Snap-frozen Samples Research Articles

Hepatitis A Virus Infection in Cynomolgus Monkeys Confounds the Safety Evaluation of a Drug Candidate.

In a 3-month toxicity study in cynomolgus monkeys at a European contract laboratory, animals were infected with HAV, initially resulting in hepatic injury being incorrectly attributed to the test compound. Elevated serum ALT/AST/GLDH (5- to 10-fold) were noted in individual animals from all groups including controls, with no apparent dose, exposure, or time-related relationship. Liver histopathology revealed minimal to slight inflammatory cell accumulation in periportal zones of most animals, and minimal to slight hepatocyte degeneration/necrosis in 10/42 animals from all groups. As these findings were more pronounced in 6 drug-treated animals, including 2/6 in the low dose group, the draft report concluded: "treatment-related hepatotoxicity at all dose levels precluded determination of a NOAEL." However, the unusual pattern of hepatotoxicity suggested a factor other than drug exposure might have caused the hepatic effects. Therefore, snap-frozen liver samples were tested for hepatitis viruses using a PCR method. Tests for hepatitis B, C, and E virus were negative; however, 20/42 samples were positive for hepatitis A virus (HAV). Infection was strongly associated with increased serum ALT/GLDH, and/or hepatocyte degeneration/necrosis. Re-evaluation of the study in light of these data concluded that the hepatic injury was not drug-related. A subsequent 6-month toxicology study in HAV-vaccinated cynomolgus monkeys confirmed the absence of hepatotoxicity. Identification of HAV infection supported progression of the drug candidate into later clinical trials. Although rarely investigated, subclinical HAV infection has occasionally been reported in laboratory primates, including those used for toxicology studies and it may be more prevalent than the literature indicates.

INNV-36. ULTRA-FAST DEEP-LEARNED CNS TUMOR CLASSIFICATION USING NANOPORE-SEQUENCING DURING SURGERY

Abstract BACKGROUND Preoperative imaging and intraoperative histological analysis provide important guidance during CNS tumor surgery but are not always precise enough. Also, the diagnosis of CNS tumors increasingly requires a combination of morphological and molecular analysis. Illumina DNA methylation array analysis provides information on hundreds of thousands of methylation sites genome-wide but is relatively time-consuming and expensive. Nanopore sequencing enables real-time generation of an increasingly precise methylation profile. AIM: To exploit the potential of nanopore sequencing as a tool for intraoperative molecular diagnostics of CNS tumors. METHODS We developed Sturgeon, a patient-agnostic, transfer-learned neural network approach. Sturgeon leverages publicly available Illumina 450K DNA methylation profiles to simulate millions of nanopore sequencing experiments, which were used to train and validate classifier models. Subsequently, snap-frozen archival samples of 50 CNS tumors were analyzed, as well as fresh tumor samples of an additional 25 CNS tumors intraoperatively. RESULTS Sturgeon delivers models capable of classifying 81 different CNS tumor types based on relatively sparse, randomly distributed nanopore sequencing data. Sturgeon was able to suggest within 40 minutes after the start of sequencing the correct diagnosis with high confidence in 45 out of 50 archival CNS tumor samples; in the remaining cases Sturgeon abstained from a diagnosis. In the real-life scenario, in 18 out of 25 cases (72%) the suggested diagnosis was correct, and in 7 cases the required confidence threshold was not reached (mostly because of low tumor cell percentage). Importantly, the Sturgeon models are portable, work across patients and sequence depths, and require less than a minute of computation on a laptop CPU. CONCLUSIONS Intraoperative nanopore sequencing can assist neurosurgical decision making and opens avenues for starting tumor-specific therapy already during operation. Furthermore, this diagnostic approach has the potential to reduce the costs of methylation profiling analysis and speed-up the multidisciplinary decision-making process.

