Molecular Panel Research Articles

Molecular screening of XY SRY-negative sex reversal cases in horses revealed anomalies in amelogenin testing.

Male-to-female sex reversal in horses is a developmental disorder in which phenotypic females have a male genetic constitution. Male-to-female sex reversal is the second most common genetic sex abnormality, after X chromosome monosomy. All male-to-female sex reversal cases studied to date have been found to be infertile. Therefore, a screening test is particularly useful in laboratories doing DNA genotyping in horses. Our laboratory has tested > 209,000 horses for parentage using a panel of microsatellite markers and the sex marker gene amelogenin (AMEL). Suspect XY sex reversal cases are reported females with a male profile by AMEL testing. After routine genotyping, 49 cases were detected and further tested using the sex-determining region Y (SRY) gene, confirming the XY SRY-negative genotype of suspect sex reversal cases. When some inconsistencies arose in the initial result, a molecular panel of X- and Y-linked markers was analyzed for these samples. Of the 49 cases, 33 were confirmed as XY SRY-negative. The remaining 16 cases were identified as false-positives as a result of anomalies of AMEL testing in horses.

Be aware of Shiga-toxin 2f-producing Escherichia coli: case report and false-negative results with certain rapid molecular panels

We report a hemolytic uremic syndrome (HUS) case due to Stx2f-producing E. coli illustrating the diagnostic difficulty of this Shiga-toxin subtype. Clinicians should be aware of limits of certain rapid molecular panels that are increasingly being used and may play a role in underestimating the global burden of such infections.

Abstract 3301: Immune monitoring for immuno-oncology applications

Abstract The immunotherapy revolution has spurred the development of many new drugs and drug regimens for patient treatment. A key challenge is to identify the factors that drive patient toxicities and responses to treatment, with a particularly acute need for predictive biomarkers that can discriminate patients destined to respond and fail treatment. Technologies to interrogate immune cells are now readily available, but important gaps remain in their application, which limit full realization of the promise of precision oncology. One such technology, high parameter flow cytometry, represents a gold-standard for immune monitoring; however, antibody panel design and lack of standardization represent important application gaps that hinder broader use. Here, we present a new tool for panel design, Color Wheel, which builds antibody panels optimized for performance on the user's instrument. We used Color Wheel to develop six panels (cell lineage, immune checkpoint, checkpoint ligand, regulatory T-cell, B-cell, cytokine) that comprehensively interrogate the tumor microenvironment. We demonstrate the value of these panels in identifying differences between disease types, treatment responses, and patient groups in a variety of cancer settings. Furthermore, we have developed the immune checkpoint panel into a dried, ready to use (single test-per-tube) format to lower the barriers to access high parameter flow cytometry technology, and to drive workflow efficiency and assay standardization. We demonstrate excellent concordance between the dried and liquid (wet) versions of the high parameter multicolor panel. A second technology, molecular cytometry, represents an exciting new approach to high dimensional immune analysis. The technology is capable of measuring at least 102 proteins simultaneously per cell, alongside 400 mRNA targets. An important application gap for this technology is lack of data indicating the sequencing depth needed to resolve cell types in an experiment. Here, we present results from a molecular cytometry experiment sequenced deeply, and then bioinformatically sub-sampled at different levels to identify the minimum level of sequencing needed for clear identification of cells, which can serve as a reference guide for users to save time and cost in their experiments. From the perspective of workflow efficiency, we also present preliminary performance data generated using a dried version of molecular cytometry panel(s). Finally, our presentation includes an analysis of the rationale for choosing an immune monitoring technology, providing some guidelines that match study type and goals, cost, timelines, complexity, and labor with various single cell technologies. Citation Format: Woodrow E. Lomas, Guo-Jian Gao, Na Li, Suraj Saksena, Pratip K. Chattopadhyay. Immune monitoring for immuno-oncology applications [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 3301.

Infección invasiva por Listeria monocytogenes en un paciente con síndrome de Evans.

