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Introduction: Traumatic brain injury (TBI) accounts for a significant amount of death and disability worldwide and the majority of this burden affects individuals in low-and-middle income countries. Despite this, considerable geographical differences have been reported in the care of TBI patients. On this background, we aim to provide a comprehensive international picture of the epidemiological characteristics, management and outcomes of patients undergoing emergency surgery for traumatic brain injury (TBI) worldwide., Methods and Analysis: The Global Neurotrauma Outcomes Study (GNOS) is a multi-centre, international, prospective observational cohort study. Any unit performing emergency surgery for TBI worldwide will be eligible to participate. All TBI patients who receive emergency surgery in any given consecutive 30-day period beginning between 1st of November 2018 and 31st of December 2019 in a given participating unit will be included. Data will be collected via a secure online platform in anonymised form. The primary outcome measures for the study will be 14-day mortality (or survival to hospital discharge, whichever comes first). Final day of data collection for the primary outcome measure is February 13th. Secondary outcome measures include return to theatre and surgical site infection., Ethics and Dissemination: This project will not affect clinical practice and has been classified as clinical audit following research ethics review. Access to source data will be made available to collaborators through national or international anonymised datasets on request and after review of the scientific validity of the proposed analysis by the central study team., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

Purpose: Acute graft-versus-host disease (aGVHD) remains a significant complication of allogeneic hematopoietic cell transplantation (HCT) and limits its broader application. The ability to predict grade II to IV aGVHD could potentially mitigate morbidity and mortality. To date, researchers have focused on using snapshots of a patient (eg, biomarkers at a single time point) to predict aGVHD onset. We hypothesized that longitudinal data collected and stored in electronic health records (EHRs) could distinguish patients at high risk of developing aGVHD from those at low risk., Patients and Methods: The study included a cohort of 324 patients undergoing allogeneic HCT at the University of Michigan C.S. Mott Children's Hospital during 2014 to 2017. Using EHR data, specifically vital sign measurements collected within the first 10 days of transplantation, we built a predictive model using penalized logistic regression for identifying patients at risk for grade II to IV aGVHD. We compared the proposed model with a baseline model trained only on patient and donor characteristics collected at the time of transplantation and performed an analysis of the importance of different input features., Results: The proposed model outperformed the baseline model, with an area under the receiver operating characteristic curve of 0.659 versus 0.512 ( P = .019). The feature importance analysis showed that the learned model relied most on temperature and systolic blood pressure, and temporal trends (eg, increasing or decreasing) were more important than the average values., Conclusion: Leveraging readily available clinical data from EHRs, we developed a machine-learning model for aGVHD prediction in patients undergoing HCT. Continuous monitoring of vital signs, such as temperature, could potentially help clinicians more accurately identify patients at high risk for aGVHD.

The detection and quantification of biomarkers have numerous applications in biological research and medicine. The most widely used methods to detect nucleic acids require amplification via the polymerase chain reaction (PCR). However, errors arising from the imperfect copying fidelity of DNA polymerases, limited specificity of primers, and heat-induced damage reduce the specificity of PCR-based methods, particularly for single-nucleotide variants. Furthermore, not all analytes can be amplified efficiently. While amplification-free methods avoid these pitfalls, the specificity of most such methods is strictly constrained by probe binding thermodynamics, which for example hampers detection of rare somatic mutations. In contrast, single-molecule recognition through equilibrium Poisson sampling (SiMREPS) provides ultraspecific detection with single-molecule and single-nucleotide sensitivity by monitoring the repetitive interactions of a fluorescent probe with surface-immobilized targets. In this review, we discuss SiMREPS in comparison with other analytical approaches, and describe its utility in quantifying a range of nucleic acids and other analytes., Competing Interests: Declaration of interest The University of Michigan has filed patent applications related to the single-molecule kinetic fingerprinting approach described herein, on which A.J.B, M.T. and N.G.W. are co-inventors. A.J.B., M.T., and N.G.W. are co-founders of a startup company, aLight Sciences LLC, which seeks to commercialize this technology.

We analyzed micro-RNAs (miRs) as possible diagnostic biomarkers for relevant comorbidities prior to and prognostic biomarkers for mortality following hematopoietic cell transplantation (HCT). A randomly selected group of patients (n = 36) were divided into low-risk (HCT-comorbidity index [HCT-CI] score of 0 and survived HCT) and high-risk (HCT-CI scores ≥ 4 and deceased after HCT) groups. There were 654 miRs tested and 19 met the pre-specified significance level of p < 0.1. In subsequent models, only eight miRs maintained statistical significance in regression models after adjusting for baseline demographic factors; miRs-374b and -454 were underexpressed, whereas miRs-142-3p, -191, -424, -590-3p, -29c, and -15b were overexpressed among high-risk patients relative to low-risk patients. Areas under the curve for these eight miRs ranged between 0.74 and 0.81, suggesting strong predictive capacity. Consideration of miRs may improve risk assessment of mortality and should be further explored in larger future prospective studies.

Whether cell forces or extracellular matrix (ECM) can impact genome integrity is largely unclear. Here, acute perturbations (∼1 h) to actomyosin stress or ECM elasticity cause rapid and reversible changes in lamin-A, DNA damage, and cell cycle. The findings are especially relevant to organs such as the heart because DNA damage permanently arrests cardiomyocyte proliferation shortly after birth and thereby eliminates regeneration after injury including heart attack. Embryonic hearts, cardiac-differentiated iPS cells (induced pluripotent stem cells), and various nonmuscle cell types all show that actomyosin-driven nuclear rupture causes cytoplasmic mis-localization of DNA repair factors and excess DNA damage. Binucleation and micronuclei increase as telomeres shorten, which all favor cell-cycle arrest. Deficiencies in lamin-A and repair factors exacerbate these effects, but lamin-A-associated defects are rescued by repair factor overexpression and also by contractility modulators in clinical trials. Contractile cells on stiff ECM normally exhibit low phosphorylation and slow degradation of lamin-A by matrix-metalloprotease-2 (MMP2), and inhibition of this lamin-A turnover and also actomyosin contractility are seen to minimize DNA damage. Lamin-A is thus stress stabilized to mechano-protect the genome., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Extracellular RNAs (exRNAs) in biofluids have attracted great interest as potential biomarkers. Although extracellular microRNAs in blood plasma are extensively characterized, extracellular messenger RNA (mRNA) and long non-coding RNA (lncRNA) studies are limited. We report that plasma contains fragmented mRNAs and lncRNAs that are missed by standard small RNA-seq protocols due to lack of 5' phosphate or presence of 3' phosphate. These fragments were revealed using a modified protocol ("phospho-RNA-seq") incorporating RNA treatment with T4-polynucleotide kinase, which we compared with standard small RNA-seq for sequencing synthetic RNAs with varied 5' and 3' ends, as well as human plasma exRNA Analyzing phospho-RNA-seq data using a custom, high-stringency bioinformatic pipeline, we identified mRNA/lncRNA transcriptome fingerprints in plasma, including tissue-specific gene sets. In a longitudinal study of hematopoietic stem cell transplant patients, bone marrow- and liver-enriched exRNA genes were tracked with bone marrow recovery and liver injury, respectively, providing proof-of-concept validation as a biomarker approach. By enabling access to an unexplored realm of mRNA and lncRNA fragments, phospho-RNA-seq opens up new possibilities for plasma transcriptomic biomarker development., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Purpose: Radiation-induced cardiac toxicity (RICT) is an increasingly well-appreciated source of morbidity and mortality in patients receiving thoracic radiotherapy (RT). Currently available methods to predict RICT are suboptimal. We investigated circulating microRNAs (c-miRNAs) as potential biomarkers of RICT in patients undergoing definitive RT for non-small-cell lung cancer (NSCLC)., Methods: Data from 63 patients treated on institutional trials were analyzed. Prognostic models of grade 3 or greater (G3 +) RICT based on pre-treatment c-miRNA levels ('c-miRNA'), mean heart dose (MHD) and pre-existing cardiac disease (PCD) ('clinical'), and a combination of these ('c-miRNA + clinical') were developed. Elastic net Cox regression and full cross validation were used for variable selection, model building, and model evaluation. Concordance statistic (c-index) and integrated Brier score (IBS) were used to evaluate model performance., Results: MHD, PCD, and serum levels of 14 c-miRNA species were identified as jointly prognostic for G3 + RICT. The 'c-miRNA and 'clinical' models yielded similar cross-validated c-indices (0.70 and 0.72, respectively) and IBSs (0.26 and 0.28, respectively). However, prognostication was not improved by combining c-miRNA and clinical factors (c-index 0.70, IBS 0.28). The 'c-miRNA' and 'clinical' models were able to significantly stratify patients into high- and low-risk groups of developing G3 + RICT. Chi-square testing demonstrated a marginally significantly higher prevalence of PCD in patients with high- compared to low-risk c-miRNA profile (p = 0.09), suggesting an association between some c-miRNAs and PCD., Conclusions: We identified a pre-treatment c-miRNA signature prognostic for G3 + RICT. With further development, pre- and mid-treatment c-miRNA profiling could contribute to patient-specific dose selection and treatment adaptation.

