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Hepatic cell death occurs in response to diverse stimuli such as chemical and physical damage. The exposure of intracellular contents such as DNA during necrosis induces a severe inflammatory response that has yet to be fully explored therapeutically. Here, we sought means to neutralize the ability of extracellular DNA to induce deleterious tissue inflammation when drug‐induced liver injury had already ensued. DNA exposure and inflammation were investigated in vivo in drug‐induced liver injury using intravital microscopy. The necrotic DNA debris was studied in murine livers in vivo and in DNA debris models in vitro by using a positively charged chemokine‐derived peptide (MIG30; CXCL9[74‐103]). Acetaminophen‐induced liver necrosis was associated with massive DNA accumulation, production of CXC chemokines, and neutrophil activation inside the injured tissue. The MIG30 peptide bound the healthy liver vasculature and, to a much greater extent, to DNA‐rich necrotic tissue. Moreover, MIG30 bound extracellular DNA directly in vivo in a charge‐dependent manner and independently of glycosaminoglycans and chemokines. Post‐treatment of mice with MIG30 reduced mortality, liver damage, and inflammation significantly. These effects were not observed with a control peptide that does not bind DNA. Mechanistically, MIG30 inhibited the interaction between DNA and histones, and promoted the dissociation of histones from necrotic debris. MIG30 also inhibited the pro‐inflammatory effect of CpG DNA, as measured by a reduction in CXCL8 production, indicating that MIG30 disturbs the ability of DNA to induce hepatic inflammation. Conclusion: The use of DNA‐binding peptides reduces necrotic liver injury and inflammation, even at late timepoints., Marques et al. used a small chemokine‐derived positively‐charged peptide (MIG30) to neutralize the deleterious effects of extracellular DNA during drug‐induced liver injury. Treatment with MIG30 rescued mice from injury, inflammation and mortality by binding DNA avidly, dissociating it from histones and reducing the pro‐inflammatory activity of CpG DNA.

Prioritizing genes for translation to therapeutics for common diseases has been challenging. Here, we propose an approach to identify drug targets with high probability of success by focusing on genes with both gain of function (GoF) and loss of function (LoF) mutations associated with opposing effects on phenotype (Bidirectional Effect Selected Targets, BEST). We find 98 BEST genes for a variety of indications. Drugs targeting those genes are 3.8-fold more likely to be approved than non-BEST genes. We focus on five genes (IGF1R, NPPC, NPR2, FGFR3, and SHOX) with evidence for bidirectional effects on stature. Rare protein-altering variants in those genes result in significantly increased risk for idiopathic short stature (ISS) (OR = 2.75, p = 3.99 × 10−8). Finally, using functional experiments, we demonstrate that adding an exogenous CNP analog (encoded by NPPC) rescues the phenotype, thus validating its potential as a therapeutic treatment for ISS. Our results show the value of looking for bidirectional effects to identify and validate drug targets., Prioritising genes as potential drug targets is challenging and often unsuccessful once testing efficacy in humans. Here, the authors propose an approach to identifying drug targets that uses evidence from gain- or loss-of-function mutations associated with bidirectional effects on phenotypes.

ObjectivesTo explore posttranslational modifications (PTMs), including proteolytic activation, multimerization, complex formation and citrullination of gelatinases, in particular of gelatinase B/MMP-9, and to detect in gelatin-Sepharose affinity-purified synovial fluids, the presence of specific MMP proteoforms in relation to arthritis.MethodsLatent, activated, complexed and truncated gelatinase-A/MMP-2 and gelatinase B/MMP-9 proteoforms were detected with the use of zymography analysis to compare specific levels, with substrate conversion assays, to test net proteolytic activities and by Western blot analysis to decipher truncation variants. Citrullination was detected with enhanced sensitivity, by the use of a new monoclonal antibody against modified citrullines.ResultsAll MMP-9 and MMP-2 proteoforms were identified in archival synovial fluids with the use of zymography analysis and the levels of MMP-9 versus MMP-2 were studied in various arthritic diseases, including rheumatoid arthritis (RA). Secondly, we resolved misinterpretations of MMP-9 levels versus proteolytic activities. Thirdly, a citrullinated, truncated proteoform of MMP-9 was discovered in archival RA synovial fluid samples and its presence was corroborated as citrullinated hemopexin-less MMP-9 in a small prospective RA sample cohort.ConclusionSynovial fluids from rheumatoid arthritis contain high levels of MMP-9, including its truncated and citrullinated proteoform. The combination of MMP-9 as analyte and its PTM by citrullination could be of clinical interest, especially in the field of arthritic diseases.

