Early Events Research Articles

P28: Early adverse events and loneliness among older people with depression

Object:Loneliness is experienced by many older people and associated with depressive symptoms. Childhood adverse events have been found to be a predictor of loneliness in young adults. We would like to investigate whether childhood traumatic events are associated with loneliness in older people with depression. We also explored what kinds of traumatic events are more related to loneliness and whether family support will modify the association of interest.Methods:Older adults (≧60 years) with history of major depressive disorder were enrolled from outpatient clinics. Participants cannot be diagnosed as dementia. They received a series of questionnaires, including cumulative illness rating scales, Hamilton depressive and anxiety rating scales (HDRS and HARS), Geriatric depression scale(GDS), Childhood Trauma Questionnaire(CTQ), Chinese version of the family adaptation, partnership, growth, affection and resolve(APGAR), and Mini-mental status examination.Results:Fifty-four participants were enrolled. The average age was 68.8 years and female predominance (83.7%) with mean educational year of 10.4. Univariate analysis was performed first using LS score as dependent variable, and only those variables with p value less than 0.2 were put into multiple linear regression. In the multiple regression model, age, gender, education, GDS, HDAS were entered as covariates and CTQ was treated as independent variable. The results showed that CTQ score was positively associated with LS (ß=0.565, p<0.001). The family support, presented as APGAR score, was not modified the results. In the explore analysis, physical neglect, emotional abuse, emotional neglect also showed positively associated with loneliness in the participants; however, physical abuse and sexual abuse did not have the same findings.Conclusion:Childhood adverse events may be positively associated with loneliness in older people with depressive disorder after adjusting for confounders. We should pay more attention on the childhood traumatic events in these patients. In addition, physical neglect, emotional abuse, emotional neglect rather than physical abuse and sexual abuse seemed to have more impact on the loneliness in these participants. However, some limitations, such as small sample size, recall bias of childhood events, difficulty of recall physical and sexual abuse, and other latent confounders, should be considered before making a final conclusion.

Role of microglial extracellular vesicles in the propagation of early synaptic dysfunction in Alzheimer’s disease

AbstractBackgroundSynaptic dysfunction is an early mechanism in Alzheimer’s disease (AD) which involves progressively larger areas of the brain over time. However, how it starts and propagates is unknown. We hypothesised that large extracellular vesicles (EVs) released by microglia may be responsible for these early events in AD.MethodLarge EVs carrying Aβ peptides (Aβ‐EVs) were obtained from primary microglia exposed to Aβ42. Optical manipulation was combined with time lapse imaging to study EV‐neuron interaction. Then, EVs were stereotaxically injected into naïve mice entorhinal cortex (EC), a vulnerable region in AD, to evaluate: i) long‐term potentiation (LTP) in EC and its main target region, the dentate gyrus of the hippocampus (DG), through field potential extracellular recordings in slices; ii) possible alterations in cortico‐hippocampal network activity by chronic EEG recordings; iii) progressive memory impairment in behavioural tasks. Finally, taking advantage of high‐resolution accurate‐mass spectrometry SWATH™‐MS, we investigated the molecular differences between Aβ‐ and ctrl‐EVs (from cells not exposed to Aβ). The influence of mesenchymal stem cell (MSC) indirect co‐colture with microglia primed with Aβ on cell phenotype, EVs and functions is also being explored.ResultAβ‐EVs were able to move along the axonal surface more efficiently than ctrl‐EVs and predominantly in an anterograde direction, altering dendritic spine morphology in vitro. LTP was impaired in the EC 1h after Aβ‐EV (but not ctrl‐EV) local injection, while 24h later LTP was impaired also in the DG, indicating a spreading of synaptic dysfunction between connected regions. This translated into network alterations and progressive memory deficits. Interestingly, Aβ42 alone was unable to spread alterations. Aβ‐EVs limited in their motion could neither propagate impairment nor cause network abnormalities, thus implicating Aβ‐EV extracellular motion in these processes. Proteomic analysis provided insights into the molecular mechanisms underneath Aβ‐EV motion at the neuronal surface.ConclusionOur data provide evidence of the involvement of microglial EVs in early synaptic dysfunction in AD, through a new mechanism ‐ extracellular motion at the neuronal surface ‐ not implying EV internalization, paving the way for novel therapeutic strategies. Reference: Gabrielli et al., Brain, 2022 https://doi.org/10.1093/brain/awac083 Fundings: 2018‐AARF‐588984, Horizon2020#874721PREMSTEM

Ovarian cancer relies on the PDGFRβ-fibronectin axis for tumorsphere formation and metastatic spread.

High-grade serous ovarian cancer (HGSOC) is the deadliest gynecological malignancy. The most common form of metastatic spread of HGSOC is transcoelomic dissemination. In this process, detached cells from the primary tumor aggregate as tumorspheres and promote the accumulation of peritoneal ascites. This represents an early event in HGSOC development and is indicative of poor prognosis. In this study, based on tumorspheres isolated from ascitic liquid samples from HGSOC patients, ovarian cancer spheroid 3D cultures, and in vivo models, we describe a key signal for tumorsphere formation in HGSOC. We report that platelet-derived growth factor receptor beta (PDGFRβ) is essential for fibronectin-mediated cell clustering of ovarian cancer cells into tumorspheres. This effect is mediated by the kinase NUAK family SNF1-like kinase 1 (NUAK1) and blocked by PDGFRβ pharmacological or genetic inhibition. In the absence of PDGFRβ, ovarian cancer cells can be provided with fibronectin by cancer-associated fibroblasts to generate chimeric spheroids. This work provides new insights that uncover potential targets to prevent peritoneal dissemination, the main cause of advanced disease in HGSOC patients.

