Supplementary Figures 1-11. Supplementary Figure 1. Sample and analyses diagram. Supplementary Figure 2. Aggregate mutation spectrum of Guatemalan cervical tumors. Supplementary Figure 3. Highly amplified regions of chromosome (Chr.) 2, 6 and 10 in tumor A4. Supplementary Figure 4. Chromosome breakpoints in cervical tumors. Supplementary Figure 5. PIK3CA mutation and pathology. Supplementary Figure 6. Co-occurring mutations in the PIK3CA and PTEN genes. Supplementary Figure 7. Sanger sequence concordance of mutant read percentage. Supplementary Figure 8. Mutation Fraction of PIK3CA mutations. Supplementary Figure 9. Age range associations with PIK3CA mutation and HPV type. Supplementary Figure 10. PIK3CA and HPV E6 and E7 mRNA expression. Supplementary Figure 11. Model of cervical cancer progression.

Supplementary Figures and legends

Supplementary Tables 1-6. Supplemental Table 1. Primers used in Ion Torrent sequencing of HPV. Supplemental Table 2. Predicted somatic mutations in PIK3CA discovered by targeted sequencing of cervical tumors from Mexico, Venezuela, and Guatemala. Supplementary Table 3. Predicted somatic mutations in 5 candidate genes discovered by targeted sequencing of cervical tumors. Supplementary Table 4. Association of HPV type and PIK3CA mutation. Supplementary Table 5. PIK3CA mutations in histologic subtype in this and other published studies Supplementary Table 6. Fig.3A_B_C raw data

Despite recent advances, hypoplastic left heart syndrome (HLHS) patients subsequent to the Fontan still have significant morbidity and mortality. Some require heart transplant due to systemic ventricular dysfunction. Limited data exist on timing for transplant referral. This study aims to correlate systemic ventricular strain by echocardiography to transplant-free survival. HLHS patients who had Fontan palliation at our institution were included. Patients were divided into: 1) Required transplant or experienced mortality (composite end point); 2) Did not require transplant or survived. For those who experienced the composite endpoint, the last echocardiogram prior to the composite outcome was used, while for those who did not experience the composite endpoint the last echocardiogram obtained was used. Several qualitative and quantitative parameters were analyzed with focus on strain parameters. Ninety-five patients with HLHS Fontan palliation were identified. Sixty-six had adequate images and eight (12

Introduction: In the era of big data, gene-set pathway analyses derived from multi-omics are exceptionally powerful. When preparing and analyzing high-dimensional multi-omics data, the installation process and programing skills required to use existing tools can be challenging. This is especially the case for those who are not familiar with coding. In addition, implementation with high performance computing solutions is required to run these tools efficiently. Methods: We introduce an automatic multi-omics pathway workflow, a point and click graphical user interface to Multivariate Single Sample Gene Set Analysis (MOGSA), hosted on the Cancer Genomics Cloud by Seven Bridges Genomics. This workflow leverages the combination of different tools to perform data preparation for each given data types, dimensionality reduction, and MOGSA pathway analysis. The Omics data includes copy number alteration, transcriptomics data, proteomics and phosphoproteomics data. We have also provided an additional workflow to help with downloading data from The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium and preprocessing these data to be used for this multi-omics pathway workflow. Results: The main outputs of this workflow are the distinct pathways for subgroups of interest provided by users, which are displayed in heatmaps if identified. In addition to this, graphs and tables are provided to users for reviewing. Conclusion: Multi-omics Pathway Workflow requires no coding experience. Users can bring their own data or download and preprocess public datasets from The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium using our additional workflow based on the samples of interest. Distinct overactivated or deactivated pathways for groups of interest can be found. This useful information is important in effective therapeutic targeting.

