Multiregion whole-exome sequencing of intraductal papillary mucinous neoplasms reveals frequent somatic KLF4 mutations predominantly in low-grade regions.

OBJECTIVE: Intraductal papillary mucinous neoplasms (IPMNs) are non-invasive precursor lesions that can progress to invasive pancreatic cancer and are classified as low-grade or high-grade based on the morphology of the neoplastic epithelium. We aimed to compare genetic alterations in low-grade and high-grade regions of the same IPMN in order to identify molecular alterations underlying neoplastic progression. DESIGN: We performed multiregion whole exome sequencing on tissue samples from 17 IPMNs with both low-grade and high-grade dysplasia (76 IPMN regions, including 49 from low-grade dysplasia and 27 from high-grade dysplasia). We reconstructed the phylogeny for each case, and we assessed mutations in a novel driver gene in an independent cohort of 63 IPMN cyst fluid samples. RESULTS: Our multiregion whole exome sequencing identified KLF4, a previously unreported genetic driver of IPMN tumorigenesis, with hotspot mutations in one of two codons identified in >50% of the analyzed IPMNs. Mutations in KLF4 were significantly more prevalent in low-grade regions in our sequenced cases. Phylogenetic analyses of whole exome sequencing data demonstrated diverse patterns of IPMN initiation and progression. Hotspot mutations in KLF4 were also identified in an independent cohort of IPMN cyst fluid samples, again with a significantly higher prevalence in low-grade IPMNs. CONCLUSION: Hotspot mutations in KLF4 occur at high prevalence in IPMNs. Unique among pancreatic driver genes, KLF4 mutations are enriched in low-grade IPMNs. These data highlight distinct molecular features of low-grade and high-grade dysplasia and suggest diverse pathways to high-grade dysplasia via the IPMN pathway.

Intraductal Papillary Mucinous Neoplasms Arise From Multiple Independent Clones, Each With Distinct Mutations.

BACKGROUND & AIMS: Intraductal papillary mucinous neoplasms (IPMNs) are lesions that can progress to invasive pancreatic cancer and constitute an important system for studies of pancreatic tumorigenesis. We performed comprehensive genomic analyses of entire IPMNs to determine the diversity of somatic mutations in genes that promote tumorigenesis. METHODS: We microdissected neoplastic tissues from 6-24 regions each of 20 resected IPMNs, resulting in 227 neoplastic samples that were analyzed by capture-based targeted sequencing. Somatic mutations in genes associated with pancreatic tumorigenesis were assessed across entire IPMN lesions, and the resulting data were supported by evolutionary modeling, whole-exome sequencing, and in situ detection of mutations. RESULTS: We found a high prevalence of heterogeneity among mutations in IPMNs. Heterogeneity in mutations in KRAS and GNAS was significantly more prevalent in IPMNs with low-grade dysplasia than in IPMNs with high-grade dysplasia (P < .02). Whole-exome sequencing confirmed that IPMNs contained multiple independent clones, each with distinct mutations, as originally indicated by targeted sequencing and evolutionary modeling. We also found evidence for convergent evolution of mutations in RNF43 and TP53, which are acquired during later stages of tumorigenesis. CONCLUSIONS: In an analysis of the heterogeneity of mutations throughout IPMNs, we found that early-stage IPMNs contain multiple independent clones, each with distinct mutations, indicating their polyclonal origin. These findings challenge the model in which pancreatic neoplasms arise from a single clone. Increasing our understanding of the mechanisms of IPMN polyclonality could lead to strategies to identify patients at increased risk for pancreatic cancer.

Combined circulating tumor DNA and protein biomarker-based liquid biopsy for the earlier detection of pancreatic cancers.

The earlier diagnosis of cancer is one of the keys to reducing cancer deaths in the future. Here we describe our efforts to develop a noninvasive blood test for the detection of pancreatic ductal adenocarcinoma. We combined blood tests for KRAS gene mutations with carefully thresholded protein biomarkers to determine whether the combination of these markers was superior to any single marker. The cohort tested included 221 patients with resectable pancreatic ductal adenocarcinomas and 182 control patients without known cancer. KRAS mutations were detected in the plasma of 66 patients (30%), and every mutation found in the plasma was identical to that subsequently found in the patient's primary tumor (100% concordance). The use of KRAS in conjunction with four thresholded protein biomarkers increased the sensitivity to 64%. Only one of the 182 plasma samples from the control cohort was positive for any of the DNA or protein biomarkers (99.5% specificity). This combinatorial approach may prove useful for the earlier detection of many cancer types.