OBJECTIVE: Juvenile idiopathic arthritis (JIA) is the most common rheumatic disease in childhood. The predominant subtypes, oligoarticular and polyarticular JIA, are traditionally considered to be autoimmune diseases with a central role for T cells and autoantibodies. Mounting evidence suggests an important role for neutrophils in JIA pathogenesis. We undertook this study to investigate the phenotypic features of neutrophils present in the blood and inflamed joints of patients. METHODS: JIA synovial fluid (SF) and parallel blood samples from JIA patients and healthy children were collected. SF-treated neutrophils from healthy donors and pleural neutrophils from patients with pleural effusion were investigated as controls for SF exposure and extravasation. Multicolor flow cytometry panels allowed for in-depth phenotypic analysis of neutrophils, focusing on the expression of adhesion molecules, activation, and maturation markers and chemoattractant receptors. Multiplex technology was used to quantify cytokines in plasma and SF. RESULTS: SF neutrophils displayed an activated, hypersegmented phenotype with decreased CD62L expression, up-regulation of adhesion molecules CD66b, CD11b, and CD15, and down-regulation of CXCR1/2. An elevated percentage of CXCR4-positive neutrophils was detected in SF from patients. Pleural neutrophils showed less pronounced maturation differences. Strikingly, significant percentages of SF neutrophils showed a profound up-regulation of atypical neutrophil markers, including CXCR3, intercellular adhesion molecule 1, and HLA-DR. CONCLUSION: Our data show that neutrophils in inflamed joints of JIA patients have an activated phenotype. This detailed molecular analysis supports the notion that a complex intertwining between these innate immune cells and adaptive immune events drives JIA. ispartof: ARTHRITIS & RHEUMATOLOGY vol:73 issue:5 pages:896-897 ispartof: location:United States status: published

Circulating free DNA in plasma is an alternative source of tumor-derived DNA that can be a surrogate for tissue epidermal growth factor receptor (EGFR) testing. We evaluated the analytical performance of the cobas® EGFR Mutation Test v2 (cobas test), a real-time polymerase chain reaction assay designed to detect defined EGFR gene mutations in plasma from patients with advanced non-small cell lung cancer (NSCLC). We used K2-ethylenediaminetetraacetic acid plasma samples from NSCLC patients and healthy donors (HDs), along with cell line DNA. Results from a complete technical performance evaluation are described, including a comparison between NSCLC and HD plasma to support the use of surrogate samples and an independent confirmation of the limit of detection (LoD). The cobas test reported an overall percent agreement of approximately 88% for plasma samples when compared with a next-generation sequencing method. The LoD for all EGFR mutations was ≤ 100 copies/mL for plasma samples. An external study confirmed the LoD for exon 19 deletion, L858R, and T790M at ≤ 100 copies/mL using samples derived from NSCLC patient specimens. The cobas test showed linearity between at least 50 and 10,000 copies/mL for plasma samples. An internal repeatability study reported a correct call accuracy of 99.2% for plasma samples. The performance of the cobas test is equivalent when using sheared or intact cell line DNA diluted into either HD plasma or NSCLC patient plasma. The cobas test is a sensitive, robust, and accurate assay that delivers reproducible results.