Identification of a Novel Mechanism Fostering the Alterations of Brain Energy Metabolism: a Link Between AD and T2DM

AbstractBackgroundBrain insulin resistance (bIR) is associated with mitochondrial stress, failure of energy metabolism, synaptic loss and ultimately neurodegeneration both in Alzheimer’s disease (AD) and Type 2‐Diabetes Mellitus (T2DM). Our group previously demonstrated that loss of BVR‐A is an early event triggering bIR in AD. Hence, we tested the hypothesis that reduced BVR‐A levels link bIR and mitochondrial defects resulting in AD‐like neuropathology in T2DM.MethodAlterations of insulin signalling, autophagic flux, oxidative stress levels and AD hallmarks were analyzed in the hippocampus of wild‐type and the goto‐kakizaki (GK) model of T2DM rats. Hippocampal mitochondrial function was evaluated by measuring oxygen consumption rate (OCR), mitochondrial complexes, mitochondrial unfolded protein response (UPRmt) and oxidative stress levels. These data were correlated with peripheral metabolic measurements (fasting glucose, insulin and OGTT) and cognitive tasks (spatial memory). To confirm the role of BVR‐A, similar analyses were performed in the hippocampus of WT and BVR‐A KO mice fed a high fat diet (HFD, 60% kcal from fat) for 1, 3 or 8 weeks. Additional mechanistic insights were gained by evaluating mitochondrial function (sea‐horse) and the above‐mentioned intracellular pathways in response to insulin, in neuronal cells lacking BVR‐A.ResultGK rats displayed a diabetic phenotype and impaired spatial memory. Reduced BVR‐A levels along with IRS1 hyper‐activation and loss of Akt‐mediated inhibition of GSK3ß were observed in the hippocampus, consistent with the regulatory role for BVR‐A. As result, hyperactive GSK3ß accumulated in hippocampal mitochondria fostering their impairment characterized by reduced OCR and activation of UPRmt. Similar alterations in BVR‐A KO mice and in neuronal cells lacking BVR‐A were observed, reinforcing the role for BVR‐A in regulating mitochondrial bioenergetics in response to insulin. HFD regimen promoted worse outcomes in BVR‐A KO mice with respect to WT. Remarkably, gender‐differences were observed whereby female mice show earlier and greater alterations with respect to male mice.ConclusionThese results suggest that early BVR‐A loss triggers bIR and the hyper‐action of GSK3ß, that, in turn, drives the development of mitochondrial stress in T2DM brain. These alterations accelerate the impairment of energy metabolism and development of AD‐like neuropathology in T2DM.

A multi‐omics approach to investigate the synapse dysfunction in Alzheimer’s disease

AbstractBackgroundSynapse dysfunction is an early event for Alzheimer’s disease (AD) progression. Synapse dysfunctions are caused by multiple cellular and pathologic factors such as Amyloid beta, p‐tau, inflammation and aging. However, exact molecular mechanism of synapse dysfunction is largely unknown in AD. Therefore, to understand the molecular basis of synapse dysfunction in AD, we conducted a high throughput multi‐omics analysis of synaptosome fraction in AD and cognitively normal postmortem brain samples.MethodSynaptosomes were extracted from healthy control (HC) postmortem brain (n = 14) and AD postmortem brains (n = 27). Next, we performed miRNAs and mRNAs HiSeq analysis on synaptosomal RNAs. We also used HC (n = 10) and AD (n = 10) synaptosome samples for mass spectroscopy proteomic analysis. We conducted the synaptosomal miRNAs and mRNA sequencing and mass spec analysis on the same groups of samples. Further, we conducted a high throughput multi‐comics analysis to understand the molecular interaction of deregulated synapse miRNAs, mRNAs and proteins at AD synapse.ResultMiRNA HiSeq analysis showed the significant deregulation of several miRNAs in synaptosome fraction in AD vs HC. Our study also identified the several novel potential candidate miRNAs those were deregulated in AD vs HC synaptosomes. Similarly, several protein coding genes and novel transcript were also found to be deregulated in AD vs HC synaptosomes. Further, mass spec analysis showed significant deregulation of several synaptic proteins in AD vs HC synaptosomes. Further, to understand the molecular interaction of synapse miRNAs‐mRNA‐proteins and their deregulation in AD, we conducted high throughput multi‐Omics analysis. Study revealed the involvement of omics targets in several biological process and molecular function such as signal transduction, protein binding, GABAergic synapse and synaptic vesicle cycle etc.ConclusionOur study unveiled the synapse centered novel omics candidates those could be potential therapeutic targets for synapse dysfunction in AD.

Sex differences in plasma p‐tau181 accumulation is associated with subsequent tau‐PET signal

AbstractBackgroundPET and post‐mortem studies show that women consistently exhibit higher medial‐temporal and neocortical tau compared to men, independent of age or diagnostic status. What remains unclear is whether such sex differences are also observed in the plasma biomarkers and related to future tau‐PET deposition. We examined baseline and longitudinal phosphorylated‐tau (plasma p‐tau181‐UGOT), which was derived prior to a tau‐PET scan, in order to determine whether changes in p‐tau181 are associated with higher regional tau‐PET in women relative to men.MethodWe selected 313 participants (clinically normal, n = 169; MCI/dementia, n = 144) from ADNI (Female% = 146(46%); AgeMean = 71(±7)years; APOEe4 = 105(34%); Ab+ = 162(52%)) with at least two timepoints of plasma p‐tau181‐UGOT measured in ADNI1/2/GO (time‐pointsMean = 3.7(±1)). Cross‐sectional Ab‐PET and tau‐PET (Flortaucipir) scan were collected in ADNI3 ((time‐difference from baseline plasmaMean = 7.1y(±1.9)). Two a priori tau‐PET partial volume corrected (PVC) regions were selected: entorhinal (EC) and inferior temporal (IT). We used linear regression to examine the influence of baseline p‐tau181 and sex on subsequent tau‐PET, adjusting for time gap between plasma‐PET and tau‐PET. Linear mixed‐effects models probed how retrospective change in plasma tau were associated with regional tau‐PET and sex, covarying for neocortical Ab‐PET, diagnosis, APOEe4 and age. We also examined interactions between sex and variables of interest.ResultBaseline p‐tau181 was strongly associated (b = 0.86 and b = 0.53) with subsequent tau‐PET, although sex did not display main effects in the model, nor did sex moderate the plasma‐PET association. Longitudinal p‐tau181 was not associated with subsequent tau‐PET. Women with accumulating p‐tau181 exhibited higher subsequent tau‐PET burden in EC and IT regions than men (p = 0.04 and p = 0.03, respectively; Figure 1). Higher‐order interactions between sex and tau‐PET with APOEe4, diagnosis or Ab‐PET were not significant, although, women diagnosed with MCI/dementia and elevated Ab seemed to drive the interaction (Figure 2).ConclusionElevated tau‐PET burden in women may be more strongly associated with prior increases in plasma p‐tau181 relative to men. Given the pre‐established association between p‐tau181 and Ab, this could be initial evidence that early events related to the deposition of Ab predict later cortical tau deposition in a sex‐specific manner. Given the novelty of examining longitudinal plasma measures, further investigation is necessitated beyond this preliminary evidence.

