Expanding the Clinical Utility of Targeted RNA Sequencing Panels beyond Gene Fusions to Complex, Intragenic Structural Rearrangements.

Gene fusions are a form of structural rearrangement well established as driver events in pediatric and adult cancers. The identification of such events holds clinical significance in the refinement, prognostication, and provision of treatment in cancer. Structural rearrangements also extend beyond fusions to include intragenic rearrangements, such as internal tandem duplications (ITDs) or exon-level deletions. These intragenic events have been increasingly implicated as cancer-promoting events. However, the detection of intragenic rearrangements may be challenging to resolve bioinformatically with short-read sequencing technologies and therefore may not be routinely assessed in panel-based testing. Within an academic clinical laboratory, over three years, a total of 608 disease-involved samples (522 hematologic malignancy, 86 solid tumors) underwent clinical testing using Anchored Multiplex PCR (AMP)-based RNA sequencing. Hematologic malignancies were evaluated using a custom Pan-Heme 154 gene panel, while solid tumors were assessed using a custom Pan-Solid 115 gene panel. Gene fusions, ITDs, and intragenic deletions were assessed for diagnostic, prognostic, or therapeutic significance. When considering gene fusions alone, we report an overall diagnostic yield of 36% (37% hematologic malignancy, 41% solid tumors). When including intragenic structural rearrangements, the overall diagnostic yield increased to 48% (48% hematologic malignancy, 45% solid tumor). We demonstrate the clinical utility of reporting structural rearrangements, including gene fusions and intragenic structural rearrangements, using an AMP-based RNA sequencing panel.

Real-Time PCR and Droplet Digital PCR Are Accurate and Reliable Methods To Quantify Pseudomonas syringae pv. actinidiae Biovar 3 in Kiwifruit Infected Plantlets.

Pseudomonas syringae pv. actinidiae is the etiological agent of kiwifruit canker disease, causing severe economic losses in kiwifruit production areas around the world. Rapid diagnosis, understanding of bacterial virulence, and rate of infection in kiwifruit cultivars are important in applying effective measures of disease control. P. syringae pv. actinidiae load in kiwifruit is currently determined by a labor-intense colony counting method with no high-throughput and specific quantification method being validated. In this work, we used three alternative P. syringae pv. actinidiae quantification methods in two infected kiwifruit cultivars: start of growth time, quantitative PCR (qPCR), and droplet digital PCR (ddPCR). Method performance in each case was compared with the colony counting method. Methods were validated using calibration curves obtained with serial dilutions of P. syringae pv. actinidiae biovar 3 (Psa3) inoculum and standard growth curves obtained from kiwifruit samples infected with Psa3 inoculum. All three alternative methods showed high correlation (r > 0.85) with the colony counting method. qPCR and ddPCR were very specific, sensitive (5 × 102 CFU/cm2), highly correlated to each other (r = 0.955), and flexible, allowing for sample storage. The inclusion of a kiwifruit biomass marker increased the methods' accuracy. The qPCR method was efficient and allowed for high-throughput processing, and the ddPCR method showed highly accurate results but was more expensive and time consuming. While not ideal for high-throughput processing, ddPCR was useful in developing accurate standard curves for the qPCR method. The combination of the two methods is high-throughput, specific for Psa3 quantification, and useful for research studies (e.g., disease phenotyping and host-pathogen interactions).

Genomic and outcome analyses of Ph-like ALL in NCI standard-risk patients: a report from the Children's Oncology Group.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL; BCR-ABL1-like ALL) in children with National Cancer Institute (NCI) intermediate- or high-risk (HR) ALL is associated with poor outcome. Ph-like ALL is characterized by genetic alterations that activate cytokine receptor and kinase signaling and may be amenable to treatment with tyrosine kinase inhibitors. The prevalence, outcome, and potential for targeted therapy of Ph-like ALL in standard-risk (SR) ALL is less clear. We retrospectively analyzed a cohort of 1023 SR childhood B-ALL consecutively enrolled in the Children's Oncology Group AALL0331 clinical trial. The Ph-like ALL gene expression profile was identified in 206 patients, and 67 patients with either BCR-ABL1 (n = 6) or ETV6-RUNX1 (n = 61) were excluded from downstream analysis, leaving 139 of 1023 (13.6%) as Ph-like. Targeted reverse transcription polymerase chain reaction assays and RNA-sequencing identified kinase-activating alterations in 38.8% of SR Ph-like cases, including CRLF2 rearrangements (29.5% of Ph-like), ABL-class fusions (1.4%), JAK2 fusions (1.4%), an NTRK3 fusion (0.7%), and other sequence mutations (IL7R, KRAS, NRAS; 5.6%). Patients with Ph-like ALL had inferior 7-year event-free survival compared with non-Ph-like ALL (82.4 ± 3.6% vs 90.7 ± 1.0%, P = .0022), with no difference in overall survival (93.2 ± 2.4% vs 95.8 ± 0.7%, P = .14). These findings illustrate the significant differences in the spectrum of kinase alterations and clinical outcome of Ph-like ALL based on presenting clinical features and establish that genomic alterations potentially targetable with approved kinase inhibitors are less frequent in SR than in HR ALL.