While GWAS of common diseases has delivered thousands of novel genetic findings, prioritizing genes for translation to therapeutics has been challenging. Here, we propose an approach to resolve that issue by identifying genes that have both gain of function (GoF) and loss of function (LoF) mutations associated with opposing effects on phenotype (Bidirectional Effect Selected Targets, BEST). Bidirectionality is a desirable feature of the best targets because it implies both a causal role on the phenotype in one direction and that modulating the target activity might be safe and therapeutically beneficial in the other.We used height, a highly heritable trait and a model of complex diseases, to test our approach. Using 34,231 individuals with exome sequence data and height, we identified five genes (IGF1R, NPPC, NPR2, FGFR3, and SHOX) with evidence for bidirectional effects on stature. Rare protein-altering variants significantly increased risk for idiopathic short stature (ISS) (OR=2.75, p= 3.99×10−8). These genes are key members of the only two pathways successfully targeted for short stature: Growth Hormone/Insulin-like growth factor 1 axis and C-type Natriuretic peptide (CNP) for Achondroplasia, a monogenic form of dwarfism. We assayed a subset of NPR2 mutations and identified those with elevated (GoF) and diminished (LoF) activity and found that a polygenic score for height modulates the penetrance of pathogenic variants. We also demonstrated that adding exogenous CNP (encoded by NPPC) rescues the NPR2 haploinsufficiency molecular phenotype in a CRISPR-engineered cell line, thus validating its potential therapeutic treatment for inherited forms of short stature. Finally, we found that these BEST targets increase the probability of success in clinical trials above and beyond targets with other genetic evidence. Our results show the value of looking for bidirectional effects to identify and validate drug targets.

Laboratory testing for thrombophilia is complicated but essential for diagnosis. In 2017, the cobas® Factor II and Factor V Test (cobas F2F5 test) was launched for use with the cobas z 480 analyzer. This qualitative polymerase chain reaction test enables multiplex Factor II and Factor V testing with flexible reporting and workflow efficiency. Here, we report the results from studies investigating the performance of the cobas F2F5 test. Technical performance verification, clinical validation, external laboratory performance, and workflow comparison studies were performed. Fresh and frozen whole-blood and genomic DNA (gDNA) samples were tested, and several manual and automated DNA isolation methods were used. Bidirectional Sanger sequencing was used to verify genotypes identified by the cobas F2F5 test. One hundred percent agreement between the cobas F2F5 test and Sanger sequencing was observed for all genotypes. An external laboratory using remnant clinical samples also yielded 100% agreement between cobas F2F5 test results and their routine testing method. The cobas F2F5 test reduced the total sample processing time compared with the LightCycler® 1.2 platform (98.6 vs 420.2 min; 96 samples). Hemoglobin, extraction buffer, and ethanol contamination of the gDNA sample can lead to invalid results. The cobas F2F5 test has a high degree of accuracy for identification of Factor II and Factor V genotypes. This multiplex testing with short sample processing time can reduce handling errors and increase efficiency. Both manual and automated DNA isolation methods can be used with the cobas F2F5 test. Electronic supplementary material The online version of this article (10.1007/s11239-018-1745-8) contains supplementary material, which is available to authorized users.

// C. Allison Stewart 1 , Pan Tong 2 , Robert J. Cardnell 1 , Triparna Sen 1 , Lerong Li 2 , Carl M. Gay 1 , Fatemah Masrorpour 1 , You Fan 1 , Rasha O. Bara 1 , Ying Feng 3 , Yuanbin Ru 3 , Junya Fujimoto 4 , Samrat T. Kundu 1 , Leonard E. Post 3 , Karen Yu 3 , Yuqiao Shen 3 , Bonnie S. Glisson 1 , Ignacio Wistuba 4 , John V. Heymach 1 , Don L. Gibbons 1 , Jing Wang 2 and Lauren Averett Byers 1 1 Department of Thoracic Head & Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 2 Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 3 BioMarin Pharmaceutical, San Rafael, CA 94901, USA 4 Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA Correspondence to: Lauren Averett Byers, email: lbyers@mdanderson.org Keywords: SCLC, SLFN11, ATM, EMT, PARP inhibitor Received: December 10, 2016 Accepted: January 19, 2017 Published: February 15, 2017 ABSTRACT Small cell lung cancer (SCLC) is one of the most aggressive forms of cancer, with a 5-year survival