Multi-institutional Validation Study of Pancreatic Cyst Fluid Protein Analysis for Prediction of High-risk Intraductal Papillary Mucinous Neoplasms of the Pancreas.

OBJECTIVE: Preliminary work by our group suggested that proteins within the pancreatic cyst fluid (CF) may discriminate degree of IPMN dysplasia. We sought to externally validate these markers and determine whether their inclusion in a preoperative clinical nomogram could increase diagnostic accuracy. SUMMARY BACKGROUND DATA: IPMN is the most common radiographically identifiable precursor to pancreatic cancer; however, the timing and frequency of its malignant progression are unknown, and there are currently no reliable preoperative tests that can determine the grade of dysplasia in IPMN. METHODS: Clinical and radiographic data, as well as CF samples, were obtained from 149 patients who underwent resection for IPMN at 1 of 3 institutions. High-risk disease was defined as the presence of high-grade dysplasia or invasive carcinoma. Multianalyte bead array analysis (Luminex) of CF was performed for 4 protein markers that were previously associated with high-risk disease. Logistic regression models were fit on training data, with and without adjustment for a previously developed clinical nomogram and validated with an external testing set. The models incorporating clinical risk score were presented graphically as nomograms. RESULTS: Within the group of 149 resected patients, 89 (60%) had low-risk disease, and 60 (40%) had high-risk disease. All 4 CF markers (MMP9, CA72-4, sFASL, and IL-4) were overexpressed in patients with high-risk IPMN (P < 0.05). Two predictive models based on preselected combinations of CF markers had concordance indices of 0.76 (Model-1) and 0.80 (Model-2). Integration of each CF marker model into a previously described clinical nomogram leads to increased discrimination compared with either the CF models or nomogram alone (c-indices of 0.84 and 0.83, respectively). CONCLUSIONS: This multi-institutional study validated 2 CF protein marker models for preoperative identification of high-risk IPMN. When combined with a clinical nomogram, the ability to predict high-grade dysplasia was even stronger.

Targeted DNA Sequencing Reveals Patterns of Local Progression in the Pancreatic Remnant Following Resection of Intraductal Papillary Mucinous Neoplasm (IPMN) of the Pancreas.

OBJECTIVE: The aim of this study was to characterize patterns of local progression following resection for pancreatic intraductal papillary mucinous neoplasms (IPMN) using targeted next-generation sequencing (NGS). BACKGROUND: Progression of neoplastic disease in the remnant pancreas following resection of IPMN may include development of a new IPMN or ductal adenocarcinoma (PDAC). However, it is not clear whether this progression represents recurrence of the same neoplasm or an independent second neoplasm. METHODS: Targeted-NGS on genes commonly mutated in IPMN and PDAC was performed on tumors from (1) 13 patients who developed disease progression in the remnant pancreas following resection of IPMN; and (2) 10 patients who underwent a resection for PDAC and had a concomitant IPMN. Mutations in the tumors were compared in order to determine the relationship between neoplasms. In parallel, clinical and pathological characteristics of 260 patients who underwent resection of noninvasive IPMN were reviewed to identify risk factors associated with local progression. RESULTS: We identified 3 mechanisms underlying local progression in the remnant pancreas: (1) residual microscopic disease at the resection margin, (2) intraparenchymal spread of neoplastic cells, leading to an anatomically separate but genetically related recurrence, and (3) multifocal disease with genetically distinct lesions. Analysis of the 260 patients with noninvasive IPMNs showed that family history of pancreatic cancer (P = 0.027) and high-grade dysplasia (HGD) (P = 0.003) were independent risk factors for the development of an IPMN with HGD or an invasive carcinoma in the remnant pancreas. CONCLUSIONS: Using NGS, we identify distinct mechanisms for development of metachronous or synchronous neoplasms in patients with IPMN. Patients with a primary IPMN with HGD or with positive family history are at an increased risk to develop subsequent high-risk neoplasms in the remnant pancreas.

Duodenal Involvement is an Independent Prognostic Factor for Patients with Surgically Resected Pancreatic Ductal Adenocarcinoma.

