Flex System Research Articles

Immunocorrective effects of Tricor fenofibrate in the treatment of the early and intermediate stages of age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of irreversible visual impairment, blindness and disability in the elderly. Treatment of end-stage AMD is extremely limited and requires invasive interventions. Currently, there is an active search for remedies aimed at preventing the degenerative process in the retina in the early stages of the disease. AMD is a multifactorial pathology, has common mechanisms with diseases associated with aging, metabolic shifts and hemodynamic disorders. The study of the effects of drugs already used to correct these disorders in the general clinic may also be of interest to ophthalmologists. The aim of the work was to study the dynamics of inflammatory markers of activation of the vascular endothelium and cytokines of the immune response in the tear fluid (TF) of patients with initial and intermediate stages of AMD against the background of the use of Tricor fenofibrate. 65 people were examined, divided into three groups according to the AREDS classification: group I – 20 people with early AMD (AREDS2), group II – 16 patients with intermediate AMD (AREDS3), 29 healthy elderly people without ophthalmopathology entered the age control group (AREDS1 – risk group). Patients of groups I and II received Tricor at a dose of 145 mg once a day for 9 months. Tear fluid (TF) was taken twice: before and immediately after the completion of the course of treatment. The determination of sICAM-1, sVCAM-1, sE-, sP-selectin and MCP-1/CCL2 in CS was performed by flow cytometry using a self-constructed multiplex panel from compatible simplex test kits Human FlowCytomixTM Simplex (Bender MedSystem GmbH, Germany); IL-1β, IL-2 IL-6 TNFα was determined within the framework of the Human Flow CytomixTM 15 Flex system (Bender MedSystem GmbH, Germany). Data processing was performed in the FlowCytomix Pro v 6.0 package (Bender Med Systems GmbH, Germany). According to the results of the study, a significant effect of Tricor fenofibrate was found on the initially high levels of local production of IL-1β, IL-2, IL-6, MCP-1/CCL2 and sICAM-1 in the AREDS2 group, which indicated a direct anti-inflammatory, vasoprotective effect in the treatment of the initial stage of AMD; a similar effect of the drug, but less pronounced, noted in the AREDS3 group. The ophthalmological examination data also indicated stabilization of visual functions and the clinical picture of the fundus, improvement of intraocular blood circulation in patients of the main groups. Thus, the use of Tricor can help prevent the degenerative process in the retina in the early stages of AMD development.

Sign To Speech Conversion And Home Automation Control Using Smart Gloves

In today's world, there are many new technologies changing the way we do things. One interesting technology is the home automation system. It helps people control things in their homes from far away, making life more comfortable, saving money, and being easy to use. But some people find it a bit tricky to use these systems well. To make it simpler for everyone, we can use something called a flex sensor-based home automation system. This system lets you control things by moving your hands, which is really helpful for older people or those who might find it a bit hard to move. It's also great for people who haven't had much learning. And if we add voice help, it becomes even easier. This way, people who are in bed or dealing with physical problems can use it too. This makes technology not just cool but helpful for everyone in different situations. In this paper, we explore the integration of flex sensors onto hand gloves to facilitate a dynamic interaction between hand movements and technological outputs. The flex sensors, akin to miniature potentiometers, are strategically affixed to the fingers, registering changes in value corresponding to the bending action. As the finger bends, the sensor's resistance alters, influencing the output in an inversely proportional manner. This innovative system allows for nuanced control, where specific angles of finger bending lead to calibrated adjustments in output, demonstrating a responsive and intuitive interface between human gestures and technology. The implementation of flex sensor- based gloves introduces a versatile means of capturing and translating hand movements into actionable data. The project leverages the concept of resistance modulation to precisely interpret the degree of finger bending, creating a reliable framework for diverse applications such as gesturecontrolled devices or assistive technologies for individuals with limited mobility. The abstracted communication between human hand gestures and technology opens avenues for accessible and intuitive interfaces, promising potential applications across various domains.

A Prospective Study to Evaluate the Prevalence of Microsatellite Instability in Endometrial Carcinoma by using Immunohistochemistry for Mismatch Repair Proteins as a Surrogate Marker

Abstract Aim Use of immunohistochemistry for mismatch repair (MMR) proteins to identify the prevalence of microsatellite instability (MSI) in cases of endometrial carcinoma and its subsequent correlation with various histopathological parameters. Materials and Methods The expression of MMR proteins, viz PMS2, MLH1, MSH2, and MSH6, were assessed in 114 endometrial cancer cases by immunohistochemistry using Dako EnVision FLEX system, on paraffin blocks of tumor tissue fixed in 10% formalin. Results We studied 114 endometrial cases for MMR protein expression, of which the majority were of endometrioid histologic subtype (n = 93, 81.6%), whereas the remainder comprised serous carcinoma (n = 12, 10.5%), clear cell carcinoma (n = 1, 0.9%), carcinosarcoma (n = 5, 4.4%), and dedifferentiated uterine carcinoma (n = 3, 2.6%). Twenty-one (18%) of these cases were found to be deficient for MMR proteins, of which 20 were of endometrioid histologic subtype and only 1 was dedifferentiated uterine carcinoma. Loss of MMR protein expression occurred in pairs of either PMS2 and MLH1 or MSH2 and MSH6. Conclusion MSI is one of the major molecular pathways contributing to tumorigenesis in endometrial carcinomas. Immunohistochemistry for MMR proteins is a highly sensitive and cost-effective alternative for molecular testing for MSI. It is also a great tool for screening patients for Lynch syndrome. Immunohistochemical testing for MMR should be offered to all patients of endometrial cancers.

