Search

Background: Checkpoint inhibitor immunotherapy or immuno-oncology (IO) treatment in refractory cervical cancer yielded an objective response rate (ORR) of 12% in tumors expressing the programmed cell death ligand-1 (PD-L1) in the KEYNOTE-158 phase II study. We hypothesized that the positive response might be associated with the level of PD-L1 expression and/or the tumor mutation burden (TMB). We also aimed to analyze if responses could be associated with platinum sensitivity.Methods: This is a retrospective study of all consecutive patients with cervical cancer who received pembrolizumab or nivolumab.Results: Ten patients were identified. Median age was 64.5 years old (range 48–80). The response rate was 70% and the median duration of response was 21.0 months (range 1.8–26.7) after 20.7 months of follow-up (range 2.0–31.0). The response rate was 80% in patients with PD-L1 combined positive score (CPS) ≥ 10, and 75% in patients with tumor mutation burden (TMB) ≥ 10 mut/Mb. The mean progression-free survival (PFS) for the entire cohort was 20.2 months (95% CI 12.0–28.5). Seven patients had treatment for >12 months (range 14.6–31.0). Five patients were platinum-sensitive and 5 patients were platinum-resistant at the time of immunotherapy, and the response rate was similar regardless of platinum sensitivity.Conclusions: The positive response to IO treatment in advanced cervical cancer in this study was higher than published, and a possible association with the level of PD-L1 expression and the TMB level was suggested. A PD-L1 CPS score ≥ 10 or TMB ≥ 10 may be biomarkers to correlate with response, which should be explored in large studies.

e21658 Background: First-line treatment of lung cancer patients with EGFR mutation with early generation tyrosine kinase inhibitors (TKIs) renders a median progression-free survival (mPFS) of 11 months (m), and for the patients who subsequently develop the T790M mutation, osimertinib provides an additional mPFS of 10 m. First-line treatment with osimertinib showed mPFS of 18.9 m and none developed T790M at the time of progression (FLAURA trial). We hypothesized that the mPFS for patients who developed T790M mutation might be longer than those who did not and the prognosis and outcome might be different between these two groups. Methods: This is a retrospective study on patients who were diagnosed with metastatic, EGFR-mutated NSCLC and treated at Maimonides Cancer Center between 1/1/2011 and 9/30/2019. Patients were divided into two cohorts: those who did not develop T790M mutation (group 1), and those who did (including 1 patient who had de novo mutation, group 2). Results: Fifty-six patients were identified, with median age of 70 years (range 40-93). All patients had adenocarcinoma. Thirty-nine (70%) were Chinese. There were 31 (55%) exon 19 deletion, 22 (39%) L858R, 1 E709A, 1 G719S and 1 de novo T790M mutations. Forty-five patients (80%) never developed T790M (group 1), 10 (18%) did and 1 had primary mutation (group 2). The median time to developing T790M mutation was 22 m. In group 1, 8 patients received first-line osimertinib, while other 37 received erlotinib (23), gefitinib (4) and afatinib (10). Among the 37 patients, 12 were switched to osimertinib in subsequent lines, 5 due to toxicity and 7 due to progression. In group 2, 8 patients received osimertinib at identification of T790M and 1 for de novo mutation. At 22 m median follow up, the mPFS for group 1 was 14 m (95% CI 9.3-19); the mPFS for group 2 was 21.6 m (95% CI 7.4-49.1) up to the time of identification of T790M, and 31.2 m (95% CI 4.9-45.4) until disease progression after osimertinib. The mPFS of the entire cohort was 17.1 m (95% 11.1-25.9), and mOS was 22.0 m (95% 15.6-29.6). The mOS was 19.0 m (95% CI 11.4 – 25.2), and 39.9 m (95% CI 13.3 – 62.1) for group 1 and 2 respectively. Conclusions: Patients who developed the T790M mutation appeared to have a longer PFS than those who did not and the mean duration to detection of T790M was 22 m. The development of T790M may confer a slower growth pattern and longer PFS. This question should be further explored in larger studies.

