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Platinum-based chemotherapy, including cisplatin, carboplatin, and oxaliplatin, is prescribed to 10-20% of all cancer patients. Unfortunately, platinum resistance develops in a significant number of patients and is a determinant of clinical outcome. Extensive research has been conducted to understand and overcome platinum resistance, and mechanisms of resistance can be categorized into several broad biological processes, including (1) regulation of drug entry, exit, accumulation, sequestration, and detoxification, (2) enhanced repair and tolerance of platinum-induced DNA damage, (3) alterations in cell survival pathways, (4) alterations in pleiotropic processes and pathways, and (5) changes in the tumor microenvironment. As a resource to the cancer research community, we provide a comprehensive overview accompanied by a manually curated database of the >900 genes/proteins that have been associated with platinum resistance over the last 30 years of literature. The database is annotated with possible pathways through which the curated genes are related to platinum resistance, types of evidence, and hyperlinks to literature sources. The searchable, downloadable database is available online at http://ptrc-ddr.cptac-data-view.org.

Background Conventional HER2-targeting therapies improve outcomes for patients with HER2-positive breast cancer (BC), defined as tumors showing HER2 protein overexpression by immunohistochemistry and/or ERBB2 gene amplification determined by in situ hybridization (ISH). Emerging HER2-targeting compounds show benefit in some patients with neither HER2 protein overexpression nor ERBB2 gene amplification, creating a need for new assays to select HER2-low tumors for treatment with these compounds. We evaluated the analytical performance of a targeted mass spectrometry-based assay for quantifying HER2 protein in formalin-fixed paraffin-embedded (FFPE) and frozen BC biopsies. Methods We used immunoaffinity-enrichment coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM-MS) to quantify HER2 protein (as peptide GLQSLPTHDPSPLQR) in 96 frozen and 119 FFPE BC biopsies. We characterized linearity, lower limit of quantification (LLOQ), and intra- and inter-day variation of the assay in frozen and FFPE tissue matrices. We determined concordance between HER2 immuno-MRM-MS and predicate immunohistochemistry and ISH assays and examined the benefit of multiplexing the assay to include proteins expressed in tumor subcompartments (e.g., stroma, adipose, lymphocytes, epithelium) to account for tissue heterogeneity. Results HER2 immuno-MRM-MS assay linearity was ≥103, assay coefficient of variation was 7.8% (FFPE) and 5.9% (frozen) for spiked-in analyte, and 7.7% (FFPE) and 7.9% (frozen) for endogenous measurements. Immuno-MRM-MS-based HER2 measurements strongly correlated with predicate assay HER2 determinations, and concordance was improved by normalizing to glyceraldehyde-3-phosphate dehydrogenase. HER2 was quantified above the LLOQ in all tumors. Conclusions Immuno-MRM-MS can be used to quantify HER2 in FFPE and frozen BC biopsies, even at low HER2 expression levels.

Summary Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor patient survival. Toward understanding the underlying molecular alterations that drive PDAC oncogenesis, we conducted comprehensive proteogenomic analysis of 140 pancreatic cancers, 67 normal adjacent tissues, and 9 normal pancreatic ductal tissues. Proteomic, phosphoproteomic, and glycoproteomic analyses were used to characterize proteins and their modifications. In addition, whole-genome sequencing, whole-exome sequencing, methylation, RNA sequencing (RNA-seq), and microRNA sequencing (miRNA-seq) were performed on the same tissues to facilitate an integrated proteogenomic analysis and determine the impact of genomic alterations on protein expression, signaling pathways, and post-translational modifications. To ensure robust downstream analyses, tumor neoplastic cellularity was assessed via multiple orthogonal strategies using molecular features and verified via pathological estimation of tumor cellularity based on histological review. This integrated proteogenomic characterization of PDAC will serve as a valuable resource for the community, paving the way for early detection and identification of novel therapeutic targets.

Despite advances in proteomic technologies, clinical translation of plasma biomarkers remains low, partly due to a major bottleneck between the discovery of candidate biomarkers and downstream costly clinical validation studies. Due to a dearth of multiplexable assays, generally only a few candidate biomarkers are tested, and the validation success rate is accordingly low. Here, we demonstrate the capability of internal standard triggered-parallel reaction monitoring (IS-PRM) to prioritize candidate biomarkers for validation studies. A 5,176-plex assay coupling immunodepletion and fractionation with IS-PRM was developed and implemented in human plasma to quantify peptides representing 1,314 breast cancer biomarker candidates. Compared to prior approaches using data-dependent analysis, IS-PRM showed improved sensitivity (912 vs 295 proteins quantified) and precision (CV 0.1 vs 0.27) enabling rank-ordering of candidate biomarkers for validation studies. The assay greatly expands capabilities for quantification of large numbers of proteins and is well suited for prioritization of viable candidate biomarkers.

Immunotherapies are revolutionizing cancer care, producing durable responses and potentially cures in a subset of patients. However, response rates are low for most tumors, grade 3/4 toxicities are not uncommon, and our current understanding of tumor immunobiology is incomplete. While hundreds of immunomodulatory proteins in the tumor microenvironment shape the anti-tumor response, few of them can be reliably quantified. To address this need, we developed a multiplex panel of targeted proteomic assays targeting 52 peptides representing 46 proteins using peptide immunoaffinity enrichment coupled to multiple reaction monitoring-mass spectrometry. We validated the assays in tissue and plasma matrices, where performance figures of merit showed over 3 orders of dynamic range and median inter-day CVs of 5.2% (tissue) and 21% (plasma). A feasibility study in clinical biospecimens showed detection of 48/52 peptides in frozen tissue and 38/52 peptides in plasma. The assays are publicly available as a resource for the research community.

Chimeric antigen receptor (CAR)-modified T cell therapy is effective in treating lymphomas, leukemias, and multiple myeloma in which the tumor cells have high amounts of target antigen. However, achieving durable remission in patients and extending this therapy to patients with solid tumors will require CAR T cells that can eliminate tumors with a low density of target antigen. Although CARs were designed to mimic T cell receptor (TCR) signaling, TCRs are at least 100-fold more sensitive to antigen. To design a CAR with improved antigen sensitivity, we compared TCR and CAR signaling in primary human T cells in response to ROR1, a transmembrane neurotrophic tyrosine kinase that is associated with B-cell chronic lymphocytic leukemia. Phosphoproteomic analysis revealed that key T cell signaling proteins—such as CD3δ, CD3ε, and CD3γ, which comprise a T cell coreceptor, and the TCR adaptor protein LAT—were either not phosphorylated or were only weakly phosphorylated by CAR stimulation. Modifying the CAR sequence to better engage CD3ε and LAT (specifically, BB/ζ CARs containing CD3ε or GRB2 domains) resulted in enhanced T cell activation against tumor cells with a low density of antigen, in both culture and in vivo models of lymphoma, leukemia, and breast cancer. These CARs represent examples of alterations in receptor design that were guided by in-depth interrogation of T cell signaling.

Simple Summary Functionality of the cellular DNA damage response (DDR) network affects risk for developing cancer, and the DDR is also a target of cancer therapies. Thus, it is important that we have reliable laboratory methods for determining the activity of this network. We describe the development and analytical validation of a targeted mass spectrometry-based 51-plex assay (DDR-2) for measuring proteins and post-translational modifications related to the DDR. The findings demonstrate identification of potential novel pharmacodynamic biomarkers. Abstract The ATM serine/threonine kinase (HGNC: ATM) is involved in initiation of repair of DNA double-stranded breaks, and ATM inhibitors are currently being tested as anti-cancer agents in clinical trials, where pharmacodynamic (PD) assays are crucial to help guide dose and scheduling and support mechanism of action studies. To identify and quantify PD biomarkers of ATM inhibition, we developed and analytically validated a 51-plex assay (DDR-2) quantifying protein expression and DNA damage-responsive phosphorylation. The median lower limit of quantification was 1.28 fmol, the linear range was over 3 orders of magnitude, the median inter-assay variability was 11% CV, and 86% of peptides were stable for storage prior to analysis. Use of the assay was demonstrated to quantify signaling following ionizing radiation-induced DNA damage in both immortalized lymphoblast cell lines and primary human peripheral blood mononuclear cells, identifying PD biomarkers for ATM inhibition to support preclinical and clinical studies.