High-Resolution Spatial Lipidomic Profiling of the Kidney During Periods of Cyst Expansion in a Mouse Model of ADPKD

Introduction: The progression of renal pathology in autosomal dominant polycystic kidney disease (ADPKD) involves a complex series of changes including renal cyst formation, interstitial cyst expansion, tubular atrophy, and fibrosis. Changes to in tissue metabolism and cyst fluid metabolite composition have been shown in ADPKD models, however it has been diffcult to discretely localize these compounds to specific regions of the kidney. Further, very little is known about lipid profiles within the kidney during ADPKD. We hypothesized that spatial lipidomic analysis during periods of cyst expansion would reveal new understanding of changes in lipid metabolism during this critical window of pathology. In this study we focused on changes in the mcwPkd1nl/nl mouse, a hypomorphic ADPKD model that rapidly develops a cystic phenotype. Methods: Kidneys were collected from female cystic mcwPkd1nl/nl mice at postnatal days 7, 14 and 28, representing periods preceding, during and at maximal cyst expansion, respectively. Control kidneys were collected from female wild-type littermates at postnatal day 7 and 28. Tissues were rapidly cryoembedded for 10 mm sectioning onto Intellislides (BrukerDaltonik). Slides were scanned on a Reflecta MF-5000 scanner and sprayed with 7 mg/mL CHCA in 70% Acetonitrile, 30% water and 0.1% TFA using a TM Sprayer Model M3 (HTX Technologies, LLC). Mass spec imaging data was acquired with a Bruker timsTOF Flex system (BrukerDaltonik) with a Smartbeam 3D laser at 10kHz and 10 μm resolution. Imaging data analysis was performed in SCiLS Lab software (version 2023b, Bruker). Calculations were performed on data normalized to total ion count (TIC). Spatial segmentation analysis of data was performed using an unsupervised bisecting k-means algorithm and resulting clusters were investigated by ROC. Lipid annotation was performed the Lipid Species annotation tool with MSDial VS68 spectral library in Metaboscape (version 2022b, Bruker). Results: There were vast global differences between lipid composition of wild-type and cystic kidneys at postnatal day 28. When comparing cystic to wild-type kidneys phosphoinositol and ceremide lipid species were decreased and other metabolites, such as thiophene-3-carboxylate and benzothiazine-2-carbonitrile, were increased at both PN7 and PN28. Further numerous lipid compounds were identified only within cystic fluid that have not been previously annotated. Conclusions: The results of this study, for the first time, reveals alterations in lipid molecule profiles throughout the cystic kidney in tandem with morphological changes. This is a major step forward in understanding how lipid metabolism changes in response to cyst expansion during the progression of ADPKD, as well as revealing molecular changes that precede cellular pathology. This work has been supported by Children’s Wisconsin Foundation and Children’s Research Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Validation of improved automated nucleic acid extraction methods for direct detection of polioviruses for global polio eradication

Polioviruses (PV), the main causative agent of acute flaccid paralysis (AFP), are positive-sense single-stranded RNA viruses of the family Picornaviridae. As we approach polio eradication, accurate and timely detection of poliovirus in stool from AFP cases becomes vital to success for the eradication efforts. Direct detection of PV from clinical diagnostic samples using nucleic acid (NA) extraction and real-time reverse transcriptase polymerase chain reaction (rRT-PCR) instead of the current standard method of virus isolation in culture, eliminates the long turn-around time to diagnosis and the need for high viral titer amplification in laboratories. An essential component of direct detection of PV from AFP surveillance samples is the efficient extraction of NA. Potential supply chain issues and lack of vendor presence in certain areas of the world necessitates the validation of multiple NA extraction methods. Using retrospective PV-positive surveillance samples (n=104), two extraction kits were compared to the previously validated Zymo Research Quick-RNA™ Viral Kit. The Roche High Pure Viral RNA Kit, a column-based manual extraction method, and the MagMaX™ Pathogen RNA/DNA kit used in the automated Kingfisher Flex system were both non-inferior to the Zymo kit, with similar rates of PV detection in pivotal rRT-PCR assays, such as pan-poliovirus (PanPV), poliovirus serotype 2 (PV2), and wild poliovirus serotype 1 (WPV1). These important assays allow the identification and differentiation of PV genotypes and serotypes and are fundamental to the GPLN program. Validation of two additional kits provides feasible alternatives to the current piloted method of NA extraction for poliovirus rRT-PCR assays.