Summary High-throughput sequencing can enhance the analysis of aptamer libraries generated by the Systematic Evolution of Ligands by EXponential enrichment. Robust analysis of the resulting sequenced rounds is best implemented by determining a ranked consensus of reads following the processing by multiple aptamer detection algorithms. While several such approaches have been developed to this end, their installation and implementation is problematic. We developed AptCompare, a cross-platform program that combines six of the most widely used analytical approaches for the identification of RNA aptamer motifs and uses a simple weighted ranking to order the candidate aptamers, all driven within the same GUI-enabled environment. We demonstrate AptCompare’s performance by identifying the top-ranked candidate aptamers from a previously published selection experiment in our laboratory, with follow-up bench assays demonstrating good correspondence between the sequences’ rankings and their binding affinities. Availability and implementation The source code and pre-built virtual machine images are freely available at https://bitbucket.org/shiehk/aptcompare. Supplementary information Supplementary data are available at Bioinformatics online.

Unrestrained receptor tyrosine kinase (RTK) signaling and epigenetic deregulation are root causes of tumorigenesis. We establish linkage between these processes by demonstrating that aberrant RTK signaling unleashed by oncogenic HRas(G12V) or loss of negative feedback through Sprouty gene deletion remodels histone modifications associated with active typical and super-enhancers. However, although both lesions disrupt the Ras-Erk axis, the expression programs, enhancer signatures, and transcription factor networks modulated upon HRas(G12V) transformation or Sprouty deletion are largely distinct. Oncogenic HRas(G12V) elevates histone 3 lysine 27 acetylation (H3K27ac) levels at enhancers near the transcription factor Gata4 and the kinase Prkcb, as well as their expression levels. We show that Gata4 is necessary for the aberrant gene expression and H3K27ac marking at enhancers, and Prkcb is required for the oncogenic effects of HRas(G12V)-driven cells. Taken together, our findings demonstrate that dynamic reprogramming of the cellular enhancer landscape is a major effect of oncogenic RTK signaling.

Systems biology approaches to the integrative study of cells, organs and organisms offer the best means of understanding in a holistic manner the diversity of molecular assays that can be now be implemented in a high throughput manner. Such assays can sample the genome, epigenome, proteome, metabolome and microbiome contemporaneously, allowing us for the first time to perform a complete analysis of physiological activity. The central problem remains empowering the scientific community to actually implement such an integration, across seemingly diverse data types and measurements. One promising solution is to apply semantic techniques on a self-consistent and implicitly correct ontological representation of these data types. In this paper we describe how we have applied one such solution, based around the InterMine data warehouse platform which uses as its basis the Sequence Ontology, to facilitate a systems biology analysis of virulence in the apicomplexan pathogen $Toxoplasma~gondii$, a common parasite that infects up to half the worlds population, with acute pathogenic risks for immuno-compromised individuals or pregnant mothers. Our solution, which we named `toxoMine', has provided both a platform for our collaborators to perform such integrative analyses and also opportunities for such cyberinfrastructure to be further developed, particularly to take advantage of possible semantic similarities of value to knowledge discovery in the Omics enterprise. We discuss these opportunities in the context of further enhancing the capabilities of this powerful integrative platform., To appear in the Proceedings of the 2016 IEEE Tenth International Conference on Semantic Computing (ICSC 2016)

Pathogenic New World hemorrhagic fever mammarenaviruses (NWM) utilize Glycoprotein 1 (GP1) to target the apical domain of the human transferrin receptor (hTfR) for facilitating cell entry. However, the conservation between their GP1s is low. Considering this and the slow evolutionary progression of mammals compared to viruses, therapeutic targeting of hTfR provides an attractive avenue for cross-strain inhibition and diminishing the likelihood of escape mutants. Aptamers present unique advantages for the development of inhibitors to vial entry, including ease of synthesis, lack of immunogenicity, and potentially cold-chain breaking solutions to diseases endemic to South America. Here, recognizing that in vivo competition with the natural ligand, transferrin (Tf), likely drove the evolution of GP1 to recognize the apical domain, we performed competitive in vitro selections against hTfR-expressing cells with supplemented Tf. The resultant minimized aptamer, Waz, binds the apical domain of the receptor and inhibits infection of human cells by recombinant NWM in culture (EC50 ≃400 nmol/l). Aptamer multimerization further enhanced inhibition >10-fold (EC50 ≃30 nmol/l). Together, our results highlight the ability to use a competitor to bias the outcome of a selection and demonstrate how avidity effects can be leveraged to enhance both aptamer binding and the potency of viral inhibition.