BACKGROUND: The current staging system for pancreatic ductal adenocarcinoma (PDAC) includes information about size and local extension of the primary tumor (T stage). The value of incorporating any local tumor extension into pancreatic staging systems has been questioned because it often is difficult to evaluate tumor extension to the peri-pancreatic soft tissues and because most carcinomas of the head of the pancreas infiltrate the intra-pancreatic common bile duct. This study sought to evaluate the prognostic implications of having PDAC with local tumor extension. METHODS: A single-institution, prospectively collected database of 1128 patients who underwent surgical resection for PDAC was queried to examine the prognostic significance of extra-pancreatic tumor involvement ("no involvement," "duodenal involvement," and "extensive involvement"; e.g., gastric, colon or major vein involvement). RESULTS: The median overall survival for the patients without extra-pancreatic involvement was 26 months versus 19 months for the patients with duodenal involvement and 16 months for the patients with extensive involvement (p < 0.001). In the multivariable analysis, duodenal and extensive involvement independently predicted increased risk of death compared with no involvement (hazard ratio [HR] 1.30; 95% confidence interval [CI] 1.08-1.57 and 1.78; 95% CI 1.25-2.55, respectively). A multivariable model combining duodenal and extensive extra-pancreatic involvement, tumor grade, lymph node ratio, and other prognostic features had the highest c-index (0.67). CONCLUSIONS: Inclusion of duodenal involvement in the staging of PDAC adds independent prognostic information.

A combination of molecular markers and clinical features improve the classification of pancreatic cysts.

BACKGROUND & AIMS: The management of pancreatic cysts poses challenges to both patients and their physicians. We investigated whether a combination of molecular markers and clinical information could improve the classification of pancreatic cysts and management of patients. METHODS: We performed a multi-center, retrospective study of 130 patients with resected pancreatic cystic neoplasms (12 serous cystadenomas, 10 solid pseudopapillary neoplasms, 12 mucinous cystic neoplasms, and 96 intraductal papillary mucinous neoplasms). Cyst fluid was analyzed to identify subtle mutations in genes known to be mutated in pancreatic cysts (BRAF, CDKN2A, CTNNB1, GNAS, KRAS, NRAS, PIK3CA, RNF43, SMAD4, TP53, and VHL); to identify loss of heterozygozity at CDKN2A, RNF43, SMAD4, TP53, and VHL tumor suppressor loci; and to identify aneuploidy. The analyses were performed using specialized technologies for implementing and interpreting massively parallel sequencing data acquisition. An algorithm was used to select markers that could classify cyst type and grade. The accuracy of the molecular markers was compared with that of clinical markers and a combination of molecular and clinical markers. RESULTS: We identified molecular markers and clinical features that classified cyst type with 90%-100% sensitivity and 92%-98% specificity. The molecular marker panel correctly identified 67 of the 74 patients who did not require surgery and could, therefore, reduce the number of unnecessary operations by 91%. CONCLUSIONS: We identified a panel of molecular markers and clinical features that show promise for the accurate classification of cystic neoplasms of the pancreas and identification of cysts that require surgery.

Time to progression of pancreatic ductal adenocarcinoma from low-to-high tumour stages.

OBJECTIVE: Although pancreatic ductal adenocarcinoma is considered a rapidly progressive disease, mathematical models estimate that it takes many years for an initiating pancreatic cancer cell to grow into an advanced stage cancer. In order to estimate the time it takes for a pancreatic cancer to progress through different tumor, node, metastasis (TNM) stages, we compared the mean age of patients with pancreatic cancers of different sizes and stages. DESIGN: Patient age, tumour size, stage and demographic information were analysed for 13,131 patients with pancreatic ductal adenocarcinoma entered into the National Cancer Institute's Surveillance, Epidemiology and End Results (SEER) database. Multiple linear regression models for age were generated, adjusting for patient ethnicity, gender, tumour location and neoplastic grades. RESULTS: African-American ethnicity and male gender were associated with an earlier age at diagnosis. Patients with stage I cancers (mean age 64.8 years) were on average 1.3 adjusted years younger at diagnosis than those with stage IV cancers (p=0.001). Among patients without distant metastases, those with T1 stage cancers were on average 1.06 and 1.19 adjusted years younger, respectively, than patients with T3 or T4 cancers (p=0.03 for both). Among patients with stage IIB cancers, those with T1/T2 cancers were 0.79 adjusted years younger than those with T3 cancers (p=0.06). There was no significant difference in the mean adjusted age of patients with stage IA versus stage IB cancers. CONCLUSIONS: These results are consistent with the hypothesis that once pancreatic ductal adenocarcinomas become detectable clinically progression from low-stage to advanced-stage disease is rapid.

