Compartmental Analysis Research Articles

Abstract 2822: Single-cell evaluation to identify tumor-stroma niches driving the transition from in situ to invasive breast cancer

Abstract Introduction: Ductal carcinoma in situ is a non-invasive lesion of the breast that comprises ~20% of newly diagnosed breast cancers in the United States. Prospectively distinguishing indolent from aggressive lesions has been a major clinical challenge. To begin addressing these challenges, we use an experimentally tractable mouse model of breast cancer in which invasive (but rarely in situ) lesions display architectural and morphological abnormalities while also being heavily immune-infiltrated. Methods: To explore the tumor-stroma niches that evolve as breast cancer progresses, we are undertaking single cell RNA sequencing and imaging mass cytometry to identify tumor and immune cells present within in situ (early) versus invasive (advanced) lesions, and to characterize shifts in phenotype and tumor-stroma niches that may occur as breast cancer transitions from in situ to invasive disease. Results: Single cell analyses revealed major cell populations within the tumor compartment of both early and advanced disease, including luminal- and basal-like populations, as well as an additional minor population that appears to represent a unique intermediate between luminal and basal states. Early stage lesions were comprised primarily of the major luminal population, whereas advanced lesions exhibited a significant expansion of intermediate and basal populations, suggesting that breast cancer cells may undergo a transition from luminal-like to basal-like phenotype during progression to invasive disease. Analysis of the immune compartments of early versus advanced disease also revealed that the relative neutrophil/MDSC/S100A8+ population increased as lesions advanced, while B cells, NK cells, and some T cells decreased. Furthermore, the immune populations of advanced lesions exhibited widespread upregulation of IL-17 targets, suggesting that the IL-17 pathway may drive shifts in the immune microenvironmental to support disease progression, particularly the increase in neutrophils/MDSCs. Additionally, given a previously reported role of IL-17 to alter epithelial stemness, increased IL-17 signaling in advanced lesions may also be driving the potential luminal-to-basal transition of malignant cells. Conclusion: Taken together, these findings suggest that a subset of malignant epithelial cells may transition from luminal-like to basal-like cells, and that this transition may be driven and/or maintained by a commensurate increase in IL-17 signaling that both directly impacts tumor cells and also establishes a tumor-supportive microenvironment. Citation Format: Vidya C. Sinha, Mingchu Xu, Amanda L. Rinkenbaugh, Xinhui Zhou, Xiaomei Zhang, Helen Piwnica-Worms. Single-cell evaluation to identify tumor-stroma niches driving the transition from in situ to invasive breast cancer [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 2822.

Vitamin A Absorption Efficiency Determined by Compartmental Analysis of Postprandial Plasma Retinyl Ester Kinetics in Theoretical Humans

ABSTRACTBackgroundBetter methods are needed for determining vitamin A absorption efficiency in humans to support development of dietary recommendations and to improve the accuracy of predictions of vitamin A status.ObjectivesWe developed and evaluated a method for estimating vitamin A absorption efficiency based on compartmental modeling of theoretical data on postprandial plasma retinyl ester (RE) kinetics.MethodsWe generated data on plasma RE and retinol kinetics (30 min to 8 h or 56 d, respectively) after oral administration of labeled vitamin A for 12 theoretical adults with a range of values assigned for vitamin A absorption (55–90%); we modeled all data to obtain best-fit values for absorption and other parameters using Simulation, Analysis, and Modeling software. We then modeled RE data only (16 or 10 samples), with or without added random error, and compared assigned to predicted absorption values. We also compared assigned values to areas under RE response curves (RE AUCs).ResultsWe confirmed that a unique value for vitamin A absorption cannot be identified by modeling plasma retinol tracer kinetics. However, when RE data were modeled, predicted vitamin A absorptions were within 1% of assigned values using data without error and within 12% when 5% error was included. When the sample number was reduced, predictions were still within 13% for 10 of the 12 subjects and within 23% overall. Assigned values for absorption were not correlated with RE AUC (P = 0.21).ConclusionsWe describe a feasible and accurate method for determining vitamin A absorption efficiency that is based on compartmental modeling of plasma RE kinetic data collected for 8 h after a test meal. This approach can be used in a clinical setting after fasting subjects consume a fat-containing breakfast meal with a known amount of vitamin A or a stable isotope label.