GENETIC VARIANTS ASSOCIATED WITH BLOOD PRESSURE PHENOTYPES IN OLDER ADULTS

Abstract African Americans (AAs) suffer from higher rates of uncontrolled HTN even when receiving commonly prescribed classes of antihypertensive medications. Variation in blood pressure (BP) in older adults is known to be associated with a variety of cardiovascular endpoints, yet our understanding of underlying mechanisms is limited. This study aimed to determine if HTN genetic variants influence blood pressure variation within a population of AA older adults taking antihypertensives. Community-dwelling older adults from the Translational Approaches to Personalized Health (TAPH) cohort were recruited to enroll in a 24-hour ambulatory BP monitoring (ABPM) phenotyping study. The Quant Studio 12-K Flex system was used for genotyping participants for three previously established CACNA1C and CACNB2 genetic risk alleles (rs758173, rs2228645, and rs2357790). 69 participants had ABPM data that passed the minimum 80% successful readings threshold: age=68□6, Female=50.3%, Black=73%, White=21%, and Other=6%. ABPM revealed 50.7% controlled HTN, 27.5% Sustained uncontrolled HTN, 14.5 % White coat uncontrolled HTN and 7.2% Masked uncontrolled HTN. ANOVA and Linear regression models found that the CACNB2 genetic risk allele rs2228645 broadly impacts ABPM Phenotypes (C/C SBP= 120.8+/-12.9 mmHg, C/T SBP= 125.73+/-13.5, and T/T SBP= 140+/-34.8 mmHg, p=0.011), mean awake 24-hour SBP (C/C SBP= 124+/-12.7 mmHg, C/T SBP= 133.5+/-14.8, and T/T SBP= 143.7+/-25.6 mmHg, p=0.012), and mean sleep SBP (C/C SBP= 114+/-16 mmHg, C/T SBP= 125.4+/-15, and T/T SBP= 136+/-50 mmHg, p=0.024). CACNA1C and CACNB2 genetic variants influence blood pressure phenotypes in older AAs. Determining genetic variants that affect antihypertensive effectiveness could lead to improved antihypertensive therapy for individuals with HTN/CVD.

Robotic surgery in the treatment of oropharyngeal cancer: a case report

Background. The incidence of HpV-associated oropharyngeal squamous cell carcinoma (oscc) is steadily increasing. given the better prognosis in patients with HpV-positive cancer compared to HpV-negative cancer, attempts were made to reduce the therapeutic effect in patients with early-stage oscc to improve the quality of life of these patients. early-stage oscc can currently been treated with radiation therapy or surgery used alone or in combination. Currently, the concept of transoral surgery includes both transoral laser microsurgery and robot-assisted surgeries (da Vinci, medrobotics Flex system). Case description. We report a case of using the da Vinci robot-assisted system in the combined modality treatment of oropharyngeal cancer. The patient underwent surgery followed by chemoradiotherapy. At a follow-up of 10 months, no evidence of disease progression was found. The patient experienced no any pain on swallowing.Conclusion. The use of the da Vinci robot-assisted surgical system in the combined modality treatment of oropharyngeal cancer, especially in such a hard-to-reach area as the root of the tongue, makes it possible to better visualize and determine the boundaries of the lesion, followed by en block resection, as well as to improve functional and aesthetic results. However, careful selection of patients for this type of treatment is necessary.