Outcome of superior mesenteric-portal vein resection during pancreatectomy for borderline ductal adenocarcinoma: results of a prospective comparative study.

BACKGROUND: Approximately 20 % of patients affected by pancreatic ductal adenocarcinoma are amenable to surgical resection. Several tumours are reported as "borderline resectable" because of their proximity to the major vessels. In the effort to achieve a radical tumour removal, vein resection has been proposed, but its oncological benefits remain debated. METHODS: Our aim is to investigate morbidity, mortality and survival after pancreatectomy with vein resection. RESULTS: Forty patients underwent pancreatectomy and vein resection (group A), and 20 patients (group B) underwent bilio-enteric and/or gastro-entero bypass. In group A, cancer vein invasion was microscopically proven in 14 cases (35 %). Vein infiltration, tumour differentiation and node-positive disease were not adverse prognostic variables. No difference in survival was seen over a 1-year follow-up. After this period, group A showed significant survival benefits with a longer stabilisation of the disease (p = 0.005). Tumour-free resection margins and adjuvant chemoradiotherapy were the most important prognostic factors (p < 0.05). CONCLUSIONS: Suspicion of vein infiltration should not be a contraindication to resection. Pancreatectomy can be safely performed with an acceptable morbidity and better survival trend.

Whole-exome sequencing of neoplastic cysts of the pancreas reveals recurrent mutations in components of ubiquitin-dependent pathways.

More than 2% of adults harbor a pancreatic cyst, a subset of which progresses to invasive lesions with lethal consequences. To assess the genomic landscapes of neoplastic cysts of the pancreas, we determined the exomic sequences of DNA from the neoplastic epithelium of eight surgically resected cysts of each of the major neoplastic cyst types: serous cystadenomas (SCAs), intraductal papillary mucinous neoplasms (IPMNs), mucinous cystic neoplasms (MCNs), and solid pseudopapillary neoplasms (SPNs). SPNs are low-grade malignancies, and IPMNs and MCNs, but not SCAs, have the capacity to progress to cancer. We found that SCAs, IPMNs, MCNs, and SPNs contained 10 ± 4.6, 27 ± 12, 16 ± 7.6, and 2.9 ± 2.1 somatic mutations per tumor, respectively. Among the mutations identified, E3 ubiquitin ligase components were of particular note. Four of the eight SCAs contained mutations of the von Hippel-Lindau gene (VHL), a key component of the VHL ubiquitin ligase complex that has previously been associated with renal cell carcinomas, SCAs, and other neoplasms. Six of the eight IPMNs and three of the eight MCNs harbored mutations of RNF43, a gene coding for a protein with intrinsic E3 ubiquitin ligase activity that has not previously been found to be genetically altered in any human cancer. The preponderance of inactivating mutations in RNF43 unequivocally establish it as a suppressor of both IPMNs and MCNs. SPNs contained remarkably few genetic alterations but always contained mutations of CTNNB1, previously demonstrated to inhibit degradation of the encoded protein (ß-catenin) by E3 ubiquitin ligases. These results highlight the essential role of ubiquitin ligases in these neoplasms and have important implications for the diagnosis and treatment of patients with cystic tumors.

Perioperative management of patients undergoing pancreatic resection: implementation of a care plan in a tertiary-care center.

Evidence-based perioperative management is important for a successful outcome after pancreatic surgery. Use of protocolized pathways of care based on fast-track concepts has been shown to reduce hospital stay and contain costs. These regimens include pain control, early device removal (nasogastric tube, abdominal drains), enforced early mobilization, and early oral feeding. In this article, current evidence on perioperative management of pancreatic resections was analyzed in the attempt to implement our institutional care plan.

Clinicopathological characteristics and molecular analyses of multifocal intraductal papillary mucinous neoplasms of the pancreas.