Basal-like and classical cells coexist in pancreatic cancer revealed by single-cell analysis on biopsy-derived pancreatic cancer organoids from the classical subtype.

Pancreatic ductal adenocarcinoma (PDAC) is composed of stromal, immune, and cancerous epithelial cells. Transcriptomic analysis of the epithelial compartment allows classification into different phenotypic subtypes as classical and basal-like. However, little is known about the intra-tumor heterogeneity particularly in the epithelial compartment. Growing evidences suggest that this phenotypic segregation is not so precise and different cancerous cell types may coexist in a single tumor. To test this hypothesis, we performed single-cell transcriptomic analyses using combinational barcoding exclusively on epithelial cells from six different classical PDAC patients obtained by Endoscopic Ultrasound (EUS) with Fine Needle Aspiration (FNA). To purify the epithelial compartment, PDAC were grown as biopsy-derived pancreatic cancer organoids. Single-cell transcriptomic analysis allowed the identification of four main cell clusters present in different proportions in all tumors. Remarkably, although all these tumors were classified as classical, one cluster present in all corresponded to a basal-like phenotype. These results reveal an unanticipated high heterogeneity of pancreatic cancers and demonstrate that basal-like cells, which have a highly aggressive phenotype, are more widespread than expected.

Dietary Iron Repletion Stimulates Hepatic Mobilization of Vitamin A in Previously Iron-Deficient Rats as Determined by Model-Based Compartmental Analysis

Iron deficiency can result in hyporetinolemia and hepatic vitamin A (VA) sequestration. We used model-based compartmental analysis to determine the impact of iron repletion on VA metabolism and kinetics in iron-deficient rats. At weaning, Sprague-Dawley rats were assigned to either a VA-marginal diet (0.35mg retinol equivalent/kg) with adequate iron (35ppm, control group [CN]) or reduced iron (3ppm, iron-deficient group [ID-]), with an equivalent average body weight for each group. After 5 wk, n=4 rats from each group were euthanized for baseline measurements of VA and iron indices, and the remaining rats (n=6 CN, n=10 ID-) received an intravenous injection of 3H-labeled retinol in an emulsion as tracer to initiate the kinetic study. On day 21 after dosing, half of the ID- rats were switched to the CN diet to initiate iron repletion, referred to as the iron-repletion group (ID+). From the time of dosing, 34 serial blood samples were collected from each rat over a 92-d time course. Plasma tracer and tissue tracee data were fitted to 6- and 4-compartment models, respectively, to analyze the kinetic behavior of VA in all groups. Our mathematical model indicated that ID- rats exhibited a nearly 6-fold decrease in liver VA secretion and >4-fold reduction in whole-body VA utilization, compared with CN rats, whereas these perturbed kinetic behaviors were notably corrected in ID+rats, close to those from the CN group. Iron repletion can remove the inhibitory effect that iron deficiency exerts on hepatic mobilization of VA and restore retinol kinetic parameters to values similar to that of never-deficient CN rats. Together with improvements in iron and VA indices, our results suggest that restoration of an iron-adequate diet is sufficient to improve VA kinetics after a previous state of iron deficiency.

A Structural Analysis of Tympanic Compartments of the Middle Ear in Patients With Down's Syndrome: A Temporal Bone Study.