Pancreatic exocrine insufficiency (PEI) is a common long-term complication after pancreaticoduodenectomy (PD) and is observed in 23-80% of patients. As the postoperative mortality after PD has substantially decreased, it warrants more attention on the diagnosis and treatment of functional long-term consequences after PD. These include PEI and endocrine insufficiency that can result in significant nutritional impairment and often adversely impacts quality of life (QOL) of the patient. A PubMed search was performed for articles using key words "pancreatic exocrine insufficiency"; "pancreaticoduodenectomy"; "quality of life after pancreaticoduodenectomy"; "stool elastase"; "direct, indirect tests for pancreatic exocrine insufficiency"; "pancreatic enzyme replacement therapy." Relevant studies were shortlisted and analyzed. This review summarizes relevant studies addressing PEI following PD. We also discuss functional changes after PD, risk factors and predictive factors for postoperative PEI, clinical symptoms, direct and indirect tests for estimation of PEI, pancreatic enzyme replacement therapy (PERT), and QOL after pancreatic resection for malignancy. It was found that significant PEI occurs in most patients following PD. Fecal elastase 1 is an easy indirect test and should be performed routinely in both symptomatic and asymptomatic patients after PD. PERT should be considered in every patient after PD with the aim to improve the QOL and perhaps even their long time survival.

Stool contains DNA shed from cells of the gastrointestinal (GI) tract and has great potential as a bio-specimen for non-invasive, nucleic acid-based detection of GI diseases. Whereas methods for studying faecal microbiome DNA are plentiful, there is a lack of well-characterised procedures for stabilisation, isolation, and quantitative analysis of faecal host DNA. We report an optimised pipeline for faecal host DNA analysis from the point-of-collection to droplet digital PCR (ddPCR) absolute quantification of host-specific gene targets. We evaluated multiple methods for preservation and isolation of host DNA from stool to identify the highest performing methods. To quantify host DNA even if present in partially degraded form, we developed sensitive, human-specific short-amplicon ddPCR assays targeting repetitive nuclear genomic elements (LINE-1) and mitochondrial genes. We validated the ability of these optimised methods to perform absolute quantification of host DNA in 200 stool DNA extracts from samples that were serially collected from three healthy individuals and three hospitalised patients. These specimens allowed assessment of host DNA day-to-day variability in stool specimens with widely varying physical characteristics (i.e., Bristol scores). We further extended this approach to mouse stool analysis, to enable faecal host DNA studies in animal disease models as well.

Background: Enhanced recovery after surgery (ERAS) protocol is a multimodal, multidisciplinary and evidence-based approach to reduce surgical stress and enhance recovery in the postoperative period. This study aimed to analyze the outcome of ERAS protocol in patients after pancreaticoduodenectomy (PD)., Methods: A total of 50 consecutive patients with pancreatic/periampullary cancer who underwent PD between January 2016 to August 2017 were included in the study. As per the institute ERAS protocol, nasogastric tube (NGT) was removed on postoperative day (POD) 1 if output was less than 200 mL and oral sips were allowed; oral liquids were allowed on POD2; semisolid diet by POD3; abdominal drain was removed on POD 4 if output was less than 100 mL with no evidence of postoperative pancreatic fistula (POPF); normal diet was allowed on POD5. Discharge criteria on POD6 were afebrile, tolerating oral normal diet, pain free and no surgery related complications (defined as per the ISGPS definitions)., Results: NGT was removed on POD1 in 45 (90%) patients, abdominal drain removed by POD4 in 41 (82%) and 43 (86%) patients were discharged on POD6. There was no 30-day postoperative mortality. Three (6%) patients had delayed gastric emptying (DGE). None had postoperative hemorrhage and POPF. Readmission rate was 8%. A significant relation was found between the length of hospital stay (LOS) with age (P < 0.05) and a marginal relation between LOS and postoperative albumin (P = 0.05)., Conclusions: ERAS protocol can be safely followed in the perioperative care of patients who undergo PD. Early removal of NGT and allowing oral diet restore bowel function early. ERAS decreases the LOS and postoperative complications., (Copyright © 2018. Published by Elsevier B.V.)

Postoperative pancreatic fistula (POPF) is the most feared complication after pancreaticoduodenectomy (PD) that leads to intra-abdominal abscess, sepsis, or bleeding and remains the single most important source of morbidity and mortality after PD. To minimize this dreaded complication, various surgical techniques and modifications of pancreaticoenteric reconstruction have been proposed. However, still POPF does occur even in experienced hands. We herein describe the outcome of 150 post PD patients who underwent duct-to-mucosa (DM) pancreaticojejunostomy (PJ) using a special technique, Blumgart's "through & through" U transpancreatic sutures. The technique is described in detail. Postoperative octreotide and metoclopramide were used in all patients for 3 days. An enhanced recovery (ERAS) protocol was followed in a subset of patients. All patients were ASA grade 1 and had adenocarcinoma of the periampullary region/pancreatic head and underwent standard pylorus resecting PD after due optimization. Eighty-eight (58.7%) patients had pancreatic duct < 3 mm and pancreatic texture was soft to very soft in 112 (74.6%) patients. There was only one International Study Group of Pancreatic Surgery (ISGPS) grade C POPF with concomitant hemorrhage. Five patients developed ISGPS grade B and two grade C, delayed gastric emptying (DGE). There was no 30-day mortality. The average length of hospital stay was 7.3 ± 4.2 days with a median of 6 days in the ERAS subset of patients. Blumgart's "through & through" DMPJ technique is very helpful in reducing the POPF and other complications even in high-risk pancreas (i.e., soft with a small pancreatic duct) and is easy to learn and perform., Competing Interests: Compliance with Ethical StandardsThe authors declare that they have no conflict of interest.