B-231 Development and Evaluation of a Lyophilized Mpox Virus qPCR Assay

Abstract Background Increased cases of monkeypox virus (MPXV) have been reported to WHO from 110 countries. As of January 16, 2023, a total of 84 733 laboratory confirmed and 1354 probable cases, including 80 deaths, have been reported to WHO. The US is assessed as high-risk region. Real-time PCR, with its sensitivity and specificity, is the preferred lab test technique. Since gene mutation has been observed in MPXV, developing assays with more than one target was recommended by the FDA. Methods A lyophilized qPCR multiplex assay was developed for MPXV detection. This assay contains primers and (1) MGB probe targeting highly conserved MPXV sequence (GeneBank AF380138 F3L), (2) BHQ probe targeting non-variola orthopoxvirus (OPX) sequence, and (3) a probe targeting a selected human house-keeping gene as an internal control. Furthermore, external positive and negative controls were included. A MPXV positive clinical sample from human skin lesions was used for the assay validation. The viral copy number of the sample was determined as 1×107 copies/mL using the standard curve method. Two hundred µL of samples were processed in the KingFisherTM Flex system. The PCR reaction consisted of 5 µL of DNA sample and 20 µL reaction mix and was run on 7500 Fast Dx Real-Time PCR System (Life Technologies). The limit of detection (LoD) was determined as the concentration at which at least 95% of samples were tested positive. A total of 230 contrived samples with concentrations lower than LoD were used to determine cut-off value. Inclusivity and exclusivity studies were conducted by in silico analysis of 80 sequences of MPXV (clade I), 3026 of MPXV (clade II), 10 of camelpox virus, 93 of cowpox virus, 6 of ectromelia virus, and 116 of vaccinia virus (NCBI and GISAID accessed on 10/03/22). Clinical evaluation of the assay was conducted using 80 contrived clinical samples in unique negative skin lesion matrix, including 40 positives (five samples at 5× LOD, five at 2.5×, ten at 1.5×, and twenty at 1×), along with 40 negatives. The positive (PPA) and negative percent agreement (NPA) of the assay were calculated. The study was approved by BRANY IRB. Results The qPCR assay had a cut-off Ct value of 40, and a LoD of 500 copies/mL for both MPXV and OPX with good inclusivity and exclusivity. Clinical evaluation for MPXV yielded a PPA and NPA of 94.9% (95% CI: 83.1%–98.6%) and 100% (89.9%–100%), respectively. For OPX, the PPA and NPA were 92.3% (79.7%–97.4%) and 100% (89.9%–100%), respectively. Conclusions Both analytical and clinical performances of the developed MPXV qPCR assay were good. Lyophilized reagents contain all required components, providing longer shelf-life and easier transportation. MPXV is the only member of the OPX genus known to be circulating among humans in the US currently. This assay as designed could potentially cross-detect different non-variola OPX, such as vaccinia virus and cowpox virus. Healthcare providers should always contact the local public health authorities for more guidance. Furthermore, this study was limited to lesion swab specimens and performance evaluation with additional specimen types is warranted.

Immunohistochemical Identification and Assessment of the Location of Immunoproteasome Subunits LMP2 and LMP7 in Acquired Cholesteatoma.

Cholesteatoma, accompanied by chronic inflammatory response, is characterized by invasive growth and osteolytic activity. As specific proteasome isoforms, the immunoproteasomes serve as an important modulator of inflammatory responses. The aim of the present study was to determine the biological activity of cholesteatoma through the analysis of the expression and localization of immunoproteasome subunits of low molecule weight protein (LMP) 2 and LMP7. Cholesteatoma specimens were obtained from 15 adults who underwent ear surgery due to acquired attic cholesteatoma. Normal skin specimens were taken from retro-auricular skin incisions from the same patients. The specimens were stained with anti-LMP7 antibody, using immunohistochemistry techniques based on the binding of biotinylated secondary antibody with the enzyme-labeled streptavidin and the Envision FLEX system. In all specimens of cholesteatoma, the immunohistochemical reaction with the antibody against the LMP2 was positive, in both the cytoplasm of the cholesteatoma matrix and the perimatrix. A negative reaction with anti-LMP2 was observed in the cytoplasm and nuclei of control skin cells. A positive nuclear and cytoplasmic immunohistochemical reaction with anti-LMP7 has been demonstrated in numerous cells, in both the matrix and perimatrix of cholesteatoma. We present evidence of the presence of expressions of LMP2 and LMP7 within cholesteatoma tissue. Our results might bring new information concerning immunoproteasome-dependent pathophysiologic mechanisms in cholesteatoma.

Abstract 6778: Spatial analysis of genomic signatures on colorectal cancer pathogenesis using the GeoMx® Digital Spatial Profiler

Abstract Background: The development of colorectal cancer (CRC) is a complex process, involving multiple sequential molecular aberrations that contribute to disease progression. Understanding the immunopathogenesis of CRC particularly within the spatial context of tumor microenvironment (TME) may elucidate the genomic and biological changes to improve patient prognosis for adjuvant therapies and identifying potential drug targets. However, this can be challenging, as genomic signatures from sub-cellular populations and varying levels of immune cell infiltration in TME may be lost in bulk sequencing. In this study, we used the NanoString GeoMx® Digital Spatial Profiler (DSP) to spatially select epithelial, proximal stromal and distal stromal regions of TMEs on a total of 10 FFPE samples (5 colon cancer patients and 5 healthy donor samples) and profiled up to 1,800 genes included in the Cancer Transcriptome Atlas (CTA) panel. Methods: Selection of regions of interest (ROI) were guided by pathology assessment of H&E images and fluorescent markers (CD45, PanCK, Syto13). Tumor and stroma (proximal and distal) regions were profiled through geometric ROI selection in PanCK+ (epithelial) and CD45+ (stromal/immune) enriched areas respectively, followed by collection of indexed oligonucleotides and sequencing on NextSeq 550 Illumina instrument. Differential expression and pathways enrichment analyses was performed using R BioConductor package DESeq2 and DSP GeoMx analysis suite. A subset of samples were tested for bulk RNA expression using PanCancer IO360 panel on NanoString nCounter Flex system and data analyzed using nSolver software 3.0 and TIS algorithm App. Results: The CTA panel is designed to profile the global immune response and all aspects of tumor microenvironment biology. From differential gene expression analysis of epithelial and distal/proximal stromal ROIs, we have identified biomarkers such as SOX9, IRAK2 and NLRP3 that are specifically upregulated in CRC samples compared to the healthy cohort. Comprehensive pathways analysis revealed an over activation of NF-kB signaling, extracellular matrix organization and interferon signaling pathways in the CRC cohort. Suppression of the NF-kB signaling pathway is a potential therapeutic approach in the treatment of colon cancer. Conclusion: With this spatial study using GeoMx CTA panel, we have highlighted key genes and pathways involved in CRC in order to provide a much-needed understanding of the underlying mechanisms as well as immune signatures in the development of colon cancer. Citation Format: Xiang Li, Qinqin Zha, Brigitte Lovell, Jane Reed, Anna Juncker-Jensen, Tricia Peters, Lakshmi Chandramohan. Spatial analysis of genomic signatures on colorectal cancer pathogenesis using the GeoMx® Digital Spatial Profiler. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6778.