Listeria monocytogenes es una bacteria intracelular facultativa que tiene predilección por causar infecciones del sistema nervioso central en humanos. Cuya población de riesgo predominante es lactantes, adultos mayores y en estados de inmunosupresión. Los principales síndromes clínicos causados por Listeria monocytogenes incluyen gastroenteritis febril, infección perinatal e infecciones sistémicas marcadas por infecciones del sistema nervioso central con o sin bacteriemia. Presentamos el caso de un paciente masculino de 66 años portador de síndrome de Evans en tratamiento inmunosupresor quien acude al servicio de emergencias del Hospital San Carlos el 27 de agosto de 2019, por un cuadro agudo de fiebre, cefalea y ataque al estado general en quien se documenta síndrome de respuesta inflamatoria sistémica y meningitis bacteriana ante lo cual inicio empíricamente cefotaxime. El panel molecular y los hemocultivos fueron positivos por Listeria monocytogenes. El paciente recibió 3 semanas de antibioticoterapia con ampicilina con excelente evolución clínica y sin secuelas neurológicas.

Differential metabolic profile associated with the condition of normoalbuminuria in the hypertensive population

Background and aimAlbuminuria is an indicator of sub-clinical organ damage and a marker of cardiovascular risk and renal disease. A percentage of hypertensive patients develop albuminuria despite being under chronic suppression of the renin-angiotensin system (RAS). We previously identified urinary metabolites associated with the development of albuminuria. In this study, we searched for metabolic alterations which reflect different levels within the condition of normoalbuminuria. Patients, materials and methodsUrine from 48 hypertensive patients under chronic RAS suppression was analyzed. They were classified according to the albumin / creatinine ratio (ACR) into 3 groups: Normoalbuminuria (<10 mg / g); Normal-high (10–30 mg / g in men, or 20−40 mg / g in women); and moderately high albuminuria (microalbuminuria, 30−200 mg / g or 40−300 mg / g, respectively). The metabolome was analyzed by mass spectrometry and a correlation analysis was performed between altered metabolite levels and ACR. ResultsOxaloacetate, 3-ureidopropionate, guanidoacetate and malate show significant variation between the normo and micro groups. Additionally, these metabolites are able to differentiate between patients in the normo and high-normal range. A significant correlation between metabolites and ACR was found. Observed variations point to alterations in the energy metabolism already in patients with albuminuria in the high-normal range. ConclusionsThe association between the molecular panel consisting of 3-ureidopropionate, oxaloacetate, malate and guanidoacetate and different levels of albuminuria is confirmed. A metabolic fingerprint was also identified showing variations within the condition of normoalbuminuria allowing an earlier molecular stratification of patients.

Перспективы использования молекулярно-генетических панелей в дооперационной дифференциальной диагностике узловых образований щитовидной железы

Перспективы использования молекулярно-генетических панелей в дооперационной дифференциальной диагностике узловых образований щитовидной железы

