Characterisation of premature cell senescence in Alzheimer's disease using single nuclear transcriptomics.

Aging is associated with cell senescence and is the major risk factor for AD. We characterized premature cell senescence in postmortem brains from non-diseased controls (NDC) and donors with Alzheimer's disease (AD) using imaging mass cytometry (IMC) and single nuclear RNA (snRNA) sequencing (> 200,000 nuclei). We found increases in numbers of glia immunostaining for galactosidase beta (> fourfold) and p16INK4A (up to twofold) with AD relative to NDC. Increased glial expression of genes related to senescence was associated with greater ß-amyloid load. Prematurely senescent microglia downregulated phagocytic pathways suggesting reduced capacity for ß-amyloid clearance. Gene set enrichment and pseudo-time trajectories described extensive DNA double-strand breaks (DSBs), mitochondrial dysfunction and ER stress associated with increased ß-amyloid leading to premature senescence in microglia. We replicated these observations with independent AD snRNA-seq datasets. Our results describe a burden of senescent glia with AD that is sufficiently high to contribute to disease progression. These findings support the hypothesis that microglia are a primary target for senolytic treatments in AD.

A single nuclear transcriptomic characterisation of mechanisms responsible for impaired angiogenesis and blood-brain barrier function in Alzheimer's disease.

Brain perfusion and blood-brain barrier (BBB) integrity are reduced early in Alzheimer's disease (AD). We performed single nucleus RNA sequencing of vascular cells isolated from AD and non-diseased control brains to characterise pathological transcriptional signatures responsible for this. We show that endothelial cells (EC) are enriched for expression of genes associated with susceptibility to AD. Increased ß-amyloid is associated with BBB impairment and a dysfunctional angiogenic response related to a failure of increased pro-angiogenic HIF1A to increased VEGFA signalling to EC. This is associated with vascular inflammatory activation, EC senescence and apoptosis. Our genomic dissection of vascular cell risk gene enrichment provides evidence for a role of EC pathology in AD and suggests that reducing vascular inflammatory activation and restoring effective angiogenesis could reduce vascular dysfunction contributing to the genesis or progression of early AD.

Translocator protein is a marker of activated microglia in rodent models but not human neurodegenerative diseases.

Microglial activation plays central roles in neuroinflammatory and neurodegenerative diseases. Positron emission tomography (PET) targeting 18 kDa Translocator Protein (TSPO) is widely used for localising inflammation in vivo, but its quantitative interpretation remains uncertain. We show that TSPO expression increases in activated microglia in mouse brain disease models but does not change in a non-human primate disease model or in common neurodegenerative and neuroinflammatory human diseases. We describe genetic divergence in the TSPO gene promoter, consistent with the hypothesis that the increase in TSPO expression in activated myeloid cells depends on the transcription factor AP1 and is unique to a subset of rodent species within the Muroidea superfamily. Finally, we identify LCP2 and TFEC as potential markers of microglial activation in humans. These data emphasise that TSPO expression in human myeloid cells is related to different phenomena than in mice, and that TSPO-PET signals in humans reflect the density of inflammatory cells rather than activation state.

Distinct characteristics of microglia from neurogenic and non-neurogenic regions of the human brain in patients with Mesial Temporal Lobe Epilepsy.

The study of microglia isolated from adult human brain tissue provides unique insight into the physiology of these brain immune cells and their role in adult human brain disorders. Reports of microglia in post-mortem adult human brain tissue show regional differences in microglial populations, however, these differences have not been fully explored in living microglia. In this study biopsy tissue was obtained from epileptic patients undergoing surgery and consisted of both cortical areas and neurogenic ventricular and hippocampal (Hp) areas. Microglia were concurrently isolated from both regions and compared by immunochemistry. Our initial observation was that a greater number of microglia resulted from isolation and culture of ventricular/Hp tissue than cortical tissue. This was found to be due to a greater proliferative capacity of microglia from ventricular/Hp regions compared to the cortex. Additionally, ventricular/Hp microglia had a greater proliferative response to the microglial mitogen Macrophage Colony-Stimulating Factor (M-CSF). This enhanced response was found to be associated with higher M-CSF receptor expression and higher expression of proteins involved in M-CSF signalling DAP12 and C/EBPß. Microglia from the ventricular/Hp region also displayed higher expression of the receptor for Insulin-like Growth Factor-1, a molecule with some functional similarity to M-CSF. Compared to microglia isolated from the cortex, ventricular/Hp microglia showed increased HLA-DP, DQ, DR antigen presentation protein expression and a rounded morphology. These findings show that microglia from adult human brain neurogenic regions are more proliferative than cortical microglia and have a distinct protein expression profile. The data present a case for differential microglial phenotype and function in different regions of the adult human brain and suggest that microglia in adult neurogenic regions are "primed" to an activated state by their unique tissue environment.

