Bone Marrow Samples Research Articles

Abstract 4961: Large-scale CyTOF data modeling of leukemia patient cohorts

Abstract Introduction: Cytometry by Time-of-Flight (CyTOF) is a single-cell proteomic assay that uses antibody-lanthanide metal conjugates to tag surface or intracellular proteins, quantified via mass spectrometry. Compared to flow cytometry, CyTOF significantly reduces overlapping signals and increases the maximum protein features from 8 to 50. CyTOF data is commonly analyzed with the ‘gating’ workflow, which is the iterative process of plotting cells by two protein features to select discrete cell populations. While CyTOF can comprehensively profile nuanced cell types, gating does not scale well in the number of features. Biases in gating often arise from the forced discretization of cell populations, subjectivity of gate boundaries, and intrinsic sample-sample variation. Contemporary challenges in CyTOF data analysis can be attributed to data volume and gating bias, which can lead to oversight of disease-related cell populations while hindering reproducibility. Methods: We developed a computational platform that enables the processing and analysis of large-scale CyTOF data. Our workflow includes modular components for data harmonization, normalization, transformation, batch correction, quality control, subsampling, and dimensionality reduction via UMAP of input sample files in Flow Cytometry Standard (FCS) format. Further, additional analytical modules are currently being integrated to deploy large-scale machine learning models for cell type identification. We use the results of these models as input for multi-scale analysis, where we aim to link single-cell CyTOF data with clinical outcomes. We test our workflow on previously published datasets and apply it to new leukemia patient cohorts of blood and bone marrow samples and a variety of marker panels. Results: We tested our workflow on previously published datasets of 4.7 M peripheral blood mononuclear cells from healthy donors (N= 20) and T cells (N = 8). We observed that samples mixed well upon integration and separated into major cell types like monocytes, CD4+/CD8+ T-cells, naïve/mature B-cells, and natural killer T-cells. We then applied it to a bone marrow cohort of healthy donors (N = 10), where we observe good mixing of samples but less clear separation of cell types. Finally, we applied our workflow to 9.3 M cells from 51 chronic lymphocytic leukemia (CLL) patients and 4 healthy donors, using a lymphocytic marker panel. Here, we observed a clear resolution of a major clonal B-cell population and immune T-cells. We are in the process of applying it to Acute Myeloid Leukemia (AML) patient data with myeloid and immune marker panels. Conclusions: We developed a computational platform that facilitates the simultaneous analysis of large-scale CyTOF data, both in the number of samples and cells per sample, a variety of marker panels, while minimizing gating biases common in traditional gating analysis. Citation Format: Garth Kong, Tania Vu, Evan Lind, Olga H. Nikolova. Large-scale CyTOF data modeling of leukemia patient cohorts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4961.

Abstract 5184: Biomarker results from PEVENAZA, a randomized phase 2 study of venetoclax and azacitidine +/- pevonedistat in newly diagnosed AML patients unfit for intensive chemotherapy: Increased efficacy in a subset of patients with IDH1/2 mutations and other observations

