Longitudinal Profiling Research Articles

Phase 1 Trial of Dendritic Cell/AML Fusion Cell Vaccine after Allogeneic Stem Cell Transplant in Acute Myeloid Leukemia

Introduction We have developed a vaccine in which patient-derived AML cells are fused with donor-derived dendritic cells (DCs). Vaccination after initial remission and consolidation was shown to elicit leukemia-specific immune responses. We hypothesized that donor-derived DC/AML vaccination post-transplant would elicit the durable expansion of leukemia-specific T cells within the donor T cell repertoire to effectively protect against disease relapse. We report updated results of a phase 1 clinical trial (NCT03679650) evaluating the use of DC/AML fusion vaccine following allogeneic transplant. Methods DC/AML fusion vaccine was generated with patient leukemia blasts, cryopreserved at the time of diagnosis, and donor-derived DCs, from leukapheresis of patients who achieved hematopoietic recovery and remained in CR 25-90 days after an allogeneic transplant. DCs were differentiated via culture of adherent mononuclear cells in the presence of GM-CSF, IL-4 and TNFa. Vaccine was administered starting day 70-100 post-transplant as long as the patient did not have active GVHD requiring therapy. Two doses of the vaccine were given subcutaneously at 3 week intervals, in conjunction with 100 mcg GMCSF daily at the vaccine site for 4 days. A booster vaccine was given 30-60 days following the taper of immune suppression, in the absence of GVHD. Longitudinal profiling of the immune landscape by single cell RNA sequencing and CyTOF from peripheral blood samples obtained before and after vaccination is underway. Immunologic response of leukemia-specfic T cells is being evaluated via IFN-gamma expression following stimulation with autologous tumor lysate. OS was calculated using Kaplan Meier Curve. Results 27 participants have been enrolled with median age 59 years (range 23-74). 15 patients were transplanted with a matched unrelated donor, 10 with a matched sibling donor and two with a haplo-identical donor. One patient relapsed prior to leukapheresis and was ineligible. The median yield of leukemia cells was 218x10 6 (range 62-818x10 6)and mean viability was 97%. The median yield of DCs was 106x10 6 (range 19-182x10 6) and mean viability 81%. Fusion vaccine was successfully generated in 25 of 26 patients who underwent leukapheresis; one patient had insufficient DC. Mean fusion efficiency was 47% with viability of 79%. Mean fusion vaccine dose was 3.8 x10 6 cells. 6 patients from whom vaccine was generated did not meet eligibility to initiate vaccination due to GVHD or COVID19 infection. 19 participants initiated vaccine administration and are evaluable for toxicity and immunologic response. The most common side effects were vaccine site reactions (n=10 grade 1, n=2 grade 2). 5 patients developed GVHD that was attributed as possibly related to vaccination, at a median time of 17 days after vaccination (range 5-70 days). Acute GVHD included two events of skin grade 2, one GI grade 2, one liver and skin grade 3. Chronic GVHD included a moderate event affecting 3 organs and severe event affecting 4 organs. There were 10 additional GVHD events with a median time of 108 days post vaccination (range 7-126 days), assessed as unlikely related or unrelated to vaccine based on pre-existing GVHD and timing of onset. Median time of follow-up is 23 months post-transplant (range 11-59 months). Amongst 19 vaccinated patients, 14 remained in CR and median overall survival estimate is 49 months post transplant. 3 died of relapsed disease, 2 died of GVHD and/or infection, and 1 died of unknown cause. Preliminary results of PB CyTOF from 4 patients in remission and 1 patient that relapsed indicate higher proportions of T cells and DCs in patients in CR. Interferon expression was higher in a patient with durable remission while absent from a patient with early relapse. Conclusions Vaccination using host-derived leukemia cells and donor derived DC was safe and feasible. Adverse events post vaccination have included injection site reactions as well as acute and chronic graft versus host disease. Vaccination may be a promising strategy to reduce relapse as 14 of 19 vaccinated patients remain relapse free. Albeit this approach is limited to patients who have not had early relapse or early GVHD. Immunologic response by interferon release upon lysate stimulation, CyTOF and single cell RNA sequencing of the immune milieu will be reported.

Longitudinal profiling and tracking stability in the Singapore study of macro-angiopathy and microvascular reactivity in type 2 diabetes cohort.

