed by DNA precipitation provide rapid, effective, less laborious and truly cost-effective methods for extraction of bacterial DNA suitable for PCR studies.