Exploring differences in stakeholders' perceptions of illegal bird trapping in Cyprus.

BACKGROUND: Cyprus is recognised as a hotspot for illegal bird trapping in the Mediterranean basin. A consumer demand for the Eurasian blackcap (Sylvia atricapilla) is driving the use of non-selective trapping methods, resulting in the indiscriminate killing of millions of migratory birds. Efforts to tackle the issue have so far been characterised mostly by a top-down approach, focusing on legislation and enforcement. However, trapping levels are not decreasing and conflict between stakeholder groups is intensifying. METHODS: To understand why efforts to stop illegal bird trapping have not been effective, we used semi-structured interviews to interview 18 local bird trappers and nine representatives from the pertinent environmental non-governmental organisations (NGOs) and the governmental agencies responsible for enforcing the legislation. RESULTS: We found distinct differences between the views of the local trapping community and the environmental NGOs, particularly on why trapping is occurring and its impact on the avifauna. This disparity has contributed to misrepresentations of both sides and a high degree of conflict, which is potentially proving counterproductive to conservation interventions. In addition, it appears that trappers are a heterogeneous group, likely driven by various motivations besides profit. CONCLUSION: We argue that stakeholders interested in reducing illegal bird trapping need to develop anti-poaching strategies that aim at minimising the disparity in the views, and subsequently the conflict, acknowledging also that trappers are not a homogenous group, as often treated.

Targetable kinase gene fusions in high-risk B-ALL: a study from the Children's Oncology Group.

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a high-risk subtype characterized by genomic alterations that activate cytokine receptor and kinase signaling. We examined the frequency and spectrum of targetable genetic lesions in a retrospective cohort of 1389 consecutively diagnosed patients with childhood B-lineage ALL with high-risk clinical features and/or elevated minimal residual disease at the end of remission induction therapy. The Ph-like gene expression profile was identified in 341 of 1389 patients, 57 of whom were excluded from additional analyses because of the presence of BCR-ABL1 (n = 46) or ETV6-RUNX1 (n = 11). Among the remaining 284 patients (20.4%), overexpression and rearrangement of CRLF2 (IGH-CRLF2 or P2RY8-CRLF2) were identified in 124 (43.7%), with concomitant genomic alterations activating the JAK-STAT pathway (JAK1, JAK2, IL7R) identified in 63 patients (50.8% of those with CRLF2 rearrangement). Among the remaining patients, using reverse transcriptase polymerase chain reaction or transcriptome sequencing, we identified targetable ABL-class fusions (ABL1, ABL2, CSF1R, and PDGFRB) in 14.1%, EPOR rearrangements or JAK2 fusions in 8.8%, alterations activating other JAK-STAT signaling genes (IL7R, SH2B3, JAK1) in 6.3% or other kinases (FLT3, NTRK3, LYN) in 4.6%, and mutations involving the Ras pathway (KRAS, NRAS, NF1, PTPN11) in 6% of those with Ph-like ALL. We identified 8 new rearrangement partners for 4 kinase genes previously reported to be rearranged in Ph-like ALL. The current findings provide support for the precision-medicine testing and treatment approach for Ph-like ALL implemented in Children's Oncology Group ALL trials.

Past, present and future of respiratory research: A survey of Canadian health care professionals.

BACKGROUND: The Canadian Respiratory Health Professionals (CRHP) is the multidisciplinary health care professional group of the Canadian Lung Association. Although the CRHP has a growing number of highly qualified researchers, the landscape of their research in Canada has not been described. OBJECTIVES: To describe the level of respiratory research engagement; identify barriers and facilitators to research engagement; describe the experience and interest in developing research skills; and identify priority areas of future respiratory research among health care professionals. METHODS: An online survey of CRHP members was used to collect demographic information; barriers and facilitators to conducting research; future directions in respiratory research; and research funding and mentorship. Experience with and interest in 'upskilling' research skills were also evaluated. RESULTS: A total of 119 surveys were completed (22% response rate), of which 69 (58%) respondents were engaged in respiratory research. Reasons for not being involved in respiratory research were lack of mentorship, support and funding. The top research areas were chronic obstructive pulmonary disease (74%) and asthma (41%). The top facilitators for research engagement were amount of funding (29%) and mentorship (28%). Respondents in research positions rated their experience in research skills as high; those in nonresearch positions as low. However, both groups expressed interest in improving their research skills. CONCLUSIONS: Areas of development, such as research skills, greater funding opportunities and mentorship to increase the research capacity of health care professionals in respiratory health were identified. Health professional researchers have an important role in the national respiratory research strategy to increase interdisciplinary engagement and build collaborative teams.