Summary: A primary goal of the US National Cancer Institute's Ras initiative at the Frederick National Laboratory for Cancer Research is to develop methods to quantify RAS signaling to facilitate development of novel cancer therapeutics. We use targeted proteomics technologies to develop a community resource consisting of 256 validated multiple reaction monitoring (MRM)-based, multiplexed assays for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. As proof of concept, we quantify the response of melanoma (A375 and SK-MEL-2) and colorectal cancer (HCT-116 and HT-29) cell lines to BRAF inhibition by PLX4720. These assays replace over 60 western blots with quantitative mass-spectrometry-based assays of high molecular specificity and quantitative precision, showing the value of these methods for pharmacodynamic measurements and mechanism-of-action studies. Methods, fit-for-purpose validation, and results are publicly available as a resource for the community at assays.cancer.gov. Motivation: A lack of quantitative, multiplexable assays for phosphosignaling limits comprehensive investigation of aberrant signaling in cancer and evaluation of novel treatments. To alleviate this limitation, we sought to develop assays by using targeted mass spectrometry for quantifying protein expression and phosphorylation through the receptor tyrosine kinase, MAPK, and AKT signaling networks. The resulting assays provide a resource for replacing over 60 western blots in examining cancer signaling and tumor biology with high molecular specificity and quantitative rigor.

Amongst the pleotropic roles of transforming growth factor β (TGFβ) signaling in cancer, its impact on genomic stability is least understood. Inhibition of TGFβ signaling increases use of alternative end-joining (alt-EJ), an error-prone DNA repair process that typically functions as a ‘backup’ pathway if double strand break repair by homologous recombination or non-homologous end-joining is compromised. However, the consequences of this functional relationship on therapeutic vulnerability in human cancer remain unknown. Here, we show that TGFβ broadly controls the DNA damage response and that it suppresses alt-EJ genes that are associated with genomic instability. Mechanistically-based TGFβ and alt-EJ gene expression signatures were anti-correlated in glioblastoma, squamous cell lung cancer, and serous ovarian cancer. Consistent with error-prone repair, more of the genome was altered in tumors classified as low TGFβ and high alt-EJ, and the corresponding patients had better outcomes. Pan-cancer analysis of solid neoplasms revealed that alt-EJ genes are coordinately expressed and anti-correlated with TGFβ competency in 16 of 17 cancer types tested. Moreover, regardless of cancer type, tumors classified as low TGFβ and high alt-EJ were characterized by an insertion-deletion mutation signature containing short microhomologies and were more sensitive to genotoxic therapy. Collectively, experimental studies revealed that loss or inhibition of TGFβ signaling compromises the DNA damage response, resulting in ineffective repair by alt-EJ. Translation of this mechanistic relationship into gene expression signatures identified a robust anti-correlation that predicts response to genotoxic therapies, thereby expanding the potential therapeutic scope of TGFβ biology. ONE SENTENCE SUMMARY: The impact of TGFβ signaling on DNA repair competency is observed in pan-cancer analysis of survival after treatments that cause DNA damage.

Made available in DSpace on 2021-06-25T11:13:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Objectives: The objectives of this study were to characterize the craniofacial and airway morphology of oculo-auriculo-vertebral spectrum (OAVS) individuals using computed tomography (CT) examination. Setting and Sample Population: This sample included individuals in the age range from 5 to 14 years, consisted of a group of 18 OAVS individuals (12 females and 6 males), Pruzansky-Kaban1 IIB and III and by a paired control group matched by age and sex for comparison of morphometric and airway variables. Materials and Methods: Through the CT examination, airway analysis was performed using Dolphin Imaging® Software, and seven morphometric measurements were performed to evaluate craniofacial morphology by Materialize Mimics® Software. To compare airway and morphometric variables, the control group was used. Student's t test and Mann-Whitney U test were performed to compare differences between the groups. Results: Statistically significant differences were showed between the control and OAVS groups for the variables: total airway (TA) area, volume and MAA, RP area, RP volume, RP MAA, RG volume, RG MAA, total posterior height diff, Md incl and y-axis asymmetry. Pearson and Spearman's correlation showed mostly moderate correlations between Mand Occlusal canting AS with TA area and RP volume, Ax-Gn with TA area and Hy-C3 with TA volume. Conclusions: The OAVS's airway was altered and worse than the control group. Our results suggest that the contralateral side of OAVS individuals is unaffected; however, longitudinal assessments are needed to confirm it. Hyoid bone and postural measures play an important role in interpreting airway features of individuals with and without OAVS. Department of Pediatric Dentistry School of Dentistry São Paulo State University (UNESP) Department of Orthodontics University of São Paulo (USP) Hospital for Rehabilitation of Craniofacial Anomalies Department of Orthodontics Dentistry School of Dentistry University of Michigan Department of Orthodontics University of the Pacific Arthur A. Dugoni School of Dentistry Department of Clinical Genetics University of São Paulo (USP) Hospital for Rehabilitation of Craniofacial Anomalies Hospital for Rehabilitation of Craniofacial Anomalies University of São Paulo (USP) Department of Pediatric Dentistry School of Dentistry São Paulo State University (UNESP)

Lung squamous cell carcinoma (LSCC) remains a leading cause of cancer death with few therapeutic options. We characterized the proteogenomic landscape of LSCC, providing a deeper exposition of LSCC biology with potential therapeutic implications. We identify NSD3 as an alternative driver in FGFR1-amplified tumors and low-p63 tumors overexpressing the therapeutic target survivin. SOX2 is considered undruggable, but our analyses provide rationale for exploring chromatin modifiers such as LSD1 and EZH2 to target SOX2-overexpressing tumors. Our data support complex regulation of metabolic pathways by crosstalk between post-translational modifications including ubiquitylation. Numerous immune-related proteogenomic observations suggest directions for further investigation. Proteogenomic dissection of CDKN2A mutations argue for more nuanced assessment of RB1 protein expression and phosphorylation before declaring CDK4/6 inhibition unsuccessful. Finally, triangulation between LSCC, LUAD, and HNSCC identified both unique and common therapeutic vulnerabilities. These observations and proteogenomics data resources may guide research into the biology and treatment of LSCC.