Introduction Tumor biopsies for detecting EGFR mutations in advanced NSCLC are invasive, costly, and not always feasible for patients with late-stage disease. The clinical utility of the cobas EGFR Mutation Test v2 (Roche Molecular Systems, Inc., Pleasanton, CA) with plasma samples from patients with NSCLC at disease progression after previous EGFR tyrosine kinase inhibitor therapy was investigated to determine eligibility for osimertinib treatment. Methods Matched tumor tissue and plasma samples from patients screened for the AURA extension and AURA2 phase II studies were tested for EGFR mutations by using tissue- and plasma-based cobas EGFR mutation tests. Plasma test performance was assessed by using the cobas tissue test and a next-generation sequencing method (MiSeq [Illumina Inc., San Diego, CA]) as references. The objective response rate, measured by blinded independent central review, was assessed in patients receiving osimertinib with a plasma T790M mutation–positive status. Results During screening, 551 patients provided matched tumor tissue and plasma samples. Pooled analysis of the positive and negative percent agreements between the cobas plasma and tissue tests for detection of T790M mutation were 61% and 79%, respectively. Comparing cobas plasma test with next-generation sequencing demonstrated positive and negative percent agreements of 90% or higher. The objective response rate was 64% (95% confidence interval: 57–70) in T790M mutation–positive patients by both cobas tissue and plasma tests (evaluable for response). Conclusions The cobas plasma test detected the T790M mutation in 61% of tumor tissue T790M mutation–positive patients. To mitigate the risk of false-negative plasma results, patients with a negative plasma result should undergo a tissue test where feasible.

Aim Our objective was to assess the performance of the cobas test versus comparators for KRAS mutation status and predicting clinical response to anti-epidermal growth factor receptor (EGFR) therapy in patients with metastatic colorectal cancer (mCRC). Methods mCRC samples from 398 patients from Roche study NO16968 (XELOXA) and 82 supplemental samples were tested with the cobas® KRAS mutation test (cobas test), the therascreen® KRAS RGQ PCR kit test (therascreen test), and Sanger sequencing as the reference method for detecting mutations in codons 12/13. Results For 461 eligible samples, the cobas test, therascreen test, and sequencing had invalid results for 5.2, 10.8, and 2.6 % of specimens, respectively. Valid cobas and therascreen test results had similar KRAS mutation-positive rates (37.3 vs. 36.3 %, respectively); sequencing was 28.5 %. Positive and negative percent agreement (PPA/NPA) between the cobas test and sequencing was 96.9 % (95 % confidence interval [CI] 92.2–98.8), and 88.7 % (95 % CI 84.7–91.8), respectively. PPA/NPA between the cobas and therascreen tests was 93.3 % (95 % CI 88.1–96.3) and 96.5 % (95 % CI 93.5–98.1), respectively. Bridging analysis from NCIC-CO.17 and NCT00113763 using the cobas test yielded modeled hazard ratios for overall survival and progression-free survival (PFS) of 0.558 (95 % CI 0.422–0.752) and 0.413 (95 % CI 0.304–0.550), respectively, for cetuximab and 0.989 (95 % CI 0.778–1.299) and 0.471 (95 % CI 0.360–0.626), respectively, for panitumumab, demonstrating significant efficacy in the KRAS-negative population for PFS. Conclusion The cobas test showed similar accuracy to the therascreen test for detecting KRAS mutations and could appropriately identify mCRC patients ineligible for anti-EGFR therapy as demonstrated by bridging analysis results. Electronic supplementary material The online version of this article (doi:10.1007/s40291-016-0193-4) contains supplementary material, which is available to authorized users.