Structural insights into the activation mechanism of phosphoinositide 3-kinase alpha

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases known to regulate important cellular functions by phosphorylating the inositol ring of inositol-phospholipids (PtdIns) at 3’ position. The PI3Kα is a heterodimer and the activation of the catalytic subunit (p110α) is regulated by its regulatory subunit (p85α). The current work deals with studying the activation mechanism of the PI3Kα using multi micro-second molecular dynamic simulations. Structural changes involved in activation mechanism is studied by gradually releasing the inhibitory effects of different domains of regulatory subunit namely, n-terminal SH2 (nSH2) and inter SH2 (iSH2). The observation shows that even in the presence of n-terminal and inter SH2 domain (niSH2) of regulatory subunit, the catalytic domain has some intrinsic activation activity and the presence of c-terminal SH2 (cSH2) domain may be required for complete inhibition. The release of nSH2 domain leads to loss of interactions between iSH2 domain (regulatory subunit) and C2 and kinase domain (catalytic subunit). The study shows that early events in the activation mechanism involve the movement of the ABD domain of the catalytic subunit along with the linker region between ABD and RBD region which may lead to movement of ABD closer to the CLobe of the kinase domain. This movement is essentially as it triggers the rearrangement of CLobe especially the catalytic loop and activation loop which bring catalytic important residues closer to ATP and PIP2(phosphatidylinositol-4,5-bisphosphate). Water mediated interaction analysis reveal that water may be playing an important role in the transfer of phosphate from ATP to PIP2. The study shows that initial signal for release of inhibitory effect of the regulatory subunit might be propagated through the linker region between ABD and RBD through allosteric effect to different regions of the protein. These understanding of early events during the activation mechanism may help in the design of better therapeutic targeting PI3K.

Unveiling the role of osteosarcoma-derived secretome in premetastatic lung remodelling

BackgroundLung metastasis is the most adverse clinical factor and remains the leading cause of osteosarcoma-related death. Deciphering the mechanisms driving metastatic spread is crucial for finding open therapeutic windows for successful organ-specific interventions that may halt or prevent lung metastasis.MethodsWe employed a mouse premetastatic lung-based multi-omics integrative approach combined with clinical features to uncover the specific changes that precede lung metastasis formation and identify novel molecular targets and biomarker of clinical utility that enable the design of novel therapeutic strategies.ResultsWe found that osteosarcoma-bearing mice or those preconditioned with the osteosarcoma cell secretome harbour profound lung structural alterations with airway damage, inflammation, neutrophil infiltration, and extracellular matrix remodelling with increased deposition of fibronectin and collagens by resident stromal activated fibroblasts, favouring the adhesion of disseminated tumour cells. Systemic-induced microenvironmental changes, supported by transcriptomic and histological data, promoted and accelerated lung metastasis formation. Comparative proteome profiling of the cell secretome and mouse plasma identified a large number of proteins involved in extracellular-matrix organization, cell-matrix adhesion, neutrophil degranulation, and cytokine-mediated signalling, consistent with the observed lung microenvironmental changes. Moreover, we identified EFEMP1, an extracellular matrix glycoprotein exclusively secreted by metastatic cells, in the plasma of mice bearing a primary tumour and in biopsy specimens from osteosarcoma patients with poorer overall survival. Depletion of EFEMP1 from the secretome prevents the formation of lung metastasis.ConclusionsIntegration of our data uncovers neutrophil infiltration and the functional contribution of stromal-activated fibroblasts in ECM remodelling for tumour cell attachment as early pro-metastatic events, which may hold therapeutic potential in preventing or slowing the metastatic spread. Moreover, we identified EFEMP1, a secreted glycoprotein, as a metastatic driver and a potential candidate prognostic biomarker for lung metastasis in osteosarcoma patients.Graphical abstractOsteosarcoma-derived secreted factors systemically reprogrammed the lung microenvironment and fostered a growth-permissive niche for incoming disseminated cells to survive and outgrow into overt metastasis.Daily administration of osteosarcoma cell secretome mimics the systemic release of tumour-secreted factors of a growing tumour in mice during PMN formation;Transcriptomic and histological analysis of premetastatic lungs revealed inflammatory-induced stromal fibroblast activation, neutrophil infiltration, and ECM remodelling as early onset pro-metastatic events;Proteome profiling identified EFEMP1, an extracellular secreted glycoprotein, as a potential predictive biomarker for lung metastasis and poor prognosis in osteosarcoma patients.Osteosarcoma patients with EFEMP1 expressing biopsies have a poorer overall survival.

Automated Real-Time Monitoring of Extracellular pH to Assess Early Plant Defense Signaling.

Extracellular alkalinization mediated by the inhibition of plasma membrane-located proton pumping ATPases hallmarks the initiation of defense signaling in plant cells. Early defense responses also include depolarization of the plasma membrane, increase in cytosolic Ca2+ concentration, and an oxidative burst. Together these early signaling events lead to the activation of plant immunity. The transient alkalinization response is triggered by well-studied pathogen-derived and plant endogenous elicitors, including, for example, bacterial flagellin, fungal chitin, and tomato systemin in both model and agronomic species. Employing cell suspension cultures, extracellular alkalinization can be easily assessed by measuring the elicitor-induced pH changes of the cultivating medium. Here, we provide a protocol for an improved alkalinization assay in a system which is able to simultaneously monitor multiple samples, and fully automatically transfer customizable real-time pH records. In this system flagellin, chitin and systemin elicit robust time- and dose-dependent responses, proving a powerful tool for assessing plant early defense signaling.

Preferential selection of viral escape mutants by CD8+ T cell 'sieving' of SIV reactivation from latency.

HIV rapidly rebounds after interruption of antiretroviral therapy (ART). HIV-specific CD8+ T cells may act to prevent early events in viral reactivation. However, the presence of viral immune escape mutations may limit the effect of CD8+ T cells on viral rebound. Here, we studied the impact of CD8 immune pressure on post-treatment rebound of barcoded SIVmac293M in 14 Mamu-A*01 positive rhesus macaques that initiated ART on day 14, and subsequently underwent two analytic treatment interruptions (ATIs). Rebound following the first ATI (seven months after ART initiation) was dominated by virus that retained the wild-type sequence at the Mamu-A*01 restricted Tat-SL8 epitope. By the end of the two-month treatment interruption, the replicating virus was predominantly escaped at the Tat-SL8 epitope. Animals reinitiated ART for 3 months prior to a second treatment interruption. Time-to-rebound and viral reactivation rate were significantly slower during the second treatment interruption compared to the first. Tat-SL8 escape mutants dominated early rebound during the second treatment interruption, despite the dominance of wild-type virus in the proviral reservoir. Furthermore, the escape mutations detected early in the second treatment interruption were well predicted by those replicating at the end of the first, indicating that escape mutant virus in the second interruption originated from the latent reservoir as opposed to evolving de novo post rebound. SL8-specific CD8+ T cell levels in blood prior to the second interruption were marginally, but significantly, higher (median 0.73% vs 0.60%, p = 0.016). CD8+ T cell depletion approximately 95 days after the second treatment interruption led to the reappearance of wild-type virus. This work suggests that CD8+ T cells can actively suppress the rebound of wild-type virus, leading to the dominance of escape mutant virus after treatment interruption.