Shed skin as a source of DNA for genotyping-by-sequencing (GBS) in reptiles

Over a fifth of reptile species are classified as ‘Threatened’ and conservation efforts, especially those aimed at recovery of isolated or fragmented populations, will require genetic and genomic data and resources. Shed skins of snakes and other reptiles contain DNA; are a safe and ethical way of non-invasively sampling large numbers of individuals; and provide a simple mechanism by which to involve the public in scientific research. Here we test whether the DNA in dried shed skin is suitable for reduced representation sequencing approaches, specifically genotyping-by-sequencing (GBS). Shed skin-derived libraries resulted in fewer sequenced reads than those from snap-frozen muscle samples, and contained slightly fewer variants (70,685 SNPs versus 97,724), but this issue can easily be rectified with deeper sequencing of shed skin-derived libraries. Skin-derived libraries also have a very slight (but significantly different) profile of transitions and transversions, most likely as a result of DNA damage, but the impact of this is minimal given the large number of single nucleotide polymorphisms (SNPs) involved. SNP density tends to scale with chromosome length, and microchromosomes have a significantly higher SNP density than macrochromosomes, most likely because of their higher GC content. Overall, shed skin provides DNA of sufficient quality and quantity for the identification of large number of SNPs, but requires greater sequencing depth, and consideration of the GC richness of microchromosomes when selecting restriction enzymes.

Association between skeletal muscle mitochondrial dysfunction and insulin resistance in patients with rheumatoid arthritis: a case–control study

BackgroundInsulin resistance affects a substantial proportion of patients with rheumatoid arthritis (RA). Skeletal muscle mitochondrial dysfunction results in the accumulation of lipid intermediates that interfere with insulin signaling. We therefore sought to determine if lower oxidative phosphorylation and muscle mitochondrial content are associated with insulin resistance in patients with RA.MethodsThis was a cross-sectional prospective study of RA patients. Matsuda index from the glucose tolerance test was used to estimate insulin sensitivity. Mitochondrial content was measured by citrate synthase (CS) activity in snap-frozen muscle samples. Mitochondrial function was measured by using high-resolution respirometry of permeabilized muscle fibers and electron transport chain complex IV enzyme kinetics in isolated mitochondrial subpopulations.ResultsRA participants demonstrated lower insulin sensitivity as measured by the Matsuda index compared to controls [median 3.95 IQR (2.33, 5.64) vs. 7.17 (5.83, 7.75), p = 0.02]. There was lower muscle mitochondrial content among RA vs. controls [median 60 mU/mg IQR (45, 80) vs. 79 mU/mg (65, 97), p = 0.03]. Notably, OxPhos normalized to mitochondrial content was higher among RA vs. controls [mean difference (95% CI) = 0.14 (0.02, 0.26), p = 0.03], indicating a possible compensatory mechanism for lower mitochondrial content or lipid overload. Among RA participants, the activity of muscle CS activity was not correlated with the Matsuda index (ρ = − 0.05, p = 0.84), but it was positively correlated with self-reported (IPAQ) total MET-minutes/week (ρ = 0.44, p = 0.03) and Actigraph-measured time on physical activity (MET rate) (ρ = 0.47, p = 0.03).ConclusionsMitochondrial content and function were not associated with insulin sensitivity among participants with RA. However, our study demonstrates a significant association between muscle mitochondrial content and physical activity level, highlighting the potential for future exercise interventions that enhance mitochondrial efficiency in RA patients.

A Comparison of MGMT Testing by MSP and qMSP in Paired Snap-Frozen and Formalin-Fixed Paraffin-Embedded Gliomas.

Comparative analysis of the conventional methylation-specific PCR (MSP) vs. the quantitative MSP (qMSP) assessment of the O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status in 34 snap-frozen (SF) glioma samples was performed. The accuracy of the semi-quantitative MSP was compared with the corresponding qMSP semi-quantitative values using two semi-quantitative cut-off values (0-unmethylated and 1-weakly methylated) to discriminate methylated from unmethylated samples. In the case of the cut-off value 0, MSP test showed 80.0% sensitivity and 78.9% specificity compared to the reference qMSP analysis. However, when using the cut-off value 1, the diagnostic accuracy of the MSP test was significantly higher (85.7% sensitivity, 85.2% specificity). Fleiss' Kappa statistical analyses indicated moderate agreement (Fleiss' Kappa Coefficient = 0.509; 70.59% agreement) between MSP and qMSP semi-quantitative measurements of MGMT promoter methylation in glioma patients, justifying the conventional MSP use in diagnostics and confirming its high reliability. Further, we aimed to compare the validity of SF and formalin-fixed paraffin-embedded (FFPE) glioma samples for MGMT testing. Statistical analyses indicated moderate overall agreement of FFPE glioma samples and SF MSP semi-quantitative measurements (Fleiss' Kappa Coefficient = 0.516/0.509; 70.0% agreement) and emphasized their low reliability in the assessment of highly methylated MGMT promoter samples.

Single-nucleus RNA-Seq reveals singular gene signatures of human ductal cells during adaptation to insulin resistance.