Understanding medical students' transformative experiences of early preclinical international rural placement over a 20-year period.

INTRODUCTION: Rural placements are an important component of rural medical education programs seeking to develop rural practice pathways for medical students. These placements are usually domestic, but James Cook University in Australia developed an international rural placement program in the first half of the medical course that was funded through bursaries. This study explores how the international rural placement helped to shape the lives (personal development and learning) of the participants, using Transformational Learning Theory as a framework for identifying and describing the transformational elements, process and impact of the program. METHODS: Sixty-five students received a bursary for an international rural placement between 2001-2019. All were contacted by email and invited to participate in a short survey and a follow-up interview. Fifteen participants agreed and twelve were able to participate in individual semi-structured interviews which were recorded, transcribed and analysed using inductive thematic analysis. RESULTS: Participants reported that the bursary provided a "once in a lifetime opportunity" to "experience eye-opening and culturally rich difference". Nonetheless, some elements of the placement experience presented disorientating dilemmas that triggered deep reflections and shifts in perceptions. The bursary recipients realised that "being open-minded" allowed them "enjoy good company". They were also able to assume "outsider view which allowed reassessment of their own country" and the "isolation experiences gingered desire to right health wrongs". The triggers and mental shifts had significant impact on the bursary recipients and fostered the development of "inspirational new horizons" based on an appreciation of the "value of rural practice" and "role-modelling for life-long learning." These findings are consistent with Transformational Learning Theory. CONCLUSION: Participants in this study reported meaningful and strongly positive impacts from the experiences gained during an international rural clinical placement early in their course. They described transformative experiences which appear to contribute strongly to personal development. This finding supports maintaining opportunities for international experiences during rurally-oriented medical programs as these may impact longer term career choice.

Reactive astrocytes acquire neuroprotective as well as deleterious signatures in response to Tau and Aß pathology.

Alzheimer's disease (AD) alters astrocytes, but the effect of Aß and Tau pathology is poorly understood. TRAP-seq translatome analysis of astrocytes in APP/PS1 ß-amyloidopathy and MAPTP301S tauopathy mice revealed that only Aß influenced expression of AD risk genes, but both pathologies precociously induced age-dependent changes, and had distinct but overlapping signatures found in human post-mortem AD astrocytes. Both Aß and Tau pathology induced an astrocyte signature involving repression of bioenergetic and translation machinery, and induction of inflammation pathways plus protein degradation/proteostasis genes, the latter enriched in targets of inflammatory mediator Spi1 and stress-activated cytoprotective Nrf2. Astrocyte-specific Nrf2 expression induced a reactive phenotype which recapitulated elements of this proteostasis signature, reduced Aß deposition and phospho-tau accumulation in their respective models, and rescued brain-wide transcriptional deregulation, cellular pathology, neurodegeneration and behavioural/cognitive deficits. Thus, Aß and Tau induce overlapping astrocyte profiles associated with both deleterious and adaptive-protective signals, the latter of which can slow patho-progression.

Diverse human astrocyte and microglial transcriptional responses to Alzheimer's pathology.

To better define roles that astrocytes and microglia play in Alzheimer's disease (AD), we used single-nuclei RNA-sequencing to comprehensively characterise transcriptomes in astrocyte and microglia nuclei selectively enriched during isolation post-mortem from neuropathologically defined AD and control brains with a range of amyloid-beta and phospho-tau (pTau) pathology. Significant differences in glial gene expression (including AD risk genes expressed in both the astrocytes [CLU, MEF2C, IQCK] and microglia [APOE, MS4A6A, PILRA]) were correlated with tissue amyloid or pTau expression. The differentially expressed genes were distinct between with the two cell types and pathologies, although common (but cell-type specific) gene sets were enriched with both pathologies in each cell type. Astrocytes showed enrichment for proteostatic, inflammatory and metal ion homeostasis pathways. Pathways for phagocytosis, inflammation and proteostasis were enriched in microglia and perivascular macrophages with greater tissue amyloid, but IL1-related pathway enrichment was found specifically in association with pTau. We also found distinguishable sub-clusters in the astrocytes and microglia characterised by transcriptional signatures related to either homeostatic functions or disease pathology. Gene co-expression analyses revealed potential functional associations of soluble biomarkers of AD in astrocytes (CLU) and microglia (GPNMB). Our work highlights responses of both astrocytes and microglia for pathological protein clearance and inflammation, as well as glial transcriptional diversity in AD.