The Cancer Genomics Cloud (CGC), powered by Seven Bridges, is an NCI-funded platform that streamlines access to large cancer datasets, bioinformatic tools, and cloud computation for cancer researchers. The attributes of the CGC designed to democratize data analysis also make it ideal for training the next generation of data scientists. During the Covid-19 pandemic, it has become clear that remote/virtual learning is of great importance for workforce development, and that reducing barriers to high quality educational resources is critical for many populations. Here we present our best practices for education of bioinformatics using the CGC, taking advantage of both distributed cloud networks and platform features to enhance learning. Our best practices methods are focused on a systematic approach that takes the instructor and students through a typical bioinformatics workflow in their field of research. Briefly, the organizational structure of the CGC, known as “projects,'' contains all aspects of an analysis, including data files, tools, and tool settings. Projects have fine-grained permission settings, which allow the owner to securely share their project for viewing or editing. An instructor can generate an example analysis from start to finish then share an entire project with trainees. Both students and instructors have access to the same data on the cloud, so the teacher can pre-populate the projects with specific files, ensuring the same starting point. All members within the project can communicate, including adding notes directly on the results, allowing students to troubleshoot in real time with the teacher. Instructors can manage costs for the whole class through the CGC’s billing system. The CGC also provides Public Projects for self-guided training, where a researcher can learn by example using the data, tools, and completed tasks within the project, including detailed instructions embedded in markdown language. We have successfully used these methods to train masters students in bioinformatics at Georgetown University for three years, as well as high school students, college students, and current cancer researchers. This approach to virtual learning of bioinformatics can democratize training for the next generation of data scientists. The FAIR practices built into the CGC enables education across all levels of expertise, empowering users to drive cancer research from any stage of their career. Citation Format: Manisha Ray, Sai Lakshmi Subramanian, David Roberson, Zelia Worman, Dennis A. Dean, Soner Koc, Jack DiGiovanna, Brandi Davis-Dusenbery. The Cancer Genomics Cloud enables bioinformatics training in a hybrid educational world [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6393.

The lack of sensitive and robust behavioral assessments of pain in preclinical models has been a major limitation for both pain research and the development of novel analgesics. Here, we demonstrate a novel data acquisition and analysis platform that provides automated, quantitative, and objective measures of naturalistic rodent behavior in an observer-independent and unbiased fashion. The technology records freely behaving mice, in the dark, over extended periods for continuous acquisition of 2 parallel video data streams: (1) near-infrared frustrated total internal reflection for detecting the degree, force, and timing of surface contact and (2) simultaneous ongoing video graphing of whole-body pose. Using machine vision and machine learning, we automatically extract and quantify behavioral features from these data to reveal moment-by-moment changes that capture the internal pain state of rodents in multiple pain models. We show that these voluntary pain-related behaviors are reversible by analgesics and that analgesia can be automatically and objectively differentiated from sedation. Finally, we used this approach to generate a paw luminance ratio measure that is sensitive in capturing dynamic mechanical hypersensitivity over a period and scalable for high-throughput preclinical analgesic efficacy assessment.

Background and Purpose Many pain-triggering nociceptor neurons express TRPV1 or TRPA1, cation-selective channels with large pores that enable permeation of QX-314, a cationic analogue of lidocaine. Co-application of QX-314 with TRPV1 or TRPA1 activators can silence nociceptors. In this study, we describe BW-031, a novel more potent cationic sodium channel inhibitor, and test whether its application alone can inhibit pain associated with tissue inflammation and whether this strategy can also inhibit cough. Experimental Approach We tested the ability of BW-031 to inhibit pain in three models of tissue inflammation:- inflammation in rat paws produced by complete Freund's adjuvant or by surgical incision and a mouse ultraviolet (UV) burn model. We tested the ability of BW-031 to inhibit cough induced by inhalation of dilute citric acid in guinea pigs. Key Results BW-031 inhibited Na(v)1.7 and Na(v)1.1 channels with approximately sixfold greater potency than QX-314 when introduced inside cells. BW-031 inhibited inflammatory pain in all three models tested, producing more effective and longer-lasting inhibition of pain than QX-314 in the mouse UV burn model. BW-031 was effective in reducing cough counts by 78%-90% when applied intratracheally under isoflurane anaesthesia or by aerosol inhalation in guinea pigs with airway inflammation produced by ovalbumin sensitization. Conclusion and Implications BW-031 is a novel cationic sodium channel inhibitor that can be applied locally as a single agent to inhibit inflammatory pain. BW-031 can also effectively inhibit cough in a guinea pig model of citric acid-induced cough, suggesting a new clinical approach to treating cough.