Conventional methods for detecting small quantities of nucleic acids require amplification by the polymerase chain reaction (PCR), which necessitates prior purification and introduces copying errors. While amplification-free methods do not have these shortcomings, they are generally orders of magnitude less sensitive and specific than PCR-based methods. In this review, we provide a practical guide to a novel amplification-free method, single-molecule recognition through equilibrium Poisson sampling (SiMREPS), that provides both single-molecule sensitivity and single-base selectivity by monitoring the repetitive interactions of fluorescent probes to immobilized targets. We demonstrate how this kinetic fingerprinting filters out background arising from the inevitable nonspecific binding of probes, yielding virtually zero background signal. As practical applications of this digital detection methodology, we present the quantification of microRNA miR-16 and the detection of the mutation EGFR L858R with an apparent single-base discrimination factor of over 3 million., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Conventional techniques for detecting rare DNA sequences require many cycles of PCR amplification for high sensitivity and specificity, potentially introducing significant biases and errors. While amplification-free methods exist, they rarely achieve the ability to detect single molecules, and their ability to discriminate between single-nucleotide variants is often dictated by the specificity limits of hybridization thermodynamics. Here we show that a direct detection approach using single-molecule kinetic fingerprinting can surpass the thermodynamic discrimination limit by 3 orders of magnitude, with a dynamic range of up to 5 orders of magnitude with optional super-resolution analysis. This approach detects mutations as subtle as the drug-resistance-conferring cancer mutation EGFR T790M (a single C → T substitution) with an estimated specificity of 99.99999%, surpassing even the leading PCR-based methods and enabling detection of 1 mutant molecule in a background of at least 1 million wild-type molecules. This level of specificity revealed rare, heat-induced cytosine deamination events that introduce false positives in PCR-based detection, but which can be overcome in our approach through milder thermal denaturation and enzymatic removal of damaged nucleobases.

RNA-seq is increasingly used for quantitative profiling of small RNAs (for example, microRNAs, piRNAs and snoRNAs) in diverse sample types, including isolated cells, tissues and cell-free biofluids. The accuracy and reproducibility of the currently used small RNA-seq library preparation methods have not been systematically tested. Here we report results obtained by a consortium of nine labs that independently sequenced reference, 'ground truth' samples of synthetic small RNAs and human plasma-derived RNA. We assessed three commercially available library preparation methods that use adapters of defined sequence and six methods using adapters with degenerate bases. Both protocol- and sequence-specific biases were identified, including biases that reduced the ability of small RNA-seq to accurately measure adenosine-to-inosine editing in microRNAs. We found that these biases were mitigated by library preparation methods that incorporate adapters with degenerate bases. MicroRNA relative quantification between samples using small RNA-seq was accurate and reproducible across laboratories and methods.

We have previously shown that low frequency ultrasound can release biomarkers from cells into the murine circulation enabling an amplification and localization of the released biomarker that could be used as a blood-based method to detect cancer earlier and monitor therapy. In this study, we further demonstrate that this technique could be used for characterization of tumors and/or identification of cellular masses of unknown origin due to the release of multiple protein and nucleic acid biomarkers in cells in culture, mice and patients. We sonicated colon (LS174T) and prostate (LNCaP) cancer cell lines in culture at a low frequency of 1 MHz and show that there were several-fold changes in multiple protein and microRNA (miRNA) abundance with treatment at various intensities and time. This release was dependent on the duration and intensity of the sonication for both cell lines. Significant increased release in biomarkers was also observed following tumor sonication in living mice bearing subcutaneous LS174T cell line xenografts (for proteins and nucleic acids) and in an experimental LS174T liver tumor model (for proteins only). Finally, we demonstrated this methodology of multiple biomarker release in patients undergoing ablation of uterine fibroids using MR guided high intensity focused ultrasound. Two protein biomarkers significantly increased in the plasma after the ultrasound treatment in 21 samples tested. This proof that ultrasound-amplification method works in soft tissue tumor models together with biomarker multiplexing, could allow for an effective non-invasive method for identification, characterization and localization of incidental lesions, cancer and other disease. Pre-treatment quantification of the biomarkers, allows for individualization of quantitative comparisons. This individualization of normal marker levels in this method allows for specificity of the biomarker-increase to each patient, tumor or organ being studied.

Background: Previous studies suggest circulating, blood-based microRNAs (miRNAs) may serve as minimally invasive prostate cancer biomarkers, however there is limited data from prospective clinical trials. Here, we explore the role of candidate plasma miRNAs as potential biomarkers in the SWOG 0925 randomized phase II study of androgen deprivation combined with cixutumumab versus androgen deprivation alone in patients with new metastatic hormone-sensitive prostate cancer., Methods: Correlative biospecimens, including circulating tumor cells (CTCs) and plasma for miRNA analysis, were collected at baseline and after 12 weeks on treatment from 50 patients enrolled on SWOG 0925. Circulating microRNAs were quantified using real-time RT-PCR microRNA array that allowed specific analysis of previously identified candidate miRNAs (miR-141, miR-200a, miR-200b, miR-210, and miR-375) as well as discovery analysis to identify new candidate miRNAs. MiRNA levels were correlated to previously reported CTC counts using CellSearch® (Veridex) and with the primary study outcome of 28-week PSA response (≤0.2, 0.2 to ≤4.0, or >4.0 ng/mL), previously shown to correlate with overall survival., Results: We observed a correlation between baseline circulating miR-141, miR-200a, and miR-375 levels with baseline CTCs. Baseline miR-375 levels were associated with 28-week PSA response (≤0.2, 0.2 to ≤4.0, or >4.0 ng/mL, P = 0.007). Using ROC curve analysis, there was no significant difference between baseline miR-375 and baseline CTC in predicting 28-week PSA response (≤0.2 vs >0.2 ng/mL). To discover novel candidate miRNAs, we analyzed 365 miRNAs for association with the 28-week PSA response endpoint and identified new candidate miRNAs along with the existing candidates miR-375 and miR-200b (P = 0.0012, P = 0.0046, respectively., Conclusions: Baseline plasma miR-141, miR-200a, and miR-375 levels are associated with baseline CTC count. Baseline miR-375 was also associated with the trial endpoint of 28-week PSA response. Our results provide evidence that circulating miRNA biomarkers may have value as prognostic biomarkers and warrant further study in larger prospective clinical trials., (© 2017 Wiley Periodicals, Inc.)