Does size matter? Two new deletions in the HBB gene cause β0-thalassemia.

Most β-thalassemias are caused by mutations involving one or a limited number of nucleotides within the gene or its adjacent regions. They can be substitutions or deletions; in these cases, the loss ranges from a single nucleotide to even the entire HBB gene, so we wonder if the phenotype is due to the size of the deletion or the location of the mutation. To clarify this, we present two new deletions in the β-globin gene that cause β0-thalassemia. The hematological parameters were determined with an automated cell counter; the Hb A2 and Hb F levels were measured by performance liquid chromatography. Hemoglobins were analyzed by capillary zone electrophoresis (Sebia Capillarys Flex system) and ion-exchange HPLC (BioRad Variant II β-thalassemia Short Program). Molecular characterization was performed by automatic Sanger sequencing. The screening of common α-thalassemia point mutations and deletions in the world (21 in total) were carried out using multiplex PCR followed by reverse-hybridization with a commercial Alpha-Globin StripAssay kit. We have characterized two new mutations-(1) 1-bp deletion [CD61/62(-G)] [HBB:c.186_187delG], (2) 105-bp deletion [IVS-2-nt767-CD111] [HBB:c.316-84_333del]-and we have described, for first time in Spain, the 25-bp deletion [β nts 252 - 276 deleted] [HBB:c.93-22_95del] mutation. These mutations were classified as pathogenic by UniProt Variants confirmed according to the American College of Medical Genetics and Genomics guidelines. These mutations present a phenotype compatible with β0-thalassemia, supported by hematological parameters that correlate the degree of reduction in the synthesis of the β-globin chain. Identification of this type of mutation is important for genetic counselling of partners where both are carriers, so that they are aware of the genetic risk of having affected children, allowing them to take an informed decision about their reproductive choices.

Development of Personalized Non-Invasive Ventilation Interfaces for Neonatal and Pediatric Application Using Additive Manufacturing.

The objective of this study was to present a methodology and manufacturing workflow for non-invasive ventilation interfaces (NIV) for neonates and small infants. It aimed to procure a fast and feasible solution for personalized NIV produced in-house with the aim of improving fit and comfort for the patient. Three-dimensional scans were obtained by means of an intraoral (Trios 3) and a facial scanner (3dMd Flex System). Fusion 360 3D-modelling software was employed to automatize the design of the masks and their respective casting molds. These molds were additively manufactured by stereolithography (SLA) and fused filament fabrication (FFF) technologies. Silicone was poured into the molds to produce the medical device. In this way, patient individualized oronasal and nasal masks were produced. An automated design workflow and use of additive manufacturing enabled a fast and feasible procedure. Despite the cost for individualization likely being higher than for standard masks, a user-friendly workflow for in-house manufacturing of these medical appliances proved to have potential for improving NIV in neonates and infants, as well as increasing comfort.

Identification of SARS-CoV-2 in the vaginal fluid and cervical exfoliated cells of women with active COVID-19 infection: A pilot study.

SynopsisIn women with confirmed COVID‐19, a negative RT‐PCR test result does not rule out the possibility of a low level of SARS‐CoV‐2 in the female genital tract.

Describing Treatment of Primary Mediastinal Large B Cell Lymphoma Using Rigorously Defined Molecular Classification: A Retrospective Analysis