Repeat cognitive assessment comparing post-injury performance to a pre-injury baseline is common in concussion management. Although post-injury tests are typically administered in clinical settings, baseline tests may be conducted individually with one-on-one supervision, in a group with supervision, or without supervision. The extent to which these different test settings affect cognitive performance is not well understood. To assess if performance on the Cogstate Brief Battery (CBB) differs across these settings, tests completed individually with one-on-one supervision were compared to those taken either in a group with supervision or individually but without supervision.A crossover study design was utilized to account for any effect of individual variability or test order to provide an unbiased examination of the effect of test setting on cognitive performance. Young adult participants completed an individually supervised test either before or after also completing a group or unsupervised test.CBB scores from the same individuals were not significantly different across test settings. Effect sizes ranged in magnitude from .09 to .12 for supervised versus unsupervised tests and from .01 to .37 for individual versus group tests across CBB tasks.These results suggest that cognitive testing with the CBB in alternate settings can provide valid cognitive data comparable to data obtained during individually supervised testing.

Expectation has been found to be a major etiological factor in Post-concussion Syndrome (PCS), but research has mostly relied on selfreport. This study examines the role of diagnosis threat on actual cognitive performance on concussion screening tests. Negative performance expectations were induced in some participants by presenting research showing cognitive impairment in contact sport athletes. Other participants read neutral research findings showing no impairment in athletes. Overall, athletes in the “negative expectation” condition performed slower but more accurately than those in the “neutral expectation” condition. Negative expectations might have caused participants to hesitate before responding, allowing them to double-check their responses. However, due to a small, homogenous sample, these promising but preliminary findings should be further explored with further research.

Poly-(ADP)-ribose polymerase (PARP) was identified as a potential novel therapeutic target for small cell lung cancer (SCLC) in two key proteomic studies. These findings led to the inclusion of a SCLC cohort in a Phase I study of talazoparib (BMN 673) - currently the most potent PARP inhibitor available - with promising efficacy signals. Identifying predictors of response to PARP inhibition would bring a needed therapeutic advance in this recalcitrant disease. PARP inhibition was first demonstrated to have marked efficacy in BRCA1/2 mutant cancers that are deficient in homologous recombination (HR). Large chromosomal structural alterations characteristic of these tumors can be quantitated by three correlated HR deficiency (HRD) metrics. We sought to determine if HRD would be a relevant predictive biomarker in SCLC. Thirty-six SCLC cell lines represented in the Cancer Cell Line Encyclopedia (CCLE) were interrogated for sensitivity to talazoparib, olaparib and cisplatin. We found significant correlation between these three pharmacologic agents and sought to determine response predictors. Three different HRD metrics were computed in these 36 SCLC cell lines using publically available CCLE Affymetrix SNP 6.0 array datasets. Surprisingly, none of these three measures of HRD were discriminatory for response to talazoparib in vitro. However, we found that after controlling for HRD and PARP1 target expression, expression of a single gene, SLFN11, was significantly correlated with talazoparib sensitivity as measured by the CCLE Affymetrix U133+2 arrays (q value = 0.008). After deriving SLFN11 knock-out SCLC cell lines by CRISPR/Cas9 gene editing, we found that loss of SLFN11 conferred resistance to talazoparib (IC50 >log difference) as compared to parental SLFN11 proficient cells. We then treated several SCLC patient-derived xenograft (PDX) models with talazoparib daily and found that SLFN11 expression in vivo was necessary but not sufficient for response. None of the SLFN11 low expressing PDXs responded to therapy, whereas 3/5 (60%) of SLFN11 high expressing PDXs demonstrated significant tumor growth inhibition of 54-75% after 21-28 days of daily treatment with talazoparib 0.3mg/kg. In conclusion, we demonstrate that SLFN11 expression is correlated with response to PARP inhibition in SCLC in vitro and in vivo. HRD structural aberration scores do not predict for in vitro PARP inhibitor sensitivity, however functional HR interrogation (such as RAD51 foci recruitment) may still be a relevant discriminator in addition to ongoing in vivo analysis. SLFN11 may be a pertinent predictive biomarker in SCLC for therapeutic response to PARP inhibition with considerable clinical implications. Citation Format: Benjamin H. Lok, Ying Feng, G. Karen Yu, Eric E. Gardner, Nadeem Riaz, Yuanbin (Kevin) Ru, Elisa de Stanchina, Simon N. Powell, Jerry Shen, John T. Poirier, Charles M. Rudin. PARP inhibitor sensitivity in small cell lung cancer cell lines and patient-derived xenografts correlates with SLFN11 expression but not with structural homologous recombination deficiency. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3756.

