Female car users are reported to have a higher incidence of soft tissue neck injuries in low speed rear-end collisions than males, and they apparently take longer to recover. This paper addresses the whiplash problem by developing a biomechanical FEM (Finite Element Method) model of the 50th and the 5th percentile female cervical spines, based on the earlier published male model created at the Nottingham Trent University. This model relies on grafting a detailed biomechanical model of the neck and head onto a standard HYBRID III dummy model. The overall philosophy of the investigation was to see if females responded essentially as scaled down males from the perspective of rear end collisions. It was found that detailed responses varied significantly with gender and it became clear that females cannot be modelled as scaled-down males, thus confirming the need for separate male and female biomechanical models and a revision of car test programmes and regulations which are currently based on the average male. Further investigation is needed to quantify the gender differences and then recommendations can be made for changes to the design of car seats and head restraints in order to reduce the risk of soft tissue injury to women.

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Introduction: The FDA has approved 11 therapies for non-small cell lung cancer (NSCLC) patients diagnosed with gene fusions. Importantly, these life-saving targeted therapies remain underutilized due to a lack of cost-effective, comprehensive, and simple-to-use technologies for gene fusion detection. Our previous work using ASPYRE technology (allele-specific pyrophosphorolysis reaction; Silva et al, 2020) demonstrated detection of somatic variants at single-molecule level from DNA extracted from tumor tissue. However, DNA-based fusion detection requires tiling across large introns, which limits sensitivity particularly if breakpoints are located in repetitive regions of the genome. Here, we show that ASPYRE technology can also be utilized for highly sensitive detection of RNA fusions, scaling to over 30 fusion targets covering the main 3’ fusion partners (ALK, ROS1, RET, NTRK1, NTRK3) and from a single reverse transcription PCR (RT-PCR) amplification reaction. Methods: The ASPYRE assay consists of four sequential enzymatic stages: RT-PCR; enzymatic clean-up; combined exonuclease digestion, hybridization, pyrophosphorolysis and ligation; rolling circle isothermal amplification and detection. All targets are detected across two reaction wells using four channels on a standard real-time PCR instrument. A panel of synthetic RNA oligonucleotides comprising gene fusion boundaries was spiked into a background pool of lung-RNA, quantified using digital PCR (dPCR) and compared against a commercial reference standard to validate its suitability for assessing assay performance. RNA was extracted from formalin-fixed paraffin-embedded (FFPE) lung tissue from healthy donors to determine assay specificity, and from FFPE lung tissue from NSCLC patients with known fusions to exemplify end-to-end sample analysis. Results: We demonstrate equivalence of our in-house reference samples to commercial reference standards. Using serial dilutions of input RNA standards, we show that detection by ASPYRE is consistent with detection of single molecules as input for common ROS1, ALK, RET and NTRK3 fusions. In addition, ASPYRE was able to detect previously confirmed ROS1 and ALK fusions and MET exon skipping variants in FFPE samples. ASPYRE had high specificity, correctly confirming the absence of RNA fusions and exon skipping variants in healthy control FFPE samples. Conclusions: ASPYRE approached single-molecule sensitivity for over 30 RNA fusion targets in a single reaction. The assay required only 1 ng RNA input, with a turn-around time of <4 hours from extracted RNA to result, opening the door to routine detection of RNA fusions from limited specimens. The simplicity and ease of use of ASPYRE, combined with its rapid turnaround time and analytical performance characteristics, provides a new tool for rapid and comprehensive treatment selection in NSCLC. Citation Format: Eleanor Gray, Justyna Mordaka, Efthimia Christoforou, Kristine von Bargen, Nicola Potts, Rebecca Palmer, Barnaby Balmforth. Low-cost, simple and rapid assay for single-molecule detection of gene fusions from RNA with ASPYRE technology [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 4104.