Introduction Primary mediastinal large B cell lymphoma (PMBCL) is a rare non-Hodgkin lymphoma (NHL) with a female predominance; often presenting with a large anterior mediastinal mass. Though PMBCL has clinical and molecular features overlapping with Hodgkin lymphoma, it is a distinct entity defined by the World Health Organization classification. PMBCL is heterogeneously treated, and most patients receive front line therapy with either rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP) with radiotherapy (RT), or the more intensive etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin with rituximab (EPOCH-R) regimen. Diagnosis of PMBCL is made using clinicopathologic criteria and radiographic imaging, however gene expression profiling (GEP) studies reveal a characteristic genotypic signature distinct from diffuse large B cell lymphoma (DLBCL). Molecular classification of PMBCL using the Lymph3Cx assay from formalin-fixed paraffin-embedded tissue (FFPE) is feasible, reproducible, and highly concordant in a training and validation cohort (Mottok et al. Blood 2018). Using a multicenter cohort of patients, we sought to estimate the rate of mis-match among patients with a clinical diagnosis of PMBCL using Lymph3Cx, and describe treatment selections and outcomes for each group. Methods Patients were identified from a cohort of patients with newly diagnosed NHL from the University of Iowa and Mayo Clinic Molecular Epidemiology Resource, and the Lymphoma Epidemiology of Outcomes cohort. Patients were enrolled between 2002-2019, and included if they had clinically defined PMBCL. FFPE was retrieved from hematopathology archives of participating academic centers. All diagnoses of PMBCL were based on expert hematopathology review at the time of therapy, and all cases underwent classification by GEP using the Lymph3Cx assay. Lymph3Cx was performed in the clinical lab at the Mayo Clinic in Arizona: Contiguous unstained sections were deparaffinized and macrodissected to enrich for tumor content before RNA isolation;100-200 ng of total RNA was used in an nCounter Elements XT, hybridized, and processed the following day using the nCounter FLEX system. Raw counts were processed through the Lymph3Cx algorithm and results reported as probability of PMBCL (≥0.90 as PMBCL, ≤0.10 as DLBCL all other results "Unclear PMBCL/DLBCL") (A. Mottok et al, Blood, 2018). For cases classified as DLBCL, the Lymph2Cx cell-of-origin classifier results was reported (Scott et al, JCO, 2016). Time to event endpoints were described with Kaplan-Meier plots by groups defined by mismatch status and compared with a logrank test. Binary outcomes will be presented with 90% exact confidence intervals. Results Fifty patients were identified. Median age was 35 years (range 19-70). Sixty four percent were women. Median follow up was 47 months. Treatments included R-CHOP (44%), EPOCH-R (44%), and MACOP-B [methotrexate with leucovorin rescue, doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin] (6%), other (4%). Ten patients (20%) had events (defined as progression or death). Three patients in the entire cohort (6%) died. The Kaplan-Meier estimated survival at 47 months (median follow-up) is 92%. The Lymph3Cx assay yielded gene expression data of sufficient quality in 47/50 cases (94%, 90% CI=85.2, 98.3%). Of 47 cases clinically identified as PMBCL, 5 unclear were DLBCL/PMBCL and 1 was Germinal Center B cell subtype of DLBCL. Among these 6 patients, 4 received R-EPOCH (66%), 1 received R-CHOP (16.6%). One patient had missing treatment data. One patient had an event requiring subsequent therapy; all patients remain alive. Conclusions Using 47 patients with PMBCL defined by histology, clinical and radiographic findings, and molecular features, we demonstrate high concordance between clinical phenotype and molecular genotype of PMBCL by Lymph3Cx. Among the 6 patients not classified as PMBCL, most received R-EPOCH. Differences in outcome by mis-match status await longer follow-up and further accrual of subjects to our data base. Our data suggest molecular genotyping may have a role in mediastinal presentations of large cell lymphoma to optimize treatment decision making. Disclosures Maurer: Nanostring: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene / BMS: Research Funding; Morphosys: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees. Cerhan:BMS/Celgene: Research Funding; NanoString: Research Funding. Flowers:AbbVie: Consultancy, Research Funding; Kite: Research Funding; Burroughs Wellcome Fund: Research Funding; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Denovo Biopharma: Consultancy; Celgene: Consultancy, Research Funding; Cancer Prevention and Research Institute of Texas: Research Funding; TG Therapeutics: Research Funding; Eastern Cooperative Oncology Group: Research Funding; V Foundation: Research Funding; Bayer: Consultancy; National Cancer Institute: Research Funding; Millennium/Takeda: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Acerta: Research Funding; Spectrum: Consultancy; Pharmacyclics/Janssen: Consultancy; Karyopharm: Consultancy; OptumRx: Consultancy; Leukemia and Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; BeiGene: Consultancy. Friedberg:Acerta Pharma - A member of the AstraZeneca Group, Bayer HealthCare Pharmaceuticals.: Other; Astellas: Consultancy; Bayer: Consultancy; Kite Pharmaceuticals: Research Funding; Portola Pharmaceuticals: Consultancy; Roche: Other: Travel expenses; Seattle Genetics: Research Funding.

Subtyping of male breast cancer by PAM50 and immunohistochemistry: a pilot study of a consecutive Danish cohort.