Interphase phosphorylation of lamin-A,C depends dynamically on a cell's microenvironment, including the stiffness of extracellular matrix. However, phosphorylation dynamics is poorly understood for diseased forms such as progerin, a permanently farnesylated mutant of LMNA that accelerates aging of stiff and mechanically stressed tissues. Here, fine-excision alignment mass spectrometry (FEA-MS) is developed to quantify progerin and its phosphorylation levels in patient iPS cells differentiated to mesenchymal stem cells (MSCs). The stoichiometry of total A-type lamins (including progerin) versus B-type lamins measured for Progeria iPS-MSCs prove similar to that of normal MSCs, with total A-type lamins more abundant than B-type lamins. However, progerin behaves more like farnesylated B-type lamins in mechanically-induced segregation from nuclear blebs. Phosphorylation of progerin at multiple sites in iPS-MSCs cultured on rigid plastic is also lower than that of normal lamin-A and C. Reduction of nuclear tension upon i) cell rounding/detachment from plastic, ii) culture on soft gels, and iii) inhibition of actomyosin stress increases phosphorylation and degradation of lamin-C > lamin-A > progerin. Such mechano-sensitivity diminishes, however, with passage as progerin and DNA damage accumulate. Lastly, transcription-regulating retinoids exert equal effects on both diseased and normal A-type lamins, suggesting a differential mechano-responsiveness might best explain the stiff tissue defects in Progeria.

Droplet-based digital PCR provides high-precision, absolute quantification of nucleic acid target sequences with wide-ranging applications for both research and clinical diagnostic applications. Droplet-based digital PCR enables absolute quantification by counting nucleic acid molecules encapsulated in discrete, volumetrically defined water-in-oil droplet partitions. The current available systems overcome the previous lack of scalable and practical technologies for digital PCR implementation. Extracellular microRNAs in biofluids (plasma, serum, urine, cerebrospinal fluid, etc.) are promising noninvasive biomarkers in multiple diseases and different clinical settings (e.g., diagnosis, early diagnosis, prediction of recurrence, and prognosis). Here we describe a protocol that enables highly precise and reproducible absolute quantification of extracellular microRNAs using droplet digital PCR.

Purpose: To assess the utility of circulating serum microRNAs (c-miRNAs) to predict response to high-dose radiation therapy for locally advanced non-small cell lung cancer (NSCLC)., Methods and Materials: Data from 80 patients treated from 2004 to 2013 with definitive standard- or high-dose radiation therapy for stages II-III NSCLC as part of 4 prospective institutional clinical trials were evaluated. Pretreatment serum levels of 62 miRNAs were measured by quantitative reverse transcription-polymerase chain reaction array. We combined miRNA data and clinical factors to generate a dose-response score (DRS) for predicting overall survival (OS) after high-dose versus standard-dose radiation therapy. Elastic net Cox regression was used for variable selection and parameter estimation. Model assessment and tuning parameter selection were performed through full cross-validation. The DRS was also correlated with local progression, distant metastasis, and grade 3 or higher cardiac toxicity using Cox regression, and grade 2 or higher esophageal and pulmonary toxicity using logistic regression., Results: Eleven predictive miRNAs were combined with clinical factors to generate a DRS for each patient. In patients with low DRS, high-dose radiation therapy was associated with significantly improved OS compared to treatment with standard-dose radiation therapy (hazard ratio 0.22). In these patients, high-dose radiation also conferred lower risk of distant metastasis and local progression, although the latter association was not statistically significant. Patients with high DRS exhibited similar rates of OS regardless of dose (hazard ratio 0.78). The DRS did not correlate with treatment-related toxicity., Conclusions: Using c-miRNA signature and clinical factors, we developed a DRS that identified a subset of patients with locally advanced NSCLC who derive an OS benefit from high-dose radiation therapy. This DRS may guide dose escalation in a patient-specific manner., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Diffuse serous cystic neoplasm (SCN) associated with pancreatic neuroendocrine tumor (PNET) is a rare finding reported previously in only three patients to the best of our knowledge. We herein present one such interesting report of a diffuse serous cystic adenoma (SCA) and co-existent PNET in a 25-year old lady who presented with abdominal pain for past 6 months. A triple-phase pancreatic protocol computed tomography (CT) scan revealed multiple cysts involving the entire pancreas. The cysts were thin walled, ranging from 2 to 8 cm in width, with no calcification or central scar that was confirmed at laparotomy. A frozen section revealed a neuroendocrine tumor and she underwent total pancreatectomy. Diffuse SCA with co-existent PNET infiltrating nerve bundles of the pancreatic parenchyma was made upon histopathology further verified by chromogranin-A immunostaining. The patient is insulin dependent and doing well at 2 years of follow-up. The origin of endocrine tumors from multipotent ductular stem cells has been suggested.

Current cell-free DNA (cfDNA) next generation sequencing (NGS) precision oncology workflows are typically limited to targeted and/or disease-specific applications. In advanced cancer, disease burden and cfDNA tumor content are often elevated, yielding unique precision oncology opportunities. We sought to demonstrate the utility of a pan-cancer, rapid, inexpensive, whole genome NGS of cfDNA approach (PRINCe) as a precision oncology screening strategy via ultra-low coverage (~0.01x) tumor content determination through genome-wide copy number alteration (CNA) profiling. We applied PRINCe to a retrospective cohort of 124 cfDNA samples from 100 patients with advanced cancers, including 76 men with metastatic castration-resistant prostate cancer (mCRPC), enabling cfDNA tumor content approximation and actionable focal CNA detection, while facilitating concordance analyses between cfDNA and tissue-based NGS profiles and assessment of cfDNA alteration associations with mCRPC treatment outcomes. Therapeutically relevant focal CNAs were present in 42 (34%) cfDNA samples, including 36 of 93 (39%) mCRPC patient samples harboring AR amplification. PRINCe identified pre-treatment cfDNA CNA profiles facilitating disease monitoring. Combining PRINCe with routine targeted NGS of cfDNA enabled mutation and CNA assessment with coverages tuned to cfDNA tumor content. In mCRPC, genome-wide PRINCe cfDNA and matched tissue CNA profiles showed high concordance (median Pearson correlation = 0.87), and PRINCe detectable AR amplifications predicted reduced time on therapy, independent of therapy type (Kaplan-Meier log-rank test, chi-square = 24.9, p < 0.0001). Our screening approach enables robust, broadly applicable cfDNA-based precision oncology for patients with advanced cancer through scalable identification of therapeutically relevant CNAs and pre-/post-treatment genomic profiles, enabling cfDNA- or tissue-based precision oncology workflow optimization., Competing Interests: CONFLICTS OF INTEREST C.H., T.T., J.L. and E.K. are current or former employees of Takara Bio USA. D.H.H. has received travel support from Thermo Fisher. S.A.T has received travel support from, and had a sponsored research agreement with Compendia Bioscience/Life Technologies/ThermoFisher that provided access to the targeted sequencing panel used herein. No other aspect of this study was supported by Compendia Bioscience/Life Technologies/ThermoFisher. The University of Michigan has been issued a patent on ETS gene fusions in prostate cancer on which A.M.C. and S.A.T. are co-inventors. The diagnostic field of use has been licensed to Hologic/Gen-Probe, Inc., which has sublicensed rights to Roche/Ventana Medical Systems. S.A.T. has an unrelated sponsored research agreement with Astellas. S.A.T. has served as a consultant for and received honoraria from Roche/Ventana Medical Systems, Almac Diagnostics, Janssen, AbbVie and Astellas/Medivation. S.A.T. is a co-founder of, consultant for and Laboratory Director of Strata Oncology. The other authors have no competing interests to declare.