Early detection of Primary Immunodeficiencies Disorders (PIDDs) is of paramount importance for effective treatment and disease management. Many PIDDs would be strong candidates for newborn screening (NBS) if robust screening methods could identify patients from dried blood spots (DBS) during the neonatal period. As majority of congenital PIDDs result in the reduction or absence of specific proteins, direct quantification of these target proteins represents an attractive potential screening tool. Unfortunately, detection is often limited by the extremely low protein concentrations in blood cells and limited blood volume present in DBS. We have recently developed a robust novel method for quantification of low abundance proteins in DBS for PIDDs using peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-SRM). Here, we further generated a multiplexed Immuno-SRM panel for simultaneous screening of eight signature peptides representing five PIDD-specific and two cell-type specific proteins from DBS. In samples from 28 PIDD patients including two carriers, representing X-Linked Agammaglobulinemia (XLA), Wiskott-Aldrich Syndrome (WAS), X-Linked Chronic Granulomatous Disease (XL-CGD), DOCK8 Deficiency and ADA deficiency, peptides representing each disease are significantly reduced relative to normal controls and patient identification had excellent agreement with clinical and molecular diagnosis. Also included in the multiplex panel are cell specific markers for platelets (CD42) and Natural Killer Cells (CD56). In patients with WAS, CD42 levels were found to be significantly reduced consistent with characteristic thrombocytopenia. A patient with WAS analyzed before and after bone marrow transplant showed normalized WAS protein and platelet CD42 after treatment highlighting the ability of immuno-SRM to monitor the effects of PIDD treatment. The assay was readily reproduced in two separate laboratories with similar analytical performance and complete agreement in patient diagnosis demonstrating the effective standardized methods. A high-throughput Immuno-SRM method screens PIDD-specific peptides in a 2.5-min runtime meeting high volume NBS workflow requirements was also demonstrated in this report. This high-throughput method returned identical results to the standard Immuno-SRM PIDD panel. Immuno-SRM peptide analysis represents a robust potential clinical diagnostic for identifying and studying PIDD patients from easily collected and shipped DBS and supports a significant potential for early PIDD diagnosis through newborn screening.

Background AZD0156 and AZD6738 are potent and selective inhibitors of ataxia-telangiectasia-kinase (ATM) and ataxia-telangiectasia-mutated and Rad3-related (ATR), respectively, important sensors/signallers of DNA damage. Methods We used multiplexed targeted-mass-spectrometry to select pRAD50(Ser635) as a pharmacodynamic biomarker for AZD0156-mediated ATM inhibition from a panel of 45 peptides, then developed and tested a clinically applicable immunohistochemistry assay for pRAD50(Ser635) detection in FFPE tissue. Results We found moderate pRAD50 baseline levels across cancer indications. pRAD50 was detectable in 100% gastric cancers (n = 23), 99% colorectal cancers (n = 102), 95% triple-negative-breast cancers (TNBC) (n = 40) and 87.5% glioblastoma-multiformes (n = 16). We demonstrated AZD0156 target inhibition in TNBC patient-derived xenograft models; where AZD0156 monotherapy or post olaparib treatment, resulted in a 34–72% reduction in pRAD50. Similar inhibition of pRAD50 (68%) was observed following ATM inhibitor treatment post irinotecan in a colorectal cancer xenograft model. ATR inhibition, using AZD6738, increased pRAD50 in the ATM-proficient models whilst in ATM-deficient models the opposite was observed, suggesting pRAD50 pharmacodynamics post ATR inhibition may be ATM-dependent and could be useful to determine ATM functionality in patients treated with ATR inhibitors. Conclusion Together these data support clinical utilisation of pRAD50 as a biomarker of AZD0156 and AZD6738 pharmacology to elucidate clinical pharmacokinetic/pharmacodynamic relationships, thereby informing recommended Phase 2 dose/schedule.

Abstract: Low output syndrome significantly increases morbidity and mortality of cardiac surgery and lengthens the durations of intensive care unit and hospital stays. Its treatment by catecholamines can lead to undesirable systemic and cardiac complications. Levosimendan is a calcium sensitiser and adenosine triphosphate (ATP)-sensitive potassium channel (IK,ATP) opener agent. Due to these effects, it improves myocardium performance, does not influence adversely the balance between O2 supply and demand, and possesses cardioprotective and organ protective properties as well. Based on the scientific literature and experts’ opinions, a European recommendation was published on the perioperative use of levosimendan in cardiac surgery in 2015. Along this line, and also taking into consideration cardiac surgeon, anaesthesiologist and cardiologist representatives of the seven Hungarian heart centres and the children heart centre, the Hungarian recommendation has been formulated that is based on two pillars: literature evidence and Hungarian expert opinions. The reviewed fields are: coronary and valvular surgery, assist device implantation, heart transplantation both in adult and pediatric cardiologic practice. Orv Hetil. 2018; 159(22): 870–877.

Following preclinical optimization of B7-H3-specific CAR T cells against pediatric brain tumor models, we opened BrainChild-03 (NCT04185038), a phase 1 clinical trial of repeatedly dosed, outpatient, locoregional B7-H3-specific CAR T cells for children with recurrent/refractory central nervous system (CNS) tumors or diffuse intrinsic pontine glioma (DIPG). Here, we report the interim findings from patients enrolled on Arm C, dedicated to DIPG. The primary endpoints are feasibility and safety, with secondary endpoints of disease response. We utilize second-generation CAR T cells with a 4-1BB costimulatory domain and a methotrexate-resistant human DHFR mutein (huDHFRFS; L22F,F31S), allowing for methotrexate selection. We do not deliver conditioning chemotherapy. The first three evaluable patients with DIPG all met feasibility for generating a balanced CD4:CD8 CAR T cell product, with 3.85x109 CAR T cells generated for S005, 4.29x109 for S008, and 2.45x109 for S012, allowing for greater than 6 months of biweekly dosing for each patient. All subjects were treated at Dose Level 1 (1x107 CAR T cells). S005 received 10 doses before clinical progression greater than 2 years from diagnosis, S008 has received 10 doses and continues on therapy with decreased tumor volume, and S012 has received 5 doses and continues on study with stable disease. There have been no dose limiting toxicities (DLT). 3/3 patients exhibited post infusion fever, headache, and elevated serum CRP but no evidence of cytokine release syndrome (CRS) or systemic CAR T cells. 0/3 patients required PICU admissions. In the cerebrospinal fluid (CSF), 2/3 patients have had elevations of cytokines such as CXCL10 and CCL2, as well as circulating CSF CAR T cells. Advanced serial patient CSF proteomic and transcriptomic profiling are underway. Ultimately, this report provides preliminary evidence that outpatient locoregional B7-H3 CAR T cells for children with DIPG may be feasible and tolerable.

Lung cancer is the deadliest cancer worldwide, mainly due to its advanced stage at the time of diagnosis. A non-invasive method for its early detection remains mandatory to improve patients&rsquo, survival. Plasma levels of 351 proteins were quantified by Liquid Chromatography-Parallel Reaction Monitoring (LC-PRM)-based mass spectrometry in 128 lung cancer patients and 93 healthy donors. Bootstrap sampling and least absolute shrinkage and selection operator (LASSO) penalization were used to find the best protein combination for outcome prediction. The PanelomiX platform was used to select the optimal biomarker thresholds. The panel was validated in 48 patients and 49 healthy volunteers. A 6-protein panel clearly distinguished lung cancer from healthy individuals. The panel displayed excellent performance: area under the receiver operating characteristic curve (AUC) = 0.999, positive predictive value (PPV) = 0.992, negative predictive value (NPV) = 0.989, specificity = 0.989 and sensitivity = 0.992. The panel detected lung cancer independently of the disease stage. The 6-protein panel and other sub-combinations displayed excellent results in the validation dataset. In conclusion, we identified a blood-based 6-protein panel as a diagnostic tool in lung cancer. Used as a routine test for high- and average-risk individuals, it may complement currently adopted techniques in lung cancer screening.