Otitis media with effusion is a highly prevalent condition with DS. Knowledge of the volume of the tympanic compartments and the area of the tympanic isthmus might be important to find out the pathogenesis of highly prevalent otitis media with effusion in those patients. We compared the volume of the epitympanum, mesotympanum, and the areas of the tympanic isthmus and tympanic orifice of eustachian tube in temporal bones from patients with DS. We also investigated the eustachian tube histopathologically. The mean volume of the epitympanum and the mesotympanum was significantly smaller in the DS group than the control group. We found no significant difference in the mean diameter of the protympanic opening and tympanic orifice between the two groups. The mean narrowest area of the aerated and bony tympanic isthmus also was not significantly different between the two groups. An immature development of eustachian tube and cartilage was seen. We found mesenchyme remaining at the epitympanum and/or mesotympanum in all specimens in the DS group, and in five specimens in the control group. In the presence of the small middle ear, poorly developed eustachian tube, and tensor muscle, a vicious circle occurs, making otitis media with effusion difficult to resolve.

Dynamic Capture and Release of Endoplasmic Reticulum Exit Sites by Golgi Stacks in Arabidopsis.

SummaryProtein transport from the endoplasmic reticulum (ER) to Golgi stacks is mediated by the coat protein complex COPII, which is assembled at an ER subdomain called ER exit site (ERES). However, the dynamic relationship between ERESs and Golgi stacks is unknown. Here, we propose a dynamic capture-and-release model of ERESs by Golgi stacks in Arabidopsis thaliana. Using variable-angle epifluorescence microscopy with high-temporal-resolution imaging, COPII-component-bound ERESs were detected as punctate structures with sizes of 300–500 nm. Some punctate ERESs are distributed on ER tubules and sheet rims, whereas others gather around a Golgi stack in an ER-network cavity to form a beaded-ring structure. Free ERESs that wander into an ER cavity are captured by a Golgi stack in a cytoskeleton-independent manner. Then, they are released by the Golgi stack for recycling. The dynamic ERES cycling might contribute to efficient transfer of de novo synthesized cargo proteins from the ER to Golgi stacks.

Hermite collocation method for obtaining the chaotic behavior of a nonlinear coupled system of FDEs

This paper is devoted to introduce an efficient solver using the Hermite collocation technique (HCT), of the coupled system of fractional differential equations (FDEs). The given systems are of basic importance in modeling various phenomena like Cascades and Compartment Analysis, Pond Pollution, Home Heating, Chemostats, and Microorganism Culturing, Nutrient Flow in an Aquarium, Biomass Transfer, Forecasting Prices, Electrical Network, Earthquake Effects on Buildings. The proposed method reduces the system of FDEs to a system of algebraic equations in the coefficients of the expansion using the Hermite polynomials. The introduced method is computer oriented and provides highly accurate solution. To demonstrate the efficiency of the method, two examples are solved and the results are displayed graphically. Finally, we convert the presented coupled systems from the case of its standard form to a first-order ordinary differential equations to compare the obtained numerical solutions with those solutions using the fourth-order Runge–Kutta method (RK4).

Research and analysis of modern space heating technologies and management for industrial buildings

The paper observes different modern space heating technologies suitable for industrial buildings. Their main working principles and applications are researched related to their energy efficiency. The problems about heating of large scale spaces in industry are observed. A compartment analysis between the technologies is carried out with their advantages and disadvantages.

The “Super-Child” Approach Is Applied To Estimate Retinol Kinetics and Vitamin A Total Body Stores in Mexican Preschoolers

Background:Retinol isotope dilution (RID) and model-based compartmental analysis are recognized techniques for assessing vitamin A (VA) status. Recent studies have shown that RID predictions of VA total body stores (TBS) can be improved by using modeling and that VA kinetics and TBS in children can be effectively studied by applying population modeling (“super-child” approach) to a composite data set. Objectives:The objectives were to model whole-body retinol kinetics and predict VA TBS in a group of Mexican preschoolers using the super-child approach and to use model predictions of RID coefficients to estimate TBS by RID in individuals. Methods:Twenty-four healthy Mexican children (aged 3–6 y) received an oral dose (2.96 µmol) of [13C10]retinyl acetate in corn oil. Blood samples were collected from 8 h to 21 d after dosing, with each child sampled at 4 d and at 1 other time. Composite data for plasma labeled retinol compared with time were analyzed using a 6-component model to obtain group retinol kinetic parameters and pool sizes. Model-predicted TBS was compared with mean RID predictions at 4 d; RID estimates at 4 d were compared with those calculated at 7–21 d. Results:Model-predicted TBS was 1097 µmol, equivalent to ~2.4 y-worth of VA; using model-derived coefficients, group mean RID-predicted TBS was 1096 µmol (IQR: 836–1492 µmol). TBS at 4 d compared with a later time was similar (P = 0.33). The model predicted that retinol spent 1.5 h in plasma during each transit and recycled to plasma 13 times before utilization. Conclusions:The super-child modeling approach provides information on whole-body VA kinetics and can be used with RID to estimate TBS at any time between 4 and 21 d postdose. The high TBS predicted for these children suggests positive VA balance, likely due to large-dose VA supplements, and warrants further investigation.