Adaptation to increased insulin demand is mediated by β cell proliferation and neogenesis, among other mechanisms. Although it is known that pancreatic β cells can arise from ductal progenitors, these observations have been limited mostly to the neonatal period. We have recently reported that the duct is a source of insulin-secreting cells in adult insulin-resistant states. To further explore the signaling pathways underlying the dynamic β cell reserve during insulin resistance, we undertook human islet and duct transplantations under the kidney capsule of immunodeficient NOD/SCID-γ (NSG) mouse models that were pregnant, were insulin-resistant, or had insulin resistance superimposed upon pregnancy (insulin resistance + pregnancy), followed by single-nucleus RNA-Seq (snRNA-Seq) on snap-frozen graft samples. We observed an upregulation of proliferation markers (e.g., NEAT1) and expression of islet endocrine cell markers (e.g., GCG and PPY), as well as mature β cell markers (e.g., INS), in transplanted human duct grafts in response to high insulin demand. We also noted downregulation of ductal cell identity genes (e.g., KRT19 and ONECUT2) coupled with upregulation of β cell development and insulin signaling pathways. These results indicate that subsets of ductal cells are able to gain β cell identity and reflect a form of compensation during the adaptation to insulin resistance in both physiological and pathological states.

Decreased Expression of Estrogen Receptors Is Associated with Tumorigenesis in Papillary Thyroid Carcinoma.

Papillary thyroid carcinomas (PTC), which is derived from thyroid follicular cells, is the most commonly differentiated thyroid cancer with sex disparity. However, the role of estrogen receptors (ERs) in the pathogenesis of PTC remains unclear. The present study aimed to determine the association of ER mRNA expression levels with clinicopathologic features in PTC. To that aim, the mRNA levels of ESR1 (ERα66), ESR1 (ERα36), ESR2, and G-protein-coupled estrogen receptor 1 (GPER1) in snap-frozen tissue samples from PTCs and adjacent normal thyroid tissues were determined using quantitative reverse transcription polymerase chain reaction (RT-qPCR), and the correlation between ER mRNA expression levels and clinicopathologic features was analyzed. The expression of ERα66, ERα36, ERβ, and GPER1 was lower in PTC specimens than in adjacent normal thyroid tissues. Moreover, low GPER1 expression was associated with extrathyroidal extension. There was no obvious difference in expression of ERs between PTC specimens from male and female patients. In conclusion, our findings highlight the importance of ERs in PTC tumorigenesis.

The Placental Innate Immune System Is Altered in Early-Onset Preeclampsia, but Not in Late-Onset Preeclampsia.

Preeclampsia is a severe placenta-related pregnancy disorder that is generally divided into two subtypes named early-onset preeclampsia (onset <34 weeks of gestation), and late-onset preeclampsia (onset ≥34 weeks of gestation), with distinct pathophysiological origins. Both forms of preeclampsia have been associated with maternal systemic inflammation. However, alterations in the placental immune system have been less well characterized. Here, we studied immunological alterations in early- and late-onset preeclampsia placentas using a targeted expression profile approach. RNA was extracted from snap-frozen placenta samples (healthy n=13, early-onset preeclampsia n=13, and late-onset preeclampsia n=6). The expression of 730 immune-related genes from the Pan Cancer Immune Profiling Panel was measured, and the data were analyzed in the advanced analysis module of nSolver software (NanoString Technology). The results showed that early-onset preeclampsia placentas displayed reduced expression of complement, and toll-like receptor (TLR) associated genes, specifically TLR1 and TLR4. Mast cells and M2 macrophages were also decreased in early-onset preeclampsia compared to healthy placentas. The findings were confirmed by an immunohistochemistry approach using 20 healthy, 19 early-onset preeclampsia, and 10 late-onset preeclampsia placentas. We conclude that the placental innate immune system is altered in early-onset preeclampsia compared to uncomplicated pregnancies. The absence of these alterations in late-onset preeclampsia placentas indicates dissimilar immunological profiles. The study revealed distinct pathophysiological processes in early-onset and late-onset preeclampsia placentas and imply that a tailored treatment to each subtype is desirable.