Platelet Reduction after Aortic Bioprosthesis Implantation: Results from the PORTRAIT Study

Background: Platelet count reduction (PR) is a common but unclear phenomenon that occurs after aortic bioprosthesis valve implantation (bio-AVR). This study aimed to investigate the occurrence and clinical impact of PR in patients receiving stented, rapid deployment (RDV), or stentless bioprostheses. Methods: 1233 adult bio-AVR patients were enrolled. Platelet count variation, early post-operative adverse events, and in-hospital mortality were analysed. Results: 944 patients received a stented valve, an RDV was implanted in 218 patients, and 71 patients had a stentless bioprosthesis. In all groups, the platelet count at discharge was lower than the baseline values (p < 0.001). The percentage of PR was 27% in the stented group, 56% in the RDV group, and 55% in the stentless group. A higher platelet reduction, reaching the minimum platelet value, was observed in the RDV (mean: −30.84, standard error (SE): 5.91, p < 0.001) and stentless (mean: 22.54, SE: 9.10, p = 0.03) groups compared to the stented group. A greater PR occurred as the size of the bioprosthesis increased in RDV (p = 0.01), while platelet count variation was not directly proportional to the stented bioprosthesis size (p < 0.001). PR was not affected by cardiopulmonary bypass (mean: −0.00, SE: 0.001, p = 0.635) or cross-clamp (mean: −0.00, SE: 0.002, p = 0.051) times in any of the groups. RDV subjects experienced more in-hospital adverse events. PR was found to be associated with ischemic strokes in the overall population. Conclusions: Bio-AVR is associated with significant but transient PR. RDV patients more likely experience significant PR and related adverse clinical events. PR is associated with ischemic strokes, regardless of the bioprosthesis type.

Feasibility of Extracorporeal Delivery of Fusosomes to Generate CAR T Cells In Vivo

Fusosomes are viral vectors pseudotyped with modified paramyxovirus envelopes targeting specific cell types. A CD8-targeted fusosome delivering a CD19CAR transgene has the potential to provide an off-the-shelf therapeutic approach to generate in vivo CD19-directed chimeric antigen receptor (CAR) T cells in patients. For an in vivo gene delivery platform there are several approaches for administration that could be considered including direct intravenous (IV) administration or extracorporeal delivery (ECD). With ECD, patients would first undergo apheresis, then fusosomes would be retained with patient peripheral blood mononuclear cells (PBMCs) in the apheresis bag for a brief period of time before the entire contents of the bag is IV delivered into the same patient. Here, we demonstrate the feasibility of this approach to generate in vivo CAR T cells in a clinical setting with a mock infusion and discuss the translation of these findings to direct IV administration. A healthy donor was apheresed to collect PBMCs using standard apheresis equipment. No further cell enrichment, separation, or washing was performed. CD8-targeted fusosome formulated in an isotonic cryoprotective buffer was administered into the apheresis bag by gravity via an infusion set at a dose of ~0.5 IU/cell, gently mixed and retained for 30 min. The contents of the bag were mock infused into sample collection bags at a controlled flow rate using an infusion pump. Samples were collected before and after fusosome addition, after 30 min retention, and every 30 min during the mock infusion. Cell counts and viability were measured by complete blood count (CBC) and flow cytometry analyses. Fusosome binding to CD8 + T cells was measured by flow cytometry. Cytokines and complement components in supernatants were measured by ELISA or MSD. Residual fusosome in the supernatant was measured by ddPCR. Transduction of CD8 + T cells was measured by flow cytometry and vector copy number (VCN) analysis after PBMCs were washed, activated with CD3/CD28 beads, and cultured for 8 days. Early transcription events were measured by ddPCR using specific amplicons targeting different DNA species generated during reverse transcription. The CD8-targeted fusosome was successfully deliveredusing aseptic techniques to a freshly collected apheresis bag.PBMC viability stayed high (>%95) throughout the process. There were no major changes in white blood cell, platelet, or red blood cell levels as measured by CBC analysis. There were no changes in CD8 + T cell phenotype throughout the time course. Fusosome binding to CD8 + T cells was detected as early as fusosome addition to the apheresis bag (~5 min) and increased over the course of the process. Analysis of vector genomes in the supernatants indicated presence of unbound residual fusosome. Exposure of PBMC to fusosome did not induce cytokine secretion or complement activation throughout the process. Early events of transduction of cells could be detected by analysis of reverse transcription intermediates. Analysis of cultured cells via both flow cytometry and VCN analysis indicated generation of CD8 + CAR T cells upon short-term exposure to fusosome. In vivo delivery ofCD8-targeted fusosome encoding a CD19CAR transgene enables a novel CAR T cell approach. This study demonstrated that administration of fusosomes via ECD is operationally feasible in a clinical setting and scale as a novel method of administration to generate CD8 + CD19CAR T cells in vivo. ECD setup also served as an in vitro pharmacokinetic model for direct IV infusion. In scenarios where in vivo exposure is limited due to very rapid clearance (e.g. few minutes), ECD has the benefit of reducing the volume of distribution and bringing T cells and fusosomes in close proximity for efficient gene delivery. The findings of study suggest that with rapid binding of fusosomes to CD8 + T cell leading to early transcription events, and an in vivo half-life of more than 30 min, fusosome may generate CAR T cells successfully via direct IV administration.