Abstract Background: Many newly diagnosed patients with AML are ineligible for intensive chemotherapy due to pre-existing comorbidities and older age. VIALE-A, a phase-3 trial with venetoclax (ven) and azacitidine (aza) established this combination as a new standard of care (SOC) for this patient population. The addition of pevonedistat (pevo) to this combination was evaluated in the same patient population in the current trial (NCT04266795). Here we present results of biomarker analyses from baseline and on-treatment bone marrow samples. Methods: Molecular mutational analysis was performed using a next generation sequencing (NGS) panel on baseline bone marrow samples. Combining this data with cytogenetics allowed centralized assignment of risk category based on ELN2017 guidelines. Longitudinal bone marrow aspirate samples were also examined by multi-color flow cytometry for expression of select surface antigen expression and to identify AML leukemic stem cells (LSCs) from the blast population. Results: Eleven genes were selected for analyses based on prevalence and scientific interest, including DNMT3A, ASXL1, TET2, FLT3, and IDH1/2. In contrast to other genes and ELN risk categories, we observed a statistically significant increase (p-value 0.013) in CR+CRi rates for IDH1/2 mutant patients when treated with pevo + ven + aza (95%, n=21) compared to ven+aza control arm (62%, n=21). In addition, we evaluated the prevalence of CD33, CD123 and CLEC12a, targets of current AML developmental therapeutics, in both the blast and LSC populations at baseline. The median (n=120 pts) % blast cells expressing CD33, CD123, or CLEC12a were 89.8%, 63.2%, and 66.0%, respectively. The median (n=78 pts) % LSCs expressing CD33, CD123, or CLEC12a were 48.2%, 90.9%, and 32.0%, respectively. Importantly, we did not see any significant difference in median fluorescence intensity (MFI) or % expression in any of the three proteins after treatment with ven and aza (+/- pevo). Finally, no strong associations were observed between expression levels and either mutations or ELN risk categories. Conclusions: Analyses of biomarker samples as part of the PEVENAZA trial resulted in several insights for not only the pevonedistat program but also for other AML therapeutics in development. The observation that newly diagnosed patients with IDH1/2 mutations may benefit from the addition of pevo to ven+aza is certainly hypothesis-generating but would require further follow-up with higher patient numbers. In addition, baseline CD33, CD123, and CLEC12a expression levels were in line with published studies. Here we have also shown they remain similar after SOC treatment (ven+aza) supporting the development of agents that target these proteins in both first and second line settings for AML. Citation Format: Radha Ramesh, Xiang Fang, Shuli Li, Sharon Friedlander, Farhad Sedarati, Lionel Adès, Nicholas Short, Cristina Papayannidis, Tammie Yeh. Biomarker results from PEVENAZA, a randomized phase 2 study of venetoclax and azacitidine +/- pevonedistat in newly diagnosed AML patients unfit for intensive chemotherapy: Increased efficacy in a subset of patients with IDH1/2 mutations and other observations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5184.

Abstract 2548: Retrospective prediction of clinical response to standard-of-care therapies in acute myeloid leukemia by an ex vivo drug sensitivity assay using cryopreserved primary samples

Abstract Background: Assessing drug efficacy in primary acute myeloid leukemia (AML) patient cells is crucial for pinpointing drug targets and rationally selecting therapies for patients. We have previously shown that our flow cytometry-based predictive precision medicine platform (PPMP) predicts patient outcomes for standard-of-care (SOC) AML treatments using fresh blood or bone marrow samples with high precision. Here, we wanted to test the feasibility and efficacy of the PPMP on cryopreserved patient-derived leukemic samples. However, cryopreserved leukemic samples are known for exhibiting low viability and cell recovery, rendering their practical utilization often challenging. In this study, we adapt an existing methodology for cryopreservation for patient-derived leukemic cells to investigate the potential of cryopreserved samples as an alternative to fresh samples for ex vivo prediction of treatment outcomes on our PPMP. Methods: We screened freshly isolated white blood cells from AML patients on the PPMP to assess their response to multiple SOC single agents and combinations. The assay was also run without drug treatments immediately after receiving the fresh sample (≤ 2 days from draw) to establish a phenotype baseline. The cryopreserved samples were processed using a similar protocol, with post-thaw culture times of either 24 or 48 hours in two types of cytokine-containing media. A comparative analysis between untreated fresh and cryopreserved samples determined the optimal post-thaw culture time for cryopreserved samples. Once culture time was optimized, we screened cryopreserved samples on the PPMP with identical SOC drug conditions as the fresh samples and compared the response of leukemic blasts to drug treatment as well as impacts on other phenotypic markers. Results: Based on our preliminary data, we found that culturing cryopreserved cells for 24 hours post-thaw was sufficient to maintain a minimum cell viability of 80% while closely maintaining a phenotype that recapitulated that of the corresponding fresh samples. In response to drug treatments within the ex vivo assays, preliminary data support a correlation (Decitabine single agent, R2=0.81) between the response profiles of cryopreserved and fresh patient samples as gauged from AUC calculations. Conclusion: While the preferred method for screening is to use fresh patient samples, many practical constraints make it challenging or unfeasible. The ability of Notable Lab’s high-throughput platform to use cryopreserved samples opens the doors to enhanced retrospective studies leveraging clinically annotated data from biobanks or clinical trials. These retrospective studies provide a substantial reduction in the time needed to conclude such investigations. Citation Format: Lokesh Patil, Christine J. Gu, Wade Anderson, Nicola Long, Kara Johnson, Cristina Tognon, Markus D. Lacher. Retrospective prediction of clinical response to standard-of-care therapies in acute myeloid leukemia by an ex vivo drug sensitivity assay using cryopreserved primary samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2548.