Abstract 38: Molecular determinants of drug response in TNBC cell lines

Abstract Triple-negative breast cancer (TNBC) is one of the deadliest forms of breast cancer due to limited treatment options beyond conventional chemotherapy. TNBC constitutes approximately 10-20% of total breast cancer cases in the US; however, TNBC tends to be more aggressive, higher grade, and have a poorer prognosis than other forms of breast cancer. While oncogenic aberrations have been identified in TNBC, such as the PI3K/AKT/mTOR pathway that is misregulated in ~25% of tumors, there is a general lack of predictive markers of therapeutic efficacy to identify subsets of patients most likely to positively respond to a given targeted or conventional therapy. Simply relying on mutation status is undependable, as some patients without PI3K mutations respond to PI3K inhibitors and some patients with PI3K mutations do not respond. Thus, there is a critical need for biomarkers of drug efficacy in TNBC to help design precision medicine clinical trials based on systematically derived predictive biomarkers. To fill this gap, we devised a strategy to identify biomarkers of drug efficacy in TNBC. Using 23 TNBC cell lines, drug sensitivity scores (DSS3) were determined across a panel of investigational drugs and drugs approved for other indications. Molecular readouts were generated for each cell line using RNA sequencing, RNA targeted panels, DNA sequencing, and functional proteomics. DSS3 values were correlated with molecular readouts using an FDR-corrected significance cutoff of p* &amp;lt; 0.05 and yielded molecular determinant panels that predict anti-TNBC efficacy. Molecular determinant panels were obtained from 12 drugs that were prioritized on the basis of their efficacy. Six molecular determinant panels were obtained by correlating DSS3 with molecular readouts. We found that coinhibiting correlated pathways led to robust synergy across many cell lines. Together, our work presents an integrated method to identify biomarkers of drug efficacy in TNBC, where DNA predictions correlate poorly with drug response. Furthermore, we outline a framework to identify optimal companion drugs for combination therapy. Citation Format: Nathan M. Merrill, Eric J. Lachacz, Nathalie M. Vandecan, Peter J. Ulintz, Li Wei Bao, John P. Lloyd, Aki Morikawa, Sofia D. Merajver, Matthew B. Soellner. Molecular determinants of drug response in TNBC cell lines [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 38.

A Novel de novo Frameshift Mutation in the BCL11A Gene in a Patient with Intellectual Disability Syndrome and Epilepsy

Intellectual disability syndrome (IDS) associated with a hereditary persistence of fetal haemoglobin (HbF), also known as Dias-Logan syndrome, is commonly characterised by psychomotor developmental delay, intelectual disability, language delay, strabismus, thin upper lip, abnormalities of external ears, microcephaly, downslanting palpebral fissures. Sporadically, autism spectrum disorders and blue sclerae in infancy have been reported in IDS. Rarely, IDS-affected patients present with epilepsy and/or epileptic syndromes. It has been shown that a haploinsufficiency of the B cell leukaemia/lymphoma 11A gene (BCL11A) is responsible for IDS. Herein, we identified a novel de novo frameshift deletion (c.271delG; p.E91Afs*2) in the BCL11A gene in a boy affected with IDS. Interestingly, this heterozygous loss-of-function BCL11A mutation was also associated with a generalised idiopathic epilepsy and severe language delay observed in the patient. Moreover, our study showed that the combination of molecular genetic analyses with the monitoring of HbF was essential to make the final diagnosis of Dias-Logan syndrome. Because our patient suffered from well-controlled epilepsy, we propose to include the BCL11A gene in routinely used molecular genetic epilepsy-related gene panels. Additionally, many of the clinical features of IDS overlap with symptoms observed in patients with suspected alcohol spectrum disorders. Therefore, we also suggest monitoring HbF levels in patients with these syndromes to further facilitate clinical diagnosis.

Update on pediatric lung transplantation: mir-ando into the mechanisms of chronic lung allograft dysfunction in children.

Lung transplantation (LTx) is a worthwhile treatment for children with end-stage lung diseases who have no practicable medical or surgical solutions. But the long-term survival remains the lowest in all solid-organ transplant, with a median survival of 5.7 years, limited by the onset of chronic lung allograft dysfunction (CLAD). This reviews a recent publication in pediatric patients that focuses on translational regulation by microRNA. The mechanisms that cause transplanted lung allografts have been difficult to identify. This review discusses pertinent findings in the first and largest observational prospective study of pediatric lung transplant recipients. The review discusses the relevance of microRNA that distinguish stable patients from those who can be predicted to display graft dysfunction on a molecular panel. The article under review detected highly specific and sensitive markers of both acute rejection and CLAD in pediatric LTx recipients. With the use of next-generation sequencing techniques, biomarkers may soon provide the basis for earlier detection of graft function and stimulate development of therapeutic interventions to impact outcomes and survival. The review touches on the relevance of these findings and how future research can build on them.