Comparative Analysis of Methods for Analyzing Clinical Results of Immuno-Oncology Therapy with Delayed Treatment Effects

Introduction: CAR-T therapy is a groundbreaking therapy that involves modifying patient's T cells to recognize and combat cancer cells. This therapy requires a period of CAR-T manufacture following apheresis, delaying the actual receipt of the CAR-T. During this period the patient may receive bridging therapy. CAR-T therapies are increasingly being studied in randomized controlled clinical trials and delays potentially affect the proportional hazards assumption, reduce log-rank test statistical power, and complicate reporting and interpretation. To address this, we assessed available statistical methods for analyzing immune-oncology outcomes, specifically CAR-T therapy's delayed treatment effects, with a focus on current and emerging tools to improve analysis and reporting. Methods: We systematically reviewed and compared existing and emerging tools to evaluate survival analysis methods suitable for assessing outcomes in CAR-T studies. These methods included standard, stratified, and weighted log-rank tests, cox proportional hazards (time dependent), parametric survival (weibull, etc.), time-dependent covariates, landmark analysis, joint modeling and restricted mean survival time (RMST). We simulated trial results, highlighted features, underlying assumptions, and interpretability. Results: Results were based on a simulated dataset (N=240) and revealed distinct characteristics and implications of each method used to analyze data (Table). Standard log-rank tests, which give equal weight to all time points and assumes proportional hazards, resulted in less discerning outcome measurements compared to weighted techniques such as censored weighted estimation/constant piecewise weighted (CWE/CPW) and Fleming-Harrington which do not assume proportional hazards and can be weighted towards early or late events. RMST summarized average time until progression, which may be a more intuitive measure for clinicians and patients and was presented along with other analytical techniques. Cox proportional hazards modeling provided ratios to quantify and compare efficacy across different time points. Parametric survival models captured survival dynamics and offered precise estimation of survival probabilities at specific intervals. Time-dependent covariates accounted for time-varying factors influencing treatment response, while landmark analysis evaluated treatment efficacy beyond a specific duration and was used in combination with other analytical techniques. Joint modeling simultaneously assessed treatment effects on both longitudinal and survival outcomes, accounting for their interdependencies. Stratification identified patient subgroups with differential treatment responses and outcomes, enabling precise reporting of categorical CAR-T treatment approaches. Conclusions: Our comparative analysis of analytical techniques underscores the critical role of selecting an appropriate approach in the context of CAR-T therapies. Conventional methods such cox regression and the standard log-rank test, while useful in most contexts, may underestimate key outcomes associated with CAR-T therapy due to violations of proportional hazard assumption. Weighted techniques, which can be weighted towards early or late events, may provide a more nuanced picture, and better accommodate CAR-T therapies. Similarly, the RMST offers an intuitive average time to progression, offering complementary insights to other methods. These findings highlight the importance of careful method selection in survival analysis for CAR-T therapy studies. Rigorous, tailored analytical approaches will be crucial in accurately determining the efficacy of these promising therapies and informing future clinical study designs. By considering these methods, researchers can gain deeper insights regarding CAR-T therapy and improve the design and analysis of clinical trials, ultimately advancing the understanding and application of this transformative therapy.

Early outcomes of one-anastomosis gastric bypass in the elderly population at high-volume bariatric centers.

The increasing rate of obesity and life expectancy will lead to increasing numbers of bariatric procedures in the elderly. We aim to report the early (≤ 30days) adverse events of One-Anastomosis Gastric Bypass (OAGB) in this patient population. Assuta Bariatric Centers in Israel. Retrospective review of perioperative OAGB outcomes between elderly group (≥ 65years) and non-elderly group (18-64years) at high-volume bariatric centers between January 2017-December 2021. Operative time, length of stay (LOS), and overall ≤ 30days complication rates, as ranked by the Clavien-Dindo Classification (CDC) were compared. There were 6618 patients (non-elderly group) and 104 (elderly group) who underwent OAGB. Gender and preoperative BMI were comparable between the two age groups. The elderly group had significantly higher rate of ischemic heart disease and chronic renal failure. The number of patients with LOS ≥ 3days was significantly higher in the elderly group [19.4% (n = 20) vs. 6.6% (n = 331), respectively; p < 0.001]. The total early adverse events were higher in the elderly group with no statistical significance [7.7% (n = 8) vs. 3.8% (n = 250), respectively; p = 0.062]. The rate of minor and major adverse events and reoperation rate was comparable between the two groups. The rate of readmissions was significantly higher in the elderly group 5.8% (n = 6) vs. 1.9% (n = 124), respectively p = 0.015. There was 0.06% mortality (n = 2) in the non-elderly group. OAGB is a relatively safe metabolic and bariatric surgery for elderly obese patients with early (≤ 30days) morbidity rates similar to the non-elderly population.

Interplay between Inflammatory Microenvironment and RUNX1-Mediated Transcriptomic Changes Drives Defective Hematopoiesis in Familial Platelet Disorder

Background: Familial platelet disorder (FPD) is caused by germline loss-of-function mutations in RUNX1 an essential transcription factor regulating hematopoiesis. The absence of functional RUNX1 leads to mild-to-moderate thrombocytopenia, platelet dysfunction, and increased bleeding in FPD patients. In ~40% of cases, the patients progress to overt leukemia at median age of 33 years through unknown mechanisms. FPD patients also present with skin allergy and eczema which are associated with systematic elevation of cytokines. Given the well-established connection between inflammation and leukemia initiation and progression, we sought to investigate whether inflammatory stress play a role in regulating abnormal hematopoiesis and eventually cause clonal evolution in FPD. Methods: We acquired bone marrow cells from FPD patients who had not developed leukemia yet. We then compared FPD bone marrow hematopoietic stem and progenitors (HSPCs) with healthy progenitors using in vitro differentiation and colony formation assays. For comprehensive mechanistic evaluation, we performed 10x single-cell RNA sequencing (scRNA-seq) on FPD (n=10) and healthy (n=4) bone marrow cells, analyzing &amp;gt;100,000 cells using Seruat package. We evaluated cytokine levels in bone marrow fluid and peripheral blood using 65-plex Luminex assay. The impact of the inflammatory cytokines and intervention strategies were tested using in vitro differentiation and colony formation assays. The results were validated in vivo using mice with germline Runx1 mutation. Results: We identified that FPD progenitors are impaired in megakaryocytic (&amp;gt;2.0-fold, p&amp;lt;0.05) and erythroid differentiation with increased myeloid differentiation (&amp;gt;1.7-fold, p&amp;lt;0.05) compared to healthy control. In addition, FPD HSPCs show increased myeloid colony formation (&amp;gt;1.6-fold, p&amp;lt;0.05) and serial replating ability. scRNA-seq identified 18 cell clusters and pseudotime analysis revealed higher percentage of FPD cells differentiating towards monocytes, supporting enhanced myeloid differentiation. The pathway enrichment analysis of differentially expressed genes in HSC cluster revealed upregulation of inflammatory (TNFα via NF-kB) and pro-survival pathways (PI3K/AKT/mTOR). Accordingly, cytokine profiling revealed upregulation of numerous cytokines and chemokines in FPD compared to healthy samples including TNFα, CXCL8, CCL24, CCL2, and IL1β (&amp;gt;2.5 to 1000-fold, p&amp;lt;0.05), secreted by progenitors, monocytes, and MSCs. Further, under inflammatory stress mediated by TNFα, CXCL8, CCL24, CCL2, and IL1β, FPD HSPCs exhibited resistant to growth suppression and have increased myeloid colony formation. However, pharmacological inhibition of individual cytokines only partially rescued the differentiation defects, possibly due to compensatory effects of each cytokine. Phospho-flow analysis revealed that increased cytokine levels leads to the activation of PI3K/mTOR and JAK signaling that we also found upregulated using scRNA-seq analysis. Interestingly, inhibition of mTORi (rapamycin and AZD2014), PI3Ki (idelalisib), and JAKi (ruxolitinib) showed an increase in percentage of megakaryocytes (up to 2-fold, p&amp;lt;0.05) and a decrease in monocytes (up to 0.6-fold change, p&amp;lt;0.05). Also, inhibition of these pathways in FPD reduced cytokines levels. In line with human FPD data, in vivo treatment of Runx1 R188Q/+ mouse with ruxolitinib suppressed myeloid differentiation with no effects on megakaryocytes, while rapamycin resulted in significantly increased platelet activation compared to untreated mice (&amp;gt;1.7-fold, p&amp;lt;0.05) and a reduction of monocytes (&amp;lt;0.5-fold, p&amp;lt;0.05), suggesting mTOR inhibition might be an effective intervention strategy. Conclusion: FPD HSPCs manifest defective megakaryopoiesis with increased myelopoiesis. The elevated autocrine and paracrine inflammatory stress is an early event in FPD, driving myeloid differentiation. This inflammatory stress mediates activation of PI3K/mTOR and JAK signaling, leading to augmented myeloid differentiation. Consequently, the observed FPD phenotypes are the result of combined transcriptional changes and inflammatory stress. Targeting inflammatory and pro-survival signaling at an early stage might mitigate hematopoietic defects and delay disease evolution in FPD. Our results advance mechanistic understanding and hold significant clinical implications.