The Singapore Study of Macro-Angiopathy and microvascular Reactivity in Type 2 Diabetes (SMART2D) is a prospective cohort study which was started in 2011 to investigate the effect of risk factors on vascular function and diabetes-related complications in Asians. We aimed to compare the longitudinal change in risk factors by accounting for batch effect and assess the tracking stability of risk factors over time in patients recruited for SMART2D. In this study, we (1) described batch effect and its extent across a heterogenous range of longitudinal data parameters; (2) mitigated batch effect through statistical approach; and (3) assessed the tracking stability of the risk factors over time. A total of 2258 patients with type 2 diabetes mellitus (T2DM) were recruited at baseline. The study adopted a three-wave longitudinal design with intervals of 3years between consecutive waves. The changes in a few selected risk factors were assessed after calibration, assuming patients with similar demographic and anthropometry profile had similar physiology. The tracking pattern of the risk factors was determined with stability coefficients derived from generalised estimating equations. The medians of the longitudinal differences in risk factors between the waves were mostly modest at <10%. Larger increases in augmentation index (AI), aortic systolic blood pressure (BP) and aortic mean BP were consistently observed after calibration. The medians of the longitudinal differences in AI, aortic systolic BP and aortic mean BP between the waves were <2% before calibration, but increased slightly to <5% after calibration. Most of the risk factors had moderate to high tracking stability. Muscle mass and serum creatinine were among those with relatively high tracking stability. The longitudinal differences in parameters between the waves were overall modest after calibration, suggesting that calibration may attenuate longitudinal differences inflated by non-biological factors such as systematic drift due to batch effect. Changes of the hemodynamic parameters are robust over time and not entirely attributable to age. Our study also demonstrated moderate to high tracking stability for most of the parameters.

557P Longitudinal plasma proteomic profiling of EML4-ALK positive lung cancer receiving ALK-TKIs therapy

557P Longitudinal plasma proteomic profiling of EML4-ALK positive lung cancer receiving ALK-TKIs therapy

Longitudinal Profiling of Tumor RNA Expression Signatures Reveal Key Biological Features Associated with Response to Neoadjuvant Stereotactic Body Radiation Therapy in High-Risk Prostate Cancer

Stereotactic body radiation therapy (SBRT) is a safe and effective treatment for localized prostate cancer (PCa). PREPARE-SBRT is a clinical trial testing the safety of neoadjuvant MRI-guided SBRT for men with high-risk localized PCa. We leveraged paired samples from pre-treatment biopsy and irradiated prostatectomy (RP) specimens to evaluate transcriptomic changes in irradiated tumors at acute time points following neoadjuvant SBRT. Tumor RNA expression profiles were generated using Decipher GRID by Veracyte on 12 subjects with paired pre- and post-SBRT tissues (n = 24). Descriptive statistics using gene expression profiles describing key biological features [DNA damage & repair (DDR), tumor proliferation, suppressed immune, activated immune, tumor microenvironment (TME)] and an exploratory analysis of RT sensitivity score with binary classification as sensitive or resistant were reported. A control cohort of transcriptomic profiles of 803 matched untreated biopsy and matching RP samples from the same subjects were used to control for signature differences attributable to sample type. Key tumor biology signatures most frequently observed were DDR (15/24), TME (11/24) and tumor proliferation (11/24). Signatures associated with tumor proliferation were disproportionately represented in pre-treatment samples (10/11) whereas TME-associated signatures were enriched predominantly in irradiated RP samples (8/11). Collectively, immune-related immune signatures skewed towards immune activation. All 3 samples annotated with suppressed immune signatures were from pre-treatment specimens whereas 75% (6/8) of samples annotated with activated immune status were from irradiated specimens. Additionally, conversion from suppressed to activated immune status was observed in 2 of 3 subjects (66%). In total, 42% of specimens (10/24) were designated as radio-resistant by RT sensitivity score. Among 8 baseline specimens annotated with RT resistant status, 75% of subjects (6/8) converted to RT sensitive status after neoadjuvant SBRT. Interestingly, in the two subjects with persistent radio-resistant status in pre- and post-samples there was associated with upregulation of TGF-β or PI3K-AKT pathway activation signatures. Pre- and post-SBRT transcriptomic signatures were heterogeneous and dynamic in a cohort of 12 patients with high-risk localized PCa highlighting the importance of studying longitudinal changes in individual patients. These data highlight an opportunity to leverage tumor RNA expression profiles to personalize patient and treatment selection and augment radiation response assessment. gov ID (NCT03663218).

Multi-Omic Candidate Screening for Markers of Severe Clinical Courses of COVID-19.