Background: Small cell lung cancer (SCLC) is an aggressive subtype of lung cancer, accounting for ∼15% of all lung cancer cases in US. Talazoparib (BMN 673) is a potent, specific PARP1/2 inhibitor that has demonstrated significant clinical activity in ovarian and breast cancer patients with deleterious germline BRCA1/2 mutations (ASCO 2014). Antitumor responses were also reported in SCLC patients treated with talazoparib (ASCO 2014). However, molecular determinants for SCLC response to talazoparib treatment have not been determined. Method: in vitro: 38 human SCLC cell lines were screened for their sensitivity towards single-agent treatment with talazoparib or cisplatin. Sensitive vs. resistant cell lines were categorized based on median GI50 of 38 cell lines (as measured by generational growth inhibition approach) and 90% experimental maximum inhibition. Bioinformatics analysis was carried out to search for genomic features associated with talazoparib sensitivity, using CCLE genomic profiling data. in vivo: 12 human SCLC patient-derived xenograft (PDX) tumor models were established in immunocompromised mice. These PDX models were treated with either vehicle or talazoparib, and tumor growth was monitored by standard methods. Untreated tumor samples were collected from mice for molecular analyses (Myriad HRD assay, Foundation Medicine gene mutation analysis, reverse-phase protein array (RPPA), and Western blotting) to survey for talazoparib sensitivity biomarkers. Result: The 38 SCLC lines showed a wide range of sensitivity to talazoparib (GI50 ranging from 2 nM to >2000 nM, with median GI50 = 56 nM) and cisplatin treatment (GI50 ranging from 10 nM to >10,000 nM). Sensitivity towards talazoparib and cisplatin are well correlated (Pearson r = 0.748). Differential gene expression analysis suggests sensitivity to talazoparib correlated well with high SLFN11 expression in SCLC lines (nominal p-value < 0.001). The in vitro sensitivity results were confirmed in several cell line-derived xenograft models. In the SCLC PDX tumor screen, 3 of 12 PDX models showed tumor regression during talazoparib treatment with one complete response; 3 of 12 PDX tumors exhibited stable disease (SD)-like responses, while the remainder were resistant to talazoparib treatment. Gene mutation analysis indicates that all 12 SCLC PDX tumors have TP53 and/or RB1 mutations as expected for SCLC, but a correlation between mutations in the homologous recombination DNA repair genes (e.g. BRCA1, BRCA2, ATM) and response to talazoparib was not established in these models. Furthermore, no apparent relationship was found between talazoparib response and the Myriad HRD score in these SCLC PDX models, with all HRD scores being below the cutoff score associated with response to platinum treatment in breast and ovarian cancer. Protein expression analysis of PDX tumors is currently underway, and data will be presented at the meeting. Citation Format: Ying Feng, Karen Yu, Robert Cardnell, Yuanbin (Kevin) Ru, Evelyn Wang, Jing Wang, Leonard E. Post, Lauren A. Byers, Yuqiao (Jerry) Shen. Talazoparib predictive biomarker analysis in human small cell lung cancer cells and PDX tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A37.