RNA-Seq for the detection of gene fusions in solid tumors: development and validation of the JAX FusionSeq™ 2.0 assay.

Whole transcriptome sequencing (RNA-Seq) has gained prominence for the detection of fusions in solid tumors. Here, we describe the development and validation of an in-house RNA-Seq-based test system (FusionSeq™ 2.0) for the detection of clinically actionable gene fusions, in formalin-fixed paraffin-embedded (FFPE) specimens, using seventy tumor samples with varying fusion status. Conditions were optimized for RNA input of 50 ng, shown to be adequate to call known fusions at as low as 20% neoplastic content. Evaluation of assay performance between FFPE and fresh-frozen (FF) tissues exhibited little to no difference in fusion calling capability. Performance analysis of the assay validation data determined 100% accuracy, sensitivity, specificity, and reproducibility. This clinically developed and validated RNA-Seq-based approach for fusion detection in FPPE samples was shown to be on par if not superior to off-the-shelf commercially offered assays. With gene fusions implicated in a variety of cancer types, offering high-quality, low-cost molecular testing services for FFPE specimens will serve to best benefit the patient and the advancement of precision medicine in molecular oncology. KEY MESSAGES: A custom RNA-Seq-based test system (FusionSeq™ 2.0) for the detection of clinically actionable gene fusions, Evaluation of assay performance between FFPE and fresh-frozen (FF) tissues exhibited little to no difference in fusion calling capability. The assay can be performed with low RNA input and neoplastic content. Performance characteristics of the assay validation data determined 100% accuracy, sensitivity, specificity, and reproducibility.

Evaluation of indicators supporting reproducibility and transparency within cardiology literature.

OBJECTIVES: It has been suggested that biomedical research is facing a reproducibility issue, yet the extent of reproducible research within the cardiology literature remains unclear. Thus, our main objective was to assess the quality of research published in cardiology journals by assessing for the presence of eight indicators of reproducibility and transparency. METHODS: Using a cross-sectional study design, we conducted an advanced search of the National Library of Medicine catalogue for publications in cardiology journals. We included publications published between 1 January 2014 and 31 December 2019. After the initial list of eligible cardiology publications was generated, we searched for full-text PDF versions using Open Access, Google Scholar and PubMed. Using a pilot-tested Google Form, a random sample of 532 publications were assessed for the presence of eight indicators of reproducibility and transparency. RESULTS: A total of 232 eligible publications were included in our final analysis. The majority of publications (224/232, 96.6%) did not provide access to complete and unmodified data sets, all 229/232 (98.7%) failed to provide step-by-step analysis scripts and 228/232 (98.3%) did not provide access to complete study protocols. CONCLUSIONS: The presentation of studies published in cardiology journals would make reproducing study outcomes challenging, at best. Solutions to increase the reproducibility and transparency of publications in cardiology journals is needed. Moving forward, addressing inadequate sharing of materials, raw data and key methodological details might help to better the landscape of reproducible research within the field.

Molecular profiling of gynecologic cancers for treatment and management of disease - demonstrating clinical significance using the AMP/ASCO/CAP guidelines for interpretation and reporting of somatic variants.

Molecular features of gynecologic cancers have been investigated in comprehensive studies, but correlation of these molecular signatures with clinical significance for precision medicine is yet to be established. Towards this end, we evaluated 95 gynecologic cancer cases submitted for testing using The JAX ActionSeq™ NGS panel. Molecular profiles were studied and compared to TCGA datasets to identify similarities and distinguishing features among subtypes. We identified 146 unique clinically significant variants (Tier I and II) across 45 of the 212 genes (21%), in 87% (83/95) of cases. TP53, PTEN, ARID1A, PIK3CA and ATM were the most commonly mutated genes; CCNE1 and ERBB2 amplifications were the most frequently detected copy-number alterations. PARP inhibitors were among the most commonly reported drug class with clinical trials, consistent with the frequency of DNA damage-response pathway mutations in our cohort. Overall, our study provides additional insight into the molecular profiles of gynecologic cancers, highlighting regulatory pathways involved, raising the potential implications for targeted therapeutic options currently available.

