Prospective, Multi-Institutional, Real-Time Next-Generation Sequencing of Pancreatic Cyst Fluid Reveals Diverse Genomic Alterations That Improve the Clinical Management of Pancreatic Cysts.

BACKGROUND & AIMS: Next-generation sequencing (NGS) of pancreatic cyst fluid is a useful adjunct in the assessment of patients with pancreatic cyst. However, previous studies have been retrospective or single institutional experiences. The aim of this study was to prospectively evaluate NGS on a multi-institutional cohort of patients with pancreatic cyst in real time. METHODS: The performance of a 22-gene NGS panel (PancreaSeq) was first retrospectively confirmed and then within a 2-year timeframe, PancreaSeq testing was prospectively used to evaluate endoscopic ultrasound-guided fine-needle aspiration pancreatic cyst fluid from 31 institutions. PancreaSeq results were correlated with endoscopic ultrasound findings, ancillary studies, current pancreatic cyst guidelines, follow-up, and expanded testing (Oncomine) of postoperative specimens. RESULTS: Among 1933 PCs prospectively tested, 1887 (98%) specimens from 1832 patients were satisfactory for PancreaSeq testing. Follow-up was available for 1216 (66%) patients (median, 23 months). Based on 251 (21%) patients with surgical pathology, mitogen-activated protein kinase/GNAS mutations had 90% sensitivity and 100% specificity for a mucinous cyst (positive predictive value [PPV], 100%; negative predictive value [NPV], 77%). On exclusion of low-level variants, the combination of mitogen-activated protein kinase/GNAS and TP53/SMAD4/CTNNB1/mammalian target of rapamycin alterations had 88% sensitivity and 98% specificity for advanced neoplasia (PPV, 97%; NPV, 93%). Inclusion of cytopathologic evaluation to PancreaSeq testing improved the sensitivity to 93% and maintained a high specificity of 95% (PPV, 92%; NPV, 95%). In comparison, other modalities and current pancreatic cyst guidelines, such as the American Gastroenterology Association and International Association of Pancreatology/Fukuoka guidelines, show inferior diagnostic performance. The sensitivities and specificities of VHL and MEN1/loss of heterozygosity alterations were 71% and 100% for serous cystadenomas (PPV, 100%; NPV, 98%), and 68% and 98% for pancreatic neuroendocrine tumors (PPV, 85%; NPV, 95%), respectively. On follow-up, serous cystadenomas with TP53/TERT mutations exhibited interval growth, whereas pancreatic neuroendocrine tumors with loss of heterozygosity of ≥3 genes tended to have distant metastasis. None of the 965 patients who did not undergo surgery developed malignancy. Postoperative Oncomine testing identified mucinous cysts with BRAF fusions and ERBB2 amplification, and advanced neoplasia with CDKN2A alterations. CONCLUSIONS: PancreaSeq was not only sensitive and specific for various pancreatic cyst types and advanced neoplasia arising from mucinous cysts, but also reveals the diversity of genomic alterations seen in pancreatic cysts and their clinical significance.

A Combined DNA/RNA-based Next-Generation Sequencing Platform to Improve the Classification of Pancreatic Cysts and Early Detection of Pancreatic Cancer Arising From Pancreatic Cysts.

OBJECTIVE: We report the development and validation of a combined DNA/RNA next-generation sequencing (NGS) platform to improve the evaluation of pancreatic cysts. BACKGROUND AND AIMS: Despite a multidisciplinary approach, pancreatic cyst classification, such as a cystic precursor neoplasm, and the detection of high-grade dysplasia and early adenocarcinoma (advanced neoplasia) can be challenging. NGS of preoperative pancreatic cyst fluid improves the clinical evaluation of pancreatic cysts, but the recent identification of novel genomic alterations necessitates the creation of a comprehensive panel and the development of a genomic classifier to integrate the complex molecular results. METHODS: An updated and unique 74-gene DNA/RNA-targeted NGS panel (PancreaSeq Genomic Classifier) was created to evaluate 5 classes of genomic alterations to include gene mutations (e.g., KRAS, GNAS, etc.), gene fusions and gene expression. Further, CEA mRNA ( CEACAM5 ) was integrated into the assay using RT-qPCR. Separate multi-institutional cohorts for training (n=108) and validation (n=77) were tested, and diagnostic performance was compared to clinical, imaging, cytopathologic, and guideline data. RESULTS: Upon creation of a genomic classifier system, PancreaSeq GC yielded a 95% sensitivity and 100% specificity for a cystic precursor neoplasm, and the sensitivity and specificity for advanced neoplasia were 82% and 100%, respectively. Associated symptoms, cyst size, duct dilatation, a mural nodule, increasing cyst size, and malignant cytopathology had lower sensitivities (41-59%) and lower specificities (56-96%) for advanced neoplasia. This test also increased the sensitivity of current pancreatic cyst guidelines (IAP/Fukuoka and AGA) by >10% and maintained their inherent specificity. CONCLUSIONS: PancreaSeq GC was not only accurate in predicting pancreatic cyst type and advanced neoplasia but also improved the sensitivity of current pancreatic cyst guidelines.

"Invisible" pancreatic masses identified by EUS by the "ductal cutoff sign".

Making a tissue diagnosis of pancreatic adenocarcinoma is best accomplished by EUS and fine-needle aspiration (FNA) of the lesion. Typically, a dark, or "hypoechoic" mass will be seen, which presents an obvious target for FNA. For small lesions, computerized tomography (CT) may be negative, but the lesion is still almost always seen on EUS imaging. Rarely, a pancreatic mass will appear isoechoic on EUS imaging. We report three "invisible" pancreatic masses identified only by a cutoff in the pancreatic duct (PD) and/or common bile duct (CBD). No mass, isoechoic or otherwise, was seen. EUS-FNA was performed in the area of ductal narrowing, with a positive identification of adenocarcinoma in these cases.