Pregnancy and Lactation Alter Vitamin A Metabolism and Kinetics in Rats under Vitamin A-Adequate Dietary Conditions

Abstract Objectives We investigated the impact of pregnancy and lactation on vitamin A (VA) metabolism and kinetics in rats, hypothesizing that this changed physiological status would perturb whole-body VA kinetics. Such information may be informative for future dietary recommendations. Methods Ten female rats (7 wk of age) and 6 male rats (9 wk of age) were fed an AIN-93 G diet upon arrival. After 1 week of acclimation, female rats received an oral dose of 3H-labeled retinol as the tracer to initiate the kinetic study. On d 21 after dosing (when 3H-retinol was expected to reach a log-linear terminal slope), 6 female rats were mated and checked daily for a vaginal plug to determine the date of pregnancy. On the day of delivery, litter size was adjusted to 10 pups/dam. Serial blood samples were collected from each female rat at 27–28 time points after dose administration until dams and pups were euthanized on d 14 of lactation. Hematocrit was measured, plasma tracer level was determined, and plasma fraction of dose vs. time was plotted. Model-based compartmental analysis will be applied to the plasma tracer data to develop VA kinetic models. Results All mated female rats became pregnant (pregnant group, PG, n = 6). Non-mated female rats were studied as non-pregnant controls (CN, n = 4). No difference was observed in hematocrit between PG and CN rats, suggesting no significant change in plasma volume expansion. Before breeding, plasma tracer response profiles were similar to CN rats. However, a consistent decline in plasma tracer levels was observed in PG rats during the middle of pregnancy, followed by a rise in late pregnancy, whereas such a change did not occur in CN rats. Moreover, during lactation, PG rats exhibited a steeper terminal slope compared to CN rats, indicating a more rapid utilization of VA in these lactating rats. Conclusions Pregnancy and lactation resulted in altered VA metabolism and kinetics in rats. Further analysis using mathematical modeling will explore the changes in kinetic parameters that underlie the perturbations we have observed in VA kinetics. Funding Sources National Institutes of Health.

Validation of a Method for Estimating Vitamin A Absorption Efficiency Based on Compartmental Analysis of Post-Prandial Plasma Retinyl Ester Kinetics

Abstract Objectives Because good methods are not available to estimate vitamin A (VA) absorption, we evaluated an approach based on modeling retinyl ester response to an oral VA dose. Methods We generated data for 12 theoretical subjects, assigning values for VA absorption, stores, and kinetic parameters; we used WinSAAM (Simulation, Analysis and Modeling software) to simulate data (without and with 5% average error) for plasma chylomicron retinyl esters (RE) and retinol versus time (30 min to 8 h or 56 d, respectively) after ingestion of labeled VA; next we fit data to a previously-published 9-component model for VA metabolism to obtain “known” values for VA absorption. Then RE data only were modeled for each subject using a robust (n = 16 times) vs truncated sampling schedule (n = 10) and model-predicted absorptions were compared to known values. Areas under the plasma RE response curves (AUCs) were also calculated and compared to known absorption values. Results Known values for VA absorption based on modeling all data with error ranged from 54 – 92% (mean, 72%), VA stores from 160 – 1775 μmol, and chylomicron t1/2 from 6 – 12 min. Using the full sampling scheme for RE, the ratio of model-predicted to known absorption ranged from 0.927 – 1.06 (mean, 0.997); using the truncated scheme, the ratio was 0.814 – 1.13 (mean, 0.973). AUCs were not significantly correlated with known values for VA absorption (R2 = 0.112; P > 0.05), presumably because absorption and chylomicron catabolism are occurring simultaneously. Conclusions By modeling chylomicron RE tracer data after ingestion of labeled VA, absorption efficiency was estimated accurately using error-free data; using data with 5% error, estimates were within 10% of known values (full sampling) or within 20% (truncated). If subjects, after an overnight fast, consume a breakfast containing some fat and a known amount of VA, then no tracer is required to estimate VA absorption using this modeling approach. By incorporating a population-based design, the method could be used in children. Funding Sources Supported by Bill & Melinda Gates Foundation (Project Number OPP1115464) and HarvestPlus (BH183438).