Molecular profiling of CNS tumors for the treatment and management of disease.

The World Health Organization (WHO) has defined more than 130 distinct central nervous system (CNS) tumor entities, of which glioblastoma is the most fatal primary brain tumor. However, the correlation of the molecular signatures of glioblastoma with clinical significance for precision medicine is not well-known. How, and to what extent these variants may affect clinical decision making remains uncertain. Here, we evaluate 48 glioblastomas submitted for testing using the JAX ActionSeq™ Next-generation sequencing (NGS) panel. We identified 131 clinically significant variants (Tier I and II) across 30 of the 212 genes (14%). TP53, EGFR, PTEN, IDH1 were the most commonly mutated genes; EGFR, CDK4 amplifications, and CDKN2A deletion were the most frequently detected copy-number alterations. CDK4/6 and PI3K inhibitors were among the most commonly reported drug class with FDA approved therapies and investigational therapies, which is consistent with the frequencies of these genes in our cohort. Overall, our study established the molecular profiles of glioblastoma based on the 2017 joint consensus guidelines by AMP/ASCO/CAP and provides the potential implications for targeted therapeutic options currently available.

Mutation Yield of a Custom 212-Gene Next-Generation Sequencing Panel for Solid Tumors: Clinical Experience of the First 260 Cases Tested Using the JAX ActionSeq™ Assay.

OBJECTIVE: The study aimed to retrospectively evaluate the positive yield rate of a custom 212-gene next-generation sequencing (NGS) panel, the JAX ActionSeq™ assay, used in molecular profiling of solid tumors for precision medicine. METHODS: We evaluated 261 cases tested over a 24-month period including cancers across 24 primary tissue types and report on the mutation yield in these cases. RESULTS: Thirty-three of the 261 cases (13%) had no detectable clinically significant variants. In the remaining 228 cases (87%), we identified 550 clinically significant variants in 88 of the 212 genes, with four of fewer clinically significant variants being detected in 62 of 88 genes (70%). TP53 had the highest number of variants (125), followed by APC (47), KRAS (47), ARID1A (20), PIK3CA (20) and EGFR (18). There were 38 tier I and 512 tier II variants, with two genes having only a tier I variant, seven genes having both a tier I and tier II variant, and 79 genes having at least one tier II variant. Overall, the ActionSeq™ assay detected clinically significant variants in 42% of the genes included in the panel (88/212), 68% of which (60/88) were detected in more than one tumor type. CONCLUSIONS: This study demonstrates that of the genes with documented involvement in cancer, only a limited number are currently clinically significant from a therapeutic, diagnostic and/or prognostic perspective.

Technical and Regulatory Considerations for Taking Liquid Biopsy to the Clinic: Validation of the JAX PlasmaMonitorTM Assay.

The standard of care in oncology has been genomic profiling of tumor tissue biopsies for the treatment and management of disease, which can prove to be quite challenging in terms of cost, invasiveness of procedure, and potential risk for the patient. As the number of available drugs in oncology continues to increase, so too does the demand for technologies and testing applications that can identify genomic alterations targetable by these new therapies. Liquid biopsies that use a blood draw from the diseased patient may offset the many disadvantages of the invasive procedure. However, as with any new technology or finding in the clinical field, the clinical utility of an analytical test such as that of the liquid biopsy has to be established. Here, we review the clinical testing space for liquid biopsy offerings and elucidate the technical and regulatory considerations to develop such an assay, using our recently validated PlasmaMonitorTM test.

Genomic Profiling of Two Histologically Distinct Rare Urothelial Cancers in a Clinical Setting to Identify Potential Therapeutic Options for Treatment and Management of Disease.