SUMMARYThe lack of robust quantifiable behavioral assessments of pain in pre-clinical models, especially of ongoing pain, has been a major limitation for both pain neurobiology research and development of novel analgesics. Current methods typically require extensive animal-human observer interaction and are often followed by manual, thus subjective, scorings, which are susceptible to bias and variability, and are labor-intensive and time-consuming. Here we demonstrate that, by leveraging modern machine vision and machine learning tools, quantifiable features of the spontaneous behavior, or the “body language” of an animal recorded in a dark and observer-free environment from a bottom-up view, can be directly used to automatically detect and infer both the internal state of pain and analgesia in an animal, and from surface contact measurements the presence of mechanical hypersensitivity, in an objective manner. This approach minimizes animal handling, reduces numbers of animals needed and the potential for biased assessment, making it a useful tool for exploring the biology of pain and other neurological disorders, one readily scalable for pre-clinical drug efficacy assessment.

BACKGROUND Failure of the pulmonary valve autograft (PVA) after the Ross procedure (RP) has discouraged its widespread use and has led to modifications or alternatives to the procedure. We sought to analyze whether certain technical modifications could improve results of the RP in children. METHODS Sixty-nine patients (median age, 12 years; range, 0.25-17.9) underwent the RP between January 1996 and December 2018. A concomitant Konno procedure was performed on 20 of 69 patients (29%). Prior interventions included balloon valvuloplasty in 30 (44%) and/or surgical valvuloplasty in 39 (57%). Technical modifications included using the native aortic root for external annuloplasty, implanting the autograft using uniplanar horizontal sutures through the aortic wall, normalizing the sinotubular junction, and wrapping the native root remnant around the PVA. RESULTS Operative mortality was 1 of 69 patients (1.5%), with no late deaths. No patient had neoaortic valvar stenosis and 7 of 68 (10%) had mild regurgitation on discharge echocardiogram. At latest follow-up (median, 9.4 years; range, 0.4-21.3) there was no significant change in Z scores of annulus, sinus, or sinotubular junction diameters when compared with those at discharge. Three patients (4.4%) required late autograft replacement, 2 PVA repair, and 2 resection of a pseudoaneurysm. Actuarial freedom from PVA replacement was 87% at 20 years. Freedom from right ventricular outflow tract catheter reintervention or reoperation was 83% and 80%, respectively. CONCLUSIONS Technical modifications of the RP used in this cohort might successfully prolong the life of the PVA without compromising its growth, an important advantage in pediatric patients. (Ann Thorac Surg 2021;112:1997-2004) (c) 2021 by The Society of Thoracic Surgeons

Objective To evaluate risk factors for pediatric posttonsillectomy hemorrhage (PTH) and the need for transfusion using a national database. Study Design Retrospective cohort study. Setting The study was conducted using the Pediatric Health Information System (PHIS) database. Methods Children <= 18 years who underwent tonsillectomy with or without adenoidectomy (T +/- A) between 2004 and 2015 were included. We evaluated the risk of PTH requiring cauterization according to patient demographics, comorbidities, indication for surgery, medications, year of surgery, and geographic region. Results Of the 551,137 PHIS patients who underwent T +/- A, 8735 patients (1.58%) experienced a PTH. The risk of PTH increased from 1.33% (95% confidence interval [CI]: 1.15%, 1.53%) in 2010 to 1.91% (95% CI: 1.64%, 2.24%) in 2015 (P < .001). Older age (>= 12 vs <5 years old: adjusted odds ratio [aOR] 3.17; 95% CI: 2.86, 3.52), male sex (aOR 1.11; 95% CI: 1.05, 1.17), medical comorbidities (aOR 1.18; 95% CI: 1.08, 1.29), recurrent tonsillitis (aOR 1.15; 95% CI: 1.07, 1.24), and intensive care unit admission (aOR 1.74; 95% CI: 1.55, 1.95) were significantly associated with an increased risk of PTH. Use of ibuprofen (aOR 1.36; 95% CI: 1.22, 1.52), ketorolac (aOR 1.39; 95% CI: 1.14, 1.69), anticonvulsant (aOR 1.23; 95% CI: 1.03, 1.76), and antidepressants (aOR 1.35; 95% CI: 1.03, 1.76) were also associated with an increased risk of PTH. The need for blood transfusion was 2.1% (181/8735). Conclusion The incidence of PTH increased significantly between 2011 and 2015, and ibuprofen appears to be one contributing factor. Given the benefits of ibuprofen, it is unclear whether this increased risk warrants a change in practice.