Multi-omic single-cell analysis of antigen-presenting compartment reveals a unique functional signature for head and neck squamous cell carcinoma.

e15179 Background: Immune studies in head and neck squamous cell carcinoma (HNSCC) are lacking. Studies in the past decade have highlighted the biological significance and the distinct functional properties of immune cell subsets that are resident in non-lymphoid tissues. While tissue-resident memory T cells (TRM) have been well characterized, much less is known about the human myeloid compartment in HNSCC, which includes professional antigen-presenting cells (APCs) such as dendritic cells (DCs) and macrophages, both of which are critical for shaping the local T cell response. This is particularly relevant in the context of anti-tumor immune responses, which are currently a major focus for therapeutic intervention in HNSCC by either checkpoint inhibitory blockade or adoptive T cell therapy. Methods: A combination of multi-omic single cell RNA sequencing (sc-RNAseq) and 30-parameter fluorescent flow cytometry were used to define the APC compartment in 7 human head and neck squamous cell carcinoma (HNSCC) samples and 4 normal gingival tissue samples as references. Importantly, we performed parallel profiling of the adaptive and innate T cell compartment to elucidate the relationship between APCs and local TRM cells that have been designated as critical players for immune responses in solid tumor tissue. Results: Several novel myeloid phenotypes and an altered composition of the APC compartment in HNSCC relative to normal gingival tissues. APCs expressing pro-inflammatory cytokines such as IL-1b and IL-6 showed reduced abundance, while previously unknown APC subsets defined by the expression of a unique chemokine profile were found to infiltrate HNSCC tissue. Furthermore, our multi-omic approach allowed for profiling of the protein surface phenotype in these transcript-defined clusters, opening up avenues for future therapeutic targeting of tumor-specific antigen-presenting cells. Conclusions: A novel myeloid phenotype and APC compartment were observed in HNSCC when compared to normal gingival tissues. A multi-omic assay identified tumor-specific APCs that may represent future therapeutic targets.

Subcellular partitioning of Kaiso (ZBTB33) as a biomarker to predict overall breast cancer survival.