Severe coronavirus disease 2019 (COVID-19) disease courses are characterized by immuno-inflammatory, thrombotic, and parenchymal alterations. Prediction of individual COVID-19 disease courses to guide targeted prevention remains challenging. We hypothesized that a distinct serologic signature precedes surges of IL-6/D-dimers in severely affected COVID-19 patients. We performed longitudinal plasma profiling, including proteome, metabolome, and routine biochemistry, on seven seropositive, well-phenotyped patients with severe COVID-19 referred to the Intensive Care Unit at the German Heart Center. Patient characteristics were: 65 ± 8 years, 29% female, median CRP 285 ± 127 mg/dL, IL-6 367 ± 231 ng/L, D-dimers 7 ± 10 mg/L, and NT-proBNP 2616 ± 3465 ng/L. Based on time-series analyses of patient sera, a prediction model employing feature selection and dimensionality reduction through least absolute shrinkage and selection operator (LASSO) revealed a number of candidate proteins preceding hyperinflammatory immune response (denoted ΔIL-6) and COVID-19 coagulopathy (denoted ΔD-dimers) by 24-48 h. These candidates are involved in biological pathways such as oxidative stress/inflammation (e.g., IL-1alpha, IL-13, MMP9, C-C motif chemokine 23), coagulation/thrombosis/immunoadhesion (e.g., P- and E-selectin), tissue repair (e.g., hepatocyte growth factor), and growth factor response/regulatory pathways (e.g., tyrosine-protein kinase receptor UFO and low-density lipoprotein receptor (LDLR)). The latter are host- or co-receptors that promote SARS-CoV-2 entry into cells in the absence of ACE2. Our novel prediction model identified biological and regulatory candidate networks preceding hyperinflammation and coagulopathy, with the most promising group being the proteins that explain changes in D-dimers. These biomarkers need validation. If causal, our work may help predict disease courses and guide personalized treatment for COVID-19.

Dynamic lipidome alterations associated with human health, disease and ageing

Lipids can be of endogenous or exogenous origin and affect diverse biological functions, including cell membrane maintenance, energy management and cellular signalling. Here, we report >800 lipid species, many of which are associated with health-to-disease transitions in diabetes, ageing and inflammation, as well as cytokine–lipidome networks. We performed comprehensive longitudinal lipidomic profiling and analysed >1,500 plasma samples from 112 participants followed for up to 9 years (average 3.2 years) to define the distinct physiological roles of complex lipid subclasses, including large and small triacylglycerols, ester- and ether-linked phosphatidylethanolamines, lysophosphatidylcholines, lysophosphatidylethanolamines, cholesterol esters and ceramides. Our findings reveal dynamic changes in the plasma lipidome during respiratory viral infection, insulin resistance and ageing, suggesting that lipids may have roles in immune homoeostasis and inflammation regulation. Individuals with insulin resistance exhibit disturbed immune homoeostasis, altered associations between lipids and clinical markers, and accelerated changes in specific lipid subclasses during ageing. Our dataset based on longitudinal deep lipidome profiling offers insights into personalized ageing, metabolic health and inflammation, potentially guiding future monitoring and intervention strategies.

Multiplexed transcriptomic profiling of the fate of human CAR T cells in vivo via genetic barcoding with shielded small nucleotides.

The design of chimeric antigen receptor (CAR) T cells would benefit from knowledge of the fate of the cells in vivo. This requires the permanent labelling of CAR T cell products and their pooling in the same microenvironment. Here, we report a cell-barcoding method for the multiplexed longitudinal profiling of cells in vivo using single-cell RNA sequencing (scRNA-seq). The method, which we named shielded-small-nucleotide-based scRNA-seq (SSN-seq), is compatible with both 3' and 5' single-cell profiling, and enables the recording of cell identity, from cell infusion to isolation, by leveraging the ubiquitous Pol III U6 promoters to robustly express small-RNA barcodes modified with direct-capture sequences. By using SSN-seq to track the dynamics of the states of CAR T cells in a tumour-rechallenge mouse model of leukaemia, we found that a combination of cytokines and small-molecule inhibitors that are used in the ex vivo manufacturing of CAR T cells promotes the in vivo expansion of persistent populations of CD4+ memory T cells. By facilitating the probing of cell-state dynamics in vivo, SSN-seq may aid the development of adoptive cell therapies.

Blood Proteome Profiling Reveals Biomarkers and Pathway Alterations in Fragile X PM at Risk for Developing FXTAS.

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder associated with the FMR1 premutation. Currently, it is not possible to determine when and if individual premutation carriers will develop FXTAS. Thus, with the aim to identify biomarkers for early diagnosis, development, and progression of FXTAS, along with associated dysregulated pathways, we performed blood proteomic profiling of premutation carriers (PM) who, as part of an ongoing longitudinal study, emerged into two distinct groups: those who developed symptoms of FXTAS (converters, CON) over time (at subsequent visits) and those who did not (non-converters, NCON). We compared these groups to age-matched healthy controls (HC). We assessed CGG repeat allele size by Southern blot and PCR analysis. The proteomic profile was obtained by liquid chromatography mass spectrometry (LC-MS/MS). We identified several significantly differentiated proteins between HC and the PM groups at Visit 1 (V1), Visit 2 (V2), and between the visits. We further reported the dysregulated protein pathways, including sphingolipid and amino acid metabolism. Our findings are in agreement with previous studies showing that pathways involved in mitochondrial bioenergetics, as observed in other neurodegenerative disorders, are significantly altered and appear to contribute to the development of FXTAS. Lastly, we compared the blood proteome of the PM who developed FXTAS over time with the CSF proteome of the FXTAS patients recently reported and found eight significantly differentially expressed proteins in common. To our knowledge, this is the first report of longitudinal proteomic profiling and the identification of unique biomarkers and dysregulated protein pathways in FXTAS.