AbstractBackground:Tetralogy of Fallot is a congenital heart defect diagnosed in infancy. Assessment of right ventricular size and function is important for evaluation of patients with tetralogy of Fallot, but these quantitative measures are challenging by echocardiography. This study evaluates a semi-automated software (EchoInsight®, Epsilon Imaging) by comparing its measures to manual measures in children with tetralogy of Fallot.Methods:Echocardiographic measurements were performed using manual techniques and semi-automated software. Right ventricular measurements included end-diastolic and end-systolic area, fractional area change, chamber dimensions, and tricuspid annular plane systolic excursion. Reliability, correlation, and agreement between manual and semi-automated measures were assessed.Results:Echocardiograms for 46 patients were analysed. Intra- and inter-observer reliabilities for semi-automated measures were good with intraclass correlation coefficients all over 0.95 and 0.85, respectively. There was high correlation between manual and semi-automated methods for areas and dimensions (r = 0.91–0.98). Tricuspid annular plane systolic excursion measures and fractional area change also correlated, albeit less strongly. The semi-automated measurements of end-systolic and end-diastolic area were a 20 and 47% higher than manual methods, respectively.The semi-automated method yielded a relative 52% lower fractional area change compared to the manual method.Conclusions:The semi-automated software generates quantitative right ventricular measures in children with tetralogy of Fallot with good reliability and good correlation with manual methods for all measures, but with significant difference between manual and semi-automated techniques for area and functional measures. The specific right ventricular geometry in tetralogy of Fallot children may be why, compared to normal anatomy, greater differences were observed between the two techniques.

Background: Despite improvements in the clinical management of hypoplastic left heart syndrome patients with Fontan palliation, this patient population is highly vulnerable to complications and mortality. Many will require heart transplant due to either inefficiencies of systemic ventricular dysfunction. Limited data exists to help determine an appropriate time for transplant referral. This study aims to determine if systemic ventricular strain by echocardiography would predict transplant free survival. Methods: Hypoplastic left …

The BioCompute Object (BCO) standard is an IEEE standard (IEEE 2791-2020) designed to facilitate the communication of next-generation sequencing data analysis with applications across academia, government agencies, and industry. For example, the Food and Drug Administration (FDA) supports the standard for regulatory submissions and includes the standard in their Data Standards Catalog for the submission of HTS data. We created the BCO App to facilitate BCO generation in a range of computational environments and, in part, to participate in the Advanced Track of the precisionFDA BioCompute Object App-a-thon. The application facilitates the generation of BCOs from both workflow metadata provided as plaintext and from workflow contents written in the Common Workflow Language. The application can also access and ingest task execution results from the Cancer Genomics Cloud (CGC), an NCI funded computational platform. Creating a BCO from a CGC task significantly reduces the time required to generate a BCO on the CGC by auto-populating workflow information fields from CGC workflow and task execution results. The BCO App supports exporting BCOs as JSON or PDF files and publishing BCOs to both the CGC platform and to GitHub repositories.

HPV16 causes half of cervical cancers worldwide; for unknown reasons, most infections resolve within two years. Here, we analyze the viral genomes of 5,328 HPV16-positive case-control samples to investigate mutational signatures and the role of human APOBEC3-induced mutations in viral clearance and cervical carcinogenesis. We identify four de novo mutational signatures, one of which matches the COSMIC APOBEC-associated signature 2. The viral genomes of the precancer/cancer cases are less likely to contain within-host somatic HPV16 APOBEC3-induced mutations (Fisher's exact test, P = 6.2 x 10(-14)), and have a 30% lower nonsynonymous APOBEC3 mutation burden compared to controls. We replicate the low prevalence of HPV16 APOBEC3-induced mutations in 1,749 additional cases. APOBEC3 mutations also historically contribute to the evolution of HPV16 lineages. We demonstrate that cervical infections with a greater burden of somatic HPV16 APOBEC3induced mutations are more likely to be benign or subsequently clear, suggesting they may reduce persistence, and thus progression, within the host.

Although multiple diseases of the respiratory system cause cough, there are few effective treatments for this common condition. We previously developed a strategy to treat pain and itch via the long-lasting inhibition of nociceptor sensory neurons with QX-314, a cationic sodium channel blocker that selectively enters only into activated nociceptors by permeating through the endogenous TRPV1 and TRPA1 large pore ion channels they express. In this study we design and characterize BW-031, a novel cationic compound with ∼6-fold greater potency than QX-314 for inhibiting sodium channels when introduced inside cells and with minimal extracellular activity. We show that inhalation of aerosolized BW-031 effectively inhibits citric acid-induced cough in an allergic inflammation guinea pig cough model. These data support the use of charged sodium channel blockers for the selective inhibition of airway sensory neurons with activated large pore channels as a novel targeted therapy for treating cough.