Genomic and Transcriptomic Characterization of Relapsed SCLC Through Rapid Research Autopsy.

INTRODUCTION: Relapsed SCLC is characterized by therapeutic resistance and high mortality rate. Despite decades of research, mechanisms responsible for therapeutic resistance have remained elusive owing to limited tissues available for molecular studies. Thus, an unmet need remains for molecular characterization of relapsed SCLC to facilitate development of effective therapies. METHODS: We performed whole-exome and transcriptome sequencing of metastatic tumor samples procured from research autopsies of five patients with relapsed SCLC. We implemented bioinformatics tools to infer subclonal phylogeny and identify recurrent genomic alterations. We implemented immune cell signature and single-sample gene set enrichment analyses on tumor and normal transcriptome data from autopsy and additional primary and relapsed SCLC data sets. Furthermore, we evaluated T cell-inflamed gene expression profiles in neuroendocrine (ASCL1, NEUROD1) and non-neuroendocrine (YAP1, POU2F3) SCLC subtypes. RESULTS: Exome sequencing revealed clonal heterogeneity (intertumor and intratumor) arising from branched evolution and identified resistance-associated truncal and subclonal alterations in relapsed SCLC. Transcriptome analyses further revealed a noninflamed phenotype in neuroendocrine SCLC subtypes (ASCL1, NEUROD1) associated with decreased expression of genes involved in adaptive antitumor immunity whereas non-neuroendocrine subtypes (YAP1, POU2F3) revealed a more inflamed phenotype. CONCLUSIONS: Our results reveal substantial tumor heterogeneity and complex clonal evolution in relapsed SCLC. Furthermore, we report that neuroendocrine SCLC subtypes are immunologically cold, thus explaining decreased responsiveness to immune checkpoint blockade. These results suggest that the mechanisms of innate and acquired therapeutic resistances are subtype-specific in SCLC and highlight the need for continued investigation to bolster therapy selection and development for this cancer.

Cross-platform transcriptional profiling identifies common and distinct molecular pathologies in Lewy body diseases.

Parkinson's disease (PD), Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB) are three clinically, genetically and neuropathologically overlapping neurodegenerative diseases collectively known as the Lewy body diseases (LBDs). A variety of molecular mechanisms have been implicated in PD pathogenesis, but the mechanisms underlying PDD and DLB remain largely unknown, a knowledge gap that presents an impediment to the discovery of disease-modifying therapies. Transcriptomic profiling can contribute to addressing this gap, but remains limited in the LBDs. Here, we applied paired bulk-tissue and single-nucleus RNA-sequencing to anterior cingulate cortex samples derived from 28 individuals, including healthy controls, PD, PDD and DLB cases (n = 7 per group), to transcriptomically profile the LBDs. Using this approach, we (i) found transcriptional alterations in multiple cell types across the LBDs; (ii) discovered evidence for widespread dysregulation of RNA splicing, particularly in PDD and DLB; (iii) identified potential splicing factors, with links to other dementia-related neurodegenerative diseases, coordinating this dysregulation; and (iv) identified transcriptomic commonalities and distinctions between the LBDs that inform understanding of the relationships between these three clinical disorders. Together, these findings have important implications for the design of RNA-targeted therapies for these diseases and highlight a potential molecular "window" of therapeutic opportunity between the initial onset of PD and subsequent development of Lewy body dementia.

Understanding Older Adults' Memory Distortion in the Light of Stereotype Threat.