Molecular profiling of urothelial cancers for therapeutic and prognostic potential has been very limited due to the absence of cancer-specific targeted therapies. We describe here 2 clinical cases with a histological diagnosis of an invasive sarcomatoid and a poorly differentiated carcinoma favoring urothelial with some neuroendocrine differentiation, two of the rarer types of urothelial cancers, which were evaluated for mutations in 212 genes for single-nucleotide variants and copy-number variants and 53 genes for fusions associated with solid tumors. In both cases, we identified variants in 2 genes, ARID1A and CDKN2A, indicative of the role of dysregulation of chromatin remodeling and cell cycle control as being common features of bladder cancer, consistent with the proposed model of tumorigenesis in these rare, highly aggressive pathological subtypes. The presence of a KRAS mutation in the poorly differentiated cancer and a TP53 mutation in the sarcomatoid tumor is indicative of a distinctive profile and adds a potential layer of molecular stratification to these rarer histological subtypes. We present a comparative analysis of the histological, clinical, and molecular profile of both cases and discuss the potential to delineate these tumors at the molecular level keeping in mind the possible therapeutic implications.

Achilles tendon biomechanics in response to acute intense exercise.

Achilles tendinopathy is a common disorder and is more prevalent in men. Although differences in tendon mechanics between men and women have been reported, understanding of tendon mechanics in young active people is limited. Moreover, there is limited understanding of changes in tendon mechanics in response to acute exercise. Our purpose was to compare Achilles tendon mechanics in active young adult men and women at rest and after light and strenuous activity in the form of repeated jumping with an added load. Participants consisted of 17 men and 14 women (18-30 years) who were classified as being at least moderately physically active as defined by the International Physical Activity Questionnaire. Tendon force/elongation measures were obtained during an isometric plantarflexion contraction on an isokinetic dynamometer with simultaneous ultrasound imaging of the Achilles tendon approximate to the soleus myotendinous junction. Data were collected at rest, after a 10-minute treadmill walk, and after a fatigue protocol of 100 toe jumps performed in a Smith machine, with a load equaling 20% of body mass. We found greater tendon elongation, decreased stiffness, and lower Young's modulus only in women after the jumping exercise. Force and stress were not different between groups but decreased subsequent to the jumping exercise bout. In general, women had greater elongation and strain, less stiffness, and a lower Young's modulus during plantarflexor contraction. These data demonstrate differences in tendon mechanics between men and women and suggest a potential protective mechanism explaining the lower incidence of Achilles tendinopathy in women.

Measuring Achilles tendon mechanical properties: a reliable, noninvasive method.

The purpose of this technical report is to describe a cost-effective and highly reliable methodology to measure mechanical and material properties of the Achilles tendon. Subjects are positioned on an isokinetic dynamometer time synchronized to a diagnostic ultrasound device. A tendon fascicle distal to the soleus is visualized during a ramped isometric maximal plantarflexion contraction. Excursion of the fascicle and tendon torque output yield a force-elongation curve in which mechanical characteristics and material properties are derived. Excellent intrasession and intersession reliabilities were observed for both the dynamometer (intraclass correlation coefficient [ICC] 0.99, 0.95) and excursion (ICC 0.99, 0.93) measures. Practical applications for this methodology include examination of training regimes for optimal tendon adaptation and rehabilitation in the presence of tendinopathy.

Developing a clinical informatics system which supports a model of partnership among caregivers, patients, and families.

Partnership Care Delivery is an evidence-based practice model being developed at this institution. It emphasizes interdisciplinary collaboration, in which all members of the healthcare team work with the patient as active participants in care processes. In order to achieve true partnership among disciplines, and to engage the patient and family, it was important to create supportive clinical informatics. The new system will provide access to scientifically sound information and empower synergy of interdisciplinary teams united as partners with patients and families through shared information.

Transforming the fall-injury risk assessment tool from paper to electronic.

In order to ensure the highest possible safety for our patients, we identified a need to introduce a falls risk assessment tool with high sensitivity, specificity and reliability within our institution. A comparative research study was conducted which evaluated several instruments against actual patient falls. Based on the findings a new instrument was selected and converted to an electronic format compatible with existing nursing informatics systems. We are now testing the use of this version of the instrument for its ability to guide appropriate interventions that prevent falls.