Acute myelogenous leukemia (AML) frequently relapses after complete remission (CR), necessitating improved detection and phenotypic characterization of treatment-resistant residual disease. In this work, we have optimized droplet digital PCR to broadly measure mutated alleles of recurrently mutated genes in CR marrows of AML patients at levels as low as 0.002% variant allele frequency. Most gene mutations persisted in CR, albeit at highly variable and gene-dependent levels. The majority of AML cases demonstrated residual aberrant oligoclonal hematopoiesis. Importantly, we detected very rare cells (as few as 1 in 15,000) that were genomically similar to the dominant blast populations at diagnosis and were fully clonally represented at relapse, identifying these rare cells as one common source of AML relapse. Clinically, the mutant allele burden was associated with overall survival in AML, and our findings narrow the repertoire of gene mutations useful in minimal residual disease-based prognostication in AML. Overall, this work delineates rare cell populations that cause AML relapse, with direct implications for AML research directions and strategies to improve AML therapies and outcome.

Periampullary carcinomas are a group of rare lesions around the ampulla of Vater including distal bile duct and duodenum and are very different from pancreatic ductal adenocarcinoma clinically and pathologically, but the molecular alterations in these tumours are less known . Genetic alterations of the KRAS oncogenes, tumour suppressor genes p53 , p16 and MADH4 ( SMAD4/DPC4 ) and genome maintenance genes (MLHI, MSH2) are commonly altered in pancreatic adenocarcinoma and have also been described in periampullary cancers, although at lower frequencies. To understand the molecular characteristics of non-pancreatic periampullary carcinomas, ampullary cancers can now be further defined accurately into their intestinal and pancreatobiliary subtypes using histomolecular profiling. KRAS mutation, which occurs in most pancreatic cancers, is found to occur less frequently in ampullary (42-52%), biliary (22-23%) and duodenal cancers (32-35%). Mutations are also found in tumour suppressor genes (p53) and are associated with transformation of adenomas and low-grade carcinomas into high-grade carcinomas. Loss of DPC4 occurs late in ampullary carcinogenesis. This study summarizes the current knowledge in molecular aberrations in non-pancreatic periampullary cancers., Competing Interests: Conflict of InterestThe authors declare that they have no conflict of interest., (© Indian Association of Surgical Oncology 2017.)

Current biomarkers for sepsis are limited by their non-specificity, short half-life, and insensitive response to therapy. Recently, we have demonstrated that citrullinated histone H3(CitH3) is released into the blood from neutrophil extracellular traps(NETs) in response to severe infection, and CitH3 may be a potential biomarker for sepsis. In the present study, we found that NET components were released in mouse models of both lipopolysaccharide(LPS)-induced shock (LPSS) and hemorrhagic shock (HS). To further quantify CitH3 in the NETs, we established a CitH3 specific enzyme-linked immunosorbent assay. Circulating CitH3 was found to be elevated only in LPSS but not in HS. Importantly, blood CitH3 was detected 30 minutes after LPS insult, and remained elevated for 24 hours (period of the highest mortality). Treatment of endotoxic mice with YW3-56, a peptidylarginine deiminase-2/4 inhibitor, significantly diminished levels of CitH3 in the blood. Interleukin-1β did not respond to LPS early, and interleukin-1β and interleukin-6 fluctuated although they responded to treatment. Procalcitonin reacted to LPS insult late. Compared to CitH3, these biomarkers were non-specifically induced in LPSS and HS. Collectively, our results demonstrate that YW3-56 protects animals from LPSS, and CitH3 is a reliable biomarker due to its early appearance, specificity, duration, and response to therapeutic intervention.

Purpose: Noninvasive drug biomarkers for the early assessment of tumor response can enable adaptive therapeutic decision-making and proof-of-concept studies for investigational drugs. Circulating tumor DNA (ctDNA) is released into the circulation by tumor cell turnover and has been shown to be detectable in urine. Experimental Design: We tested the hypothesis that dynamic changes in EGFR activating (exon 19del and L858R) and resistance (T790M) mutation levels detected in urine could inform tumor response within days of therapy for advanced non-small cell lung cancer (NSCLC) patients receiving osimertinib, a second-line third-generation anti-EGFR tyrosine kinase inhibitor. Results: Eight of nine evaluable NSCLC patients had detectable T790M-mutant DNA fragments in pretreatment baseline samples. Daily monitoring of mutations in urine indicated a pattern of intermittent spikes throughout week 1, suggesting apoptosis with an overall decrease in fragment numbers from baselines to day 7 preceding radiographic response assessed at 6 to 12 weeks. Conclusions: These findings suggest drug-induced tumor apoptosis within days of initial dosing. Daily sampling of ctDNA may enable early assessment of patient response and proof-of-concept studies for drug development. The modeling of tumor lysis through the day-to-day kinetics of ctDNA released into the blood and then into the urine is demonstrated in this proof-of-concept study in lung cancer patients receiving anti-EGFR tyrosine kinase inhibitors. This strategy may determine the specific clonal populations of cells which undergo apoptosis within the first week of therapy. This has important implications for developing combinational strategies to address inter- and intralesional heterogeneity and characterizing residual disease after initial drug exposure. Clin Cancer Res; 23(16); 4716-23. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Keap1-Nrf2 pathway is continuously involved in the cytoprotection from oxidative stress generated due to various factors either extrinsic or intrinsic in origin. This role of Nrf2 in the response to oxidative stress is well established. Following oxidative insult, Nrf2 mediates the regulation of the inducible expression of cytoprotective genes. The level and functional capacity of Nrf2 is regulated at the post-transcriptional level, mainly through its association with an actin-associated protein, Keap1. Various studies reported that any discrepancy from their routine may lead to promotion of tumor as well. So there is need to explore their role in cytoprotection and tumor promotion if any. This review is an attempt to critically analyze the available data that may lighten up the present knowledge and unveil the new regime for cancer prevention and treatment., (Copyright © 2017. Published by Elsevier B.V.)