OBJECTIVE: To examine the clinicopathologic features and clonal relationship of multifocal intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. BACKGROUND: Intraductal papillary mucinous neoplasms are increasingly diagnosed cystic precursor lesions of pancreatic cancer. Intraductal papillary mucinous neoplasms can be multifocal and a potential cause of recurrence after partial pancreatectomy. METHODS: Thirty four patients with histologically documented multifocal IPMNs were collected and their clinicopathologic features catalogued. In addition, thirty multifocal IPMNs arising in 13 patients from 3 hospitals were subjected to laser microdissection followed by KRAS pyrosequencing and loss of heterozygosity (LOH) analysis on chromosomes 6q and 17p. Finally, we sought to assess the clonal relationships among multifocal IPMNs. RESULTS: We identified 34 patients with histologically documented multifocal IPMNs. Synchronous IPMNs were present in 29 patients (85%), whereas 5 (15%) developed clinically significant metachronous IPMNs. Six patients (18%) had a history of familial pancreatic cancer. A majority of multifocal IPMNs (86% synchronous, 100% metachronous) were composed of branch duct lesions, and typically demonstrated a gastric-foveolar subtype epithelium with low or intermediate grades of dysplasia. Three synchronous IPMNs (10%) had an associated invasive cancer. Molecular analysis of multiple IPMNs from 13 patients demonstrated nonoverlapping KRAS gene mutations in 8 patients (62%) and discordant LOH profiles in 7 patients (54%); independent genetic alterations were established in 9 of the 13 patients (69%). CONCLUSIONS: The majority of multifocal IPMNs arise independently and exhibit a gastric-foveolar subtype, with low to intermediate dysplasia. These findings underscore the importance of life-long follow-up after resection for an IPMN.

GNAS codon 201 mutations are uncommon in intraductal papillary neoplasms of the bile duct.

BACKGROUND: Activating point mutations of GNAS at codon 201 have been detected in approximately two thirds of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. Intraductal papillary neoplasms of the bile ducts (IPNBs) morphologically resemble pancreatic IPMNs. This study sought to assess the mutational status of GNAS at codon 201 in IPNBs. METHODS: Thirty-four patients were included. DNA from microdissected IPNBs was subjected to a polymerase chain reaction and ligation method for the detection of GNAS mutations at codon 201 and of KRAS mutations at codon 12. Mutational status was compared with clinical and pathologic data. RESULTS: The IPNBs had a median diameter of 3.5 cm and were located intrahepatically (n= 6), extrahepatically (n= 13), both intra- and extrahepatically (n= 4) or in the gallbladder (intracystic papillary neoplasms, n= 11). Most exhibited pancreatobiliary differentiation (n= 20), high-grade dysplasia (n= 26) and an associated adenocarcinoma (n= 20). Analysis of GNAS codon 201 identified only one mutant sample in a multifocal intestinal subtype intrahepatic IPNB with high-grade dysplasia. Six lesions harboured a KRAS codon 12 mutation. CONCLUSIONS: GNAS codon 201 mutations are uncommon in IPNBs, by contrast with pancreatic IPMNs. More comprehensive molecular profiling is needed to uncover the pathways involved in IPNB development.

The tumour immune microenvironment and microbiome of pancreatic intraductal papillary mucinous neoplasms.

Pancreatic intraductal papillary mucinous neoplasms (IPMNs) have gained substantial attention because they represent one of the only radiographically identifiable precursors of invasive pancreatic ductal adenocarcinoma. Although most of these neoplasms have low-grade dysplasia and will remain indolent, a subset of IPMNs will progress to invasive cancer. The role of the immune system in the progression of IPMNs is unclear, but understanding its role could reveal the mechanism of neoplastic progression and targets for immunotherapy to inhibit progression or treat invasive disease. The available evidence supports a shift in the immune composition of IPMNs during neoplastic progression. Although low-grade lesions contain a high proportion of effector T cells, high-grade IPMNs, and IPMNs with an associated invasive carcinoma lose the T-cell infiltrate and are characterised by a predominance of immunosuppressive elements. Several possible therapeutic strategies emerge from this analysis that are unique to IPMNs and its microbiome.

Presence of pancreatic intraepithelial neoplasia in the pancreatic transection margin does not influence outcome in patients with R0 resected pancreatic cancer.