Urinary tumor DNA MRD analysis to identify responders to neoadjuvant immunotherapy in muscle-invasive bladder cancer.

Bladder preservation is a viable option for some muscle-invasive bladder cancer (MIBC) patients, but an effective noninvasive biomarker test to accurately identify promising candidates is lacking. Here we present the clinical application of a novel tissue-agnostic, urine-based minimal residual disease (MRD) assay in the neoadjuvant setting for personalized disease surveillance and actionable target identification to facilitate bladder-sparing treatment approaches. The urinary tumor DNA (utDNA) analysis was evaluated in an investigator-initiated phase I trial RJBLC-I2N003 in which 20 patients diagnosed with resectable MIBC were treated presurgically with the PD-1 inhibitor toripalimab followed by radical cystectomy (RC). We showed that neoadjuvant toripalimab therapy was feasible, safe, and induced a 40% rate (8/20) of pathological complete response. Longitudinal utDNA profiling outperformed radiographic assessment and conventional biomarkers to predict the pathologic outcome of immune checkpoint blockade. In addition to detecting 3 exceptional responders with molecular MRD-negative status, we identified 7 other individuals characterized for utDNA clearance and 4 harboring FGFR3 mutants, all of whom (60%, 12/20) could have postponed or avoided RC. These findings demonstrate the safety and efficacy of neoadjuvant toripalimab, and suggest the immense potential of noninvasive utDNA MRD testing to guide tailored decision-making with regard to bladder preservation and change the current treatment paradigm for MIBC patients.

Longitudinal metabolomics analysis reveals the acute effect of cysteine and NAC included in the combined metabolic activators.

Growing evidence suggests that the depletion of plasma NAD+ and glutathione (GSH) may play an important role in the development of metabolic disorders. The administration of Combined Metabolic Activators (CMA), consisting of GSH and NAD+ precursors, has been explored as a promising therapeutic strategy to target multiple altered pathways associated with the pathogenesis of the diseases. Although studies have examined the therapeutic effect of CMA that contains N-acetyl-l-cysteine (NAC) as a metabolic activator, a system-wide comparison of the metabolic response to the administration of CMA with NAC and cysteine remains lacking. In this placebo-controlled study, we studied the acute effect of the CMA administration with different metabolic activators, including NAC or cysteine with/without nicotinamide or flush free niacin, and performed longitudinal untargeted-metabolomics profiling of plasma obtained from 70 well-characterized healthy volunteers. The time-series metabolomics data revealed the metabolic pathways affected after the administration of CMAs showed high similarity between CMA containing nicotinamide and NAC or cysteine as metabolic activators. Our analysis also showed that CMA with cysteine is well-tolerated and safe in healthy individuals throughout the study. Last, our study systematically provided insights into a complex and dynamics landscape involved in amino acid, lipid and nicotinamide metabolism, reflecting the metabolic responses to CMA administration containing different metabolic activators.

Multi-omics profiling of CSF from spinal muscular atrophy type 3 patients after nusinersen treatment: a 2-year follow-up multicenter retrospective study

Spinal muscular atrophy (SMA) is a neurodegenerative disorder caused by mutations in the SMN1 gene resulting in reduced levels of the SMN protein. Nusinersen, the first antisense oligonucleotide (ASO) approved for SMA treatment, binds to the SMN2 gene, paralogue to SMN1, and mediates the translation of a functional SMN protein. Here, we used longitudinal high-resolution mass spectrometry (MS) to assess both global proteome and metabolome in cerebrospinal fluid (CSF) from ten SMA type 3 patients, with the aim of identifying novel readouts of pharmacodynamic/response to treatment and predictive markers of treatment response. Patients had a median age of 33.5 [29.5; 38.25] years, and 80% of them were ambulant at time of the enrolment, with a median HFMSE score of 37.5 [25.75; 50.75]. Untargeted CSF proteome and metabolome were measured using high-resolution MS (nLC-HRMS) on CSF samples obtained before treatment (T0) and after 2 years of follow-up (T22). A total of 26 proteins were found to be differentially expressed between T0 and T22 upon VSN normalization and LIMMA differential analysis, accounting for paired replica. Notably, key markers of the insulin-growth factor signaling pathway were upregulated after treatment together with selective modulation of key transcription regulators. Using CombiROC multimarker signature analysis, we suggest that detecting a reduction of SEMA6A and an increase of COL1A2 and GRIA4 might reflect therapeutic efficacy of nusinersen. Longitudinal metabolome profiling, analyzed with paired t-Test, showed a significant shift for some aminoacid utilization induced by treatment, whereas other metabolites were largely unchanged. Together, these data suggest perturbation upon nusinersen treatment still sustained after 22 months of follow-up and confirm the utility of CSF multi-omic profiling as pharmacodynamic biomarker for SMA type 3. Nonetheless, validation studies are needed to confirm this evidence in a larger sample size and to further dissect combined markers of response to treatment.

