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.

Acute and Chronic Pancreatitis Disease Prevalence, Classification, and Comorbidities: A Cohort Study of the UK BioBank.

INTRODUCTION: Pancreatitis is a complex syndrome that results from many etiologies. Large well-characterized cohorts are needed to further understand disease risk and prognosis. METHODS: A pancreatitis cohort of more than 4,200 patients and 24,000 controls were identified in the UK BioBank (UKBB) consortium. A descriptive analysis was completed, comparing patients with acute (AP) and chronic pancreatitis (CP). The Toxic-metabolic, Idiopathic, Genetic, Autoimmune, Recurrent, and severe pancreatitis and Obstructive checklist Version 2 classification was applied to patients with AP and CP and compared with the control population. RESULTS: CP prevalence in the UKBB is 163 per 100,000. AP incidence increased from 21.4/100,000 per year from 2001 to 2005 to 48.2/100,000 per year between 2016 and 2020. Gallstones and smoking were confirmed as key risk factors for AP and CP, respectively. Both populations carry multiple risk factors and a high burden of comorbidities, including benign and malignant neoplastic disorders. DISCUSSION: The UKBB serves as a rich cohort to evaluate pancreatitis. Disease burden of AP and CP was high in this population. The association of common risk factors identified in other cohort studies was confirmed in this study. Further analysis is needed to link genomic risks and biomarkers with disease features in this population.

Complex Genetics in Pancreatitis: Insights Gained From a New Candidate Locus Panel.

OBJECTIVES: Chronic pancreatitis is the end stage of a pathologic inflammatory syndrome with multiple etiological factors, including genetic. We hypothesized that some pancreatitis etiology originates in pancreatic acinar or duct cells and requires both injury and compensatory mechanism failure. METHODS: One hundred pancreatitis patients were assessed using a DNA sequencing panel for pancreatitis. Cooccurrence of variants within and between genes was measured. Gene coexpression was confirmed via published single-cell RNA sequencing. RESULTS: One hundred and twenty-one variants were identified in 2 or more patients, 15 of which were enriched compared with reference populations. Single cell RNA-sequencing data verified coexpression of GGT1, CFTR, and PRSS1 in duct cells, PRSS1, CPA1, CEL, CTRC, and SPINK1 in acinar cells, and UBR1 in both. Multiple-risk variants with injury/stress effects (CEL, CFTR, CPA1, PRSS1) and impaired cell protection (CTRC, GGT1, SPINK1, UBR1) cooccur within duct cells, acinar cells, or both. CONCLUSIONS: Pancreatitis is a complex disorder with genetic interactions across genes and cell types. These findings suggest a new, non-Mendelian genetic risk/etiology paradigm where a combination of nonpathogenic genetic risk variants in groups of susceptibility genes and injury/dysfunction response genes contribute to acquired pancreatic disease.

Introduction and Validation of a Novel Acute Pancreatitis Digital Tool: Interrogating Large Pooled Data From 2 Prospectively Ascertained Cohorts.

OBJECTIVES: Acute pancreatitis (AP) is a sudden onset, rapidly evolving inflammatory response with systemic inflammation and multiorgan failure (MOF) in a subset of patients. New highly accurate clinical decision support tools are needed to allow local doctors to provide expert care. METHODS: Ariel Dynamic Acute Pancreatitis Tracker (ADAPT) is a digital tool to guide physicians in ordering standard tests, evaluate test results and model progression using available data, propose emergent therapies. The accuracy of the severity score calculators was tested using 2 prospectively ascertained Acute Pancreatitis Patient Registry to Examine Novel Therapies in Clinical Experience cohorts (pilot University of Pittsburgh Medical Center, n = 163; international, n = 1544). RESULTS: The ADAPT and post hoc expert-calculated AP severity scores were 100% concordant in both pilot and international cohorts. High-risk criteria of all 4 severity scores at admission were associated with moderately-severe or severe AP and MOF (both P < 0.0001) and prediction of no MOF was 97.8% to 98.9%. The positive predictive value for MOF was 7.5% to 14.9%. CONCLUSIONS: The ADAPT tool showed 100% accuracy with AP predictive metrics. Prospective evaluation of ADAPT features is needed to determine if additional data can accurately predict and mitigate severe AP and MOF.

Platform for Quantitative Evaluation of Spatial Intratumoral Heterogeneity in Multiplexed Fluorescence Images.

We introduce THRIVE (Tumor Heterogeneity Research Interactive Visualization Environment), an open-source tool developed to assist cancer researchers in interactive hypothesis testing. The focus of this tool is to quantify spatial intratumoral heterogeneity (ITH), and the interactions between different cell phenotypes and noncellular constituents. Specifically, we foresee applications in phenotyping cells within tumor microenvironments, recognizing tumor boundaries, identifying degrees of immune infiltration and epithelial/stromal separation, and identification of heterotypic signaling networks underlying microdomains. The THRIVE platform provides an integrated workflow for analyzing whole-slide immunofluorescence images and tissue microarrays, including algorithms for segmentation, quantification, and heterogeneity analysis. THRIVE promotes flexible deployment, a maintainable code base using open-source libraries, and an extensible framework for customizing algorithms with ease. THRIVE was designed with highly multiplexed immunofluorescence images in mind, and, by providing a platform to efficiently analyze high-dimensional immunofluorescence signals, we hope to advance these data toward mainstream adoption in cancer research. Cancer Res; 77(21); e71-74. ©2017 AACR.

Pointwise mutual information quantifies intratumor heterogeneity in tissue sections labeled with multiple fluorescent biomarkers.

BACKGROUND: Measures of spatial intratumor heterogeneity are potentially important diagnostic biomarkers for cancer progression, proliferation, and response to therapy. Spatial relationships among cells including cancer and stromal cells in the tumor microenvironment (TME) are key contributors to heterogeneity. METHODS: We demonstrate how to quantify spatial heterogeneity from immunofluorescence pathology samples, using a set of 3 basic breast cancer biomarkers as a test case. We learn a set of dominant biomarker intensity patterns and map the spatial distribution of the biomarker patterns with a network. We then describe the pairwise association statistics for each pattern within the network using pointwise mutual information (PMI) and visually represent heterogeneity with a two-dimensional map. RESULTS: We found a salient set of 8 biomarker patterns to describe cellular phenotypes from a tissue microarray cohort containing 4 different breast cancer subtypes. After computing PMI for each pair of biomarker patterns in each patient and tumor replicate, we visualize the interactions that contribute to the resulting association statistics. Then, we demonstrate the potential for using PMI as a diagnostic biomarker, by comparing PMI maps and heterogeneity scores from patients across the 4 different cancer subtypes. Estrogen receptor positive invasive lobular carcinoma patient, AL13-6, exhibited the highest heterogeneity score among those tested, while estrogen receptor negative invasive ductal carcinoma patient, AL13-14, exhibited the lowest heterogeneity score. CONCLUSIONS: This paper presents an approach for describing intratumor heterogeneity, in a quantitative fashion (via PMI), which departs from the purely qualitative approaches currently used in the clinic. PMI is generalizable to highly multiplexed/hyperplexed immunofluorescence images, as well as spatial data from complementary in situ methods including FISSEQ and CyTOF, sampling many different components within the TME. We hypothesize that PMI will uncover key spatial interactions in the TME that contribute to disease proliferation and progression.