Work-related thumb disorders in South African physiotherapists treating musculoskeletal conditions using manual therapy techniques.

RESEARCH QUESTION: What is the prevalence of and factors associated with work-related thumb problems (WRTP) in South African physiotherapists treating musculoskeletal conditions using manual therapy techniques? DESIGN: A cross-sectional, descriptive study design was used and data were collected using two Internet-based questionnaires. PARTICIPANTS: The sample size calculated for the study was 284 using 95% confidence levels and a 5% margin of error. There were 395 participants that were included in the study. OUTCOME MEASURES: The variables measured included demographic, employment, educational and occupational factors. RESULTS: The lifetime prevalence of WRTP in the physiotherapists was 65.3%. The manual techniques that were significantly associated with WRTP in the respondents who reported thumb problems were all grades of transverse glides applied to the spine as well as grade II-IV unilateral and central posterior-anterior pressures to the spine. The factors that remained significantly associated with WRTP in all 395 respondents after regression analysis were the cervical treatment of up to six patients a day and hyperextension > 30° of the non-dominant interphalangeal (IP) joint of the thumb. CONCLUSION: This study confirms that a high percentage of physiotherapists using manual therapy techniques to treat musculoskeletal conditions are experiencing WRTP. RECOMMENDATIONS: The development of a valid and reliable WRTP screening tool is needed to aid in the identification of physiotherapists at risk and thus in the primary prevention of WRTP. A longitudinal study which follows newly qualified physiotherapists is recommended to investigate a possible cause-effect relationship and preventative strategies for WRTP in physiotherapists.

Effects of seed cryopreservation, stratification and scarification on germination for five rare species of pitcher plants.

BACKGROUND: Habitat loss and over collection have caused North American pitcher plants to become rare, including U.S. federally endangered Sarracenia alabamensis and S. oreophila, and S. leucophylla, S. psittacina and S. purpurea spp. venosa, endangered in several states. OBJECTIVE: To develop reliable seed cryopreservation protocols for endangered Sarracenia species enabling similar germination percentages before and after storage in liquid nitrogen (LN) either in vivo or using in vitro tools. METHODS: Seed germination pre- and post-cryopreservation were compared following seed drying with germination in soil, aseptic environment with wet filter paper or enriched medium, and using scarification or stratification for dormancy removal. RESULTS: After cryostorage, germination in vitro (1/6- or 1/3-strength MS medium) increased compared to germination on peat moss. Germination pre- and post-cryopreservation was similar for S. alabamensis and S. oreophila when seeds were stratified and grown in vitro. S. leucophylla and S. psittacina also showed high germination after cryopreservation when germinated on medium following stratification. CONCLUSION: Rapid liquid nitrogen exposure and rewarming induced seed coat cracking that damaged seeds, likely allowing internal damage during acid scarification and microbial entry during germination in non-sterile environments.

Vaccinia virus DNA ligase recruits cellular topoisomerase II to sites of viral replication and assembly.

Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drug-resistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase IIbeta. One can prevent the interaction by introducing a C(11)-to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase IIalpha/beta in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase IIalpha and IIbeta antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases IIalpha/beta in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.

Removal Tools are Faster and Produce Less Force and Torque on the Helmet Than Cutting Tools During Face-Mask Retraction.

OBJECTIVE: To investigate the retraction time, forces, and torques applied to the football helmet during removal of the face mask with different face-mask removal tools. DESIGN AND SETTING: Subjects retracted the face mask of a football helmet mounted to a force platform in a laboratory setting. They removed a standard face mask by cutting or removing (or both) the lateral plastic loop straps using 4 different tools: the Trainer's Angel (TA), FM Extractor (FM), power screwdriver (SD), and Quick Release System (QR) in a counterbalanced fashion. SUBJECTS: Eighteen certified athletic trainers participated in this study. MEASUREMENTS: We started measuring time when the subject picked up the tool and ended when the face mask was in a fully retracted position. Maximum forces and torques were measured from the force platform during the retraction process. RESULTS: The SD and QR retracted the face mask significantly faster than the TA and FM. Forces producing superior-inferior translation were least with the SD. The SD and QR produced less lateral translation and rotation and lateral flexion moment than the TA and FM. The FM produced less torque in the lateral flexion moment than the TA. CONCLUSIONS: Tools that removed the loop straps (SD, QR) were faster and produced less force and torque on the helmet than the tools that cut through the loop straps (TA, FM).