RAS genes are frequently mutated in cancer and have for decades eluded effective therapeutic attack. The National Cancer Institute’s RAS Initiative has a focus on understanding pathways and discovering therapies for RAS-driven cancers. Part of these efforts is the generation of novel reagents to enable the quantification of RAS network proteins. Here we present a dataset describing the development, validation (following consensus principles developed by the broader research community), and distribution of 104 monoclonal antibodies (mAbs) enabling detection of 27 phosphopeptides and 69 unmodified peptides from 20 proteins in the RAS network. The dataset characterizes the utility of the antibodies in a variety of applications, including Western blotting, immunoprecipitation, protein array, immunohistochemistry, and targeted mass spectrometry. All antibodies and characterization data are publicly available through the CPTAC Antibody Portal, Panorama Public Repository, and/or PRIDE databases. These reagents will aid researchers in discerning pathways and measuring expression changes in the RAS signaling network., Design Type(s)disease analysis objectiveMeasurement Type(s)immunocapture • immunoprecipitation • antibody • in situ immunoassay • ex situ immunoassayTechnology Type(s)western blot analysis • liquid chromatography-tandem mass spectrometry • immuno-MRM • immunohistochemistry • RPPA protein profiling assayFactor Type(s)Sample Characteristic(s)Homo sapiens • MCF-10A cell • BxPC-3 cell • NCI cell • breast • ovary • colon • lung • A549 cell • NCI-H1792 cell • HeLa cell • HEK293 Machine-accessible metadata file describing the reported data (ISA-Tab format)

Genomic characterization has allowed for the differentiation of different tumor types based upon the abundance of gene transcripts. However, owing to the many layers of regulation between transcript and the post-translationally modified protein, the functional moiety of the cell, it has been challenging to extrapolate biology from these transcriptional differences. We hypothesized that a comparative analysis of the proteome and phosphoproteome across 7 childhood brain tumors would yield a deeper understanding of the differences in their functional biology. We performed tandem mass tag labeling and triple mass spectrometry of 226 fresh frozen tumor samples collected in a single institution representing the histologic diagnoses of: high grade astrocytoma (27), low grade astrocytoma (97), ganglioglioma (20), ependymoma (32), medulloblastoma (22), atypical teratoid rhabdoid tumor (12), and craniopharyngioma (16). Among these samples were 22 pairs from pre/post recurrence. Across this sample set, we quantified 9155 proteins and 13632 phospho sites. After data preprocessing including normalization, batch correction and missing value imputation, we performed consensus clustering and showed that protein profiles can effectively distinguish major histology types. Additional regression based differential analyses revealed groups of proteins and pathways showing distinct activity patterns across different histologic types. Further leveraging the WGS data, we characterized the functional impact of mutation and fusion features of the glial tumors. Specifically, focusing on the largest cohort, low grade glioma, we analyzed the proteomic ramifications of BRAF status: V600E, BRAF-fusion and wild type. Moreover, from the primary and recurrent tumor pairs, we discovered the upregulation of proteins associated with immune evasion in more advanced LGG tumors. The incorporation of the proteomic dimension into large scale efforts at tumor characterization adds functional insight that can help drive translational efforts.

Platinum-based DNA cross-linking agents are widely used anti-cancer drugs. Tumor resistance to platinum compounds is a major determinant of patient survival, including in high grade serous ovarian cancer (HGSOC). Remarkably, despite >30 years of literature on platinum responses in human cancer, none of these findings is used clinically as a predictive biomarker to stratify patients for platinum resistance, nor exploited therapeutically to treat platinum-resistant disease. Thus, understanding mechanisms of platinum resistance is an urgent goal, both to identify predictive biomarkers of platinum response (to spare patients with platinum-resistant tumors futile platinum therapy) and to develop efficacious therapies for platinum-resistant disease. To better understand mechanisms underlying platinum resistance in HGSOC, we performed comprehensive, dynamic (+/-carboplatin), multiomic profiling of DNA, RNA, protein and post-translational modifications (phosphorylation, ubiquitination, acetylation) to identify the cellular networks that respond to platinum treatment and associate with platinum resistance in three HGSOC intra-patient cell line pairs (PEA1S/PEA2R, PEO1S/PEO4R, PEO14S/PEO23R). The cell line pairs were derived from ascites or pleural effusions (Langdon et al., 1988) from three patients both before (PEA1S, PEO1S, PEO14S) and after (PEA2R, PEO4R, PEO23R, respectively) their tumors became clinically platinum resistant (i.e., in vivo development of resistance). The molecular profiles revealed extensive responses to carboplatin and differential responses between platinum-sensitive and platinum-resistant cells. Higher oxidative phosphorylation and fatty acid oxidation (FAO) pathway expression were observed in the platinum-resistant cells, which was further validated in patient-derived xenograft (PDX) models. We show that pharmacologic inhibition or CRISPR knockout of CPT1A, which represents a rate limiting step of FAO, sensitize HGSOC cells to platinum. Thus, FAO, and CPT1A in particular, represent a candidate therapeutic target to overcome platinum resistance in HGSOC. Citation Format: Hong Wang, Dongqing Hugang, Shrabanti Chowdhury, Sara Savage, Richard Ivey, Jacob Kennedy, Jeffrey Whiteaker, Chenwei Lin, Xiaonan Hou, Catherine Huntoon, Uliana Voytovich, Zahra Shire, Qing Yu, Steven Gygi, Andrew Hoofnagle, Zachary Herbert, Anna Calinawan, Larry Karnitz, S. John Weroha, Scott Kaufmann, Bing Zhang, Pei Wang, Michael Birrer, Amanda Paulovich. Multiomic analysis identifies CPT1A and fatty acid oxidation as a potential therapeutic target in platinum-refractory high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1426.

We present a proteogenomic study of 108 human papilloma virus (HPV)-negative head and neck squamous cell carcinomas (HNSCCs). Proteomic analysis systematically catalogs HNSCC-associated proteins and phosphosites, prioritizes copy number drivers, and highlights an oncogenic role for RNA processing genes. Proteomic investigation of mutual exclusivity between FAT1 truncating mutations and 11q13.3 amplifications reveals dysregulated actin dynamics as a common functional consequence. Phosphoproteomics characterizes two modes of EGFR activation, suggesting a new strategy to stratify HNSCCs based on EGFR ligand abundance for effective treatment with inhibitory EGFR monoclonal antibodies. Widespread deletion of immune modulatory genes accounts for low immune infiltration in immune-cold tumors, whereas concordant upregulation of multiple immune checkpoint proteins may underlie resistance to anti-programmed cell death protein 1 monotherapy in immune-hot tumors. Multi-omic analysis identifies three molecular subtypes with high potential for treatment with CDK inhibitors, anti-EGFR antibody therapy, and immunotherapy, respectively. Altogether, proteogenomics provides a systematic framework to inform HNSCC biology and treatment.

Wilson's Disease (WD), a copper transport disorder caused by a genetic defect in the ATP7B gene, has been a long time strong candidate for newborn screening (NBS), since early interventions can give better results by preventing irreversible neurological disability or liver cirrhosis. Several previous pilot studies measuring ceruloplasmin (CP) in infants or children showed that this marker alone was insufficient to meet the universal screening for WD. WD results from mutations that cause absent or markedly diminished levels of ATP7B. Therefore, ATP7B could serve as a marker for the screening of WD, if the protein can be detected from dried blood spots (DBS). This study demonstrates that the immuno-SRM platform can quantify ATP7B in DBS in the picomolar range, and that the assay readily distinguishes affected cases from normal controls (p < 0.0001). The assay precision was

Immunoaffinity enrichment of peptides coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM) enables highly specific, sensitive, and precise quantification of peptides and post-translational modifications. Major obstacles to developing a large number of immuno-MRM assays are the poor availability of monoclonal antibodies (mAbs) validated for immunoaffinity enrichment of peptides and the cost and lead time of developing the antibodies de novo. Although many thousands of mAbs are commercially offered, few have been tested for application to immunoaffinity enrichment of peptides. In this study we tested the success rate of using commercially available mAbs for peptide immuno-MRM assays. We selected 105 commercial mAbs (76 targeting non-modified “pan” epitopes, 29 targeting phosphorylation) to proteins associated with the DNA damage response network. We found that 8 of the 76 pan (11%) and 5 of the 29 phospho-specific mAbs (17%) captured tryptic peptides (detected by LC-MS/MS) of their protein targets from human cell lysates. Seven of these mAbs were successfully used to configure and analytically characterize immuno-MRM assays. By applying selection criteria upfront, the results indicate that a screening success rate of up to 24% is possible, establishing the feasibility of screening a large number of catalog antibodies to provide readily-available assay reagents.