Abstract 3100: Interactive analysis of drug testing data for functional precision oncology with Breeze 2.0

Abstract High throughput screening (HTS) is used to screen large numbers of small molecules in cell-based assays to predicting their potential therapeutic response and when applying approved drug libraries using HTS it becomes drug repurposing. The Drug Sensitivity and Resistance Testing (DSRT) platform established at FIMM has been extensively used in testing panels of approved and investigational drugs on cells, both conventional cell lines and primary cell models derived from cancer patient samples. The results help in deepen our understanding of drug efficacy in individual patients and across specific disease to guide future patient stratification. Breeze 2.0 is an open access, web-based platform for interactive analysis of drug screening data in real time. Breeze accepts raw data in a simple tabular format and generates detailed quality control metrics, plots, dose-response curve fits, drug response quantification in form of IC50/EC50/AUC and Drug Sensitivity Scores (DSS). Besides tabular results, Breeze generates interactive, informative, and intuitive visualizations, such as plate heatmaps, scatterplots, barplots, clustered heatmaps, circular plots, for further analysis. Based on manually-curated published datasets available through Breeze 2.0, users can instantly compare their drug response patterns with the existing DSRT profiles from patient samples, as well as cell lines. Importantly, Breeze 2.0 has integrated curated reference DSRT data tested on bone marrow samples from healthy control individuals, which plays a crucial role in understanding cancer-selective drug response by de-prioritizing generally toxic compounds. The end-users can select multiple healthy controls or relevant patient/cell line samples and choose the drugs of interest to visualize the overlaid dose responses, as well as response quantification metrics. Breeze 2.0 is built in R, PHP, MySQL and is freely accessible at https://breeze.fimm.fi/v2 Citation Format: Swapnil Potdar, Filipp Ianevski, Aleksandr Ianevski, Ziaurrehman Tanoli, Krister Wennerberg, Brinton Seashore-Ludlow, Olli Kallioniemi, Päivi Östling, Tero Aittokallio, Jani Saarela. Interactive analysis of drug testing data for functional precision oncology with Breeze 2.0 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3100.

Abstract 5178: Guided by a predictive ex vivo test: Bringing the PLK1 inhibitor volasertib back into the clinic for venetoclax-HMA relapsed/refractory acute myeloid leukemia patients