OP0240 A MULTIMODAL MASS SPECTROMETRY APPROACH REVEALS SPECIFIC CARTILAGE MOLECULAR PROFILES ASSOCIATED TO TYPE 2 DIABETIC PATIENTS

Background:Osteoarthritis (OA) is mainly characterized by the progressive deterioration of articular cartilage. Recent studies support that type 2 diabetes (TD2) is a risk factor to develop OA [1, 2]. However, the molecular cartilage profile of patients combining these two diseases remains unclear, and a better understanding of the different OA phenotypes should be considered for the development of personalized medicine.Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is used to investigate the bimolecular distribution of proteins, lipids or metabolites through thein-situanalysis of tissue sections. Bottom-up proteomics focuses on the relative quantification of proteins. The combination of both technologies could be considered to reveal specific molecular profiles and help for patient classification.Objectives:The main goal of this study is to apply a multimodal mass spectrometry approach on cartilage to reveal specific lipidomic and proteomic profiles associated to TD2 patients.Methods:Human cartilages from OA (na=10) and OA/TD2 human patients (nb=10) were obtained from donors undergoing total knee joint replacement. Cartilage punches of 8*8mm were sectioned at 12 µm thickness for MALDI-MSI and bottom-up proteomics.For MALDI-MSI experiments (na=6; nb=6), norharmane matrix was sprayed over the samples for the detection of lipids. Experiments were then performed in positive ion polarity at 50 µm of lateral resolution using a RapifleX MALDI Tissue-typer instrument. LipostarMSI and in-house ChemomeTricks toolbox for MATLAB software were used for data processing and analysis.For bottom-up proteomics experiment (na=10; nb=10), proteins were extracted, separated using SDS-PAGE and digested prior to liquid chromatography separation coupled to an orbitrap MS Q-Exactive HF mass spectrometer. Proteome Discoverer, enrichR and Reactome software were used for data processing and analysis.Results:MALDI-MSI showed overall differences between OA and OA/TD2 patients based on their specific lipidomic profiles. In particular, sphingomyelin and phosphatidylcholine species were significantly more abundant in OA patients whereas lysolipids such as lysophosphatidylcholine species were mainly present in OA/TD2 patients, providing therefore phenotype-specific OA molecular panels. Additionally, we observed that phosphatidylcholine and sphingomyelin species were more present in the superficial layer of the cartilage whereas lysophosphatidylcholine species were more abundant in the deep layer (Fig. 1A, B).Proteomics experiments applied on cartilage enables the quantification of 114 proteins. Among those, 73 were overexpressed in OA samples whereas 41 were overexpressed in OA/TD2 patients. Among the differentially regulated proteins (Fig. 1C), phospholipase A2 was increased in the diabetic cohort, in line with the elevated level of lysolipids found in the imaging data. Our results also involved the fatty acid omega oxidation and the fatty acid biosynthesis pathways as relevant to explain this deregulation of the lipid metabolism.Conclusion:MALDI–MSI combined with proteomics experiments showed different profiles between OA and OA/TD2 patients and could be employed for patient classification.

Aneuploidy in targeted endoscopic biopsies outperforms other tissue biomarkers in the prediction of histologic progression of Barrett's oesophagus: A multi-centre prospective cohort study.

BackgroundThe cancer risk in Barrett's oesophagus (BO) is difficult to estimate. Histologic dysplasia has strong predictive power, but can be missed by random biopsies. Other clinical parameters have limited utility for risk stratification. We aimed to assess whether a molecular biomarker panel on targeted biopsies can predict neoplastic progression of BO.Methods203 patients with BO were tested at index endoscopy for 9 biomarkers (p53 and cyclin A expression; aneuploidy and tetraploidy; CDKN2A (p16), RUNX3 and HPP1 hypermethylation; 9p and 17p loss of heterozygosity) on autofluorescence-targeted biopsies and followed-up prospectively. Data comparing progressors to non-progressors were evaluated by univariate and multivariate analyses using survival curves, Cox-proportional hazards and logistic regression models.Findings127 patients without high-grade dysplasia (HGD) or oesophageal adenocarcinoma (OAC) at index endoscopy were included, of which 42 had evidence of any histologic progression over time. Aneuploidy was the only predictor of progression from non-dysplastic BO (NDBO) to any grade of neoplasia (p = 0.013) and HGD/OAC (p = 0.002). Aberrant p53 expression correlated with risk of short-term progression within 12 months, with an odds ratio of 6.0 (95% CI: 3.1–11.2). A panel comprising aneuploidy and p53 had an area under the receiving operator characteristics curve of 0.68 (95% CI: 0.59–0.77) for prediction of any progression.InterpretationAneuploidy is the only biomarker that predicts neoplastic progression of NDBO. Aberrant p53 expression suggests prevalent dysplasia, which might have been missed by random biopsies, and warrants early follow up.