Marrow-derived macrophages are highly phagocytic, but whether they can also traffic into solid tumors and engulf cancer cells is questionable, given the well-known limitations of tumor-associated macrophages (TAMs). Here, SIRPα on macrophages from mouse and human marrow was inhibited to block recognition of its ligand, the "marker of self" CD47 on all other cells. These macrophages were then systemically injected into mice with fluorescent human tumors that had been antibody targeted. Within days, the tumors regressed, and single-cell fluorescence analyses showed that the more the macrophages engulfed, the more they accumulated within regressing tumors. Human-marrow-derived macrophages engorged on the human tumors, while TAMs were minimally phagocytic, even toward CD47-knockdown tumors. Past studies had opsonized tumors in situ with antibody and/or relied on mouse TAMs but had not injected SIRPα-inhibited cells; also, unlike past injections of anti-CD47, blood parameters remained normal and safe. Consistent with tumor-selective engorge-and-accumulate processes in vivo, phagocytosis in vitro inhibited macrophage migration through micropores that mimic features of dense 3D tissue. Accumulation of SIRPα-inhibited macrophages in tumors favored tumor regression for 1-2 weeks, but donor macrophages quickly differentiated toward non-phagocytic, high-SIRPα TAMs. Analyses of macrophages on soft (like marrow) or stiff (like solid tumors) collagenous gels demonstrated a stiffness-driven, retinoic-acid-modulated upregulation of SIRPα and the mechanosensitive nuclear marker lamin-A. Mechanosensitive differentiation was similarly evident in vivo and likely limited the anti-tumor effects, as confirmed by re-initiation of tumor regression by fresh injections of SIRPα-inhibited macrophages. Macrophage motility, phagocytosis, and differentiation in vivo are thus coupled., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The cancer community understands the value of blood profiling measurements in assessing and monitoring cancer. We describe an effort among academic, government, biotechnology, diagnostic, and pharmaceutical companies called the Blood Profiling Atlas in Cancer (BloodPAC) Project. BloodPAC will aggregate, make freely available, and harmonize for further analyses, raw datasets, relevant associated clinical data (e.g., clinical diagnosis, treatment history, and outcomes), and sample preparation and handling protocols to accelerate the development of blood profiling assays., (© 2017 Authors Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Health information technology (HIT) has great potential for increasing patient engagement. Pediatric hematopoietic cell transplantation (HCT) is a setting ripe for using HIT but in which little research exists. "BMT Roadmap" is a web-based application that integrates patient-specific information and includes several domains: laboratory results, medications, clinical trial details, photos of the healthcare team, trajectory of transplant process, and discharge checklist. BMT Roadmap was provided to 10 caregivers of patients undergoing first-time HCT. Research assistants performed weekly qualitative interviews throughout the patient's hospitalization and at discharge and day 100 to assess the impact of BMT Roadmap. Rigorous thematic analysis revealed 5 recurrent themes: emotional impact of the HCT process itself; critical importance of communication among patients, caregivers, and healthcare providers; ways in which BMT Roadmap was helpful during inpatient setting; suggestions for improving BMT Roadmap; and other strategies for organization and management of complex healthcare needs that could be incorporated into BMT Roadmap. Caregivers found the tool useful and easy to use, leading them to want even greater access to information. BMT Roadmap was feasible, with no disruption to inpatient care. Although this initial study is limited by the small sample size and single-institution experience, these initial findings are encouraging and support further investigation., (Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: We developed and beta-tested a patient-centered medication management application, PresRx optical character recognition (OCR), a mobile health (m-health) tool that auto-populates drug name and dosing instructions directly from patients' medication labels by OCR., Materials and Methods: We employed a single-subject design study to evaluate PresRx OCR for three outcomes: (1) accuracy of auto-populated medication dosing instructions, (2) acceptability of the user interface, and (3) patients' adherence to chronic medications., Results: Eight patients beta-tested PresRx OCR. Five patients used the software for ≥6 months, and four completed exit interviews (n = 4 completers). At baseline, patients used 3.4 chronic prescription medications and exhibited moderate-to-high adherence rates. Accuracy of auto-populated information by OCR was 95% for drug name, 98% for dose, and 96% for frequency. Study completers rated PresRx OCR 74 on the System Usability Scale, where scores ≥70 indicate an acceptable user interface (scale 0-100). Adherence rates measured by PresRx OCR were high during the first month of app use (93%), but waned midway through the 6-month testing period (78%). Compared with pharmacy fill rates, PresRx OCR underestimated adherence among completers by 3%, while it overestimated adherence among noncompleters by 8%., Discussion: Results suggest smartphone applications supporting medication management are feasible and accurately assess adherence compared with objective measures. Future efforts to improve medication-taking behavior using m-health tools should target specific patient populations and leverage common application programming interfaces to promote generalizability., Conclusions: Our medication management application PresRx OCR is innovative, acceptable for patient use, and accurately tracks medication adherence.

Purpose To compare the abilities of three pulsed focused ultrasound regimes (that cause tissue liquefaction, permeabilization, or mild heating) to release tumor-derived microRNA into the circulation in vivo and to evaluate release dynamics. Materials and Methods All rat experiments were approved by the University of Washington Institutional Animal Care and Use Committee. Reverse-transcription quantitative polymerase chain reaction array profiling was used to identify candidate microRNA biomarkers in a rat solid tumor cell line. Rats subcutaneously grafted with these cells were randomly assigned among three pulsed focused ultrasound treatment groups: (a) local tissue liquefaction via boiling histotripsy, (b) tissue permeabilization via inertial cavitation, and (c) mild (<10°C) heating of tissue, as well as a sham-treated control group. Blood specimens were drawn immediately prior to treatment and serially over 24 hours afterward. Plasma microRNA was quantified with reverse-transcription quantitative polymerase chain reaction, and statistical significance was determined with one-way analysis of variance (Kruskal-Wallis and Friedman tests), followed by the Dunn multiple-comparisons test. Results After tissue liquefaction and cavitation treatments (but not mild heating), plasma quantities of candidate biomarkers increased significantly (P value range, <.0001 to .04) relative to sham-treated controls. A threefold to 32-fold increase occurred within 15 minutes after initiation of pulsed focused ultrasound tumor treatment, and these increases persisted for 3 hours. Histologic examination confirmed complete liquefaction of the targeted tumor area with boiling histotripsy, in addition to areas of petechial hemorrhage and tissue disruption by means of cavitation-based treatment. Conclusion Mechanical tumor tissue disruption with pulsed focused ultrasound-induced bubble activity significantly increases the plasma abundance of tumor-derived microRNA rapidly after treatment. © RSNA, 2016 Online supplemental material is available for this article.

Extracellular vesicles (EVs) contain a wide range of RNA types with a reported prevalence of non-coding RNA. To date a comprehensive characterization of the protein coding transcripts in EVs is still lacking. We performed RNA-Sequencing (RNA-Seq) of 2 EV populations and identified a small fraction of transcripts that were expressed at significantly different levels in large oncosomes and exosomes, suggesting they may mediate specialized functions. However, these 2 EV populations exhibited a common mRNA signature that, in comparison to their donor cells, was significantly enriched in mRNAs encoding E2F transcriptional targets and histone proteins. These mRNAs are primarily expressed in the S-phase of the cell cycle, suggesting that they may be packaged into EVs during S-phase. In silico analysis using subcellular compartment transcriptome data from the ENCODE cell line compendium revealed that EV mRNAs originate from a cytoplasmic RNA pool. The EV signature was independently identified in plasma of patients with breast cancer by RNA-Seq. Furthermore, several transcripts differentially expressed in EVs from patients versus controls mirrored differential expression between normal and breast cancer tissues. Altogether, this largest high-throughput profiling of EV mRNA demonstrates that EVs carry tumor-specific alterations and can be interrogated as a source of cancer-derived cargo.