Somatic mutations have been extensively characterized in breast cancer, but the effects of these genetic alterations on the proteomic landscape remain poorly understood. Here we describe quantitative mass-spectrometry-based proteomic and phosphoproteomic analyses of 105 genomically annotated breast cancers, of which 77 provided high-quality data. Integrated analyses provided insights into the somatic cancer genome including the consequences of chromosomal loss, such as the 5q deletion characteristic of basal-like breast cancer. Interrogation of the 5q trans-effects against the Library of Integrated Network-based Cellular Signatures, connected loss of CETN3 and SKP1 to elevated expression of epidermal growth factor receptor (EGFR), and SKP1 loss also to increased SRC tyrosine kinase. Global proteomic data confirmed a stromal-enriched group of proteins in addition to basal and luminal clusters, and pathway analysis of the phosphoproteome identified a G-protein-coupled receptor cluster that was not readily identified at the mRNA level. In addition to ERBB2, other amplicon-associated highly phosphorylated kinases were identified, including CDK12, PAK1, PTK2, RIPK2 and TLK2. We demonstrate that proteogenomic analysis of breast cancer elucidates the functional consequences of somatic mutations, narrows candidate nominations for driver genes within large deletions and amplified regions, and identifies therapeutic targets.

We report a comprehensive proteogenomics analysis, including whole-genome sequencing, RNA sequencing, and proteomics and phosphoproteomics profiling, of 218 tumors across 7 histological types of childhood brain cancer: low-grade glioma (n = 93), ependymoma (32), high-grade glioma (25), medulloblastoma (22), ganglioglioma (18), craniopharyngioma (16), and atypical teratoid rhabdoid tumor (12). Proteomics data identify common biological themes that span histological boundaries, suggesting that treatments used for one histological type may be applied effectively to other tumors sharing similar proteomics features. Immune landscape characterization reveals diverse tumor microenvironments across and within diagnoses. Proteomics data further reveal functional effects of somatic mutations and copy number variations (CNVs) not evident in transcriptomics data. Kinase-substrate association and co-expression network analysis identify important biological mechanisms of tumorigenesis. This is the first large-scale proteogenomics analysis across traditional histological boundaries to uncover foundational pediatric brain tumor biology and inform rational treatment selection.

We performed a comprehensive proteogenomic analysis across seven childhood brain tumors for a deeper understanding of their functional biology. Whole genome sequencing, RNAseq, quantitative proteomic profiling and phosphoproteomics were performed on 219 fresh frozen tumor samples representing the histologic diagnoses of: low grade astrocytoma (93), ependymoma (32), high grade astrocytoma (26), medulloblastoma (22), ganglioglioma (18), craniopharyngioma (16) and atypical teratoid rhabdoid tumor (12). Unsupervised clustering analysis based on proteomics data reveals eight clusters with distinct protein profiles and pathway activities. While some clusters coincide with histologic diagnoses, a couple of clusters appear to be a mixture of different diagnoses, including one cluster consisting of “aggressive” tumors characterized by poor survival and high stemness scores. By integrating proteomic data with RNAseq and WGS data, we characterize the impact of mutations (H3K27M, BRAFV600E, BRAF fusion) and CNVs upon the proteome across various diagnoses. Multiomics based kinase-substrate association analysis and co-expression network analysis reveal targetable active kinase networks within these tumors. Proteomic data reveals unique biology associated with H3K27M mutation status in HGG and BRAF aberrations in LGG. Characterization of the tumor microenvironment through deconvolution analyses based on multi-omics data reveals 5 distinct tumor clusters associated with different populations of infiltrating immune cells and the relative activity of the immune system based upon the expression of pro-inflammation or immunosuppressive markers. This study reports the first large-scale deep comprehensive proteogenomic analysis crossing traditional histologic boundaries to uncover foundational pediatric brain tumor biology including functional insight that helps drive translational efforts.

SUMMARY The integration of mass spectrometry-based proteomics with next-generation DNA and RNA sequencing profiles tumors more comprehensively. Here this “proteogenomics” approach was applied to 122 treatment-naive primary breast cancers accrued to preserve post-translational modifications, including protein phosphorylation and acetylation. Proteogenomics challenged standard breast cancer diagnoses, provided detailed analysis of the ERBB2 amplicon, defined tumor subsets that could benefit from immune checkpoint therapy, and allowed more accurate assessment of Rb status for prediction of CDK4/6 inhibitor responsiveness. Phosphoproteomics profiles uncovered novel associations between tumor suppressor loss and targetable kinases.Acetylproteome analysis highlighted acetylation on key nuclear proteins involved in the DNA damage response and revealed cross-talk between cytoplasmic and mitochondrial acetylation and metabolism. Our results underscore the potential of proteogenomics for clinical investigation of breast cancer through more accurate annotation of targetable pathways and biological features of this remarkably heterogeneous malignancy., In Brief Breast cancer is a highly heterogeneous disease with variable outcomes and subtype-driven treatment approaches, making precision medicine a considerable challenge. Proteogenomic analyses of 122 primary breast cancers provide insights into clinically relevant biology, including cell cycle dysregulation, tumor immunogenicity, aberrant metabolism, and heterogeneity in therapeutic target expression., Graphical Abstract

Although high-grade serous ovarian cancers (HGSOC) are highly chemosensitive with an 85% initial response rate to platinum-based chemotherapy, 15% of patients are “exceptional nonresponders,” with platinum-refractory tumors that remain stable or progress during treatment. Unfortunately, we have no predictive biomarkers to identify refractory patients up front, and they receive futile chemotherapy through which most patients become too ill to be eligible for clinical trials. Hence, no progress has been made in treating these deadly tumors. The goal of this study is to identify mechanisms of platinum refractoriness to: i) predict refractory HGSOCs up front and ii) identify potential new drug targets in refractory disease to point to desperately needed new therapeutic approaches. Of note, 80-90% of patients who are initially platinum responsive will relapse and develop platinum-resistant disease, and it is possible that findings in platinum-refractory tumors might also provide insights into platinum-resistant tumors. Our NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC)-funded approach combines genomic and proteomic (“proteogenomic”) analyses of both preclinical models (0, 8, and 24 hours post-platinum exposure) and treatment-naïve human tumors. For preclinical models, we studied a well-characterized collection of patient-derived xenograft (PDX) models (10 sensitive, 10 refractory), as well as intrapatient HGSOC cell line pairs derived from patients before and after the development of platinum resistance. For the PDX models, proteogenomic profiling included RNASeq, WES, global proteomics, and phosphoproteomics at all 3 time points (0, 8, 24 hours). For cell line models (3 sensitive, 3 resistant), proteogenomic profiling was performed at all 3 timepoints (0, 8, 24 hours), and experiments were performed in complete biologic triplicate. Analyses included RNASeq, WES, global proteomics, phosphoproteomics, ubiquitin proteome, acetylated proteome, and pTyr. A large collection of 275 human HGSOCs (an approximate equal balance of platinum sensitive and refractory tumors) is currently undergoing proteomic profiling, and genomic profiles (WGS, RNASeq) will be performed on a subset. In parallel, we have performed a comprehensive review of 31 years of published work on platinum responses of human cancers, identifying ~700 genes implicated in the response and scoring each gene with respect to strength of the published evidence. Using a Bayesian approach, we are integrating the curated candidates from the literature with our empirical proteogenomic datasets to identify a candidate signature for detecting platinum-refractory disease prior to chemotherapy. We are also performing gene-regulatory network analysis to identify potential drivers of chemo response. NextGen, targeted, multiplex, multiple reaction monitoring mass spectrometry-based assays are being developed to quantify proteins in the signature for validation studies, using independent patient cohorts. This abstract is also being presented as Poster A62. Citation Format: Jacob J. Kennedy, Shrabanti Chowdhury, Sara R. Savage, Xiaonan Hou, Catherine J. Huntoon, Richard G. Ivey, Qing Yu, Chenwei Lin, Dongqing Huang, Lei Zhao, Uliana J. Voytovich, Regine M. Schoenherr, Zahra Shire, Steven J. Skates, Jeffrey R. Whiteaker, Andrew N. Hoofnagle, Samuel C. Mok, Bing Zhang, Larry M. Karnitz, S. John Weroha, Steven P. Gygi, Scott H. Kaufmann, Pei Wang, Michael J. Birrer, Amanda G. Paulovich. Proteogenomic approach to identify mechanisms of platinum refractoriness in high-grade serous ovarian cancers [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr PR02.