3534 Background: The epigenetic transcriptional regulator, Kaiso (ZBTB33) has been identified as a member of the C2H2 zinc finger proteins containing a BTB/POZ -zinc finger family of transcription factors that are implicated in development of cancer. Although, our understanding of clinical relevance of subcellular distribution (cytoplasmic/nuclear) Kaiso in the growth and survival of human Breast cancer (BC) is limited. Methods: We examined a cohort of 555 BC patients who underwent surgery for their primary BC in Greenville, NC using AI and SM approach. Results: The sub-classification BC shows, cytoplasmic Kaiso is differentially enriched in ER- BC (p=0.001) compared nuclear Kaiso (p=0.8) and is significantly enriched in the more aggressive classes LumB (p=0.0017), HER2+ (p=0.05) and TNBC (p=6.1e-07) with respect to LumA BC patients. Additionally, the survival analysis of different compartments of Kaiso demonstrates that high cytoplasmic Kaiso (HR = 16.29 (7.6 – 34.8), p = 5.5e−13) is much more predictive of poor survival compared to nuclear Kaiso (HR = 2.83 (2.02 – 3.8), p = 6.1e−11). At gene expression level, ZBTB33 mRNA levels do not correlate with either nuclear (Spearman correlation: -0.03157, p= 0.7267) or cytoplasmic levels (Spearman correlation: -0.03526, p= 0.6962) of Kaiso. Surprisingly, ZBTB33 mRNA abundance is predictive of poor overall BC survival as demonstrated in two independent publicly available BC cohorts Metabric (HR = 2.14 (1.49 − 3.08), p = 2.7e−05) and Gyorffy B et al. (HR = 1.81 (1.55 − 2.12), p = 2.5e−14). Nuclear and cytoplasmic levels of Kaiso do not show significant differences based on race p=0.27 and p=0.1 respectively. Conclusions: Our data suggest subcellular distribution of high Kaiso is associated with poor prognosis of BC survival and subcellular localizations of Kaiso may play differential biological roles in BC prognosis.

Pharmacokinetics of bioactive components after oral administration of Bojungikgi-tang in Korean subjects

The purpose of this study to establish the safety and efficacy data of Bojungikgi-tang (BJIGT) soft extract by determining the clinical pharmacokinetics (PKs) of its main ingredients and their active metabolites after oral administration. A randomized, open-label, single-dose, single-center study on 12 healthy Korean male subjects was conducted. The plasma concentration of the active ingredients in BJIGT soft extract was determined in UPLC-MS/MS. Phoenix WinNonlin (version 8.1, Pharsight®, a Certara™ Company, Princeton, NJ, USA) was used to conduct compartmental and non-compartmental (NCA) analyses to assess PK parameters. The PK parameters of ginsenoside Rb1 (Rb1) and glycyrrhizin (GL) were well described with 1-compartment analysis without lag time, and the 1-compartment model with combined transit compartment model and first-order absorption was used to evaluate the parameters of glycyrrhetinic acid (GLA). PK parameters of Rb1, GL and GLA including the clearance (CL/F), the volume of distribution (V/F), the rate of absorption (Ka), the maximum concentration (Cmax), time to reach maximum concentration (Tmax), the area under the curve of a plasma concentration versus time profile (AUC0-inf), and the elimination half-life (T1/2) were successfully estimated. This is the first report to evaluate the PKs of major active ingredients and their metabolites after oral administration of BJIGT soft extract to Korean subjects.

Assessing CD8+ cytotoxic T-cell dysfunction in obesity and its implications in anti-PD-1 blockade therapy

Abstract In chronic conditions, notably cancers, CD8+ cytotoxic T cells gradually lose their effector functions due to constant exposure to antigens and consequent upregulation of inhibitory receptors like PD-1. In this regard, PD-1 blockade therapy was shown to bolster cytotoxic T-cell response and improve disease prognosis in some cases. PD-1 mediated T cell dysfunction is also involved in obesity-induced tumorigenesis. In this study, we used a molecular cytometry approach to better understand the disturbances caused by obesity in immune cells. A detailed single-cell analysis of various cell compartments revealed a unique population of CD8+ cytotoxic T cells in the epididymal fat of mice consuming a high fat diet for 16 weeks. These cells showed high expression of proteins and/or mRNA transcripts for various inhibitory receptors, including PD-1. Based on these results, we created a 28-color panel for high-throughput cell analysis at multiple time points over the 16-week period, using a BD FACSymphony™. The frequency of CD8+PD-1+ T cells in the fat correlated with the numbers of inflammatory cells, suggesting that the dysfunctional phenotype resulted from a growth in inflammation over time. The functional state of the CD8+PD-1+ cells was also examined by challenging these cells with or without PD-1 blockade. Taken together the data delineated a refined application for a broad analysis of immune cells in the fat and provided insights into the effects of obesity-induced T-cell dysfunction in mice. For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 laser product. BD, the BD Logo, FACSymphony is trademark of Becton, Dickinson and Company. © 2019 BD and its subsidiaries. All rights reserved.