RNA aptamers are small oligonucleotide molecules whose composition and resulting folded structure enable them to bind with high affinity and high selectivity to target ligands and therefore hold great promise as potential therapeutic drugs. Functional aptamers are selected from a large, randomized initial library in a process known as SELEX (systematic evolution of ligands by exponential enrichment). This is an iterative process involving numerous rounds of binding, elution, and amplification against a specific target substrate. During each iteration -- or round of selection -- we enrich for the species with the highest binding affinity to the target. After multiple rounds, we ideally have an enriched aptamer library suitable for subsequent investigation. Modern techniques employ massively parallel sequencing, enabling the generation of large libraries (~106 sequences) in a matter of hours for each round of selection. As RNA is single-stranded, covariance models (CMs) are ideal for representing motifs in their secondary structures, allowing us to discover patterns within functional aptamer populations following each round. CMs have been implemented in Infernal, a program that infers RNA alignments based on RNA sequence and structure. Calibrating a single CM in Infernal can take several hours and is a significant performance bottleneck for our work. However, as each CM calculation is itself independently determined and requires defined processing and memory resources, their computation in parallel offers a potential solution to this problem. In this paper, we describe using the Open Science Grid (OSG) to facilitate the identification of aptamer motifs by running CM calibrations and refinements in parallel across up to ten OSG clients. We use the Simple API for Grid Applications (SAGA) to interface with OSG and manage job submissions and file transfers. When run in parallel, our results show a significant speed up, constrained by typical latencies and QoS associated with nominal OSG usage. Our work demonstrates the ability of SAGA and the OSG to assist in parallelizing solutions to complex sequencing-based biomedical challenges.

The use of sequencing technologies has revolutionized the field of genomics, allowing us to study structural and functional variations within the genome to base pair level. These technologies can also be used to probe the associated epigenome, where DNA-binding proteins alter the structural integrity of the genome, restricting or enabling localized gene expression in a heritable fashion. By using assays that identify the binding location of these proteins, so called 'epigenetic marks' can be used to discover and correlate molecular functions and phenotypes being studied. As such 'epigenetic marks' are inherently plastic in their nature, being easily perturbed by environmental stimuli, they are a compelling and important area of study in the context of human development and diseases. One of the most commonly studied epigenetic marks is DNA methylation: attachment of the methyl group to cytosines in CpG dinucleotides -- an occurrence where two cytosine nucleotides are immediately followed by two guanine nucleotides in tandem. This modification can directly block the binding of regulatory proteins to that specific location thus effectively 'silencing' transcriptional activities. There are roughly 2.8 million such CpG loci in a human genome, making it an excellent target for performing a genome-wide methylation assay using sequencing technologies.

The purposes of the study were to compare the performance of ten representative focus measures in the presence of nondefocus aberrations and to evaluate their applicability to the eye. For fixed amounts of nondefocus aberrations, the amount of defocus was changed to generate a series of blurred images from which focus measure curves were derived. In the presence of small amounts of nondefocus aberrations, all focus measures showed unimodal and monotonic behavior, although there were large differences in their sensitivity to defocus and effective ranges. There were breakdowns in monotonicity and unimodality for some focus measures when applied to data from human eyes, while other focus measures could detect the shift in the best-focus plane in the blurred image series resulting from spherical aberration.

Comprehensive evaluation of retinal image quality requires that light scatter as well as optical aberrations be considered. In investigating how retinal image degradation affects eye growth in the chick model of myopia, we developed a simple method based on Shack-Hartmann images for evaluating the effects of both monochromatic aberrations and light scatter on retinal image quality. We further evaluated our method in the current study by applying it to data collected from both normal chick eyes and albino eyes that were expected to show increased intraocular light scatter. To analyze light scatter in our method, each Shack-Hartmann dot is treated as a local point spread function (PSF) that is the convolution of a local scatter PSF and a lenslet diffraction PSF. The local scatter PSF is obtained by de-convolution, and is fitted with a circularly symmetric Gaussian function using nonlinear regressions. A whole-eye scatter PSF also can be derived from the local scatter PSFs for the analyzed pupil. Aberrations are analyzed using OSA standard Zernike polynomials, and aberration-related PSF calculated from reconstructed wavefront using fast Fourier transform. Modulation transfer functions (MTFs) are computed separately for aberration and scatter PSFs, and a whole-eye MTF is derived as the product of the two. This method was applied to 4 normal and 4 albino eyes. Compared to normal eyes, albino eyes were more aberrated and showed greater light scatter. As a result, overall retinal image degradation was much greater in albino eyes than in normal eyes, with the relative contribution to retinal image degradation of light scatter compared to aberrations also being greater for albino eyes.