Abstract INTRODUCTION: Volasertib, a polo-like kinase 1 (PLK1) inhibitor, has a roughly 30% CR/CRi response rate in de novo acute myeloid leukemia (AML) patients when combined with low-dose cytarabine. However, its development was halted due in part to toxicity in a Phase 3 study. We have built a flow cytometry-based, high-throughput predictive precision medicine platform to forecast clinical responses to various treatments. Here, we present our design plan for a Phase 2a clinical trial with volasertib in relapsed/refractory (R/R) AML using a modified protocol to reduce toxicity and the development of a companion diagnostic to predict responders in a follow-up Phase 2b study. METHODS: We will initiate an open-label clinical trial in AML with volasertib in venetoclax-hypomethylating agent (HMA) R/R patients while co-developing a companion diagnostic to selectively enroll predicted responders in a follow-up, Phase 2b, study. Primary and secondary objectives will focus on efficacy/safety of intravenous volasertib and ex vivo responses, respectively. To reduce the toxicity seen in the Phase 3 study, specific strategies like tailored dosing will be implemented. To develop the clinical trial assay, peripheral blood or bone marrow samples were collected from AML patients and treated ex vivo with volasertib for 3 days. Surviving blast cells were enumerated by flow cytometry. Using a dynamic separation model restricted to the 35% most sensitive samples, we estimated optimal blast cutoffs for volasertib sensitivity. RESULTS: In single-agent experiments with 41 primary AML samples (31 de novo, 9 R/R, 1 with disease status unclear), we observed dose-response profiles consistent with volasertib’s role as a cell cycle inhibitor. Irrespective of the disease status, median and mean EC50 values were about 10 nM (1st-3rd quartile from 7-12 nM). To identify an optimal concentration of volasertib for patient stratification, we compared the ex vivo resistant blast fractions at each concentration with the areas under the dose-response curves (AUCs), assuming that in our case, AUCs most accurately reflect clinical responses. At 31.6 and 100 nM, the Pearson correlation coefficients were highest (r > 0.8), suggesting that these are optimal stratifying doses. With these concentrations and corresponding blast cutoff values, 32-33% of the de novo and 25% of the R/R patients would have been predicted to be responders were they given volasertib, assuming a positive predictive value of 100%. CONCLUSION: Volasertib’s EC50 values clustered within a narrow range, suggesting that EC50 may not be a suitable metric to stratify patients into responders and nonresponders, in contrast to AUCs and residual blast fractions at 31.6 and 100 nM of volasertib, which displayed more heterogeneous distributions. Limitations include small sample sizes, especially for the R/R group. Citation Format: Markus Daniel Lacher, Glenn Michelson, Allen Ling, Wade Anderson, Jeaneille Vinas, Christine J. Gu, Chris Leonardi, Joseph Wagner, Gabriel N. Mannis. Guided by a predictive ex vivo test: Bringing the PLK1 inhibitor volasertib back into the clinic for venetoclax-HMA relapsed/refractory acute myeloid leukemia patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5178.

Abstract 556: The Idylla™ IDH1-2 Mutation Assay Kit: A tool for mutation detection in IDH1 and IDH2 genes