Nasal and blood transcriptomic pathways underpinning the clinical response to grass pollen immunotherapy

Allergen immunotherapy (AIT) is a well-established disease-modifying therapy for allergic rhinitis, yet the fundamental mechanisms underlying its clinical effect remain inadequately understood. Gauging Response in Allergic Rhinitis to Sublingual and Subcutaneous Immunotherapy was a randomized, double-blind, placebo-controlled trial of individuals allergic to timothy grass who received 2 years of placebo (n= 30), subcutaneous immunotherapy (SCIT) (n= 27), or sublingual immunotherapy (SLIT) (n= 27) and were then followed for 1 additional year. We used yearly biospecimens from the Gauging Response in Allergic Rhinitis to Sublingual and Subcutaneous Immunotherapy study to identify molecular mechanisms of response. We used longitudinal transcriptomic profiling of nasal brush and PBMC samples after allergen provocation to uncover airway and systemic expression pathways mediating responsiveness to AIT. ClinicalTrials.gov Identifier: NCT01335139, EudraCT Number: 2010-023536-16. SCIT and SLIT demonstrated similar changes in gene module expression over time. In nasal samples, alterations included downregulation of pathways of mucus hypersecretion, leukocyte migration/activation, and endoplasmic reticulum stress (log2 fold changes -0.133 to -0.640, false discovery rates [FDRs] <0.05). We observed upregulation of modules related to epithelial development, junction formation, and lipid metabolism (log2 fold changes 0.104 to 0.393, FDRs <0.05). In PBMCs, modules related to cellular stress response and type 2 cytokine signaling were reduced by immunotherapy (log2 fold changes -0.611 to -0.828, FDRs <0.05). Expression of these modules was also significantly associated with both Total Nasal Symptom Score and peak nasal inspiratory flow, indicating important links between treatment, module expression, and allergen response. Our results identify specific molecular responses of the nasal airway impacting barrier function, leukocyte migration activation, and mucus secretion that are affected by both SCIT and SLIT, offering potential targets to guide novel strategies for AIT.

Study of the Human Microbiota Under Physiological Conditions

Profiling the human intestinal environment under physiological conditions involves characterizing the various components and factors in the gut to understand its normal functioning. This profiling can encompass multiple aspects, including the microbiome, host immune response, metabolites, and the physical environment of the intestine. Shalon and colleagues developed an ingestible device that collects samples from multiple regions of the human intestinal tract during normal digestion. The collection of 240 intestinal samples from 15 healthy individuals using the device and subsequent multi-omics analyses identified significant differences between bacteria, phages, host proteins, and metabolites in the intestines versus stool. Certain microbial taxa were differentially enriched, and prophage induction was more prevalent in the intestines than in stool. The host proteome and bile acid profiles varied along the intestines and were highly distinct from those of stool. Correlations between gradients in bile acid concentrations and microbial abundance predicted species that altered the bile acid pool through deconjugation. Furthermore, microbially conjugated bile acid concentrations exhibited amino acid-dependent trends that were not apparent in the stool. Overall, non-invasive, longitudinal profiling of microorganisms, proteins, and bile acids along the intestinal tract under physiological conditions can help elucidate the roles of the gut microbiome and metabolome in human physiology and disease. Nature. 2023 May;617(7961):581-591. doi: 10.1038/s41586-023-05989-7.

HYpofractionated, dose-redistributed RAdiotherapy with protons and photons to combat radiation-induced immunosuppression in head and neck squamous cell carcinoma: study protocol of the phase I HYDRA trial