Background: The authors investigated the durability of vaccine efficacy (VE) against human papillomavirus (HPV)16 or 18 infections and antibody response among nonrandomly assigned women who received a single dose of the bivalent HPV vaccine compared with women who received multiple doses and unvaccinated women. Methods: HPV infections were compared between HPV16 or 18-vaccinated women aged 18 to 25 years who received one (N = 112), two (N = 62), or three (N = 1365) doses, and age- and geography-matched unvaccinated women (N = 1783) in the long-term follow-up of the Costa Rica HPV Vaccine Trial. Cervical HPV infections were measured at two study visits, approximately 9 and 11 years after initial HPV vaccination, using National Cancer Institute next-generation sequencing TypeSeq1 assay. VE and 95% confidence intervals (CIs) were estimated. HPV16 or 18 antibody levels were measured in all one- and two-dose women, and a subset of threedose women, using a virus-like particle-based enzyme-linked immunosorbent assay (n =448). Results: Median follow-up for the HPV-vaccinated group was 11.3 years (interquartile range = 10.9-11.7 years) and did not vary by dose group. VE against prevalent HPV16 or 18 infection was 80.2% (95% CI = 70.7% to 87.0%) among three-dose, 83.8% (95% CI = 19.5% to 99.2%) among two-dose, and 82.1% (95% CI = 40.2% to 97.0%) among single-dose women. HPV16 or 18 antibody levels did not qualitatively decline between years four and 11 regardless of the number of doses given, although one-dose titers continue to be statistically significantly lower compared with two- and three-dose titers. Conclusion: More than a decade after HPV vaccination, single-dose VE against HPV16 or 18 infection remained high and HPV16 or 18 antibodies remained stable. A single dose of bivalent HPV vaccine may induce sufficiently durable protection that obviates the need for more doses.

AbstractHuman papillomavirus (HPV) 16 displays substantial sequence variation; four HPV16 lineages (A, B, C, and D) have been described as well as multiple sublineages. To identify molecular events associated with HPV16 carcinogenesis, we evaluated viral variation, the integration of HPV16, and somatic mutation in 96 cervical cancer samples from Guatemala. A total of 65% (62/96) of the samples had integrated HPV16 sequences and integration was associated with an earlier age of diagnosis and premenopausal disease. HPV16 integration sites were broadly distributed in the genome, but in one tumor, HPV16 integrated into the promoter of the IFN regulatory factor 4 (IRF4) gene, which plays an important role in the regulation of the IFN response to viral infection. The HPV16 D2 and D3 sublineages were found in 23% and 30% of the tumors, respectively, and were significantly associated with adenocarcinoma. D2-positive tumors had a higher rate of integration, earlier age of diagnosis, and a lower rate of somatic mutation, whereas D3-positive tumors were less likely to integrate, had later age of diagnosis, and exhibited a higher rate of somatic mutation. In conclusion, Guatemalan cervical tumors have a high frequency of very high-risk HPV16 D2 and D3 sublineages harboring distinct histology, which may help guide future therapeutic strategies to target the tumor and reduce recurrence.Significance:This study details the biological and molecular properties of the most pathogenic forms of HPV16, the cause of the majority of cervical cancers.

Background Human papillomavirus (HPV) is a common sexually transmitted infection associated with cervical cancer that frequently occurs as a coinfection of types and subtypes. Highly similar sublineages that show over 100-fold differences in cancer risk are not distinguishable in coinfections with current typing methods. Results We describe an efficient set of computational tools, rkmh, for analyzing complex mixed infections of related viruses based on sequence data. rkmh makes extensive use of MinHash similarity measures, and includes utilities for removing host DNA and classifying reads by type, lineage, and sublineage. We show that rkmh is capable of assigning reads to their HPV type as well as HPV16 lineage and sublineages. Conclusions Accurate read classification enables estimates of percent composition when there are multiple infecting lineages or sublineages. While we demonstrate rkmh for HPV with multiple sequencing technologies, it is also applicable to other mixtures of related sequences.