BACKGROUND: Margin status is one of the strongest prognosticators after resection of pancreatic ductal adenocarcinoma (PDAC). The clinical significance of pancreatic intraepithelial neoplasia (PanIN) at a surgical margin has not been established. METHODS: A total of 208 patients who underwent R0 resection for PDAC between 2004 and 2008 were selected. Intraoperative frozen section slides containing the final pancreatic parenchymal transection margin were evaluated for presence or absence, number, and grade of PanINs. Data were compared to clinicopathologic factors, including patient survival. RESULTS: PanIN lesions were present in margins in 107 of 208 patients (51.4%). Median number of PanINs per pancreatic resection margin was 1 (range, 1-11). A total of 72 patients had PanIN-1 (34.6%), 44 had PanIN-2 (21.1%), and 16 had PanIN-3 (7.2%) at their margin. Overall median survival was 17.9 (95% confidence interval, 14-21.9) months. Neither the presence nor absence of PanIN nor histological grade had any significant correlation with important clinicopathologic characteristics. There were no significant survival differences between patients with or without PanIN lesions at the resection margin or among patients with PanIN-3 (carcinoma in situ) versus lower PanIN grades. However, patients with R1 resection had a significantly worse outcome compared with patients without invasive cancer at a margin irrespective of the presence of PanIN (P=0.02). CONCLUSIONS: The presence of PanINs at a resection margin does not affect survival in patients who undergo R0 resection for PDAC. These results have significant clinical implications for surgeons, because no additional resection seems to be indicated when intraoperative frozen sections reveal even high-grade PanIN lesions.

Elevated microRNA miR-21 levels in pancreatic cyst fluid are predictive of mucinous precursor lesions of ductal adenocarcinoma.

BACKGROUND: Biomarkers for the diagnostic classification of pancreatic cysts are urgently needed. Deregulated microRNA (miRNAs) expression is widespread in pancreatic cancer. We assessed whether aberrant miRNAs in pancreatic cyst fluid could be used as potential biomarkers for cystic precursor lesions of pancreatic cancer. METHODS: Cyst fluid specimens were prospectively collected from 40 surgically resected pancreatic cysts, and small RNAs were extracted. The 'mucinous' cohort included 14 intraductal papillary mucinous neoplasms (including 3 with an associated adenocarcinoma) and 10 mucinous cystic neoplasms; the 'nonmucinous' cohort included 11 serous cystadenomas and 5 other benign cysts. Quantitative reverse transcription PCR was performed for five miRNAs (miR-21, miR-155, miR-221, miR-17-3p, miR-191), which were previously reported as overexpressed in pancreatic adenocarcinomas. RESULTS: Significantly higher expression of miR-21, miR-221, and miR-17-3p was observed in the mucinous versus nonmucinous cysts (p < 0.01), with the mean relative fold differences being 7.0-, 7.9-, and 5.4-fold, respectively. Receiver operating characteristic curves demonstrated the highest median area under the curve for miR-21, with a median specificity of 76%, at a sensitivity of 80%. CONCLUSION: This pilot study demonstrates that profiling miRNAs in pancreatic cyst fluid samples is feasible and can yield potential biomarkers for the classification of cystic lesions of the pancreas. and IAP.

Unraveling the riverine antibiotic resistome: The downstream fate of anthropogenic inputs.

River networks are one of the main routes by which the public could be exposed to environmental sources of antibiotic resistance, that may be introduced e.g. via treated wastewater. In this study, we applied a comprehensive integrated analysis encompassing mass-flow concepts, chemistry, bacterial plate counts, resistance gene quantification and shotgun metagenomics to track the fate of the resistome (collective antibiotic resistance genes (ARGs) in a microbial community) of treated wastewater in two Swiss rivers at the kilometer scale. The levels of certain ARGs and the class 1 integron integrase gene (intI1) commonly associated with anthropogenic sources of ARGs decreased quickly over short distances (2-2.5 km) downstream of wastewater discharge points. Mass-flow analysis based on conservative tracers suggested this decrease was attributable mainly to dilution but ARG loadings frequently also decreased (e.g., 55.0-98.5 % for ermB and tetW) over the longest studied distances (6.8 and 13.7 km downstream). Metagenomic analysis confirmed that ARG of wastewater-origin did not persist in rivers after 5 ~ 6.8 km downstream distance. sul1 and intI1 levels and loadings were more variable and even increased sharply at 5 ~ 6.8 km downstream distance on one occasion. While input from agriculture and in-situ positive selection pressure for organisms carrying ARGs cannot be excluded, in-system growth of biomass is a more probable explanation. The potential for direct human exposure to the resistome of wastewater-origin thus appeared to typically abate rapidly in the studied rivers. However, the riverine aquatic resistome was also dynamic, as evidenced by the increase of certain gene markers downstream, without obvious sources of anthropogenic contamination. This study provides new insight into drivers of riverine resistomes and pinpoints key monitoring targets indicative of where human sources and exposures are likely to be most acute.