BackgroundRadiotherapy (RT) is the standard of care for most advanced head and neck squamous cell carcinoma (HNSCC) and results in an unfavorable 5-year overall survival of 40%. Despite strong biological rationale, combining RT with immune checkpoint inhibitors does not result in a survival benefit. Our hypothesis is that the combination of these individually effective treatments fails because of radiation-induced immunosuppression and lymphodepletion. By integrating modern radiobiology and innovative radiotherapy concepts, the patient’s immune system could be maximally retained by (1) increasing the dose per fraction so that the total dose and number of fractions can be reduced (HYpofractionation), (2) redistributing the radiation dose towards a higher peak dose within the tumor center and a lowered elective lymphatic field dose (Dose-redistribution), and (3) using RAdiotherapy with protons instead of photons (HYDRA).MethodsThe primary aim of this multicenter study is to determine the safety of HYDRA proton- and photon radiotherapy by conducting two parallel phase I trials. Both HYDRA arms are randomized with the standard of care for longitudinal immune profiling. There will be a specific focus on actionable immune targets and their temporal patterns that can be tested in future hypofractionated immunoradiotherapy trials. The HYDRA dose prescriptions (in 20 fractions) are 40 Gy elective dose and 55 Gy simultaneous integrated boost on the clinical target volume with a 59 Gy focal boost on the tumor center. A total of 100 patients (25 per treatment group) will be recruited, and the final analysis will be performed one year after the last patient has been included.DiscussionIn the context of HNSCC, hypofractionation has historically only been reserved for small tumors out of fear for late normal tissue toxicity. To date, hypofractionated radiotherapy may also be safe for larger tumors, as both the radiation dose and volume can be reduced by the combination of advanced imaging for better target definition, novel accelerated repopulation models and high-precision radiation treatment planning and dose delivery. HYDRA’s expected immune-sparing effect may lead to improved outcomes by allowing for future effective combination treatment with immunotherapy.Trial registrationThe trial is registered at ClinicalTrials.gov; NCT05364411 (registered on May 6th, 2022).

Longitudinal transcriptome profiling of localized hormone-sensitive tumors in treatment-naïve ENACT patients with prostate cancer with and without enzalutamide (ENZA).

5026 Background: The ENACT trial (NCT02799745) randomized men with low- or intermediate-risk prostate cancer to 12 months of ENZA monotherapy followed by active surveillance (AS) for 12 months or to AS alone. Men were followed longitudinally, including by serial genome-wide expression profiling. Of 258 genomic signatures evaluated from baseline samples, increased Decipher genomic classifier score was prognostic for progression on AS; androgen receptor activity (AR-A) and PAM50 subtypes were associated with clinical benefit from ENZA, as previously reported. Here we present transcriptomic changes over time for men undergoing AS and those treated transiently with ENZA. Methods: Gene-expression profiling was performed via the Decipher GRID platform (Veracyte, Inc.) on 131 ENACT patient samples (ENZA, n=57; AS, n=74) collected at prespecified time points (screening, 12 months, and 24 months). Predefined GRID signatures were assessed for correlation with biological and clinical characteristics, including immune activity, treatment sensitivity, metastatic risk, and molecular subtypes. Statistical analysis was conducted by Cox proportional hazards models and logistic/linear regression; longitudinal changes were assessed by linear mixed-effects models. Results: Analysis of 12-month longitudinal samples showed significant transcriptomic changes in patients treated with ENZA, including markers of AR-A downregulation, decreased regulatory T-cell suppression, and increased immune activation. Many of the transcriptomic signature changes that occurred during ENZA treatment, including immune marker changes, were transient and returned to baseline values by 24 months following ENZA treatment discontinuation at 12 months. Conclusions: This exploratory biomarker analysis provides valuable data on transcriptomic changes in patients undergoing AS and those exposed to ENZA followed by cessation of treatment. Longitudinal analysis data provided insights into progression of early disease in the AS arm and possible strategies for extending ENZA activity beyond progression. Immune modulation with ENZA treatment may provide further insights for exploration of immunotherapy strategies. Clinical trial information: NCT02799745 .

Longitudinal single-cell (sc) immune profiling of cerebrospinal fluid (CSF) from melanoma patients (pts) with leptomeningeal disease (LMD) treated with intrathecal (IT) and intravenous (IV) nivolumab (N).