Exposure to the immunosuppressant, perfluorooctanoic acid, enhances the murine IgE and airway hyperreactivity response to ovalbumin.

These studies were conducted to investigate the role of dermal exposure to perfluorooctanoic acid (PFOA), a known immunosuppressant, on the hypersensitivity response to ovalbumin (OVA) in a murine model of asthma. PFOA has had widespread use as a carpet and fabric protectant. BALB/c mice were exposed dermally, on the dorsal surface of each ear, to concentrations of PFOA ranging from 0.01 to 1.5% (applied dose 0.25-50 mg/kg) for 4 days. In hypersensitivity studies, mice were also ip injected with 7.5 microg OVA and 2 mg alum on days 1 and 10 and in some studies challenged with 250 microg OVA by pharyngeal aspiration on days 17 and 26. Following exposure to PFOA, an increase in liver weights and a decrease in thymus and spleen weights and cellularities were observed. Similar immunomodulatory trends were demonstrated in mice coadministered PFOA and OVA. Compared to the OVA alone-exposed animals, an increase in total IgE was demonstrated when mice were coexposed to OVA and concentrations of PFOA ranging from 0.75 to 1.5%, while the OVA-specific IgE response peaked with 0.75% PFOA coexposure (p < or = 0.05). OVA-specific airway hyperreactivity was increased in the 1.0% PFOA coexposed group (p < or = 0.05), with an increased pleiotropic cell response characterized by eosinophilia and mucin production, in animals coexposed to concentrations of PFOA up to 1.0%, as compared to the OVA alone-exposed animals. In a murine model, PFOA was demonstrated to be immunotoxic following dermal exposure, with an enhancement of the hypersensitivity response to OVA, suggesting that PFOA exposure may augment the IgE response to environmental allergens.

Predicting the risk of recurrent adenoma and incident colorectal cancer based on findings of the baseline colonoscopy.

OBJECTIVES: The decision tree underlying current practice guidelines for post polypectomy surveillance relies on risk stratification based on predictive attributes gleaned from adenomas removed on screening colonoscopy examination. Our primary aim was to estimate the magnitude of association between baseline adenoma attributes and the risk of adenoma recurrence and invasive colorectal adenocarcinoma (CRC). Our secondary aims were to estimate the adenoma detection rate (ADR) of surveillance compared with screening colonoscopies and describe time trends in preventive colonoscopy utilization. METHODS: We used prospective analyses of retrospectively collected clinical data from electronic health records. A cohort of primary care patients eligible for colorectal cancer screening was assembled encompassing 110,452 subjects, of which 3,300 had adenomas removed on screening examination. Of those patients who had a follow-up surveillance colonoscopy (defined as a patient with a documented adenoma on prior colonoscopy) recorded during the study period, 537 had a recurrent adenoma. RESULTS: Of those recurrent adenomas, 354 had a high-risk attributes. High-risk attributes were described at >3 adenomas, at least one adenoma >10 mm in size, high-grade dysplasia, or villous features. The risk of developing invasive CRC among post polypectomy patients was significantly higher if the baseline adenomas displayed any of the following attributes: more numerous than 3 (4.3-fold higher risk, 95% confidence interval (CI) low, high 1.4, 12.9), larger than 10 mm in size (5.2-fold higher risk, 95% CI low, high 1.8, 15.1), high-grade dysplasia (13.2-fold risk, 95% CI low, high 2.8, 62.1), or villous features (7.4-fold higher risk, 95% CI low, high 2.5, 21.5). These attributes combined added a net value of 22.8% to the probability of correctly predicting CRC. There was a threefold increase in surveillance utilization relative to screening from 2005 to 2011. The ADR of surveillance (34.1%) was 1.5-fold higher than that of screening (23.1%). CONCLUSIONS: These results emphasize the need to mitigate excessive risk by performing timely surveillance colonoscopies in patients with baseline adenomas displaying high-risk attributes as recommended in practice guidelines.

Cost-effectiveness of IL28Β genotype-guided protease inhibitor triple therapy versus standard of care treatment in patients with hepatitis C genotypes 2 or 3 infection.

BACKGROUND/AIMS: Triple therapy [adding protease inhibitors to standard of care (SOC)] dramatically increases treatment response in selected patients with hepatitis C virus (HCV). Interleukin 28B (IL28Β) genotyping helps predict responsiveness in these patients; however, the economic implications of IL28Β genotyping in HCV genotype 2 or 3 infected patients are unknown. Short- and long-term costs and outcomes of SOC therapy were calculated and used to determine the cost-effectiveness thresholds for using triple therapy in HCV genotype 2 or 3 infected patients. METHODS: Costs and outcomes were calculated by conducting cohort simulations on decision trees modeling SOC and triple therapy. Quality-adjusted life expectancies and long-term costs were predicted through Markov modeling. RESULTS: For triple therapy to be cost-effective, sustained virologic response (SVR) rates must improve (depending on age) by 7.91-11.11 and 9.06-12.8% for HCV genotype 2 and 3 cohorts, respectively. When triple therapy is guided by 2 IL28Β variants, a 2.63-3.72% improvement in SVR is needed for cost-effectiveness, and when guided by only one variant, a 1.4-8.91% improvement is needed. CONCLUSIONS: Markov modeling revealed that modest increases in SVR rates from IL28Β-guided triple therapy can lead to both lower costs and better health outcomes than SOC therapy in the long run.