Numerous studies have documented the detrimental impact of age-based stereotype threat (ABST) on older adults' cognitive performance and especially on veridical memory. However, far fewer studies have investigated the impact of ABST on older adults' memory distortion. Here, we review the subset of research examining memory distortion and provide evidence for the role of stereotype threat as a powerful socio-emotional factor that impacts age-related susceptibility to memory distortion. In this review we define memory distortion as errors in memory that are associated with gist-based errors or source misattributions. Whereas, some of the reviewed experiments support the conclusion that ABST should be considered in the context of age-related differences in memory distortion, others reported little or no impact of stereotype threat. These discrepancies suggest that the role of ABST, and socio-emotional processes generally, in age-related changes in memory distortion are less clear. In this review, we argue that ABST does play an important role in age-related changes in memory distortion. We present evidence suggesting that discrepancies in the reviewed literature may be reconciled when evaluated in the context of the leading theories about stereotype threat: the Executive Resource Depletion hypothesis and the Regulatory Focus theory. We also discuss how differences in methodology and participant characteristics can account for a priori contradictory results in the literature. Finally, we propose some recommendations for researchers and practitioners when assessing memory in older adults.

Examiners' decision-making processes in observation-based clinical examinations.

BACKGROUND: Objective structured clinical examinations (OSCEs) are commonly used to assess the clinical skills of health professional students. Examiner judgement is one acknowledged source of variation in candidate marks. This paper reports an exploration of examiner decision making to better characterise the cognitive processes and workload associated with making judgements of clinical performance in exit-level OSCEs. METHODS: Fifty-five examiners for exit-level OSCEs at five Australian medical schools completed a NASA Task Load Index (TLX) measure of cognitive load and participated in focus group interviews immediately after the OSCE session. Discussions focused on how decisions were made for borderline and clear pass candidates. Interviews were transcribed, coded and thematically analysed. NASA TLX results were quantitatively analysed. RESULTS: Examiners self-reported higher cognitive workload levels when assessing a borderline candidate in comparison with a clear pass candidate. Further analysis revealed five major themes considered by examiners when marking candidate performance in an OSCE: (a) use of marking criteria as a source of reassurance; (b) difficulty adhering to the marking sheet under certain conditions; (c) demeanour of candidates; (d) patient safety, and (e) calibration using a mental construct of the 'mythical [prototypical] intern'. Examiners demonstrated particularly higher mental demand when assessing borderline compared to clear pass candidates. CONCLUSIONS: Examiners demonstrate that judging candidate performance is a complex, cognitively difficult task, particularly when performance is of borderline or lower standard. At programme exit level, examiners intuitively want to rate candidates against a construct of a prototypical graduate when marking criteria appear not to describe both what and how a passing candidate should demonstrate when completing clinical tasks. This construct should be shared, agreed upon and aligned with marking criteria to best guide examiner training and calibration. Achieving this integration may improve the accuracy and consistency of examiner judgements and reduce cognitive workload.

Characterization of Clonal Evolution in Microsatellite Unstable Metastatic Cancers through Multiregional Tumor Sequencing.

Microsatellites are short, repetitive segments of DNA, which are dysregulated in mismatch repair-deficient (MMRd) tumors resulting in microsatellite instability (MSI). MSI has been identified in many human cancer types with varying incidence, and microsatellite instability-high (MSI-H) tumors often exhibit increased sensitivity to immune-enhancing therapies such as PD-1/PD-L1 inhibition. Next-generation sequencing (NGS) has permitted advancements in MSI detection, and recent computational advances have enabled characterization of tumor heterogeneity via NGS. However, the evolution and heterogeneity of microsatellite changes in MSI-positive tumors remains poorly described. We determined MSI status in 6 patients using our previously published algorithm, MANTIS, and inferred subclonal composition and phylogeny with Canopy and SuperFreq. We developed a simulated annealing-based method to characterize microsatellite length distributions in specific subclones and assessed the evolution of MSI in the context of tumor heterogeneity. We identified three to eight tumor subclones per patient, and each subclone exhibited MMRd-associated base substitution signatures. We noted that microsatellites tend to shorten over time, and that MMRd fosters heterogeneity by introducing novel mutations throughout the disease course. Some microsatellites are altered among all subclones in a patient, whereas other loci are only altered in particular subclones corresponding to subclonal phylogenetic relationships. Overall, our results indicate that MMRd is a substantial driver of heterogeneity, leading to both MSI and subclonal divergence. IMPLICATIONS: We leveraged subclonal inference to assess clonal evolution based on somatic mutations and microsatellites, which provides insight into MMRd as a dynamic mutagenic process in MSI-H malignancies.

Early conscious prone positioning in patients with COVID-19 receiving continuous positive airway pressure: a retrospective analysis.