Migration through micron-size constrictions has been seen to rupture the nucleus, release nuclear-localized GFP, and cause localized accumulations of ectopic 53BP1-a DNA repair protein. Here, constricted migration of two human cancer cell types and primary mesenchymal stem cells (MSCs) increases DNA breaks throughout the nucleoplasm as assessed by endogenous damage markers and by electrophoretic "comet" measurements. Migration also causes multiple DNA repair proteins to segregate away from DNA, with cytoplasmic mis-localization sustained for many hours as is relevant to delayed repair. Partial knockdown of repair factors that also regulate chromosome copy numbers is seen to increase DNA breaks in U2OS osteosarcoma cells without affecting migration and with nucleoplasmic patterns of damage similar to constricted migration. Such depletion also causes aberrant levels of DNA. Migration-induced nuclear damage is nonetheless reversible for wild-type and sub-cloned U2OS cells, except for lasting genomic differences between stable clones as revealed by DNA arrays and sequencing. Gains and losses of hundreds of megabases in many chromosomes are typical of the changes and heterogeneity in bone cancer. Phenotypic differences that arise from constricted migration of U2OS clones are further illustrated by a clone with a highly elongated and stable MSC-like shape that depends on microtubule assembly downstream of the transcription factor GATA4. Such changes are consistent with reversion to a more stem-like state upstream of cancerous osteoblastic cells. Migration-induced genomic instability can thus associate with heritable changes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objectives: Optimal conditions for blood collection for circulating tumor DNA (ctDNA) are still being developed. Although both Streck and EDTA tubes are commonly used, their ability to stabilize ctDNA as a function of time and temperature post-collection has not been thoroughly studied. Additionally, the potential utility of CellSave tubes (commonly used for circulating tumor cell) for ctDNA measurements has not been studied., Design and Methods: Blood was collected into Streck, EDTA, and CellSave tubes from ten patients with metastatic breast cancer enrolled in the MI-ONCOSEQ tumor sequencing program at the University of Michigan and kept either on ice or at room temperature until plasma isolation. Plasma was processed after 2, 6, and 48h post-collection. We used droplet digital PCR (ddPCR) to quantify plasma ctDNA and wild-type DNA for six patients who had tumor tissue mutations represented in commercially available ddPCR assays., Results: ctDNA abundance was similar and stable for up to 6h in all tube types, and there was no effect of storage temperature on the yield for Streck and EDTA tubes. After 48h, however, one out of four patients with detectable ctDNA showed a ~50% decline in ctDNA in the EDTA tube, and three out of six patients showed a 2-3-fold increase in wild-type DNA in the EDTA tube., Conclusions: Streck, EDTA, and CellSave tubes showed similar performance in preserving ctDNA for up to 6h before plasma isolation. Streck and CellSave tubes more consistently stabilized ctDNA and wild-type DNA at 48h than EDTA tubes., (Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: Pancreaticoduodenectomy with superior mesenteric/portal venous resection for pancreatic ductal adenocarcinoma (PDAC) is frequently performed with no added morbidity or mortality in case of tumor abutment to the superior mesenteric or portal vein so as to obtain a margin negative resection. True histopathological portal vein invasion is found only in a small subset of such patients. The aim of this review aimed to discuss the significance of histopathological venous invasion in PDAC., Data Sources: For this review available data was searched from PubMed and analyzed. No randomized trials have been published on this topic., Results: Existing data on prognostic factors in histopathological venous invasion by PDAC are limited and recent studies indicate worse survival in this subgroup of patients. In addition, venous invasion in PDAC has been associated with large tumors, involved lymph nodes, perineural invasion and R1 resection. The survival of patients with portal venous resection but without histologic venous invasion is reportedly better than those with histopathological venous invasion; though conflicting studies do exist on the subject. Some studies also relate the depth of venous invasion to prognosis after surgical resection of PDAC., Conclusions: Frank/'histopathological' invasion of superior mesenteric/portal venous and R1 resection indicate a very poor survival. Such patients may be given the opportunity of benefit of neoadjuvant treatment.

Carcinoma of the ampulla of Vater associated with annular pancreas is a rare entity. Only seven cases have been reported so far in the English literature. We herein report one such case in a 42-year-lady who presented with progressively increasing jaundice and pain in the upper abdomen for past 2 months. A magnetic resonance cholangiopancreaticography (MRCP) and MRI revealed an annular pancreas with an ampullary mass and a dilated CBD. An upper gastrointestinal endoscopic biopsy from the ampullary mass revealed a moderately differentiated adenocarcinoma. She was diagnosed as having annular pancreas with periampullary cancer that was subsequently confirmed at laparotomy. Histological examination confirmed a complete annular pancreas with a 2 cm × 2 cm moderately differentiated adenocarcinoma of the ampulla of Vater. The management, however, remains as in any case of periampullary malignancy. This highlights the importance that obstructive jaundice in an adult patient presenting with annular pancreas may be associated with a coexisting periampullary malignancy., Competing Interests: The authors declare that they have no competing interests.

Background: Surgery (R0 resection) is the mainstay of treatment of gallbladder cancer (GBC) as GBC is relatively resistant to currently known chemotherapy and radiotherapy regimens., Aim: to assess if wedge resection of the gallbladder bed achieves an adequate oncological clearance in GBC (namely T1 and T2) and some T3 GBC with minimal liver infiltration., Patients and Methods: Patients with GBC who underwent radical cholecystectomy (en bloc cholecystectomy, wedge resection of the gallbladder fossa with a ≥2 cm rim of nonneoplastic liver tissue, and regional lymph node dissection) between October 2012 and June 2015 after obtaining informed consent., Results: Of thirty patients, mean age of 52 years, 5 had T1b, 13 T2, and 12 T3 GBC. R0 resection was achieved in all thirty GBC patients. Hepatic invasion was found in seven patients. The depth of hepatic invasion ranged from 0 to 9 mm. Follow-up ranged from a minimum of 12 to 43 months. Nineteen (63%) patients had N0 and 11 (37%) had N1 GBC. Total lymph node (TLND) count ranged from 1 to 12/patient with a median of 3. There was no local recurrence or systemic relapse of the disease., Conclusion: Wedge resection of the gallbladder bed achieves an adequate oncological clearance in early GBC. TLND counts remain poor even after a thorough standard lymph node dissection for resectable GBC.

Chylous ascites is a very rare occurrence in a patient with gallbladder cancer (GBC), and only six cases have been reported. We report here one such case in a 55-year-old lady who presented to us with upper abdominal pain for 6 months. A polypoidal gallbladder mass with minimal liver invasion but with multiple subcentimeter pericholedochal, common hepatic, mesenteric, and para-aortic lymph nodes was found on contrast-enhanced computed tomography scan. At laparotomy, the abdomen had milky fluid with engorged beaded lymphatics all over the small intestine. The abdomen was closed over a drain after a lymph node biopsy and collection of the ascetic fluid for analysis. Histopathology confirmed metastatic adenocarcinoma in the lymph node. The triglyceride levels in the ascetic fluid were elevated to 817.00 mg/dl. The patient was put on medium-chain triglyceride diet and a diuretic and recovered well. She was discharged when drain output was nil. Chylous ascites could be a result of abdominal malignancy, post surgery, cirrhosis, and disseminated infections like tuberculosis and filariasis. Treatment is primarily conservative and includes paracentesis/drainage of the peritoneal cavity supplemented by fasting, total parenteral nutrition, and/or diet modification with medium-chain triglyceride diet., Competing Interests: The authors declare that they have no competing interests.