Abstract Background: Mutations in the genes encoding isocitrate dehydrogenase (IDH1 and IDH2) are frequently detected in various cancers, including acute myeloid leukemia (~20%) and glioma (up to ~80%). They commonly involve a single amino acid substitution at codon R132 in IDH1, and R140 or R172 in IDH2 and are important for diagnosis and prognosis as well as therapeutic decisions. With advancements in molecular diagnostics and precision therapy, IDH inhibitors such as ivosidenib, vorasidenib and enasidenib are now mainstay in management of patients with a susceptible mutation. Methods: The Idylla™ IDH1-2 Mutation Assay Kit (RUO) is a fully automated qPCR assay for the qualitative detection of 15 common mutations in IDH1 (R132C/H/G/S/L) and IDH2 (R140Q/L/G/W, R172K/M/G/W/S) at codon level. The assay consists of a ready-for-use Idylla™ DNA Cartridge and an assay specific Idylla™ Vial. It runs on the Idylla™ Platform and covers the entire process from sample to result including specimen processing, real-time PCR amplification, detection, data analysis, and result reporting, within 100 minutes.Five clinical sites in US and Europe participated in an early access program to evaluate performance of the assay and establish a final classification algorithm by testing a total of 241 specimens, including FFPE, human whole blood and bone marrow samples, and extracted DNA from these sample types. Reference results were obtained using a range of established methods with different mutations in scope, such as Sanger sequencing, immunohistochemistry, NGS and qPCR. Results: Of 241 Cartridges, one invalid result was obtained, while 13 results were flagged as having low DNA input. Compared to available reference methods, overall concordance was 98.3% (235/239), 100% (140/140) and 99.6% (230/231) for IDH1 R132, IDH2 R140 and R172, respectively (see table). Conclusion: The Idylla™ IDH1-2 Mutation Assay Kit demonstrates a high concordance with established reference methods across a range of different specimen types. Reference method(s) PPA (%) NPA (%) OPA (%) MUT WT Total Idylla™ IDH1-2 Mutation Assay Kit IDH1_R132x 111 3 114 99.1 97.6 98.3 WT 1 124 125 Total 112 127 239 IDH2_R140x 10 0 10 100.0 100.0 100.0 WT 0 130 130 Total 10 130 140 IDH2_R172x 25 0 25 96.2 100.0 99.6 WT 1 205 206 Total 26 205 231 Total 148 462 610 Citation Format: Bram De Craene, Katrien Trappeniers, Ana Marcelino, Lee Robertson, Phillippe Taniere, Jo Van Dorpe, Malaïka Van der Linden, Maria Arcila, Khedoudja Nafa, Jochen K. Lennerz, Richard Craig Carson, Richard D. Ledding, Sofie Metsu. The Idylla™ IDH1-2 Mutation Assay Kit: A tool for mutation detection in IDH1 and IDH2 genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 556.

MiR-155 and miR-223 as markers of biological and clinical features of chronic lymphocytic leukemia

Introduction. Chronic lymphocytic leukemia (CLL) is a disease characterized by large individual differences both in the clinical course and in molecular patterns of expression of genes and regulatory RNAs, which can influence pathological changes. The involvement of regulatory microRNAs miR-155 and miR-223 in the pathogenesis of CLL is fairly well known, but there is insufficient information about possible fluctuations in the expression of miR-155 and miR-223 depending on the time course of pathology development and on parameters of medical treatment. Purpose – to investigate the expression of miR-155 and miR-223 in patients having CLL with different biological and clinical features and different characteristics of treatment in terms of peripheral-blood substrates (plasma, lymphocytes, and extracellular vesicles) and bone marrow. Material and Methods. This work involved samples of peripheral blood and bone marrow from 38 patients with a diagnosis of CLL from the City Hematology Center at the government-funded healthcare institution (Novosibirsk Oblast) City Clinical Hospital No. 2 from the years 2016 to 2017. Assessment of miR-155 and miR-223 expressions was carried out by reverse-transcription real-time PCR according to the TaqMan principle. Significance of differences between groups was evaluated either by the nonparametric Mann–Whitney test or by the nonparametric Kruskal–Wallis test with subsequent pairwise comparisons via the Mann–Whitney test. Results. High variation of the analyzed parameters was found. The expression levels of miR-155 and miR-223 in microvesicles of patients with unfavorable chromosomal anomalies were lower than those in patients with the chromosomal aberrations (or the normal karyotype) associated with a moderate effect on CLL prognosis. The expression level of miR-223 in peripheral blood lymphocytes of untreated patients with CLL was higher than that observed in treated patients. Conclusion. differences in the expression levels of miR-155 and miR-223 were identified depending on chromosomal aberrations and polychemotherapy. Our preliminary results will provide the basis for future larger studies on levels of microRNAs in CLL patients having specific features of the development, clinical course, and treatment of the disease.