The global pandemic of COVID-19 has challenged the management of hypoxaemic respiratory failure and strained intensive care unit resources. While prone positioning (PP) is an established therapy in mechanically ventilated patients with acute respiratory distress syndrome (ARDS), its role in conscious patients is less well defined. We retrospectively reviewed our experience of implementing early PP in a cohort of 24 patients with acute hypoxaemic respiratory failure due to COVID-19 who required support with continuous positive airway pressure (CPAP). The use of PP alongside CPAP significantly increased both the ROX index and arterial oxygen pressure:fractional inspired oxygen (PaO2:FiO2) ratio from baseline values (ROX index: 7.0±2.5 baseline vs 11.4±3.7 CPAP+PP, p<0.0001; PaO2:FiO2 ratio: 143±73 mm Hg baseline vs 252±87 mm Hg CPAP+PP, p<0.01), and the changes to both the ROX index and PaO2:FiO2 ratio remained significant 1 hour after cessation of proning. The mean duration of PP in the first 24 hours was 8±5 hours. Few complications were observed and PP was continued for a mean of 10±5 days. From our experience in a dedicated COVID-19 respiratory high care unit, PP alongside CPAP therapy was feasible, tolerated, safe and improved oxygenation. The use of conscious PP in ARDS warrants further investigation in randomised controlled trials.

To stay or go? Unpacking the decision-making process and coping strategies of International Medical Graduates practising in rural, remote, and regional Queensland, Australia.

Australia is one of many countries to rely on International Medical Graduates (IMGs) to fill general practitioner (GP) positions throughout its regional, rural, and remote (RRR) communities. Current government initiatives requiring IMGs to work for specified periods in RRR areas offer only short-term solutions. The need to improve the long-term retention of IMGs practising in RRR areas has motivated this research to improve our understanding of how IMGs make decisions about where to practise. Specifically, this study sought to: (a) identify the factors that influence an IMG's decision to remain working in RRR areas, and (b) develop a theory, grounded in the data, to explain how these factors are prioritised, evaluated and used to inform a decision to remain working in RRR areas. This study adopted a qualitative approach and employed grounded theory methods. Data collection and analysis occurred concurrently, using constant, comparative analysis, guided by theoretical sampling and data saturation. Data sources were transcripts from semi-structured interviews with IMG registrars (n = 20) and supervisors (n = 5), interviewers' notes and analytic memos. Interviewees were all currently working in RRR areas of Queensland, Australia. The analysis involved a three-phase coding process, progressing from specific, inductive coding to abstract, abductive coding. The analysis revealed that the IMG decision-making process involves a complex, dynamic, and iterative process of balancing life goals based on life stage. Many factors are considered when assessing the balance of three main life goals: satisfaction with work, family, and lifestyle. The prioritisation and balance of these life goals can vary as the IMG moves through varying work-, family-, and age-related life stages. It is hoped that having this understanding of the complexity of the IMG decision-making process, will better equip medical educators, policy makers and support service providers to tailor services to encourage IMGs to continue practising in these regions.

Efficacy of FGFR Inhibitors and Combination Therapies for Acquired Resistance in FGFR2-Fusion Cholangiocarcinoma.

The fibroblast growth factor receptor (FGFR) signaling pathway is aberrantly activated in approximately 15% to 20% of patients with intrahepatic cholangiocarcinoma. Currently, several FGFR kinase inhibitors are being assessed in clinical trials for patients with FGFR-altered cholangiocarcinoma. Despite evidence of initial responses and disease control, virtually all patients eventually develop acquired resistance. Thus, there is a critical need for the development of innovative therapeutic strategies to overcome acquired drug resistance. Here, we present findings from a patient with FGFR2-altered metastatic cholangiocarcinoma who enrolled in a phase II clinical trial of the FGFR inhibitor, infigratinib (BGJ398). Treatment was initially effective as demonstrated by imaging and tumor marker response; however, after 8 months on trial, the patient exhibited tumor regrowth and disease progression. Targeted sequencing of tumor DNA after disease progression revealed the FGFR2 kinase domain p.E565A and p.L617M single-nucleotide variants (SNV) hypothesized to drive acquired resistance to infigratinib. The sensitivities of these FGFR2 SNVs, which were detected post-infigratinib therapy, were extended to include clinically relevant FGFR inhibitors, including AZD4547, erdafitinib (JNJ-42756493), dovitinib, ponatinib, and TAS120, and were evaluated in vitro Through a proteomics approach, we identified upregulation of the PI3K/AKT/mTOR signaling pathway in cells harboring the FGFR2 p.E565A mutation and demonstrated that combination therapy strategies with FGFR and mTOR inhibitors may be used to overcome resistance to FGFR inhibition, specific to infigratinib. Collectively, these studies support the development of novel combination therapeutic strategies in addition to the next generation of FGFR inhibitors to overcome acquired resistance in patients.