Abstract A23: A second site KrasG12D mutation that impairs PI3K binding rescues embryonic lethality, abrogates myeloproliferative disease, and delays lung tumorigenesis

Abstract Phosphatidylinositol 3-kinase (PI3K) signaling is essential for RAS-driven transformation. To directly investigate the role of oncogenic K-Ras binding to PI3K in development and tumorigenesis, we generated KrasG12D/+,Y64G/+ mice that express, from the endogenous locus, a K-Ras oncoprotein that also contains a “second site” amino acid substitution at tyrosine 64 (Y64G) that disrupts the interaction between oncogenic K-Ras and PI3K. Surprisingly, KrasG12D,Y64G mice are viable, fertile, and phenotypically unremarkable, although they are born at a lower than expected Mendelian frequency (26% versus the expected 50% on a C57BL/6 strain background). As opposed to the enhanced proliferative rate observed in mouse embryonic fibroblasts (MEFs) from K-RasG12D mice, KrasG12D,Y64G MEFs exhibit wild-type rates of proliferation. Detailed analysis of the hematopoietic compartment in KrasG12D,Y64G mice reveals a reduced proportion of long-term hematopoietic stem cells, but no evidence of the aggressive myeloproliferative neoplasm observed when a conditional mutant KrasG12D is activated in hematopoietic cells. Consistent with these observations, bone marrow cells isolated from K-RasG12D,Y64G mice exhibit a normal pattern of myeloid progenitor colony growth in response to cytokine stimulation. In contrast to the lack of observed hematologic malignancy, 100% of KrasG12D,Y64G mice we examined at 1 year of age showed lung lesions, ultimately succumbing to lung tumors with a median survival of 496 days. Despite the ubiquitous KrasG12D,Y64G expression, these mice survive longer than models with mosaic, adenoviral-Cre recombinase-controlled KrasG12D (median survival of 185 days). The majority of the lung lesions that arise in KrasG12D, Y64G mice are low grade, classified pathologically as atypical lymphoid proliferation or papillary adenomas; a few adenocarcinomas are also observed. These studies reinforce the importance of oncogenic KRas-mediated activation of PI3K for transformation and demonstrate that expressing a Y64G amino acid substitution in the context of oncogenic KrasG12D normalizes cell proliferation, rescues embryonic lethality, abrogates myeloid disease, and attenuates lung tumorigenesis. Beyond the bone marrow and lung, this mutant strain is a potent genetic tool for dissecting the role of aberrant PI3K signaling in pancreatic, colon, and other tissues characterized by tumors driven by somatic KRAS mutations, and also have implications for treating human cancers with KRAS mutations. Citation Format: Jasmine C. Wong, Pedro A. Perez-Mancera, Jangkyung Kim, Kuang-Yu Jen, Scott C. Kogan, Ari J. Firestone, Eric A. Collisson, David A. Tuveson, Kevin Shannon. A second site KrasG12D mutation that impairs PI3K binding rescues embryonic lethality, abrogates myeloproliferative disease, and delays lung tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A23.

Myasthenia gravis: From single cell signatures to cancer diagnosis

Abstract Myasthenia gravis is a rare but archetypic autoimmune disease that is characterized by the autoantibody-mediated disruption of the neuromuscular junction leading to a skeletal muscle weakness. Immunomodulatory treatment options for Myasthenia gravis patients are largely unspecific, include suppression of the entire immune compartment and are often accompanied by severe side effects. In order to identify novel biomarkers for more targeted and effective therapeutic approaches, we combined high-dimensional mass and flow cytometry with supervised and unsupervised machine-learning algorithms. Analysis of the peripheral immune compartment of Myasthenia gravis patients and healthy controls revealed a cellular immune signature consisting of inflammatory memory T helper cells with a defined cytokine profile. The abundance of the identified leukocytes in the blood strongly correlated with the patients clinical disease activity, far better than auto-Ab titers. Moreover, we were able to locate T cells with the defined signature enriched in the inflamed thymus of Myasthenia gravis patients – the key organ for the induction and maintenance of the autoimmune disease. Lastly, using an unbiased pattern recognition approach, we identified lymphomas in a subset of Myasthenia gravis patients, further highlighting the potential of the applied analysis tools.