Male breast cancer (MBC) is a rare disease that is still to be fully understood. In female breast cancer, molecular subtyping by gene expression has proven its significance. In this study, we characterize a consecutive cohort of MBC patients surgically treated from 1997 to 2017, identified at our institution (N=37), and report the association between molecular subtypes found by a surrogate panel of immunohistochemical (IHC) markers, and the PAM50 signature, as well as risk of recurrence score and overall survival for the different subtypes. PAM50 subtypes were determined using the nCounter FLEX system instrument and software. The distribution of molecular subtypes according to the PAM50 signature was as follows: 56% luminal B, 39% luminal A, and 5% basal-like. None of the tumors were HER2-enriched. Using IHC surrogate markers, we found 80% luminal B-like, 15% luminal A-like, and 5% basal-like. None were HER2-positive (non-luminal). We found a strong statistical association between subtypes found by PAM50 signature and the IHC surrogate markers (p<0.001). Furthermore, we found luminal A tumors to be smaller in size compared to luminal B tumors (p=0.04). Patients with luminal A subtype tumors had the lowest ROR scores with a mean of 39, whereas patients with luminal B subtype tumors had a mean ROR score of 69. Significant worse overall survival for luminal B tumors compared to luminal A tumors was seen (p=0.02). Male breast cancer seems to be a mainly luminal disease, with luminal B being the most frequent subtype. Further studies are needed to ensure correct therapeutic strategies for this select group of patients.

Association between the rs1544410 polymorphism in the vitamin D receptor (VDR) gene and insulin secretion after gestational diabetes mellitus.

Genetic variants involved in vitamin D metabolism have been associated with diabetes and related syndromes/diseases. We wanted to investigate possible associations of polymorphisms in genes involved in vitamin D metabolism with indices of insulin resistance and insulin secretion, and also with development of diabetes after gestational diabetes mellitus (GDM). We have studied 376 women with previous GDM. Eight single nucleotide polymorphisms (SNPs) in the genes for vitamin D receptor (VDR) [rs731236, rs7975232, rs10735810, and rs1544410], vitamin D binding protein (DBP) [rs7041 and rs4588], and cytochrome P450 family 27 subfamily B member 1 (CYP27B1) [rs10877012 and rs4646536] were genotyped by TaqMan Allelic Discrimination Assay using the Quantstudio 7 Flex system. A 75-g oral glucose tolerance test (OGTT) was performed 1-2 years postpartum. The homeostasis model assessment of insulin resistance (HOMA-IR) and the disposition index [(insulinogenic index: I30/G30)/HOMA-IR] were used to calculate insulin resistance and insulin secretion, respectively. Serum samples for determination of 25(OH)D3 were collected at the time of the OGTT. Manifestation of diabetes was followed up to five years postpartum. After adjustment for BMI, age, and ethnicity, the A-allele of the VDR rs1544410 polymorphism was found to be associated with increased disposition index (difference per allele = 3.56, 95% CI: 0.4567-6.674; p = 0.03). The A-allele of the DBP rs7041 polymorphism was found to be associated with 25(OH)D3 levels (difference [in nmol/L] per allele = -5.478, 95% CI: -8.315 to -2.641; p = 0.0002), as was the T-allele of the DBP rs4588 polymorphism (OR = -6.319, 95% CI: -9.466 to -3.171; p = 0.0001). None of the SNPs were significantly associated with HOMA-IR or postpartum diabetes. This study provides evidence that the rs1544410 polymorphism of the VDR gene may be associated with increased insulin secretion in women after pregnancy complicated by GDM. Further studies in other populations are needed to confirm the results.

A rapid and reliable qPCR based system to quantitate HEK 293 residual DNA for GMP lot release in gene therapy manufacturing

Background & Aim Per regulatory requirements, gene therapeutics must limit host cell residual DNA contaminants to prevent genotoxic risk to patient. Consequently, manufacturers have traditionally outsourced testing to labs that specialize in a variety of analytical methods that measure bioprocess residuals. Real-time PCR based assays provide a viable alternative and deliver results in hours while meeting the required sensitivity. In this study, we developed a Real-time qPCR assay using Applied Biosystems 7500 Fast and Quant Studio 5 instruments with AccuSEQ software by Thermo Fisher Scientific. Methods, Results & Conclusion Method: HEK 293 genomic DNA was purified from HEK 293 viral production cells supplied by Thermo Fisher Scientific. The purified HEK 293 genomic DNA was titrated in 10 fold increments to create a 5 point linear standard curve for interpolating unknown sample concentrations. Up front sample prep was performed manually and on King Fisher Flex system using the PrepSEQ™ Residual DNA Sample Preparation kit. Gene therapy sample matrix formulations were prepared representing various stages in the bioproduction workflow. We characterized the assay sensitivity, specificity, and reproducibility. Results We found this assay has a sensitivity of 300 fg HEK 293 genomic DNA and precision of less than 5% CV when tested across multiple instruments, operators, days, and manufactured lots. Furthermore, all standard curve points conformed to linear curve fit with R2 ≥ 0.99 and PCR efficiency of 100% +/-10%. The assay detects residual HEK 293 genomic DNA in bioproduction process intermediates and final formulation samples. Conclusions A robust and reliable real-time PCR assay was developed to quantify residual host cell HEK 293 DNA and ensure gene therapeutics have minimal residual DNA contaminants. The kit offers high sensitivity, specificity, and consistent performance and enables gene therapy manufacturers to verify safety of gene therapy drug products at various stages of their workflow; from process development to final formulation lot release testing.