2015 Background: LMD is associated with an extremely poor prognosis in melanoma pts, who currently have very limited treatment options. Our group previously reported initial safety and efficacy results from the dose escalation portion of a first in human phase I/IB trial using IT and IV N (NCT03025256) in melanoma LMD pts. Here we present longitudinal sc profiling of immune cells in CSF from a subset of pts in this trial to better understand the immune composition of this unique microenvironment and features associated with treatment and outcomes. Methods: 39 CSF samples were collected longitudinally from 5 long survivors (LS; IT N number of doses received 10-92) and 5 rapid progressors (RP; IT N doses 2-9) and profiled by sc RNA sequencing. Raw FASTQ reads were aligned to the GRCh38 human reference genome using Cell Ranger v6.0.0 to generate the count matrix. All samples were aggregated by Seurat v4.3.0 and data integration was performed using Harmony v0.1.0. Cells were filtered by following criteria: (1) &gt; 20% mitochondrial content, or (2) express &lt; 100 or &gt; 2,500-5,000 genes, according to sample specific distribution. Empty droplets and doublets were identified by EmptyDrops function from the R package DropletUtils v1.18.0 and DoubletFinder v2.0.3, respectively. We applied normalization, FindVariableFeatures (n = 2,000), scaling, PCA, UMAP and FindClusters to summarize the integrated samples and determine cell clusters. Cell types were annotated based on (1) canonical marker genes using FindAllMarkers, (2) published gene expression signatures, and (3) reference-based tool using Symphony v.0.1.0 on published data: GSE164378 and GSE158803. Statistical analysis was conducted using the Wilcoxon rank-sum test. Results: A total of 10 patients were included in this pilot analysis. Five pts were LS (2 males; IT dose 5 mg, 1 pt; 10 mg, 2 pts; 20 mg, 2 pts; 2 BRAF mutant; 5 negative CSF cytology at baseline) and 5 pts were RP (3 males; IT dose 5 mg, 1 pt; 20 mg, 2 pts; 50 mg, 2 pts; 4 BRAF mutant; 4 positive CSF cytology at baseline). At baseline [prior to cycle (C)1 of IT N], LS pts had a higher percentage of NK cells (relative to total immune cells; P = 0.036) and mucosal-associated invariant T cells (relative to total T cells, P = 0.021) than RP pts. Prior to C2, LS had a significantly higher percentage of T cells (relative to all cells; P = 0.036). The abundance of immune cells and tumor cells were not different between the two groups at C3, at which point pts have received two doses of IT N and one dose of IV N. Conclusions: Exploratory sc analysis of longitudinal CSF samples of LMD pts suggest that baseline immune features may predict outcomes with IT N. Analysis of additional pts is ongoing to provide further insights.

UPS of the CNS: Longitudinal profiling of mitochondrial DNA in neuronal‐enriched packages from plasma of individuals with cognitive decline

AbstractBackgroundAlzheimer’s disease (AD) is a complex neurodegenerative disorder that disproportionately burdens Mexican Americans (MAs) due to a combination of population‐specific environmental exposures and genetic risk factors. In addition to AD, MAs have a higher prevalence of metabolic comorbidities such as type 2 diabetes (T2D) that have been implicated in accelerated age‐related cognitive impairment. Tissue‐specific mitochondrial dysfunction and oxidative stress are early and prominent features of both AD and T2D that propagate systemic inflammation as these diseases progress. Despite the shared pathophysiology, we do not fully understand the underlying molecular mechanisms linking cognitive decline and metabolic stress, especially in understudied minority populations. Mitochondrial dysfunction may serve as the biological basis of the AD health disparity in MAs who experience earlier onset of symptoms and have an overrepresentation of T2D compared to non‐Hispanic whites (NHWs). This project aims to study T2D‐related risk for cognitive impairment by assessing the packages that neurons secrete to communicate with cells in the periphery. These neuronal‐enriched exosomes (NEEs) can cross the blood‐brain barrier and are thus capable of mediating systemic inflammation in response to CNS‐sourced stress through their mitochondrial DNA (mtDNA) cargo. We hypothesize that plasma NEE mtDNA load will reflect cognitive status and T2D comorbidity over time.MethodsExosomes of neuronal origin were enriched via CD171‐based immunocapture in longitudinal plasma samples (two timepoints, 5 years apart) from 348 Texas Alzheimer’s Research and Care Consortium (TARCC) participants stratified by race/ethnicity (MA/NHW), cognitive status (AD/MCI/NC), and T2D comorbidity (+/‐). The success of NEE isolation was assessed using nanoparticle tracking analysis (NTA) and immunoblotting against CD171 and CD81 (canonical exosome surface marker). Following extraction of total DNA from NEEs, absolute mtDNA load and deletion ratio were estimated using a real‐time qPCR assay targeting sites within the minor (D‐loop) and major (ND4) arcs.ResultsHerein, we demonstrate the ability to isolate NEEs from plasma and quantify their mtDNA cargo. Resultant values were analyzed for longitudinal differences and correlation with disease status based on various neuropsychological measures.ConclusionAs the first effort to assess mtDNA in NEEs, this project provides novel insight into how T2D confers risk for AD across different populations over time.

Liquid and tissue profiling of targetable co-mutations with KRAS in non-small cell lung cancer.