Retrieval practice enhances near but not far transfer of spatial memory.

Students learn more effectively through repeated retrieval of study materials relative to repeated exposure to the materials, a phenomenon known as the testing effect or retrieval practice. This pattern has been demonstrated repeatedly with verbal materials, and more recently with visuospatial materials. The extent to which retrieval practice produces spatial memories that successfully transfer to more diverse task demands remains unknown. Transferring spatial memory to novel task demands can involve challenging orientation and perspective transformations, possibly limiting the benefits of retrieval practice for application to realistic spatial tasks. In 4 experiments, participants learned a map of a large-scale urban environment, engaging in either study practice (repeated exposure) or retrieval practice (exposure and testing). Across experiments we varied the retrieval demands of the final memory test, increasing the breadth of transfer from study to test (from near to far transfer). Final memory tests included reconstructing a map from memory (Experiment 1), judgments of relative direction from an allocentric perspective (Experiment 2), judgments of relative direction from an egocentric perspective (Experiment 3), and navigating between target landmarks within the learned environment (Experiment 4). Results demonstrated that retrieval practice enhances near to medium transfer of memory for the map itself, including accessing spatial memory from varied orientations. However, it does not assist in medium to far transfer of spatial knowledge to pointing or navigation tasks performed from an alternate perspective. Results are considered in the context of domain-specific theories of spatial memory and navigation, and domain-general theories of learning strategies and transfer. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

The Unexpected Relationship Between Retrieval Demands and Memory Performance When Older Adults Are Faced With Age-Related Stereotypes.

OBJECTIVES: In two studies, we examined the effects of age-related stereotype threat on eyewitness memory using the misinformation paradigm to (a) examine stereotype threat in the context of a more ecologically valid memory task and (b) to determine the relationship between task difficulty and susceptibility to stereotype threat. METHODS: After watching a video that depicted a crime, older and younger adult participants were presented with a written synopsis in which information consistent or inconsistent with the original event was presented. Half of the participants were then presented with information designed to activate negative stereotypes about aging. Finally, participants completed a memory test. RESULTS: In Study 1, when participants were instructed to report information from either the video or the synopsis to complete the final memory test, older adults under high stereotype threat were less accurate than those under low threat. In Study 2, when participants were required to engage in more controlled processes at retrieval and respond with only video information, older adults under stereotype threat performed as well or better than those under low threat. DISCUSSION: The results are consistent with the Regulatory Focus Model of Stereotype Threat.

Tumor heterogeneity and acquired drug resistance in FGFR2-fusion-positive cholangiocarcinoma through rapid research autopsy.

Cholangiocarcinoma is a highly aggressive and lethal malignancy, with limited treatment options available. Recently, FGFR inhibitors have been developed and utilized in FGFR-mutant cholangiocarcinoma; however, resistance often develops and the genomic determinants of resistance are not fully characterized. We completed whole-exome sequencing (WES) of 11 unique tumor samples obtained from a rapid research autopsy on a patient with FGFR-fusion-positive cholangiocarcinoma who initially responded to the pan-FGFR inhibitor, INCB054828. In vitro studies were carried out to characterize the novel FGFR alteration and secondary FGFR2 mutation identified. Multisite WES and analysis of tumor heterogeneity through subclonal inference identified four genetically distinct cancer cell populations, two of which were only observed after treatment. Additionally, WES revealed an FGFR2 N549H mutation hypothesized to confer resistance to the FGFR inhibitor INCB054828 in a single tumor sample. This hypothesis was corroborated with in vitro cell-based studies in which cells expressing FGFR2-CLIP1 fusion were sensitive to INCB054828 (IC50 value of 10.16 nM), whereas cells with the addition of the N549H mutation were resistant to INCB054828 (IC50 value of 1527.57 nM). Furthermore, the FGFR2 N549H secondary mutation displayed cross-resistance to other selective FGFR inhibitors, but remained sensitive to the nonselective inhibitor, ponatinib. Rapid research autopsy has the potential to provide unprecedented insights into the clonal evolution of cancer throughout the course of the disease. In this study, we demonstrate the emergence of a drug resistance mutation and characterize the evolution of tumor subclones within a cholangiocarcinoma disease course.