Detection of Circulating Tumor DNA in Patients with Pancreatic Cancer Using Digital Next-Generation Sequencing.

Circulating tumor DNA (ctDNA) measurements can be used to estimate tumor burden, but avoiding false-positive results is challenging. Herein, digital next-generation sequencing (NGS) is evaluated as a ctDNA detection method. Plasma KRAS and GNAS hotspot mutation levels were measured in 140 subjects, including 67 with pancreatic ductal adenocarcinoma and 73 healthy and disease controls. To limit chemical modifications of DNA that yield false-positive mutation calls, plasma DNA was enzymatically pretreated, after which DNA was aliquoted for digital detection of mutations (up to 384 aliquots/sample) by PCR and NGS. A digital NGS score of two SDs above the mean in controls was considered positive. Thirty-seven percent of patients with pancreatic cancer, including 31% of patients with stages I/II disease, had positive KRAS codon 12 ctDNA scores; only one patient had a positive GNAS mutation score. Two disease control patients had positive ctDNA scores. Low-normal-range digital NGS scores at mutation hotspots were found at similar levels in healthy and disease controls, usually at sites of cytosine deamination, and were likely the result of chemical modification of plasma DNA and NGS error rather than true mutations. Digital NGS detects mutated ctDNA in patients with pancreatic cancer with similar yield to other methods. Detection of low-level, true-positive ctDNA is limited by frequent low-level detection of false-positive mutation calls in plasma DNA from controls.

A multimodality test to guide the management of patients with a pancreatic cyst.

Pancreatic cysts are common and often pose a management dilemma, because some cysts are precancerous, whereas others have little risk of developing into invasive cancers. We used supervised machine learning techniques to develop a comprehensive test, CompCyst, to guide the management of patients with pancreatic cysts. The test is based on selected clinical features, imaging characteristics, and cyst fluid genetic and biochemical markers. Using data from 436 patients with pancreatic cysts, we trained CompCyst to classify patients as those who required surgery, those who should be routinely monitored, and those who did not require further surveillance. We then tested CompCyst in an independent cohort of 426 patients, with histopathology used as the gold standard. We found that clinical management informed by the CompCyst test was more accurate than the management dictated by conventional clinical and imaging criteria alone. Application of the CompCyst test would have spared surgery in more than half of the patients who underwent unnecessary resection of their cysts. CompCyst therefore has the potential to reduce the patient morbidity and economic costs associated with current standard-of-care pancreatic cyst management practices.

Simple Detection of Telomere Fusions in Pancreatic Cancer, Intraductal Papillary Mucinous Neoplasm, and Pancreatic Cyst Fluid.

Telomere end-to-end fusions are an important source of chromosomal instability that arise in cells with critically shortened telomeres. We developed a nested real-time quantitative PCR method for telomere fusion detection in pancreatic ductal adenocarcinomas, intraductal papillary mucinous neoplasms (IPMNs), and IPMN cyst fluids. Ninety-one pancreatic cancer cell lines and xenograft samples, 93 IPMNs, and 93 surgically aspirated IPMN cyst fluid samples were analyzed. The association between telomere shortening, telomerase activity, and telomere fusion detection was evaluated. Telomere fusions were detected in 56 of 91 pancreatic cancers (61.5%). Telomere fusion-positive cell lines had significantly shorter telomere lengths than fusion-negative lines (P = 0.003). Telomere fusions were undetectable in normal pancreas or IPMNs with low-grade dysplasia (0.0%) and were detected in IPMN with high-grade dysplasia (HGD; 48.0%) (P < 0.001). In IPMN cyst fluids, telomere fusions were more frequent in IPMNs with HGD (26.9%) or associated invasive cancer (42.9%) than IPMN with intermediate-grade dysplasia (15.4%) or low-grade dysplasia (0%) (P = 0.025). Telomerase activity levels were higher in cyst fluids with fusions than in those without (P = 0.0414). Cyst fluid telomere fusion status was an independent predictor of HGD/invasive cancer by multivariate analysis (odds ratio, 6.23; 95% CI, 1.61-28.0). Telomere fusions are detected in later stages of IPMN progression and can serve as a marker for predicting the presence of HGD and/or invasive cancer.