Relationship of high-intensity plaques on T1-weighted magnetic resonance imaging with coronary intraplaque hemorrhage: A directional coronary atherectomy study

BackgroundAlthough intraplaque hemorrhage (IPH) has been identified as a key feature of rupture-prone plaques, noninvasive imaging-based features for its detection in coronary artery have not been clearly established. The aim of this study was to investigate the relationship of the ratio between the signal intensities of coronary plaque and cardiac muscle (PMR) on non-contrast T1-weighted imaging (T1WI) in magnetic resonance with IPH in the directional coronary atherectomy (DCA) specimens. MethodsFifteen lesions from 15 patients, who underwent DCA and T1WI, were prospectively enrolled. The snap-frozen samples obtained by DCA were used for immunohistochemical staining against a protein specific to erythrocyte membranes (glycophorin A) and macrophages. The percentage of glycophorin A and macrophages was graded using a scale from 0 to 4, with higher scores indicating higher percentages. ResultsPMR showed a strong positive correlation with glycophorin A scores (ρ = 0.772, p < 0.001), whreas, there was a weak correlation between the PMR and macrophage scores (ρ = 0.626, p < 0.05). The receiver-operating characteristic curve analysis showed that the optimal PMR cutoff value for predicting glycophorin A scores ≥grade 2 (glycophorin A-positive area ≥5% of the plaque) was 1.2 (area under the curve; 0.91, 95% confidence interval; 0.73–1.00), and this PMR value had a sensitivity of 8/9 (89%), specificity of 6/6 (100%), positive predictive value of 8/8 (100%), and negative predictive value of 6/7 (86%). ConclusionsIn patients with ischemic heart disease, a high PMR on T1WI is a predictor of coronary IPH as assessed by DCA specimens.

Multiple-Organ Complement Deposition on Vascular Endothelium in COVID-19 Patients.

Increased levels of circulating complement activation products have been reported in COVID-19 patients, but only limited information is available on complement involvement at the tissue level. The mechanisms and pathways of local complement activation remain unclear. The aim of this study was to investigate the deposition of complement components in the lungs, kidneys, and liver in patients with COVID-19 patients and to determine the pathway/s of complement activation. We performed immunofluorescence analyses of autopsy specimens of lungs, kidney, and liver from 12 COVID-19 patients who died of acute respiratory failure. Snap-frozen samples embedded in OCT were stained with antibodies against complement components and activation products, IgG, and spike protein of SARS-CoV-2. Lung deposits of C1q, C4, C3, and C5b-9 were localized in the capillaries of the interalveolar septa and on alveolar cells. IgG displayed a similar even distribution, suggesting classical pathway activation. The spike protein is a potential target of IgG, but its uneven distribution suggests that other viral and tissue molecules may be targeted by IgG. FB deposits were also seen in COVID-19 lungs and are consistent with activation of the alternative pathway, whereas MBL and MASP-2 were hardly detectable. Analysis of kidney and liver specimens mirrored findings observed in the lung. Complement deposits were seen on tubules and vessels of the kidney with only mild C5b-9 staining in glomeruli, and on the hepatic artery and portal vein of the liver. Complement deposits in different organs of deceased COVID-19 patients caused by activation of the classical and alternative pathways support the multi-organ nature of the disease and the contribution of the complement system to inflammation and tissue damage.

Assessment of Biolog EcoplateTM method for functional metabolic diversity of aerotolerant pig fecal microbiota

In the last decades, gut microbiota and its role in mammal host development and health have been increasingly investigated. Metabolites produced by gut microbiota can affect intestinal homeostasis and immune system maturity and activation, and in turn, they can influence the health and growth performance of livestock. Therefore, a better understanding of the functional metabolic capability of the gut microbiota would be appreciated by the scientific community. In this study, the BiologTM Ecoplates technology was applied for studying the metabolic potential of the aerotolerant microbial community of pig fecal samples, evaluating the interference of different storage conditions and cell concentrations. The length of time for which a fecal sample maintained detectable and unchanged microbial metabolic activity was also investigated. Two assays aimed to evaluate differences in the metabolic activities between fresh and snap-frozen fecal samples at different dilutions and at different lengths of times of preservation at −80°C were carried out. The biodiversity and the predicted functionality of the entire bacterial community through a targeted metagenomic approach were also explored. The results highlighted that snap freezing of fecal samples preserved the metabolic activity of the microbial community when compared to fresh feces. Sample storage at −80 °C did not significantly affect the metabolic activity of the microbial community, which was stable for 150 days. Furthermore, the highest metabolic activity was detected with 1:2 to 1:5 dilutions of the stock suspension. BiologTM Ecoplates technology is a rapid and useful method to explore microbial communities’ metabolism in animal fecal samples contributing to investigate host animal physiology.Key points• Freezing of samples can preserve the functional activity of the aerotolerant microbial community for 150 days.• The concentration of microbial cells strongly influences metabolic activity detection.• Sequencing coupled with the BiologTMEcoplates could be a strategy to evaluate the metabolic potential of the microbiota of the fecal sample.Graphical abstract

Successful ATAC-Seq From Snap-Frozen Equine Tissues.

An assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) has become an increasingly popular method to assess genome-wide chromatin accessibility in isolated nuclei from fresh tissues. However, many biobanks contain only snap-frozen tissue samples. While ATAC-seq has been applied to frozen brain tissues in human, its applicability in a wide variety of tissues in horse remains unclear. The Functional Annotation of Animal Genome (FAANG) project is an international collaboration aimed to provide high quality functional annotation of animal genomes. The equine FAANG initiative has generated a biobank of over 80 tissues from two reference female animals and experiments to begin to characterize tissue specificity of genome function for prioritized tissues have been performed. Due to the logistics of tissue collection and storage, extracting nuclei from a large number of tissues for ATAC-seq at the time of collection is not always practical. To assess the feasibility of using stored frozen tissues for ATAC-seq and to provide a guideline for the equine FAANG project, we compared ATAC-seq results from nuclei isolated from frozen tissue to cryopreserved nuclei (CN) isolated at the time of tissue harvest in liver, a highly cellular homogenous tissue, and lamina, a relatively acellular tissue unique to the horse. We identified 20,000–33,000 accessible chromatin regions in lamina and 22–61,000 in liver, with consistently more peaks identified using CN isolated at time of tissue collection. Our results suggest that frozen tissues are an acceptable substitute when CN are not available. For more challenging tissues such as lamina, nuclei extraction at the time of tissue collection is still preferred for optimal results. Therefore, tissue type and accessibility to intact nuclei should be considered when designing ATAC-seq experiments.

Ceramide Synthase 6 Knockout Reprograms Energy Metabolism in Mice

Abstract Objectives Ceramides, a group of bioactive lipids and important signaling molecules, have been implicated in the development of cardiometabolic disease, diabetes, and cancer. Recent studies have shown that targeting Ceramide Synthases (CerS), the enzymes producing ceramides, protected from glucose intolerance and diet induced obesity. We investigated metabolomic responses to high fat diet consumption in livers and plasma of wild type (WT) and CerS6 knockout (KO) mice to obtain mechanistic understanding of the protection provided by the enzyme knockout. Methods Upon weaning, male WT and CerS6 KO mice were placed on a high fat (HFD) or control (Ctrl) diet for 16 weeks. After 14 weeks on diet, mice were placed in calorimetry cages for 48 hours. Body composition was assessed before dietary intervention and before necropsy. Plasma and snap-frozen liver samples were subjected to untargeted metabolomic analysis at Metabolon®. Results CerS6 KO mice gained significantly less weight on HFD than WT mice. Calorimetry measurements revealed that over 24 hours CerS6 KO mice did not move more than WT in either x-, y- or z-planes. Interestingly, CerS6 KO mice on a HFD consumed significantly less food, and despite the higher caloric content of the diet, they also consumed fewer calories over the 24 hour period. No differences in fat excretion between WT and CerS6 KO mice were found, measured by fecal lipid content. Calorimetry data demonstrated that on HFD, regardless of genotype, animals oxidized fat for energy. On the Ctrl diet WT mice burned a mix of substrates while CerS6 KO mice preferentially oxidized glucose for energy during the light cycle. This indicates that during the active phase of the light cycle a switch in energy source occurred in KO but not WT mice. Untargeted metabolomics revealed significant differences in intermediates of glycolysis both in liver and plasma of the KO vs WT animals. Moreover, significant increases in multiple TCA cycle metabolites in KO vs WT plasma were seen in HFD fed mice. Conclusions We found a significant shift in tissue-level and whole-body energetics in CerS6 KO mice. This shift could be responsible for the beneficial metabolic effects of targeting CerS6 when HFD is consumed. Further studies will help determine how CerS6 and ceramides influence tissue and whole body metabolism. Funding Sources This work was funded by R01 CA193782 grant to NK.