The Flex robotic system compared to transoral laser microsurgery for the resection of supraglottic carcinomas: first results and preliminary oncologic outcomes.

Transoral robotic surgery (TORS) has the potential to improve some inherent disadvantages of transoral laser microsurgery (TLM). Here, we retrospectively assessed the application of the Medrobotics Flex system for the resection of supraglottic carcinomas compared to TLM. 84 patients underwent surgery for supraglottic carcinomas with the Flex robotic system (n = 19, T-stage distribution in %: T1 42, T2 47, T3 11, T4 0) or TLM (n = 65, T-stage distribution in %: T1 40, T2 44, T3 14, T4 2). Clinical and oncologic parameters were compared. All surgeries were successfully completed with the Flex system and tracheostomy rate was 13%. For patients with adequate follow-up, 24-month disease-free survival was 71.4% (n = 5/7) after TORS compared to 64.9% (n = 24/37) after TLM. Local recurrence rates were 0% for TORS and 11% for TLM. Initial results for supraglottic carcinoma resection using the Medrobotics Flex system are encouraging with excellent local tumor control.

A case-control study of tumor necrosis factor-alpha promoter polymorphism and its serum levels in patients with chronic obstructive pulmonary disease in Kashmir, North India.

Aim:Data about polymorphism in tumor necrosis factor-alpha (TNF-α) and its serum levels in chronic obstructive pulmonary disease (COPD) are conflicting. We aimed to evaluate the association of TNF-α-308 G > A polymorphism in patients with COPD in Kashmir (North India), a high burden area and also determined the serum TNF-α levels in these patients.Materials and Methods:One hundred spirometrically confirmed COPD patients and 163 controls resident from Kashmir valley (North India) were recruited. Genotyping of the promoter region of TNF-α was carried out using polymerase chain reaction-restriction fragment length polymorphism. The serum TNF-α was quantified using the Cytometric Bead Array flex system by flow cytometry. Results were subjected to appropriate statistical treatment and P < 0.05 was considered statistically significant.Results:Ninety-one COPD patients (91%) had G/G (wild homozygous) genotype and nine patients (9%) had G/A (heterozygous) genotype. Among the control population, 150 (92%) had G/G genotype and 13 (8%) had G/A genotype. The variant allele “A” was not detected in either of the two groups. Serum levels of TNF-α were significantly higher in patients compared to control group (8.0 ± 10.1 pg/ml vs. 3.3 ± 0.42 pg/ml, respectively, P = 0.0001).Conclusion:While serum levels of TNF-α are higher in COPD patients compared to the controls, there was no difference in the prevalence of TNF-α-308 polymorphism in the ethnic Kashmiri population with COPD.

Effect of Coagulation Factor Concentrates on Markers of Endothelial Cell Damage in Experimental Hemorrhagic Shock.

Plasma-based resuscitation showed protective effects on the endothelial glycocalyx compared with crystalloid resuscitation. There is paucity of data regarding the effect of coagulation factor concentrates (CFC) on the glycocalyx in hemorrhagic shock (HS). We hypothesized that colloid-based resuscitation supplemented with CFCs offers a therapeutic value to treat endothelial damage following HS. Eighty-four rats were subjected to pressure-controlled (mean arterial pressure (MAP) 30-35 mm Hg) and lab-guided (targeted cutoff: lactate >2.2. mmol/L and base deficit > 5.5 mmol/L) HS. Animals were resuscitated with fresh frozen plasma (FFP), human albumin (HA) or Ringer's lactate (RL) and RL or HA supplemented with fibrinogen concentrate (FC) or prothrombin complex concentrate (PCC). Serum epinephrine and the following markers of endothelial damage were assessed at baseline and at the end-of-observation (120 min after shock was terminated): syndecan-1, heparan sulfate, and soluble vascular endothelial growth factor receptor 1 (sVEGFR 1). Resuscitation with FFP had no effect on sVEGFR1 compared with crystalloid-based resuscitation (FFP: 19.3 ng/mL vs. RL: 15.9 ng/mL; RL+FC: 19.7 ng/mL; RL+PCC: 18.9 ng/mL; n.s.). At the end-of-observation, syndecan-1 was similar among all groups. Interestingly, HA+FC treated animals displayed the highest syndecan-1 concentration (12.07 ng/mL). Resuscitation with FFP restored heparan sulfate back to baseline (baseline: 36 ng/mL vs. end-of-observation: 36 ng/mL). The current study revealed that plasma-based resuscitation normalized circulating heparan sulfate but not syndecan-1. Co-administration of CFC had no further effect on glycocalyx shedding suggesting a lack of its therapeutic potential. VExperimental in vivo study.