Liquid biopsy: Safety and efficacy in a tool for molecular profiling of advanced non-small cell lung cancer (NSCLC).

e21506 Background: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer with a broad spectrum of targeted therapies already available or in clinical trials. Among the NSCLC patients, 23% to 25% harbor a mutation in a gene associated with approved or emerging targeted therapy. These therapies have changed the therapeutic landscape of NSCLC with significantly improved clinical outcomes in advanced metastatic NSCLC patients. It is imperative to test for these gene alterations in order to identify patients who could potentially benefit from these efficacious targeted therapies and to avoid therapies unlikely to provide clinical benefit. A major limitation in obtaining molecular testing occurs when minimally invasive techniques are used to obtain tissue sample resulting in insufficient yield for testing. In such cases, the utilization of circulating tumor DNA (ctDNA), commonly known as liquid biopsy, has proven very beneficial. In a study utilizing ctDNA, increased detection rates were found when using ctDNA in addition to tissue testing and a &gt; 98.2% concordance rate was found. We report results of 40 NSCLC patients from our institute who had liquid biopsy with or without tissue profiling done. Methods: We molecularly profiled 40 newly diagnosed advanced NSCLC patients using both tissue and liquid biopsies. Tissue was assayed using the John Hopkins university molecular panel and liquid biopsies were performed by Biocept. Results: 14 out of 40 (35%) patients had insufficient or no tissue for molecular testing. Concordant results were found in 17 out of the 26 (65.4%) patients who had both tissue and liquid molecular testing done. Liquid Biopsy detected additional mutations in 5 (19.2%) patients which were not picked up on tissue and led to change in management in 4 patients. 12 out of 40 (30%) patients had repeat liquid biopsies done at progression of disease with new mutations detected on 4 patients revealing resistance to current treatment and change in treatment. Conclusions: Liquid Biopsy reveals high concordance rates with tissue genotyping and increases rate of detection of targetable mutations in NSCLC. It offers a safe and effective alternative when additional tissue is needed to identify genetic mutations.

MON-437 False Positive I-131 Uptake

BackgroundIodine has high specificity for thyroid tissue hence it plays very important role is management for differentiated thyroid cancer. Despite its high specificity and sensitivity, false-positive I-131 uptake could be seen on whole body scan (WBS).We are presenting a case of false positive intense uptake in lung due to bronchiectasis.Clinical casePatient is a 78-year-old Asian female who initially present with left sided 5.8 cm thyroid nodule. The fine needle aspiration was performed, and the cytology came back as Bethesda category III. Per our institute protocol a molecular mutation panel was sent, which came back with NRAS mutation.She underwent total thyroidectomy and the histology showed 6.2 cm Follicular carcinoma with extensive angioinvasion, oncocyte type. She received adjuvant 165.2 mci of RAI. Per protocol she had a one-week I-131whole body scan.There was intense abnormal uptake in left mid and upper chest. The stimulate thyroglobulin with a TSH of >100mcIU/ml was only 0.17 ng/dl with Tab negative.Patient subsequently had a PET CT which showed a faint diffuse FDG activity noted in the cystic bronchiectasis predominantly in the left apex, lingula, and right middle lobe. Patient informed us that she has history of pulmonary tuberculosis in 1970’s for which she was successfully treated in her home country. We had 10-year-old chest X-ray which showed stable cystic bronchiectasis lesion in the region of intense uptake.DiscussionAlthough I131 whole body scan has high specificity and sensitivity but physician should be aware of potential false positive uptake to avoid unnecessary intervention. In a retrospective evaluation i the most common non-thyroid conditions included were bronchiectasis, lung infection, subcutaneous injection into gluteal fatty tissue, aortic calcification, benign bone cyst, vertebral hemangioma, recent nonthyroidal surgical procedure site, rotator cuff injury, mature cystic teratoma and ovarian follicle cyst). The accumulation of the bronchial secretion is the proposed mechanism of the high false positive uptake in bronchiectasis.Endnotes i Mol Imaging Radionucl Ther 2018; 27:99–106 DOI:10.4274/mirt.37450