Non-selective Primary Human Tumor Cell Line Generation from Surgical Resections to be Paired With Flash Frozen and Paraffin Embedded Tissue: Advancements in Democratizing Translational Research Materials to Rural Institutions

Translational cancer research relies on the availability of human patient tissue demonstrating the specific disease process under investigation. Biobanks of human tissue have historically been and remain to date the primary access point for cancer research samples. Biorepositories routinely supply researchers with varying sample types for use in biomedical studies; most commonly formalin-fixed and paraffin-embedded (FFPE) tissue, or fresh snap-frozen tissue. In conjunction with preserved tissue samples, viable tumor cell lines derived from patient tissue have emerged to be a new gold standard in cancer research particularly in drug discovery and functional prognostic assays. Tissue banks providing these samples are being termed “next-generation” and are adapting to directly assist researchers by performing high throughput technical studies such as routine histology and immunostaining of donor tissue. These high quality, next-generation biorepositories are a relatively scarce resource in the broader research community in the United States and have traditionally been associated with large centralized and very well established university medical centers. This article describes the next-generation resources now available at the Edwards Comprehensive Cancer Center with its association with the Marshall University Joan C. Edwards School of Medicine in Huntington, West Virginia. This manuscript details the procedures, protocols, and success rates of the Tissue Procurement Program (TPP) to generate a growing cohort of viable primary human tumor cell lines representing varying malignancies to be used in conjunction with matched formalin-fixed and paraffin-embedded (FFPE) and snap-frozen tissue samples for comprehensive translational research.

Achieving the Best RNA Quality in Urologic Tumor Samples Intended for Transcriptome Analysis

Purpose: To conduct research on the molecular oncology, physiology, and immunology of urologic tumors requires dissociated viable samples. Improper collection compromises the quality of data attained in molecular and functional assays due to the increased quantities of degraded proteins and RNA. We sought to improve the methods for tissue collection which can avoid generating considerable loss in the viability of cells for further analyses. Materials and Methods: Fifty resected tumor samples from 35 patients were obtained with different surgical techniques and at various time points for viability and RNA quality evaluation. The degradation of RNA was evaluated by its Qubit IQ score, OD 260/280 ratio, total yield, and quantity of β-actin. Results: Snap-frozen tissue samples obtained within 30 min showed better cell viability (P &lt; 0.0001), RNA total yield (P = 0.0081), Qubit ratio (P = 0.003), OD 260/280 ratio (P = 0.4213), and quantity of β-actin (P = 0.0015). Moreover, the bladder tumor samples collected from transurethral biopsy presented more satisfied cell viability results than the ones resected by transurethral electroresection (P &lt; 0.0001). Conclusion: Tumor samples should be processed or frozen freshly within 30 min once removed from human body. Furthermore, transurethral biopsy of bladder tumor is considered a better method for collecting samples for further molecular oncology studies. The high-quality RNA produced enable researchers to conduct more reliable studies by avoiding the experimental artifacts due to the presence of cellular debris or dead cells.

Ex vivo delivery of Mirococept: A dose-finding study in pig kidney after showing a low dose is insufficient to reduce delayed graft function in human kidney.

The complement system plays a pivotal role in the pathogenesis of ischemia-reperfusion injury in solid organ transplantation. Mirococept is a potent membrane-localizing complement inhibitor that can be administered ex vivo to the donor kidney prior to transplantation. To evaluate the efficacy of Mirococept in reducing delayed graft function (DGF) in deceased donor renal transplantation, we undertook the efficacy of mirococept (APT070) for preventing ischaemia-reperfusion injury in the kidney allograft (EMPIRIKAL) trial (ISRCTN49958194). A dose range of 5-25mg would be tested, starting with 10mg in cohort 1. No significant difference between Mirococept at 10mg and control was detected; hence the study was stopped to enable a further dose saturation study in a porcine kidney model. The optimal dose of Mirococept in pig kidney was 80mg. This dose did not induce any additional histological damage compared to controls or after a subsequent 3hours of normothermic machine perfusion. The amount of unbound Mirococept postperfusion was found to be within the systemic dose range considered safe in the Phase I trial. The ex vivo administration of Mirococept is a safe and feasible approach to treat DGF in deceased donor kidney transplantation. The porcine kidney study identified an optimal dose of 80mg (equivalent to 120mg in human kidney) that provides a basis for further clinical development.

Highly Sensitive Droplet Digital PCR Method for Detection of de novo EGFR T790M Mutation in Patients with Non-Small Cell Lung Cancer.