e21201 Background: The primary objective of this study was to understand the co-occurrence of KRAS with other actionable drivers in Indian lung cancers patients, and to assess the utility of liquid biopsy in this cohort. Methods: We evaluated single nucleotide alterations, copy number alterations, and fusions in 507 tumor tissue and 1271 blood specimens with targeted NGS panels in NSCLC patients. Results: In tissue-based analysis (n = 507), KRAS mutations were detected in 68 (13.4%) samples. Of these, 63 (92.6%) samples did not show the presence of any targetable co-mutations. Targetable co-mutations were detected in 5 (7.4%), with concomitant EGFR mutations in all 5 samples(100%) and BRAF mutations in 2 samples (33.3%) In the liquid biopsy (n = 1271) analysis, KRAS mutations were observed in 94 (7.4%) samples. The liquid biopsy analysis showed higher presence of targetable co-mutations in 11.7% (n = 11). EGFR was the most common co-occurring targetable gene observed in 9 samples (81.8%). Mutations in BRAF were detected in 3 (27.3%) samples. ERBB2 Exon 20 insertion was detected in 1 sample (9.1%). No ALK, RET or ROS1 fusions were seen in our KRAS positive cohort. In the tissue cohort, one newly diagnosed adenocarcinoma case showed primary co-mutations in EGFR (Exon 19del VAF 76.8%) and KRAS (G13D VAF 10.5%), impacting utility of EGFR TKI as the first line therapy. Another EGFR Exon 19 del patient developed KRAS G12D mutation as an acquired resistance mutation post Osimertinib therapy. In the liquid biopsy cohort (n = 11), the KRAS positive cohort had four newly diagnosed patients with 3 having EGFR and 1 with BRAF co-mutation. Among the patients with prior therapy, one patient at presentations had EGFR Exon 19 deletion and developed EGFR T790M at progression on Afatinib. The baseline tissue did not have coexisting KRAS mutation. Fresh tissue biopsy was performed at the progression on Osimertinib. The noteworthy finding was the KRAS G12C (VAF 0.36%) presence in liquid biopsy but absences in the simultaneous fresh tissue sample. The EGFR Exon 19 was present at lesser frequency than KRAS in the liquid (VAF 0.23%). In the patients from liquid biopsy cohort with Osimertinib therapy (n = 4), all presented with KRAS as acquired resistance mutation. One among these also showed concomitant EGFR C797S mutation. Conclusions: KRAS mutation virtually makes it unlikely for other targetable mutations to coexist. It makes it thus compelling to further study the role of liquid biopsy with KRAS first and decide if chemotherapy with IO can be started considering the standard of care is to wait for the entire targeted panel before initiating IO. Equally its important to include KRAS in the lung panel and although small the concomitant KRAS may compromise the efficacy to TKI. Liquid biopsy is a promising alternative for detection of actionable alterations, including KRAS and can be harnessed for longitudinal profiling, monitoring tumor evolution and treatment outcome.

Molecular profiling of osteoprogenitor cells reveals FOS as a master regulator of bone non-union

Despite the advances in bone fracture treatment, a significant fraction of fracture patients will develop non-union. Most non-unions are treated with surgery since identifying the molecular causes of these defects is exceptionally challenging. In this study, compared with marrow bone, we generated a transcriptional atlas of human osteoprogenitor cells derived from healing callus and non-union fractures. Detailed comparison among the three conditions revealed a substantial similarity of callus and nonunion at the gene expression level. Nevertheless, when assayed functionally, they showed different osteogenic potential. Utilizing longitudinal transcriptional profiling of the osteoprogenitor cells, we identified FOS as a putative master regulator of non-union fractures. We validated FOS activity by profiling a validation cohort of 31 tissue samples. Our work identified new molecular targets for non-union classification and treatment while providing a valuable resource to better understand human bone healing biology.

96 Longitudinal Profiling of Carbon Nanotube-Induced Sporadic Mesothelioma Development: Defining the Adverse Outcome Pathway for Disease Progression

Abstract Exposure to asbestos causes pathological changes in the pleural cavity, including malignant mesothelioma (MPM). Length-dependent retention of asbestos fibres in the pleural cavity is crucial for disease development. Chronic inflammation induced by biopersistent asbestos fibres plays a key role in carcinogenesis. Engineered CarbonNanoTubes (CNT) are similar to long-fibre asbestos (LFA) in terms of their high aspect-ratio and thus may pose an asbestos-like inhalation hazard, with evidence from our laboratory and others showing the carcinogenic potential of Long-CNT. Asbestos-induced MPM is diagnosed most commonly at late stage; as a result, little is known about the genetic or epigenetic aberrations occurring in pre-malignant pleural lesions and whether they align with deletions/mutations commonly found in end-stage asbestos-exposed patient tumours. Using a mouse model of direct injection into the pleural cavity, we compared the molecular changes that occur at the mesothelium following injection with an occupationally-relevant dose of Long-CNT or LFA over 20-months. We show a common molecular signature induced by Long-CNT and LFA throughout disease progression eventually leading to the development of sporadic MPM. DNA methylation, gene expression profiles, as well as specific genes are similarly altered in the presence of Long-CNT and LFA, at matched longitudinal exposure times. For the first time, these data uncover the Adverse Outcome Pathway (AOP) from initial Long-CNT exposure through to induction of pre-neoplastic lesions and the development of sporadic pleural mesothelioma, replicating the pathogenesis of end-stage asbestos-induced human disease. This reinforces concerns that high aspect-ratio CNT may pose an asbestos-like hazard, including sporadic malignant mesothelioma.