Background:
Microsatellite instability-high (MSI-H) cancers are cancers with variability in short repeat sequences in numerous loci in the genome. The MSI-H phenotype is commonly a result of deficient DNA mismatch repair (dMMR), which may be caused by hereditary cancer syndromes such as Lynch Syndrome (LS). LS is characterized by MMR mutations in the germline and predisposes carriers to multiple cancers. EC is the most common extraintestinal malignancy predisposed by LS. LS screening for ECs patients is clinically useful but is a time-consuming and labour-intensive process. Checkpoint inhibitors (CPIs) such as PD-1 inhibitor pembrolizumab showed higher response in dMMR patients. Generation of frameshift neoantigens from second-hit mutations and subsequent T cell responses is hypothesized to be a contributor to the effectiveness of CPIs.
Purpose:
This study aims to select frequent frameshift neoantigens from cancer driver gene mutations, and to analyse their T cell binding properties and changes in TCR repertoire. These insights allow for further development of early diagnostic strategies and novel immunotherapies.
Methods:
A review of studies reporting somatic frameshift mutations in MSI-H ECs was conducted to shortlist the most frequent frameshift neoantigens. These will be screened against patient-derived T cells using the barcode-enabled antigen mapping T cell assay (BEAM-T) to detect the presence of compatible T cells in MSI-H tumours. The results will then be validated using bulk TCR sequencing.
Results:
The most frequent frameshift neoantigens are from RPL22 (52%), RNF43 (38%), DNA-PKcs (22%), JAK1 (21%) and PTEN (18%) among other cancer driver genes. These neoantigens will be further investigated using BEAM-T assays and TCR sequencing.
Conclusion:
In this ongoing project, frequent frameshift neoantigens were identified and will proceed into T cell assays, which will reveal neoantigen-specific T cells that can be further investigated for their effector functions, and in the development of early diagnostic strategies and novel immunotherapeutics.

Background: Ehler-Danlos syndromes (EDS) is an umbrella term describing 14 types of which 13 are monogenic with overlapping features including generalised joint hypermobility, skin and vascular fragility and generalised connective tissue friability. As DNA analysis has become the gold standard for a diagnosis of EDS, use of transmission electron microscopy (TEM) in clinical practice is decreasing. However, due to increased use of next generation sequencing, the frequency of variants of uncertain significance (VUS) is increasing as well. In certain cases of suspected monogenic EDS, TEM analysis of collagen structure may have the potential to contribute to variant classification.

Purpose: To demonstrate the added value of TEM in patients with suspicion on a monogenic EDS type.

Methods: The patient presented clinically with hyperextensible skin, skin fragility, mild atrophic scarring and generalised joint hypermobility, leading to a suspicion on monogenic EDS, in particular on classical or classical-like EDS. Whole Genome Sequencing revealed a COL5A1 variant c.3551C>T; Pro1184Leu which was classified as a cold VUS, unlikely to cause classical EDS. TEM analysis of a skin biopsy was performed to assess for abnormalities of collagen fibrils.

Results: Transmission electron microscopy studies on the patients' skin biopsy showed ultrastructural alterations in collagen fibril diameter and appearance consistent with collagen flowers. The clinical features of the patient together with the presence of abundant collagen flowers supported the suspicion on a rare EDS type in particular classical EDS or classical-like EDS despite the identified cold COL5A1 VUS. As a result, RNA sequencing in skin fibroblast is undertaken to assess for decreased expression of genes involved in monogenic EDS

Conclusions: Clinical assessment in combination with TEM analysis of collagen structure may have the potential to aid to identification and/or classification of variants in genes underlying specific rare, monogenic EDS types.

Purpose:
Medium-sized gene panels have a role in both research and diagnostic settings, particularly when they can be extended to include clinically relevant information such as microsatellite instability (MSI) and DPYD status. In conjunction with our commercial collaborator GeneFirst, we have optimised their medium-sized gene panel and tested its efficacy on a cohort of colorectal cancer (CRC) patients recruited to the Yorkshire Cancer Research funded Bowel Cancer Improvement Programme (YCRBCIP).


Methods:
We retrieved 515 formalin-fixed, paraffin embedded (FFPE) CRC resection specimens from participants recruited to the YCRBCIP, and following DNA extraction, sequenced these samples using the GeneFirst ATOM-Seq panel. The panel covers known mutational hotspots in 20 genes, with known clinical relevance in CRC, in addition to five DPYD SNPs and 29 microsatellite targets.


Results:
Analysis of known CRC driver mutation hotspots demonstrated that 19/515 (3.7%) of samples contained an NRAS mutation, 195/515 (37.9%) contained a KRAS mutation, 288/515 (55.9%) contained a TP53 mutation, 71/515 (13.6%) contained a PIK3CA mutation and 59/515 (11.5%) contained a BRAF mutation. In addition, 33/515 (6.4%) showed the presence of a DPYD SNP, indicating sensitivity to 5FU-based chemotherapy and 76/515 (14.8%) were classed as microsatellite unstable. Taking the cut-off for assay failure at a median coverage of <50X, 34/515 (6.6%) of samples failed the assay. Excluding DPYD, the variant allele frequencies (VAF) observed, ranged from 1.04-94.98. Time taken from extracted DNA to library prep completion was six hours.

Conclusions:
We have demonstrated that this novel medium-sized gene panel developed by GeneFirst, can detect the expected mutation frequencies across multiple driver genes, in a large cohort of CRC patients recruited across the Yorkshire and Humber region. Being able to also derive clinically relevant information pertaining to DPYD status and MSI status, makes this panel one which could be incorporated to either clinical diagnostic or research environments.

Background
Between the years of 2012 and 2022 there were 136 recreational diving deaths in the UK, an average of 12.4 per year, with around 3 to 5 of those occuring in the South West. The investigation of non-suspicious diving deaths falls to the Coroner, and due to the complexity of these cases they are often pushed, potentially unnecessarily, to forensic pathologists. The process requires a general understanding of the physiology of diving and how this interacts with natural disease, the interpretation of ancillary testing including toxicology and equipment testing reports, and the ability to synthesise the information into a logical sequence of events.

Purpose
This explores the processes in a non-forensic diving death investigation service in the South West of England which attempts to provide a greater level of understanding around recreational diving deaths than afforded by a routine Coronial autopsy.

Methods
With assistance from the outgoing regional diving pathology expert, we developed a service in conjunction with the HM Senior Coroner for the area filling a need in this coastal community.

Results
This includes a protocolised approach to ensure consistency and reproducibility of post mortem examination. This will also address some of the challenges and controversies within this area of practice.

Conclusions
The investigation of recreational diving deaths does not require a forensic approach, but rather a considered and informed approach incorporating knowledge of the processes involved in diving and how they interact with natural disease. With this in mind, we may be able to reduce the number of deaths certified simply as “drowning”, and improve the safety of the sport for all involved.