Genomic Characterization of Acute Myeloid Leukemia with Aberrations of Chromosome 7: A Multinational Cohort of 523 Patients

Introduction: Aberrations of chromosome 7 [abn(7)] are found in ≈ 10% of newly diagnosed acute myeloid leukemia (AML) and associate with a dismal prognosis. A large-scale comprehensive exploration of the underlying genetic heterogeneity in AML with abn(7) has yet to be performed and could add essential insights into the outcome of this poorly understood patient group. Methods: We collected diagnostic samples from 523 adult AML patients (median age 59 years) with abn(7). Whole-exome sequencing (WES) was performed to discover potentially underestimated genetic lesions in 61 paired diagnostic / remission samples. Subsequently, a gene panel including 66 genes and a SNP backbone for copy-number aberration (CNA) detection was designed and applied to the remaining 471 samples. The majority of patients (78%) were diagnosed with de novo (dn) AML, whereas 22% had secondary (s) or therapy-related (t) AML. Intensive induction treatment was administered to 80% of the patients, while 36 % underwent allogenic stem cell transplantation. Apart from 43% of patients with concomitant complex karyotype (abn(7)/CK+), 24% had -7 as a sole abnormality (-7 sole) and 13% del(7q) sole. Results: A mean of 15.3 single-nucleotide variants (SNVs) and 7.5 CNAs per patient were found by WES. Here, the most frequent recurrent SNVs were identified in TP53 (29.5%), followed by mutations (mut) in genes involved in epigenetic regulation ( DNMT3A, TET2, ASXL1, IDH2), transcription factors ( RUNX1), and genes affecting RAS-signaling ( NF1, KRAS), Figure 1. Targeted sequencing revealed 1829 SNVs with a VAF≥ 2% in 64 genes (mean: 3.8 SNVs / patient). 30% of patients harbored at least one mutation in genes located within the commonly deleted region of 7q, most frequently KMT2C, EZH2CUX1, SAMD9L, SAMD9, LUC7L2 and BRAF. The number of driver gene mutations was higher in CK- than in CK+ patients (4.5 vs. 3.3 SNVs). We found KRAS (OR 3.76, CI 1.17-16.87, P= .044) and RUNX1 (OR 3.61, CI 1.62-8.78, P= .003) mutations to be enriched in -7 sole patients, and FLT3 mutations to be associated with del(7q) sole status (OR 0.33, CI 0.13-0.82, P= .019). With respect to previously unknown lesions, a high amount of KMT2C mutations (16.6%) and recurrent alterations in FAT1 and TACC2 were discovered (6.4% each; Fig. 1). For KMT2C - located on chr7q36.1 - a total of 98 SNVs with mainly missense (73.5%) and truncating (20.4%) mutations at known cancer hotspots were noted. KMT2Cmut was associated with dnAML and AML with maturation morphology. In the entire cohort, the most common co-occurring chromosomal alterations discovered by high-resolution CNA analysis were deletions in chromosomes 5, 17, and 12. Our approach enabled the identification of small fragment deletions (≤10Mb) affecting the TP53, NF1, and ETV6 loci in 5-9% of all cases, which were missed by conventional G-banding. Cancer Cell Fraction and Bradley-Terry models were used to simulate the sequential order of genomic aberrations. While mutations in TP53 and epigenetic-related genes were early events, -7 and del(7q) were often subclonal and SNVs in NRAS, KMT2C very late events in leukemogenesis. Survival analyses in intensively-treated patients (n= 414) revealed that 61% reached complete remission, 67% relapsed, and median overall survival (OS) was 11.9 months. Abn TP53 and high WBC count were independently associated with shorter relapse-free survival (RFS). For OS, besides older age and high WBC count, we identified abn TP53, PTPN11mut, TET2mut,-5, and -18 as poor prognostic factors in multivariate analysis (Table 1). In contrast, IDH2mutconferred an independent favorable prognostic effect for RFS and OS. Notably, abn TP53 outcompeted the prognostic impact of CK+ (Table 1). Compared to TP53wt, patients with abn TP53/CK+ and abn TP53/CK- had a similar poor outcome with median RFS of 6 and 4 months (CK+/CK-, P&amp;lt;.001) and OS of 6.8 and 8.6 months (CK+/CK-, P&amp;lt;.001). In contrast to other genomic studies in myelodysplastic syndrome, we found abn TP53 to be associated with poor outcome irrespective of the single- or multihit mutation status following definitions of the latest ICC classification (Blood, 2022). Conclusion: Our results offer novel insights into the genomic landscape and clonal trajectory of AML with abn(7). This work unravels formerly underestimated genetic lesions ( KMT2Cmut) and alterations with high prognostic impact (abn TP53 and IDH2mut) for better future risk stratification.