ObjectiveThe aim of this study was to investigate the allelic relation between de novo T790M and concomitant sensitizing EGFR mutations in EGFR-TKIs naïve NSCLCs and to explore whether the formalin-fixed and paraffin-embedded (FFPE) materials affect the detection of de novo EGFR T790M mutation.MethodsSpecimens of 300 consecutive EGFR-TKI naïve NSCLCs who received surgical resection between January 2016 and June 2018 were retrospectively investigated. All the snap-frozen tumor tissues from 300 NSCLCs were screened by droplet digital PCR (ddPCR) for the detection of de novo T790M mutation. The allelic relation between de novo T790M mutation and concomitant sensitizing EGFR mutations was also investigated. Furthermore, we assessed de novo T790M mutation in paired FFPE specimens of 50 patients which included tumor tissues and paired normal lung tissues of the pretreatment NSCLCs to investigate whether FFPE materials affect the detection of de novo T790M mutation.ResultsThe de novo T790M mutation was observed in four patients which included one patient of single de novo T790M mutation and three patients of de novo T790M mutation coexisting with L858R mutation. The incidence of de novo T790M in pretreatment NSCLCs who harboring EGFR mutations was 2.9% (4/139). All the de novo T790M mutations were detected in cis with the concomitant L858R mutations for the three NSCLCs. Our ddPCR method demonstrated that the frequency of de novo T790M mutation was ranging from 0.1% to 0.5% among 90% (45/50) of the FFPE tumor samples and 92% (46/50) of the paired FFPE adjacent normal lung samples. The frequency of de novo T790M mutation in the paired snap-frozen samples was all below 0.1%.ConclusionOur study demonstrated that most de novo T790M mutations were detected in cis with concomitant sensitizing mutations for pretreatment NSCLCs. Analytical cut-off of ddPCR assay for FFPE specimens should be validated carefully considering the possibility of FFPE-derived artificial gene mutations.

Abstract 14: TRAIL inducing small molecule ONC201 prevents intestinal tumors in FAP mouse model

Abstract Colorectal cancer (CRC) is a major health problem globally. Preventing CRC in the general population, and especially in high-risk cohorts with colonic polyps, or its recurrence, is a high priority. ONC201, an orally active small molecule with robust antitumor activity, is currently in clinical trials for advanced cancers. The aim of this study was to determine the CRC preventive potential of ONC201 using the Apcmin/+ mouse model for CRC in familial adenomatous polyposis. First, the optimal and non-toxic doses of ONC201 were determined. C57BL/6J mice (n=6/group) were randomized, and five different doses of ONC201 (0-100mg/kg BW) were administered orally twice a week by gavage. After six weeks of treatment, all mice were euthanized, and tissues were analyzed for optimal dose and toxicity. Based on the body weight gain, gross organ weights at termination, blood profiling, plasma liver enzyme profile, and histopathological analysis, it was concluded that there were no toxicities associated with ONC201 within the tested dose range. For efficacy evaluation, Apcmin/+ male and female mice (n≥20) were grouped and ONC201 (0, 25 and 50 mg/kg BW) was administered by gavage, twice weekly for 14 weeks. At termination, intestinal tumors were evaluated for incidence and multiplicity to determine the efficacy of ONC201. Results indicate a strong suppressive effect of ONC201 against colonic and small intestinal (SI) tumor multiplicity in mice of both genders. Colonic tumor incidence was significantly reduced by &amp;gt;50% in ONC201-treated (50 mg/kg BW) male (40% in treated vs. 92% in control; p&amp;lt;0.0002) and female mice (30% in treated vs. 81% in control; p&amp;lt;0.001) when compared with placebo. Colonic tumor multiplicity was reduced by 68% in ONC201-treated (50 mg/kg BW) male mice (0.44 ± 0.11 in treated vs. 1.4 ± 0.14 in control; p&amp;lt;0.0001) and by 75% in female mice (0.30 ± 0.10 in treated vs. 1.19 ± 0.19 in control; p&amp;lt;0.0003). SI polyps were suppressed by up to 68% in both male mice (11.40 ± 1.19 in treated vs. 36.08 ± 2.62 in control; p&amp;lt;0.0001) and female mice (9.65 ± 1.15 in treated vs. 29.24 ± 2.51 in control; p&amp;lt;0.0001). Snap-frozen colon tumor samples were analyzed for various biomarkers. Western blot protein analysis of the tumors suggested a dose-dependent increase in TRAIL expression upon treatment with ONC201. Similar increases were observed for DR5 and cleaved caspase 8 protein expression. Cleaved caspase 7, caspase 3, FADD and p21 were found to be increased in the treated tumor samples when compared with control. Immunohistochemical analysis indicated a significant decrease in PCNA index in the ONC201-treated tumors compared with controls. Serum analysis indicated a decrease in pro-inflammatory biomarkers, such as IL1β, IL6, TNFα, G-CSF, and GM-CSF, in the ONC201-treated samples compared to controls. Overall, chronic oral administration of ONC201 demonstrated strong chemopreventive efficacy against intestinal tumorigenesis in the Apcmin/+ mouse model. (Supported by NCI HHSN 261201500038I) Citation Format: Chinthalapally V. Rao, Venkateshwar Madka, Yuting Zhang, Gopal Pathuri, Janani Panneerselvam, Nicole Stratton, Niklas K. Finnberg, Howard P. Safran, Jennifer Fox, Shizuko Sei, Elizabeth R. Glaze, Robert Shoemaker, Wafik S. El-Deiry. TRAIL inducing small molecule ONC201 prevents intestinal tumors in FAP mouse model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 14.