In the beating heart, cardiac myocytes (CMs) contract in a coordinated fashion, generating contractile wave fronts that propagate through the heart with each beat. Coordinating this wave front requires fast and robust signaling mechanisms between CMs. The primary signaling mechanism has long been identified as electrical: gap junctions conduct ions between CMs, triggering membrane depolarization, intracellular calcium release, and actomyosin contraction. In contrast, we propose here that, in the early embryonic heart tube, the signaling mechanism coordinating beats is mechanical rather than electrical. We present a simple biophysical model in which CMs are mechanically excitable inclusions embedded within the extracellular matrix (ECM), modeled as an elastic-fluid biphasic material. Our model predicts strong stiffness dependence in both the heartbeat velocity and strain in isolated hearts, as well as the strain for a hydrogel-cultured CM, in quantitative agreement with recent experiments. We challenge our model with experiments disrupting electrical conduction by perfusing intact adult and embryonic hearts with a gap junction blocker, β-glycyrrhetinic acid (BGA). We find this treatment causes rapid failure in adult hearts but not embryonic hearts-consistent with our hypothesis. Last, our model predicts a minimum matrix stiffness necessary to propagate a mechanically coordinated wave front. The predicted value is in accord with our stiffness measurements at the onset of beating, suggesting that mechanical signaling may initiate the very first heartbeats.

Introduction: PAX5 is an immunomarker of B-cell origin and useful in the diagnosis of lymphoma. There is hardly any study on PAX5 expression in Indian patients with lymphoma., Aim: To evaluate the diagnostic utility of PAX5 as an adjunct immunohistochemical marker in the diagnosis of Hodgkin Lymphoma (HL) and Non-Hodgkin Lymphoma (NHL)., Materials and Methods: Immunohistochemistry was performed against CD20, CD3, CD15, CD30, and PAX5 on formalin fixed paraffin embedded tissue of 71 cases of lymphoma and CD20, CD3 and PAX5 in control samples of reactive lymph nodes. Frequency, mean values, and percentage were calculated. Fisher's-exact test and test for analysis of variance were applied., Result: For 24 cases of HL and 47 cases of NHL, the mean age of patients was 17.6±14.8 years and 44.1±21.6 years, respectively. The male: female ratio for both HL and NHL were 1.7:1. Among NHL cases, the numbers of B-cell and T-cell types were 39/47 (83%) and 8/47 (17%), respectively. In comparison to control samples, PAX5+ expression was seen in 23/24 (95.8%) cases of HL (p=1.000) and 32/39 (82%) cases of B-NHL (p=0.0834). All the cases of T-NHL showed negative expression of PAX5 (p<0.0001). Analysis of variance between NHL, HL and control samples was statistically significant (p<0.0001)., Conclusion: PAX5 staining between control samples and cases of classical HL and B-NHL was statistically not significant, whereas, statistically significant difference was observed with T-NHL. Thus, PAX5 may be used as an adjunct marker in the diagnosis of classical HL and B-NHL.

The clinical potential of circulating tumor cells (CTCs) in managing cancer metastasis is significant. However, low CTC isolation purities from patient blood have hindered sensitive molecular assays of these rare cells. Described herein is the ultra-pure isolation of CTCs from patient blood samples and how this platform has enabled highly specific molecular (mRNA and miRNA) profiling of patient CTCs.

Digital PCR (dPCR) is gaining popularity as a DNA mutation quantification method for clinical specimens. Fragmentation prior to dPCR is required for non-fragmented genomic DNA samples; however, the effect of fragmentation on DNA analysis has not been well-studied. Here we evaluated three fragmentation methods for their effects on dPCR point mutation assay performance. Wild-type (WT) human genomic DNA was fragmented by heating, restriction digestion, or acoustic shearing using a Covaris focused-ultrasonicator. dPCR was then used to determine the limit of blank (LoB) by quantifying observed WT and mutant allele counts of the proto-oncogenes KRAS and BRAF in the WT DNA sample. DNA fragmentation by heating to 95°C, while the simplest and least expensive method, produced a high background mutation frequency for certain KRAS mutations relative to the other methods. This was due to heat-induced mutations, specifically affecting dPCR assays designed to interrogate guanine to adenine (G>A) mutations. Moreover, heat-induced fragmentation overestimated gene copy number, potentially due to denaturation and partition of single-stranded DNA into different droplets. Covaris acoustic shearing and restriction enzyme digestion showed similar LoBs and gene copy number estimates to one another. It should be noted that moderate heating, commonly used in genomic DNA extraction protocols, did not significantly increase observed KRAS mutation counts.

Background: Free radicals such as reactive oxygen species (ROS), which induce oxidative stress, are the main contributors to head and neck carcinogenesis (HNC). The present study was conducted with the aim to assess the oxidant/antioxidant status and DNA damage analysis in head and neck cancer/control patients., Materials and Methods: This prospective study was conducted on 60 patients with biopsy-proven HNC and 17 patients of head and neck disease (HND). The total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) were determined by novel automatic colorimetric methods from tissue homogenate. DNA damage analysis was determined by single cell gel electrophoresis (SCGE)., Results: The mean age of the study cohort was 46.65 ± 14.84 years for HNC patients, while it was 49.41 ± 13.00 years for HND patients. There were no significant differences found between the two groups with respect to demographic presentation except tobacco addiction. The association between oxidative stress parameters and DNA damage analysis with study group revealed the following. (A) DNA damage - tissue homogenate TOS and OSI were significantly higher in HNC subjects than in HND (16.06 ± 1.78 AU vs 7.86 ± 5.97 AU, P < 0.001; 53.00 ± 40.61 vs 19.67 ± 21.90, P < 0.01; 7.221 ± 5.80 vs 2.40 ± 2.54, P < 0.01, respectively), while TAS was significantly decreased. (B) Aggressive histological features were identified, more commonly with higher TOS and lower TAS [probability (P) = 0.002, relative risk (RR) = 11.838, 95% confidence interval CI = 2.514-55.730 and P = 0.043, RR = 0.271, 95% CI = 0.077-0.960, respectively]., Conclusion: The increase in free radicals may be the event that led to the reduction of antioxidant status in HNC, thus explaining the oxidative damage of DNA and the severity of disease. Increased OSI represents a general mechanism in its pathogenesis.

Health information technology (IT) has opened exciting avenues for capturing, delivering and sharing data, and offers the potential to develop cost-effective, patient-focused applications. In recent years, there has been a proliferation of health IT applications such as outpatient portals. Rigorous evaluation is fundamental to ensure effectiveness and sustainability, as resistance to more widespread adoption of outpatient portals may be due to lack of user friendliness. Health IT applications that integrate with the existing electronic health record and present information in a condensed, user-friendly format could improve coordination of care and communication. Importantly, these applications should be developed systematically with appropriate methodological design and testing to ensure usefulness, adoption, and sustainability. Based on our prior work that identified numerous information needs and challenges of HCT, we developed an experimental prototype of a health IT tool, the BMT Roadmap. Our goal was to develop a tool that could be used in the real-world, daily practice of HCT patients and caregivers (users) in the inpatient setting. Herein, we examined the views, needs, and wants of users in the design and development process of the BMT Roadmap through user-centered Design Groups. Three important themes emerged: 1) perception of core features as beneficial (views), 2) alerting the design team to potential issues with the user interface (needs); and 3) providing a deeper understanding of the user experience in terms of wider psychosocial requirements (wants). These findings resulted in changes that led to an improved, functional BMT Roadmap product, which will be tested as an intervention in the pediatric HCT population in the fall of 2015 (ClinicalTrials.govNCT02409121)., (Copyright © 2016 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)