Unrestrained activation of receptor tyrosine kinase (RTK) pathways drives tumorigenesis through the persistent activation of critical signaling pathways, including the Ras-ERK axis. This aberrant signaling is unleashed by mutations in signaling effectors such as Ras, as well as through disruption of feedback control by regulatory proteins such as the Sprouty family. Mutations in epigenetic regulators also commonly occur during cancer progression, modulating chromatin architecture and gene expression. While unrestrained signaling and epigenetic deregulation are root causes of tumorigenesis, the relationship between aberrant RTK signaling and chromatin modifications at cis-regulatory elements remains to be fully elucidated. We establish linkage between these processes by examining the effects of oncogenic HRasG12V or loss of feedback regulation by Sprouty on global changes in gene expression and enhancer-associated chromatin modifications. Using RNA-sequencing and ChIP-sequencing, we demonstrate that aberrant RTK signaling unleashed by oncogenic HRasG12V or Sprouty gene deletion disrupts gene expression programs and remodels histone modifications associated with active enhancers, including histone 3 lysine 27 acetylation (H3K27ac). Abolishing persistent Ras-Erk signaling through chemical inhibition of MEK activity reverses the aberrant transcriptional program and H3K27ac remodeling at deregulated enhancers. While both lesions disrupt the Ras-Erk axis, the specific target genes and enhancers modulated upon HRasG12V-transformation or Sprouty deletion are largely distinct. Specifically, oncogenic HRasG12V significantly elevates the expression and H3K27ac levels near key target genes encoding the transcription factor Gata4 and the kinase Prkcb. Gata4 is necessary for the HRasG12V expression program and coordinates H3K27ac marking at enhancers of deregulated target genes. We further show that HRasG12V-driven cells are sensitive to chemical inhibition of Prkcb, which reduces the viability and clonogenicity of HRasG12V-transformed cells. These oncogenic effects upon HRasG12V-transformation are also reduced by chemical inhibition of the chromatin regulators BET bromodomain proteins and p300/CBP, which recognize and deposit H3K27ac, respectively. Taken together, our data support a model in which dynamic reprogramming of the cellular enhancer landscape is a key effect of oncogenic RTK signaling. Furthermore, our study shows that identification of enhancers regulated by oncogenic Ras yields insight into targeting key epigenetic regulators and downstream factors that promote Ras-driven malignancies. Citation Format: Behnam Nabet, Pilib O Broin, Jaime Reyes, Kevin Shieh, Charles Y. Lin, Christine M. Will, Relja Popovic, Teresa Ezponda, James E. Bradner, Aaron A. Golden, Jonathan D. Licht. Deregulation of the Ras-Erk signaling axis modulates the enhancer landscape. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2209. doi:10.1158/1538-7445.AM2015-2209

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite that must monitor for changes in the host environment and respond accordingly; however, it is still not fully known which genetic or epigenetic factors are involved in regulating virulence traits of T. gondii. There are on-going efforts to elucidate the mechanisms regulating the stage transition process via the application of high-throughput epigenomics, genomics and proteomics techniques. Given the range of experimental conditions and the typical yield from such high-throughput techniques, a new challenge arises: how to effectively collect, organize and disseminate the generated data for subsequent data analysis. Here, we describe toxoMine, which provides a powerful interface to support sophisticated integrative exploration of high-throughput experimental data and metadata, providing researchers with a more tractable means toward understanding how genetic and/or epigenetic factors play a coordinated role in determining pathogenicity of T. gondii. As a data warehouse, toxoMine allows integration of high-throughput data sets with public T. gondii data. toxoMine is also able to execute complex queries involving multiple data sets with straightforward user interaction. Furthermore, toxoMine allows users to define their own parameters during the search process that gives users near-limitless search and query capabilities. The interoperability feature also allows users to query and examine data available in other InterMine systems, which would effectively augment the search scope beyond what is available to toxoMine. toxoMine complements the major community database ToxoDB by providing a data warehouse that enables more extensive integrative studies for T. gondii. Given all these factors, we believe it will become an indispensable resource to the greater infectious disease research community. Database URL: http://toxomine.org