To explore the biology of lung adenocarcinoma (LUAD) and identify new therapeutic opportunities, we performed comprehensive proteogenomic characterization of 110 tumors and 101 matched normal adjacent tissues (NATs) incorporating genomics, epigenomics, deep-scale proteomics, phosphoproteomics, and acetylproteomics. Multi-omics clustering revealed four subgroups defined by key driver mutations, country, and gender. Proteomic and phosphoproteomic data illuminated biology downstream of copy number aberrations, somatic mutations, and fusions and identified therapeutic vulnerabilities associated with driver events involving KRAS, EGFR, and ALK. Immune subtyping revealed a complex landscape, reinforced the association of STK11 with immune-cold behavior, and underscored a potential immunosuppressive role of neutrophil degranulation. Smoking-associated LUADs showed correlation with other environmental exposure signatures and a field effect in NATs. Matched NATs allowed identification of differentially expressed proteins with potential diagnostic and therapeutic utility. This proteogenomics dataset represents a unique public resource for researchers and clinicians seeking to better understand and treat lung adenocarcinomas.

Quantifying the many immunomodulatory proteins in the “cancer-immunity cycle” is critical for both patient selection and development of novel immunotherapies. Conventional technologies to measure proteins (IHC, ELISA, Western blot, flow cytometry) are often semiquantitative, not multiplexable, feature poor specificity, and are difficult to develop. Due to these limitations, investigators have turned to mRNA-based measurements to infer protein expression of immunomodulatory factors. However, as has been unequivocally demonstrated for human breast, colorectal, and ovarian cancers (Pubmed IDs: 27251275, 25043054, 27372738), RNA levels can be poor predictors of protein levels or activity. Thus, direct quantification of proteins is desirable. As part of the National Cancer Institute’s Cancer Moonshot, we are developing >300 multiplex assays for quantifying immunomodulatory proteins in tissue and plasma using a next-gen technology for protein quantification, based on targeted multiple reaction monitoring mass spectrometry (MRM-MS). MRM-MS complements traditional immunoassays by enabling highly specific, precise, harmonizable quantification of proteins in biospecimens, even at high multiplex levels. As proof of concept, we will present a novel multiplex assay based on next-gen MRM-MS for quantification of 47 immunomodulatory proteins in plasma and/or tumor tissue. The technology uses immunoaffinity enrichment coupled with MRM-MS (immuno-MRM assay). Assay bioanalytical validation was conducted in accordance with industry standards for use in FFPE tissue and plasma matrices and included determination of limits of detection, linear range of response, intraday and interday repeatability, stability of analytes in sample processing, and characterization of reproducibility of endogenous analyte measurement. The mean linear range was over three orders of magnitude with median intra- and interday CVs less than 10%. The characterized assays are being applied to a panel of tumor specimens from a variety of cancer types to document detection and determine minimum sample requirements. Additional assay panels are under development to provide measurements of up to 350 immunomodulatory proteins. Citation Format: Jeffrey R. Whiteaker, Lei Zhao, Richard G. Ivey, Julia Voytovich, Regine M. Schoenherr, Dongqing Huang, Jacob J. Kennedy, Amanda G. Paulovich. Multiplex quantification of immunomodulatory proteins in tissue and plasma using next-gen targeted MRM mass spectrometry [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B8.

We undertook a comprehensive proteogenomic characterization of 95 prospectively collected endometrial carcinomas, comprising 83 endometrioid and 12 serous tumors. This analysis revealed possible new consequences of perturbations to the p53 and Wnt/β-catenin pathways, identified a potential role for circRNAs in the epithelial-mesenchymal transition, and provided new information about proteomic markers of clinical and genomic tumor subgroups, including relationships to known druggable pathways. An extensive genome-wide acetylation survey yielded insights into regulatory mechanisms linking Wnt signaling and histone acetylation. We also characterized aspects of the tumor immune landscape, including immunogenic alterations, neoantigens, common cancer/testis antigens, and the immune microenvironment, all of which can inform immunotherapy decisions. Collectively, our multi-omic analyses provide a valuable resource for researchers and clinicians, identify new molecular associations of potential mechanistic significance in the development of endometrial cancers, and suggest novel approaches for identifying potential therapeutic targets.

Background: Primary immunodeficiency disorders (PIDD) comprise a group of life-threatening congenital diseases characterized by absent or impaired immune responses. Despite the fact that effective, curative treatments are available with optimal clinical outcomes when diagnosed early, newborn screening does not exist for the majority of these diseases due to the lack of detectable, specific biomarkers or validated methods for population-based screening. Peptide immunoaffinity enrichment coupled with selected reaction monitoring mass spectrometry (immuno-SRM) is a sensitive proteomic assay, involving antibody-mediated peptide capture, that allows for concurrent quantification of multiple analytes. This assay has promise for use in potential newborn screening of PIDDs that lead to diminished or absent target proteins in the majority of cases. Objective: To determine and evaluate if a multiplex assay based on immuno-SRM is able to reliably and precisely distinguish affected patients with X-linked agammaglobulinemia (XLA), Wiskott-Aldrich Syndrome (WAS), and CD3ϵ-associated severe combined immunodeficiency (SCID) from one another and from unaffected normal control dried blood spot (DBS) samples. Methods: We performed a blinded, multiplexed analysis of proteolytically-generated peptides from WASp, BTK, and CD3ϵ (for WAS, XLA, and SCID, respectively) in DBS samples from 42 PIDD patients, 40 normal adult controls, and 62 normal newborns. The peptide ATPase copper transporting protein (ATP7B) 1056 was simultaneously monitored for quality assurance purposes. Results: The immuno-SRM assays reliably quantified the target peptides in DBS and accurately distinguished affected patients from normal controls. Analysis of signature peptides found statistically significant reduction or absence of peptide levels in affected patients compared to control groups in each case (WASp and BTK: p = 0.0001, SCID: p = 0.05). Intra and inter-assay precision ranged from 11 to 22% and 11 to 43% respectively; linearity (1.39–2000 fmol peptide), and stability (≤ 0.09% difference in 72 h) showed high precision for the multiplexed assay. Inter-laboratory assay comparison showed high concordance for measured peptide concentrations, with R2 linearity ≥ 0.97 for the WASp 274, CD3ϵ 197, BTK 407, and ATP7B 1056 peptides. Conclusion: Immuno-SRM-based quantification of proteotypic peptides from WASp, BTK, and CD3ϵ in DBS distinguishes relevant PIDD cases from one another and from controls, raising the possibility of employing this approach for large-scale multiplexed newborn screening of selective PIDDs.