A highly multiplexed melt-curve assay for detecting the most prevalent carbapenemase, ESBL, and AmpC genes

Resistance to third-generation cephalosporins and carbapenems in Gram-negative bacteria is chiefly mediated by beta-lactamases including extended-spectrum beta-lactamase (ESBL), AmpC, and carbapenemase enzymes. Routine phenotypic detection methods do not provide timely results, and there is a lack of comprehensive molecular panels covering all important markers. An ESBL/carbapenemase high-resolution melt analysis (HRM) assay (SHV, TEM, CTX-M ESBL families, and NDM, IMP, KPC, VIM and OXA-48–like carbapenemases) and an AmpC HRM assay (16S rDNA control, FOX, MOX, ACC, EBC, CIT, and DHA) were designed and evaluated on 111 Gram-negative isolates with mixed resistance patterns. The sensitivity for carbapenemase, ESBL, and AmpC genes was 96.7% (95% confidence interval [CI]: 82.8–99.9%), 93.6% (95% CI: 85.7–97.9%), and 93.8% (95% CI: 82.8–98.7%), respectively, with a specificity of 100% (95% CI: 95.6–100%), 93.9% (95% CI: 79.8–99.3%), and 93.7% (95% CI: 84.5–98.2%). The HRM assays enable the simultaneous detection of the 14 most important ESBL, carbapenemase, and AmpC genes and could be used as a molecular surveillance tool or to hasten detection of antimicrobial resistance for treatment management.

Immune monitoring for immuno-oncology applications

Abstract The immunotherapy revolution has spurred the development of many new drugs and drug regimens for patient treatment. A key challenge is to identify the factors that drive patient toxicities and responses to treatment, with a particularly acute need for predictive biomarkers that can discriminate patients destined to respond and fail treatment. Technologies to interrogate immune cells are now readily available, but important gaps remain in their application, which limit the full realization of the promise of precision oncology. High parameter flow cytometry is a gold-standard but is limited by difficulty of panel design and lack of standardization. We present Color Wheel, a panel design tool, which builds optimized antibody panels based on the user’s instrument. These optimized panels have been manufactured in a dried, ready to use format to drive workflow and assay standardization. Initial results demonstrate excellent concordance between the dried and liquid versions of the high parameter multicolor panel(s). Molecular cytometry represents an exciting new approach to high dimensional immune analysis because it can measure at least 102 proteins and 400 mRNA targets simultaneously per cell. An important application gap for this technology is lack of data indicating the sequencing depth needed for adequate resolution. Here, we present results from a molecular cytometry experiment sequenced deeply, and then bioinformatically sub-sampled at different levels to identify the minimum level of sequencing needed for clear identification of cells, which can serve as a reference guide for users to save time and cost in their experiments. We also present preliminary data generated using dried version of molecular cytometry panel(s).