Abstract 4124: Patient-derived ex vivo models to test drug responses of disseminated cancer cells in various metastatic niches

Abstract Metastases arise from disseminated cancer cells (DCCs) that often lodge to distant organs long before diagnosis of the primary tumor. Unfortunately, the efficacy of (neo-)adjuvant therapies against DCCs remains uncertain. Here, we harnessed novel patient-derived ex vivo models to identify promising candidate therapies targeting DCCs and explored the impact of the unique microenvironment of metastatic target organs on the phenotypes and drug responses of DCCs. We screened disaggregated lymph node (LN) or bone marrow (BM) samples from melanoma, breast, and bladder cancer patients without distant metastasis for the presence of DCCs. Then, we expanded DCCs in patient-derived xenografts, adherent cell culture, or three-dimensional culture. LN and BM suspensions were spiked with allogeneic and autologous tumor cells to unravel the impact of different metastatic niches on DCCs. Automated high-throughput microscopy of disaggregated patient LNs was used to evaluate drug effects on DCCs in the autologous microenvironment. We established 13 cell lines from melanoma DCCs, three organoid models from breast cancer DCCs, and two organoid models from bladder cancer DCCs. The origin of each model was confirmed by assessment of histomorphology, marker expression, and genetic profiles. We screened a comprehensive panel of chemotherapeutics, targeted drugs, and antibody-drug conjugates across these in vitro models, and thereby identified compounds with the potential to eradicate DCCs. We further found that the LN and BM microenvironments differentially affected phenotypes and drug responses of cancer cells particularly when immune modulating drugs were used. Of note, ex vivo drug testing, performed directly on LN suspensions containing DCCs, revealed candidate therapeutics within a time frame acceptable for personalized treatment decisions. In conclusion, we report that the microenvironment of the specific metastatic niche impacts DCC drug responsiveness. Employing DCC-derived in vitro models in drug response assays that include specific niches may help to develop (neo)-adjuvant, personalized therapies and to discover novel therapeutics against metastasis. Citation Format: Lukas Wöhrl, Catherine Botteron, Kathrin Weidele, Steffi Treitschke, Durdam Das, Melanie Werner-Klein, Martin Hoffmann, Nataša Stojanović Gužvić, Silvia Materna-Reichelt, Florian Weber, Simon Engelmann, Roman Mayr, Miodrag Gužvić, Sebastian Haferkamp, Stephan Seitz, Christoph A. Klein, Christian Werno. Patient-derived ex vivo models to test drug responses of disseminated cancer cells in various metastatic niches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4124.

Abstract 3617: Expansion and persistence of anti-BCMA CAR T cells correlates with durability of responses in multiple myeloma patients

Abstract Background: Chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (BCMA) can induce >70% response rates in patients with relapsed/refractory multiple myeloma (RRMM). However, most patients ultimately relapse. There are two FDA-approved CAR T products: ciltacabtagene autoleucel (cilta-cel) and idecabtagene vicleucel (ide-cel), the former having much more durable responses. The mechanisms that mediate more durable responses with these CAR T cells is unknown. Previous studies have shown that specific cell states in the apheresis and infusion products are associated with long-term efficacy of anti-BCMA CAR therapy in MM and the relationship between durability of response and CAR T cell persistence remains unclear. Methods: We analyzed serial bone marrow and PBMC samples from 20 MM patients who received BCMA CAR T therapy, including cilta-cel, ide-cel, and JCARH125. Flow cytometry and single-cell RNAseq were used to evaluate the features of CAR T cells that are associated with durable clinical responses. Results: While CAR T cells were detectable but variable among patients at 1-month post infusion, the frequency of CAR T cells in bone marrow at 1-month post-infusion is positively correlated with progression free survival (PFS) in all MM patients (p=0.009). This association was also evident in patients just treated with cilta-cel (p= 0.008). By 6 months, CAR T cells could still be detected by high-throughput flowcytometry in the cilta-cel-treated patients, but CAR T cells were not detectable in most of the patients receiving the other 2 products. Cilta-cel CAR T cells predominantly possessed an effector-like surface phenotype at 1-moth post infusion in both CD8+ and CD4+ compartments in bone marrow. These CAR T cells shifted to effector memory and central memory T cell states by 6 months, respectively. For ide-cel and JCARH125, patients with more durable responses (>6mos) there was a significantly higher proportion of CAR T cells with CD8+ effector memory-like state at 1-month post infusion. Conclusions: Our data demonstrate that distinct anti-BCMA CAR T cell treatments lead to very different cellular outcomes: Cilta-cel CAR T cell treatment leads to greater expansion and persistence compared to other anti-BCMA CAR T cells. Cilta-cel CAR T cells also shift from the initial effector state to a memory phenotype. These results provide insights into features of more efficacious anti-BCMA CAR T cells that can help guide the future development of more durable treatments for MM. Citation Format: Kai Wu, Karen Law, Guy Ledergor, Chang Liu, Zenghua Fan, Vibha Gurunathan, Averey Lea, Matthew Clark, Serena Kwek, Alexander Cheung, Jeffrey Wolf, Ajai Chari, Anupuma Kumar, Justin Eyquem, Thomas Martin, Lawrence Fong. Expansion and persistence of anti-BCMA CAR T cells correlates with durability of responses in multiple myeloma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3617.