Cancer genomics research aims to advance personalized oncology by finding and targeting specific genetic alterations associated with cancers. In genome-driven oncology, treatments are selected for individual patients on the basis of the findings of tumour genome sequencing. This personalized approach has prolonged the survival of subsets of patients with cancer. However, many patients do not respond to the predicted therapies based on the genomic profiles of their tumours. Furthermore, studies pairing genomic and proteomic analyses of samples from the same tumours have shown that the proteome contains novel information that cannot be discerned through genomic analysis alone. This observation has led to the concept of proteogenomics, in which both types of data are leveraged for a more complete view of tumour biology that might enable patients to be more successfully matched to effective treatments than they would using genomics alone. In this Perspective, we discuss the added value of proteogenomics over the current genome-driven approach to the clinical characterization of cancers and summarize current efforts to incorporate targeted proteomic measurements based on selected/multiple reaction monitoring (SRM/MRM) mass spectrometry into the clinical laboratory to facilitate clinical proteogenomics.

Chimeric antigen receptors (CARs) link an antigen-recognition domain to CD28/CD3ζ or 4–1BB/CD3ζ intracellular signaling domains to redirect T cell specificity and function. T cells expressing CARs with CD28/CD3ζ or 4–1BB/CD3ζ signaling domains are effective at treating refractory B cell malignancies but exhibit differences in effector function, clinical efficacy, and toxicity that are assumed to result from the activation of divergent signaling cascades. We analyzed stimulation-induced phosphorylation events in primary human CD8(+) CD28/CD3ζ and 4–1BB/CD3ζ CAR T cells by mass spectrometry and found that both CAR constructs activated similar signaling intermediates. Stimulation of CD28/CD3ζ CARs activated faster and larger-magnitude changes in protein phosphorylation, which correlated with an effector T cell–like phenotype and function. In contrast, 4–1BB/CD3ζ CAR T cells preferentially expressed T cell memory–associated genes and exhibited sustained anti-tumor activity against established tumors in vivo. Mutagenesis of the CAR CD28 signaling domain demonstrated that the increased CD28/CD3ζ CAR signal intensity was partly related to constitutive association of Lck with this domain in CAR complexes. Our data show that CAR signaling pathways cannot be predicted solely by the domains used to construct the receptor, and that signal strength is a key determinant of T cell fate. Tailoring CAR design based on signal strength may lead to improved clinical efficacy and reduced toxicity.

The Fanconi anemia pathway is an important coordinator of DNA repair pathways and is particularly relevant to repair of DNA inter-strand crosslinks. Central to the pathway is monoubiquitination of FANCD2, requiring the function of multiple proteins in an upstream Fanconi core complex. We present development and analytical characterization of a novel assay for quantification of unmodified and monoubiquitinated FANCD2 proteoforms, based on peptide immunoaffinity enrichment and targeted multiple reaction monitoring mass spectrometry (immuno-MRM). The immuno-MRM assay is analytically characterized using fit-for-purpose method validation. The assay linear range is >3 orders of magnitude with total repeatability

Introduction: Since the celebration of the 20th anniversary of the first heart transplantation in Hungary in 2012 the emerging need for modern heart failure management via mechanical circulatory support has evolved. In May 2012 the opening of a new heart failure and transplant unit with 9 beds together with the procurement of necessary devices at Semmelweis University accomplished this need. Aim: The aim of the authors was to report their initial experience obtained in this new cardiac assist device program. Method: Since May, 2012, mechanical circulatory support system was applied in 89 cases in 72 patients. Indication for support were end stage heart failure refractory to medical treatment and acute left or right heart failure. Results: Treatment was initiated for acute graft failure after heart transplantation in 27 cases, for end stage heart failure in 24 cases, for acute myocardial infarction in 21 cases, for acute postcardiotomy heart failure in 14 cases, for severe respiratory insufficiency in 2 cases and for drug intoxication in one case. Among the 30 survivor of the whole program 13 patients were successfully transplanted. Conclusions: The available devices can cover all modalities of current bridge therapy from short term support through medium support to heart transplantation or long term support and destination therapy. These conditions made possible the successful start of a new cardiac assist device program. Orv. Hetil., 2015, 156(13), 521–527.

Made available in DSpace on 2018-11-26T22:40:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-10-01. Added 1 bitstream(s) on 2019-10-09T18:27:55Z : No. of bitstreams: 1 S0103-50532018001002054.pdf: 1875083 bytes, checksum: d412309b2dfc7b515b1a8e8b893b7ce1 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) LMF/LNNano, Brazil Immunosensors based on impedance spectroscopy for diagnosing hepatitis C are reported where the sensing units were made with the antigenic peptide PPLLESWKDPDYVPPWHG (NS5A-1) derived from the NS5A protein of the hepatitis C virus (HCV) immobilized in layer-by-layer (LbL) films with silk fibroin (SF) and deposited on gold interdigitated electrodes. The electrical response of the sensing units varied upon immersion into solutions containing the antibody anti-HCV owing to the biomolecular recognition of NS5A-1. This was associated with morphological changes on the LbL films caused by adsorption of NS5A-1 and inferred from atomic force microscopy images. Buffer solutions with different anti-HCV concentrations down to 2 ng mL(-1) could be clearly distinguished by analyzing the impedance spectroscopy data with a multidimensional projection technique. The specificity toward anti-HCV antibodies was confirmed in control experiments where no significant changes in the electrical response were measured by exposing the sensing units to solutions containing an anti-human immunodeficiency virus (HIV) antibody. The high sensitivity and selectivity of the units made with LbL films demonstrate the feasibility of measuring electrical impedance as an immunosensing strategy to detect hepatitis C. Univ Estadual Paulista, Inst Quim, BR-14800060 Araraquara, SP, Brazil Univ Fed Mato Grosso do Sul, Inst Fis, CP-549, BR-79070900 Campo Grande, MS, Brazil Univ Sao Paulo, Inst Ciencias Matemat & Comp, BR-13566590 Sao Carlos, SP, Brazil Univ Sao Paulo, Inst Fis Sao Carlos, BR-13566590 Sao Carlos, SP, Brazil Univ Fed Sao Paulo, Inst Ciencia & Tecnol, BR-12231280 Sao Jose Dos Campos, SP, Brazil Univ Estadual Paulista, Inst Quim, BR-14800060 Araraquara, SP, Brazil CAPES: 26 LMF/LNNano, Brazil: LMF 12298

Az epicardialis pacemakerek ritka, de akar eletet veszelyeztető szovődmenye a generatortelepnek a subcostalisan kialakitott zsebből a hasuregbe tortenő elvandorlasa. A szerzők 69 eves nőbeteguk esetet ismertetik, aki aspecifikus hasi panaszokkal jelentkezett a klinika surgőssegi osztalyan. Anamneziseből kiemelendő, hogy korabbi mitralisműbillentyű-műtetet kovetően kialakult III. foku atrioventricularis blokkja miatt epigastrialis pacemakert helyeztek be ugy, hogy a pacemakergeneratort a subcostalisan kialakitott intramuscularis zsebben helyeztek el. Mellkasatvilagitas igazolta, hogy a telep – eredeti helyetől messzire – a Douglas-uregbe vandorolt. A szokatlan helyre es tavolsagra vandorolt keszuleket hasi feltarasbol tavolitottak el, kozvetlenul azt kovetően, hogy uj endocardialis pacemakerrendszer kerult beultetesre. A beteg szovődmenymentesen, a 7. posztoperativ napot kovetően otthonaba tavozott. Orv. Hetil., 2015, 156(4), 154–157.