De-compositing honeycombs: Rheology-architecture relationships in melt-stretched sandwich panels

Single-material melt-stretched thermoplastic sandwich panels are a way to create environmentally-sustainable alternatives to conventional honeycomb sandwich structures. In order to efficiently develop them, it is necessary to understand the relationship between molecular structure, rheology, and panel architecture. In this study, seven polymer grades were tested: four high melt strength polypropylenes, two polycarbonates, and a linear polypropylene. The resulting honeycombs were characterized by direct measurement of web thicknesses and out-of-plane compression testing. Honeycombs manufactured from high melt strength polypropylene were better able to draw material from the melt pool into the honeycomb webs, and thus exhibited a larger fraction of the total material volume contributing to the webs than seen in honeycombs manufactured from the other polymers. Strain-hardening – or lack thereof – in the polymer melt was implicated as the primary cause of the observed differences in architecture and mechanical performance between the different grades.

Dissimilar tumor cell populations in ascitic fluid of ovarian cancer patients

Ovarian cancer is one of the most aggressive and hard-to-treat cancers. About 75% of ovarian cancer cases are detected at later stages of the disease. Ascitic fluid is promising biological material to get information about the tumor nature in ovarian cancer. Peritoneal dissemination is one of the most unfavorable factors of malignant tumor progression. However, prognostic factors associated with malignant ascites are not well understood. The aim of the study was to evaluate various tumor cell populations in ascitic fluid of ovarian cancer patients by laser multicolor flow cytometry using a molecular panel of EpCam, CD45, CD44, CD24, CD133, and N-cadherin markers. The prospective study included 16 patients aged 36 to 76 years with newly diagnosed FIGO stage Ic–IV ovarian cancer, who were admitted for treatment to the Cancer Research Institute of Tomsk National Research Medical Center. The study material included EDTAstabilized ascitic fluid sampled during laparoscopy. Various populations of ascitic tumor cells (with stemness features, with epithelial mesenchymal transition (EMT) features, without stemness and EMT features, with a combination of these features, as well as atypical / hybrid cell populations) were identified by multicolor flow cytometry on a BDFACSCanto apparatus (USA) using fluorochrome-labeled EpCam, CD45, CD44, CD24, CD133, and N-cadherin monoclonal antibodies and the BD FACSDiva software. The study revealed twelve populations of Epcam-positive cells in ascitic fluid of ovarian cancer patients. The cell composition of ascitic fluid in ovarian cancer patients is represented by a heterogeneous population. A large fraction of ascitic tumor cells are atypical / hybrid tumor cells with stemness features as well as Epcam+CD45–CD44+CD24+CD133+/-cancer stem cells, both with and without EMT features.

FOXA1 Gene Expression for Defining Molecular Subtypes of Muscle-Invasive Bladder Cancer after Radical Cystectomy.

It remains unclear how to implement the recently revealed basal and luminal subtypes of muscle-invasive bladder cancer (MIBC) into daily clinical routine and whether molecular marker panels can be reduced. The mRNA expression of basal (KRT5) and luminal (FOXA1, GATA3, KRT20) markers was measured by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and correlated to clinicopathological features, recurrence-free survival (RFS), disease-free survival (DFS), and overall survival (OS) in 80 patients with MIBC who underwent radical cystectomy. Additionally, the correlation of single markers with the basal and non-basal subtypes defined by a 36-gene panel was examined and then validated in the TCGA (The Cancer Genome Atlas) cohort. High expression of FOXA1 (p = 0.0048) and KRT20 (p = 0.0317) was associated with reduced RFS. In the multivariable analysis, only FOXA1 remained an independent prognostic marker for DFS (p = 0.0333) and RFS (p = 0.0310). FOXA1 expression (AUC = 0.79; p = 0.0007) was closest to the combined marker expression (AUC = 0.79; p = 0.0015) in resembling the non-basal subtype defined by the 36-gene panel. FOXA1 in combination with KRT5 may be used to distinguish the basal and non-basal subtypes of MIBC.