Better Predictions of Vitamin A Total Body Stores by the Retinol Isotope Dilution Method Are Possible with Deeper Understanding of the Mathematics and by Applying Compartmental Modeling

Retinol isotope dilution (RID) is a well-accepted technique for assessing vitamin A status [i.e., total body stores (TBS)]. Here, in an effort to increase understanding of the method, we briefly review RID equations and discuss their included variables and their coefficients (i.e., assumptions that account for the efficiency of absorption of an orally administered tracer dose of vitamin A, mixing of the dose with endogenous vitamin A, and loss due to utilization). Then, we focus on contributions of another technique, model-based compartmental analysis and especially the “super-person” approach, that advance the RID method. Specifically, we explain how adding this modeling component, which involves taking 1 additional blood sample from each subject, provides population-specific estimates for the RID coefficients that can be used in the equation instead of values derived from the literature; using model-derived RID coefficients results in improved confidence in predictions of TBS for both a group and its individuals. We note that work is still needed to identify the optimal time for applying RID in different groups and to quantify vitamin A absorption efficiency. Finally, we mention other contributions of modeling, including the use of theoretical data to verify the accuracy of RID predictions and the additional knowledge that model-based compartmental analysis provides about whole-body vitamin A kinetics.

Pkweb: An online application for pharmacokinetic data analysis

Abstract This research presents a web-based application, pkweb ( https://pkweb.hhra.net ), with a user-friendly interface and a range of functions for pharmacokinetic (PK) data analysis. Capabilities include simulation of PK data, which integrates 27 modules (e.g., intravenous (IV) bolus injection, IV infusion and extravascular administration) and modeling the fitness of PK data. The pkweb tool provides different solutions for various conditions, such as compartmental analysis (CA) under single dosing, non-compartmental analysis (NCA) under single dosing and CA under multiple dosing. Compared to other tools, a unique feature of this tool is the ability to set boundaries for the parameters of each model. This newly released application promises to facilitate both toxicokinetic and pharmacokinetic data analysis.

Lung compartment analysis assessed from N2 multiple-breath washout in children with cystic fibrosis.

Compartment analysis (CA) based on nitrogen multiple-breath washout (N2 MBW) has been shown to allow the assessment of specific volume and ventilation of faster- and slower-ventilating lung compartments of the lung in adults with cystic fibrosis (CF). The aim of this study was to extend previous findings into the pediatric age range. Cross-sectional multicenter observational study in children with CF and healthy controls (HC) was done with the assessment of N2 MBW and spirometry. A two-lung compartment model-based analysis (CA) was used to estimate size and function of faster- and slower-ventilating lung compartments from N2 MBW. A total of 125 children with CF and 177 HC, median age 10.8 (range, 2.8-18.9) years, were included in the analysis. CA could be calculated in 66 (53%) children with CF compared with 48 (27%) HC (P < .0001). The proportion of the slower-ventilating lung compartment was significantly smaller in children with CF (53.5%; 95% confidence interval [CI]: 51.9%-55.7%) compared with HC (62.2%; 95% CI: 59.0%-65.0%) The regional specific ventilation of the slower compartment (rVT ,slow/rFRC,slow, %) was significantly lower in children with CF (4.9%; 95% CI: 4.5-5.9) compared with HC (9.7%, 95% CI: 9.2-10.9), and showed inverse correlation to lung clearance index (r2 = -.65; P < .0001), Sacin × VT (r2 = -.36; P = .003) and Scond × VT (r2 = -.51; P < .0001). There was no significant difference in pulmonary parameters between children with CF with and without feasible CA. CA is less feasible in children than in adults and correlated to other MBW parameters. The clinical value of CA is still unclear and is yet to be established.