SUMMARY To elucidate the deregulated functional modules that drive clear cell renal cell carcinoma (ccRCC), we performed comprehensive genomic, epigenomic, transcriptomic, proteomic, and phosphoproteomic characterization of treatment-naive ccRCC and paired normal adjacent tissue samples. Genomic analyses identified a distinct molecular subgroup associated with genomic instability. Integration of proteogenomic measurements uniquely identified protein dysregulation of cellular mechanisms impacted by genomic alterations, including oxidative phosphorylation-related metabolism, protein translation processes, and phospho-signaling modules. To assess the degree of immune infiltration in individual tumors, we identified microenvironment cell signatures that delineated four immune-based ccRCC subtypes characterized by distinct cellular pathways. This study reports a large-scale proteogenomic analysis of ccRCC to discern the functional impact of genomic alterations and provides evidence for rational treatment selection stemming from ccRCC pathobiology., Graphical Abstract, In Brief Comprehensive proteogenomic characterization in 103 treatment-naive clear cell renal cell carcinoma patient samples highlights tumor-specific alterations at the proteomic level that are unrevealed by transcriptomic profiling and proposes a revised subtyping scheme based on integrated omics analysis.

Successful treatment of type A acute aortic dissection depends on the promptness of diagnostic evaluation and therapy. Fast diagnosis can be challenged by numerous complications such as myocardial ischemia, acute aortic insufficiency, and disturbances in organ perfusion and pericardial tamponade. The authors report the case history of a 72-year-old woman, who was admitted after resuscitation with ST segment elevation. Echocardiography revealed acute type A aortic dissection with signs of pericardial tamponade. An emergency operation consisting of the resection of the ascending aorta and the reconstruction of the aortic root was performed, which took six hours from admission until the end of the operation. Follow-up examinations demonstrated good left ventricular function and competent aortic valve. The authors propose that with the development of diagnostic and therapeutic options, faster and less invasive interventions will be introduced in near future for the treatment of acute aortic dissection, which may reduce the morbidity and mortality rates of this lethal illness. Orv. Hetil., 2014, 155(44), 1763–1767.

Adoption of targeted mass spectrometry (MS) approaches such as multiple reaction monitoring (MRM) to study biological and biomedical questions is well underway in the proteomics community. Successful application depends on the ability to generate reliable assays that uniquely and confidently identify target peptides in a sample. Unfortunately, there is a wide range of criteria being applied to say that an assay has been successfully developed. There is no consensus on what criteria are acceptable and little understanding of the impact of variable criteria on the quality of the results generated. Publications describing targeted MS assays for peptides frequently do not contain sufficient information for readers to establish confidence that the tests work as intended or to be able to apply the tests described in their own labs. Guidance must be developed so that targeted MS assays with established performance can be made widely distributed and applied by many labs worldwide. To begin to address the problems and their solutions, a workshop was held at the National Institutes of Health with representatives from the multiple communities developing and employing targeted MS assays. Participants discussed the analytical goals of their experiments and the experimental evidence needed to establish that the assays they develop work as intended and are achieving the required levels of performance. Using this “fit-for-purpose” approach, the group defined three tiers of assays distinguished by their performance and extent of analytical characterization. Computational and statistical tools useful for the analysis of targeted MS results were described. Participants also detailed the information that authors need to provide in their manuscripts to enable reviewers and readers to clearly understand what procedures were performed and to evaluate the reliability of the peptide or protein quantification measurements reported. This paper presents a summary of the meeting and recommendations.

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays.

We performed the first proteogenomic study on a prospectively collected colon cancer cohort. Comparative proteomic and phosphoproteomic analysis of paired tumor and normal adjacent tissues produced a catalog of colon cancer-associated proteins and phosphosites, including known and putative new biomarkers, drug targets, and cancer/testis antigens. Proteogenomic integration not only prioritized genomically inferred targets, such as copy-number drivers and mutation-derived neoantigens, but also yielded novel findings. Phosphoproteomics data associated Rb phosphorylation with increased proliferation and decreased apoptosis in colon cancer, which explains why this classical tumor suppressor is amplified in colon tumors and suggests a rationale for targeting Rb phosphorylation in colon cancer. Proteomics identified an association between decreased CD8 T cell infiltration and increased glycolysis in microsatellite instability-high (MSI-H) tumors, suggesting glycolysis as a potential target to overcome the resistance of MSI-H tumors to immune checkpoint blockade. Proteogenomics presents new avenues for biological discoveries and therapeutic development.

Multiple reaction monitoring (MRM) mass spectrometry has been successfully applied to monitor targeted proteins in biological specimens, raising the possibility that assays could be configured to measure all human proteins. We report the results of a pilot study designed to test the feasibility of a large-scale, international effort for MRM assay generation. We have configured, validated across three laboratories and made publicly available as a resource to the community 645 novel MRM assays representing 319 proteins expressed in human breast cancer. Assays were multiplexed in groups of >150 peptides and deployed to quantify endogenous analytes in a panel of breast cancer-related cell lines. The median assay precision was 5.4%, with high interlaboratory correlation (R(2) > 0.96). Peptide measurements in breast cancer cell lines were able to discriminate among molecular subtypes and identify genome-driven changes in the cancer proteome. These results establish the feasibility of a large-scale effort to develop an MRM assay resource.

The Hungarian adult heart transplant program, which started in 1992, has changed gradually in the past 20 years. After the early enthusiasm of the first cases it changed significantly and it became an organized programme. However, low donation activity and moderate referral numbers to the national transplant waiting list slowed down the process therefore, heart transplant numbers did not fulfill expectations in the early years. After a moderate increase in 2007 transplant numbers have dropped again until recently when Hungary partially joined Eurotransplant network. Excess fundamental resources allocated to cardiac transplantation by health care professionals and reorganizing transplant coordination as well as logistics forced dramatic changes in clinical management. In 2011 and 2012 major structural changes had been made at Semmelweis University. The newly established transplant intensive care unit and the initiation of mechanical circulatory support and assist device programme increased transplant numbers by 131% compared to previous years, as well as it resulted an 86.63% 30-day survival rate, hence last year was the most successful year of cardiac transplantation ever. Orv. Hetil., 2013, 154, 863–867.

Cells employ error-free or error-prone postreplication repair (PRR) processes to tolerate DNA damage. Here, we present a genome-wide screen for sensitivity to 0.001% methyl methanesulfonate (MMS). This relatively low dose is of particular interest because wild-type cells exhibit no discernible phenotypes in response to treatment, yet PRR mutants are unique among repair mutants in their exquisite sensitivity to 0.001% MMS; thus, low-dose MMS treatment provides a distinctive opportunity to study postreplication repair processes. We show that upon exposure to low-dose MMS, a PRR-defective rad18Δ mutant stalls into a lengthy G2 arrest associated with the accumulation of single-stranded DNA (ssDNA) gaps. Consistent with previous results following UV-induced damage, reactivation of Rad18, even after prolonged G2 arrest, restores viability and genome integrity. We further show that PRR pathway preference in 0.001% MMS depends on timing and context; cells preferentially employ the error-free pathway in S phase and do not require MEC1-dependent checkpoint activation for survival. However, when PRR is restricted to the G2 phase, cells utilize REV3-dependent translesion synthesis, which requires a MEC1-dependent delay and results in significant hypermutability.

We present a case of a 41 year old African-American male with a history of thromboembolic events that was found to have a SSP as a result of pulmonary infarct. Patient underwent definitive surgical intervention, lobectomy, after failure of initial management with thoracostomy tube and video-assisted thoracoscopic (VATS) with wedge resection and pleurodesis.