The 3D- Telomere Profiling Assay Identifies High Risk Smoldering Multiple Myeloma Patients with High Precision

Smoldering multiple myeloma (SMM) is an asymptomatic precursor stage of multiple myeloma (MM). According to the international Myeloma Working Group data (Blood Cancer J. 2020 Oct 16;10(10):102) the probability of progression to MM is 22% at 2 years, and 42% and 64% at 5 and 10 years, respectively. Thus, identification of patients with high risk of early progression to MM is crucial for optimal treatment management. Genomic instability is a sensitive indicator of disease progression in cancer. Telomere dysfunction is an early event in genomic instability. The 3-dimensional (3D) spatial profiling of telomeres using TeloView technology allows for quantification of telomere dysfunction and was shown to be instrumental in risk stratification of cancer patients generally, but particularly in selected hematological malignancies. Importantly, the utility of the 3D-telomere profiling was recently demonstrated in 2 clinical studies validating the utility of 3D telomere profiling as a structural biomarker to identify high risk SMM patients. The two studies achieved accuracy of &amp;gt;80% and specificity and sensitivity of over 80% &amp; 76% respectively in a training dataset study followed by a blind validation (Kumar S.et al 2023. J Clin Oncol). To present the 3D telomere profiling assay as a reliable prognostic tool in the clinic, capable to precisely identify high risk SMM patients, it is important to demonstrate the repeatability and accuracy of the assay. In this study, we conducted a retrospective analytical validation including samples of 20 SMM patients with known progression outcome. The samples of each patient were blindly processed in triplets using the 3D-telomere assay, followed by the TeloView analysis. To eliminate any validation-bias the validation was conducted with uncontrolled variables. Samples were processed blindly by different operators on different dates using different instrumentation. We interrogated the repeatability and precision of the results of each patient analyzed on 2 levels. First, we calculated coefficient of variation (CV) of telomere predictors of each patient across the three runs; and secondly, we interrogated the repeatability of patient outcome using the scoring model developed and validated in (Kumar et al. 2023, J Clin Oncol) across the three runs of each patient. We set the acceptable concordance to 80% for CVs (CV &amp;lt;20%) and the acceptable repeatability of patient-outcome as predicted by the scoring model to 80% of the patients included in the study. We report CVs across all the telomere predictors, across the 3 runs of all patients ranging between 2.3 - 17.8. Furthermore, patient outcome as high/low risk of progression was consistent in 17 out of the 20 patients in all 3 runs (85%). The results of this study demonstrate the 3D-telomere profiling as an accurate and prognostic structural biomarker. The assay shows high level of precision, repeatability, and reliability, and presents a viable solution to accurately identify high risk SMM patients. This will potentially allow the treating physicians to make confident treatment decisions for high risk SMM patients based on the results of the 3D Telomere profiling assay.

The Frequency of Clonal Hematopoiesis Prior to AML and MDS Varies Among the Different Molecularly Defined Who Subtypes

Background: AML and MDS cells accumulate mutations over years or even decades. In the current WHO 2022 classification, some mutations are listed as “defining genetic abnormalities” (WHO DGA). A long phase of clonal hematopoiesis may precede some, but not all, AML or MDS. There are multiple scenarios in which order clonal hematopoiesis, WHO DGAs, and other mutations can occur. We analyzed whole-genome sequencing data from 583 patients with AML or MDS subtypes defined by a mutation and elucidated their role in the hierarchical sequence of mutations. Aim: (1) We compared patients with AML or MDS arising from clonal hematopoiesis to those with a founder mutation in a WHO DGA gene (e.g. NPM1). (2) We analyzed which other mutations may occur before WHO DGAs. Methods and patients: We analyzed 583 patients with AML or MDS who were classified into a WHO 2022 DGA class: MDS with low blasts and SF3B1 mutation (MDS- SF3B1, n=160), MDS with biallelic TP53 inactivation (MDS-bi TP53, n=40), AML, myelodysplasia-related (AML-MR with focus on mutations, n=151), AML with NPM1 (n=171), and AML with CEBPA (n=61). Amplification-free WGS was performed (median coverage &amp;gt; 100x). Genes with known relevance to myeloid malignancies were analyzed (n=47). We assumed that the mutation with the highest variant allele frequency (VAF) evolved first in the malignant process (+/- 5%). VAF was corrected for copy number changes (including the presence of one X chromosome in males) and copy-neutral loss of heterozygosity (CN-LOH). The following variant callers were used: Strelka2 (mutations and VAF), Pindel (for FLT3-ITD), HadoopCNV (CN-LOH) and GATK4 (CNV). Results: We classified patients into three groups (Figure 1a): (A) 322/583 (55%) of malignancies originate from a cell with a WHO DGA; (B) 174/583 (30%) of malignancies originate from a clonal hematopoietic cell with a DNTM3A or TET2 mutation (DT origin); (C) the remaining 87/583 (15%) of AML and MDS originate from a cell with a different initial mutation (“other”). The frequency of patients with the highest VAF in a WHO DGA varied between subtypes: MDS-bi TP53: 88%, MDS- SF3B1: 72%, AML- CEPBA: 64%, AML-MR: 57% and AML- NPM1: 27%. The highest number of patients with a disease putatively arising from DNMT3A or TET2 mutated clonal hematopoiesis was found in AML- NPM1 (49%) and the lowest in MDS-bi TP53 (5%) (for details, see Figure 1b). The group of patients with the highest VAF in neither WHO DGA nor DNMT3A/ TET2 (“other”) accounted for 17-23% of AML and only 4% and 8% of MDS- SF3B1 andMDS-bi TP53 respectively. IDH1 or IDH2 had the highest VAF in 17/583 patients (3%), especially AML- NPM1 (n=8) and AML-MR (n=7). Mutations in the spliceosome genes had the highest VAF in 12/583 patients (2%), with the combination of SRSF2 in AML- NPM1 in particular (n=6). In the following, we compared DT origin and WHO DGA origin. The differences did not affect all AML and MDS subtypes to the same extent. Patients with AML- CEBPA and DT origin were a median of 19 years older than patients with an initial CEBPA mutation (75 vs. 56 years). In AML- NPM1, the difference in median age was 11 years (71 vs. 60 years). In both groups, patients with DT origin are also predominantly male (73% vs. 44% for AML- CEBPA and 54% vs. 45% for AML- NPM1). Age and sex differences were not observed in AML-MR, MDS- SF3B1 and MDS-bi TP53 with or without DT origin. Overall survival (OS) was shorter in AML patients with DT origin compared to WHO DGA (0.7 vs. 1.8 years; p=0.073), but not in MDS (7.4 vs. 6.1 years; p=0.669). However, in a multivariate Cox analysis, including DT origin and age, age remained the only significant factor for OS. Conclusion: Using VAF to model the hierarchy of mutation evolution shows that for the majority of patients, WHO-defining mutations are early events, supporting their role as defining genetic abnormalities and (potential) drivers. However, other initial mutations occur in DNMT3A and TET2, indicating AML or MDS arising from clonal hematopoiesis,but also IDH1/ IDH2 or spliceosome genes. How these diseases will be classified in the future should be actively discussed, and aspects such as targeted therapies (with IDH inhibitors) or a second hematologic neoplasm after clonal hematopoiesis should be considered.