Rapid Detection of Airway Pathogens in Lung Transplant Recipients

Airway infections may necessitate prompt treatment in lung transplant recipients, given their immunosuppressed status, but maybe difficult to distinguish clinically from acute rejection, a potential antecedent of chronic lung allograft dysfunction and death. Traditional culture techniques can delay the time to diagnosis particularly for bacterial infections. We hypothesized that a rapid multiplex molecular assay could improve time to diagnosis and appropriate clinical decision making in lung transplant recipients. We performed a prospective observational clinical trial (NCT03933878) investigating the efficiency and accuracy of the BioFire Pneumonia Panel in the context of post-transplant bronchoscopy. This molecular assay tests the presence and/or quantity for 26 common airway bacterial and viral pathogens in under 2 hours. Prospectively enrolled lung transplant recipients had molecular and standard of care (SOC) diagnostics in parallel. Research clinicians performed shadow clinical decision making in real time using molecular diagnostic data in addition to clinical records. Time to report and interpretation were reported as median and interquartile ranges and compared by Wilcox rank-sum test. Agreement between methods, limited to pathogens on the molecular panel, was calculated by Gwet's AC1 statistic, which is robust despite skewed distributions. For the 148 enrolled subjects, molecular results were available at 3.8 [2.8-5.1] hours following bronchoscopy, which was 12.7 [6.3-30] hours prior to SOC viral results (P <0.001) and 1.8 [1.7-2.7] days prior to SOC bacterial culture results (P <0.001). Positive molecular results were interpreted 10.2 [7.9-23] hours following bronchoscopy, which was 2.4 [0.8-3.3] days less than SOC. Agreement between assays was 0.96 (95%CI 0.93-0.99) for bacteria and 0.92 (95% CI 0.86-0.97) for viruses. Of 24 positive molecular results, 5 resulted in a shadow antibiotic recommendation, whereas SOC results lead to antimicrobial initiation for 43. Notable molecular panel deficiencies included fungal pathogens and H. parainfluenzae, accounting for 17 and 13 of the 43 actionable SOC results, respectively. This molecular diagnostic could allow more rapid initiation of antibiotics in select cases and comparable viral detection versus SOC. However, as common lung transplant pathogens are not included in this panel, it cannot replace SOC microbial cultures.

Influenza-induced acute respiratory distress syndrome during the 2010-2016 seasons: bacterial co-infections and outcomes by virus type and subtype

ObjectivesWe aimed to describe bacterial co-infections and acute respiratory distress (ARDS) outcomes according to influenza type and subtype. MethodsA retrospective observational study was conducted from 2012 to 2016 in patients admitted to the respiratory intensive care unit (ICU) of Marseille university hospital for influenza-induced ARDS. Microbiological investigations, including multiplex molecular respiratory panel testing and conventional bacteriological cultures, were performed as part of the routine ICU care on the bronchoalveloar lavage collected at admission. Bacterial co-infections, ICU mortality and respiratory function were investigated according to virus type and subtype. ResultsAmong the 45 ARDS patients included, A(H1N1)pdm09 was the most frequent influenza virus identified (28/45 A(H1N1)pdm09, eight out of 45 A(H3N2) and nine out of 45 influenza B). Bacterial co-infections involving a total of 23 bacteria were diagnosed in 16/45 patients (36%). A(H1N1)pdm09 patients presented fewer bacterial co-infections (17.9% vs. 50.0% for A(H3N2) patients and 77.8% for B patients; p < 0.01). Overall, mortality at 90 days post admission was 33.3% (15/45), and there was no significant difference between influenza type and subtype. The need for extracorporeal membrane oxygenation was more frequent for A(H1N1)pdm2009 (20/28, 71.4%) and B patients (7/9, 77.8%) than the A(H3N2) subtype (1/8, 12.5%; p < 0.01). A(H1N1)pdm09-ARDS patients were associated with fewer ventilation-free days at day 28 (median (IQR): 0 (0–8) days) compared with other influenza–ARDS patients (15 (0–25) days, p < 0.05). DiscussionIn a population of influenza-induced ARDS, A(H1N1)pdm09 was associated with fewer bacterial co-infections but poorer respiratory outcomes. These data underline the major role of A(H1N1)pdm09 subtype on influenza disease severity.