Dietary Folic Acid and Fat Alter Metabolism of Multiple Vitamins in Mice

Abstract Objectives To determine how low and high folic acid (FA) intake, combined with either low or high-fat diets, affects other vitamins in mouse liver and plasma. Methods C57BL/6NHsd mice were placed on one of six diets at weaning and maintained for 16 weeks. The diets varied in their fat content and FA levels: low fat (14% kcal from fat) vs high fat (58% kcal from fat) with 3 different FA levels- 0 ppm FA (FD), 2 ppm FA (Ctrl), 12 ppm (FS). Diets were matched for all other vitamins and minerals. Untargeted metabolomics analysis of plasma and snap-frozen liver samples was conducted at Metabolon®. Results In liver, excess dietary folic acid on a low-fat diet resulted in significantly increased levels of pantothenate, α-tocopherol, FA and several folate metabolites. When FA was over-supplemented in combination with a high fat (HF) diet, α-tocopherol was increased along with several nicotinate and pantothenate metabolites. Interestingly, the HF-FD and -FS diets demonstrated similar effects. These diets resulted in significantly decreased levels of riboflavin, thiamine, vitamin A, and vitamin B6 metabolites while increasing levels of pantetheine metabolites. In plasma, fewer changes with significant differences were observed when mice were fed HF diets. Several nicotinate metabolites were significantly elevated due to the FD diet with no change due to FS. Additionally, there were no changes in pantothenate or riboflavin in the plasma. Interestingly, the HF- FD and -FS diets induced similar responses but in opposite directions in plasma vs liver. The plasma levels of thiamine, vitamin A, and vitamin B6 metabolites were all significantly increased due to both low and high FA, whereas in the liver they were decreased. Additionally, no changes in α-tocopherol were seen in plasma, but the HF-FD diet raised γ/β-tocopherol levels over 2-fold despite equal amounts of vitamin E among all diets. Conclusions Untargeted metabolomic analysis revealed that diets with too high or too low folate affect other vitamins both in liver and plasma. These effects were further modulated by dietary fat levels. The HF-FD and -FS diets had significant impact on vitamins A, B1, B2, B3, B5, B6, B9 and E, along with their related derivatives, which may have serious implications for multiple metabolic pathways. Funding Sources NIH.

Early-Life Compartmentalization of Immune Cells in Human Fetal Tissues Revealed by High-Dimensional Mass Cytometry

The human fetal immune system must protect the infant against the sudden exposure to a large variety of pathogens upon birth. While it is known that the fetal immune system develops in sequential waves, relatively little is known about the composition of the innate and adaptive immune system in the tissues. Here, we applied high-dimensional mass cytometry to profile the immune system in human fetal liver, spleen, and intestine. With Hierarchical Stochastic Neighbor Embedding (HSNE) we distinguished 177 distinct immune cell clusters, including both previously identified and novel cell clusters. PCA analysis indicated substantial differences between the compositions of the immune system in the different organs. Through dual t-SNE we identified tissue-specific cell clusters, which were found both in the innate and adaptive compartment. To determine the spatial location of tissue-specific subsets we developed a 31-antibody panel to reveal both the immune compartment and surrounding stromal elements through analysis of snap-frozen tissue samples with imaging mass cytometry. Imaging mass cytometry reconstructed the tissue architecture and allowed both the characterization and determination of the location of the various immune cell clusters within the tissue context. Moreover, it further underpinned the distinctness of the immune system in the tissues. Thus, our results provide evidence for early compartmentalization of the adaptive and innate immune compartment in fetal spleen, liver, and intestine. Together, our data provide a unique and comprehensive overview of the composition and organization of the human fetal immune system in several tissues.