Combined Deficiency of Chromosome 7 Myeloid Tumor Suppressors Enhances Chemotherapy Resistance

Monosomy 7 and del(7q) [-7/del(7q)] are recurrent in myeloid neoplasms and associated with chemoresistance. -7/del(7q) is identified in up to half of therapy-related myeloid neoplasms (t-MN), high-risk secondary malignancies arising after prior exposure to chemotherapy or radiation. -7/del(7q) is also detected in clonal hematopoiesis, suggesting chromosome 7 aberrations can be early events in disease etiology. Despite this prevalence, the pathogenesis of -7/del(7q) in leukemogenesis remains unclear. We previously reported that deficiency of the transcription factor CUX1, a 7q-encoded tumor suppressor gene, promotes hematopoietic stem and progenitor cell drug resistance and t-MN transformation. Herein, we determined the combined impact of CUX1 loss with additional 7q tumor suppressor genes. To this end, we established a CRISPR/Cas9-based murine model of del(7q) clonal hematopoiesis and drug resistance. After targeting four 7q genes simultaneously, combined deficiency of CUX1 and the histone methyltransferase EZH2 uniquely promoted clonal outgrowth under genotoxic pressure in vivo and in vitro. Mechanistically, clonal selection is due, in part, to decreased apoptosis after chemotherapy exposure. RNA-seq in the absence of genotoxic insult revealed that Cux1 and Ezh2 loss has an additive transcriptional impact that is enriched for -7/del(7q) patient-derived gene signatures. Overall, we reveal a previously unknown genetic interaction between the 7q genes CUX1 and EZH2, supporting the concept of 7q as a contiguous gene syndrome region . A refined understanding of the molecular pathways driving del(7q) pathogenesis and drug resistance will enable development of therapies designed to counter or prevent these high-risk malignancies. In addition, we report a tractable approach for interrogating the pathogenesis of aneuploid events more broadly in cancer.

The FLIPI24 Prognostic Model Identifies Poor Outcomes in Non-Immunochemotherapy Treated Patients with Follicular Lymphoma

Background: We previously reported thatearly events in follicular lymphoma (FL), defined as progression of disease within 24 months (POD24) or early transformation to a more aggressive histology, are significantly associated with increased risk of lymphoma related death. To refine clinical management, the FLIPI24 prognostic model was developed to predict the risk of early events after starting immunochemotherapy (IC) (Maurer et al, 2022). The FLIPI24 model was found to be significantly superior to standard clinical models for prediction of both event-free survival at 24 months (EFS24) and overall survival (OS) in internal and external validation sets of patients receiving IC. We sought to evaluate the prognostic ability of the FLIPI24 model in patients with FL not receiving IC. Methods: The cohort consists of prospectively enrolled patients in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER) and the multicenter Lymphoma Epidemiology of Outcomes (LEO) Cohorts. Data were collected from medical records by standard protocol with all events verified by medical record review. Eligible patients were those diagnosed with grades 1-3A FL and initiated observation, rituximab monotherapy, radiation, or other non-IC therapy. The FLIPI24 model utilizes 5 continuous variables: age (linear 60-90 years with inflection at age 75), HGB (linear 8-17 g/dL), WBC (linear 4-11x10 9/L), LDH/ULN (linear 0.5-5), and B2M (linear 1-10 mg/L). FLIPI24 risk is grouped as follows: low risk (≤10%], low-average risk (10-15%], average risk (15-20%], high risk (20-40%], very high risk (&amp;gt;40%) for an early event. EFS was defined as time from diagnosis to progression, relapse, (re)treatment, histologic transformation, or death due to any cause. OS was defined as the time from diagnosis until death from any cause. Cox models and Kaplan Meier curves were used to evaluate association between FLIPI24 and EFS or OS. Results: 1542 patients initiating non-IC management approaches at diagnosis from 2002-2020 were identified. 51% were &amp;gt;60 years (IQR 52-70), and 49% were male. Most patients (86%) had FL grade 1-2. 61% of patients met any criteria for high tumor burden per GELF, BLNI, or GITMO based on available variables. Treatment approaches were 833 observation (54%), 315 rituximab monotherapy (20%), 186 radiation (12%), and 208 other non-IC approaches (14%). At median follow-up of 58 months, EFS24 was 73.8% (95% CI: 71.6%, 76.1%), 5-year OS was 90.9% (95% CI: 89.3%, 92.5%) and 253 (16%) patients died. The FLIPI24 score identified poor outcomes among patients with high/very high-risk score (FLIPI24 &amp;gt;20%), with median EFS of 1.8 years (95% CI, 1.3, 3.28) and 5 year OS of 65.1% (95% CI: 55.9-75.8) (Figure 1). When evaluating patient subsets, for patients with high tumor burden (defined based on available variables) and high/very high-risk score, median EFS was 1.6 years and 5 year OS was 64.6% (95% CI: 54.9%-76.1%). Among patients with low tumor burden FL and high/very high-risk score, median EFS was 2.0 years (95% CI: 0.84-NA) and 5 year OS was 66.2% (95% CI: 42.4%-100%). Patients undergoing observation with high/very high-risk score similarly had shortened median EFS of 1.53 years (95% CI: 1.25-NA) and 5 year OS of 68.9% (95% CI: 56.4%-84.1%). The FLIPI24 had superior concordance for OS (c=0.726) compared to the FLIPI (c=0.646) when evaluated in patients not treated with IC. Results were comparable in the subsets of patients observed (c=0.706 vs 0.596) and receiving rituximab monotherapy (c=0.764 vs 0.721). Conclusion: The FLIPI24 model has been previously validated to provide an individual risk score at diagnosis for the likelihood of experiencing an event within 24 months from starting IC. Here we demonstrate that a high FLIPI24 score predicts for poorer outcomes following non-IC strategies including observation, treatment with radiation, and rituximab monotherapy. The FLIPI24 has better prognostic performance for OS than FLIPI in this non-IC population. High FLIPI24 in patients not treated with IC identifies a patient population who should be considered for IC and/or novel frontline therapies when feasible.