Abstract 732: Toward optimizing CXCR4 inhibition with beta adrenergic blockade to enhance chemotherapy response in acute myeloid leukemia

Abstract Introduction: CXCR4 is a chemokine receptor for CXCL12 that plays a critical role in homing of hematopoietic stem cells (HSCs) to the bone marrow microenvironment. The CXCR4 inhibitor plerixafor is used to increase mobilization of peripheral blood stem cells in combination with filgrastim. This pathway has also been investigated for applications in hematologic malignancies such as acute myeloid leukemia (AML), including clinical trials, with the goal of disrupting AML from the protective marrow environment. These have included use of plerixafor with 7+3 or MEC, BL-8040 with cytarabine, and ulocuplumab with MEC. For the ulocuplumab trial, at the study site of the authors, CR/CRi correlated with >10% CXCR4 expression (Becker et al. ASH 2014). The objective of this preclinical study is to identify the AML patient population most likely to respond to CXCR4 inhibition, and the role of combined beta-adrenergic blockade, as the latter has been shown pre-clinically to augment mobilization of HSCs when combined with a CXCR4 inhibitor, GPC-100 (Sukhtankar et al. PLoS One, 2023). Procedures: AML patient samples (N=124 including 90 blood and 34 bone marrow samples, and 21 with both) were obtained with informed consent on an IRB approved protocol. Multicolor flow cytometry was performed with sequential gating on blasts (by FSC/SSC, CD45/SSC, CD34 or phenotype), and leukemia stem cells (LSCs:CD34+CD38−,CD123+), then analysis for CXCR4 and beta-2 adrenergic receptor (ADRB2) surface expression. Correlations between CXCR4 expression and clinical characteristics including age, gender, new diagnosis vs. relapse, ELN2022 risk, cytogenetics, FLT3/NPM1 mutation status, CR1 duration, and overall survival were performed. GraphPad Prism9 was used for statistical analysis, and Mann Whitney test (two groups) and ordinary one-way ANOVA (>2 groups) were used for comparisons. Results: The range of CXCR4 expression by AML blasts is 0.05 to 99.7% (median 13.1%; mean 16.2%), and by AML LSCs 0 to 99% (median 2%, mean 15.7%). The expression by blasts and LSCs for each patient exhibited tight correlation, R2=0.88 p=1.23 × 10e-25). There was higher % expression of CXCR4 by both blasts and LSCs for new diagnosis, as compared to relapsed, patient samples (p=0.002). There was no correlation between level of CXCR4 expression and the other clinical features. For a subset of 20 patient samples analyzed for both CXCR4 and ADBR2 expression by AML blasts, ADBR2 expression ranged from 0.4 to 20.1%. For the samples that express ADBR2, there was high level expression of CXCR4, range 35.9 to 98.7% (median 78.4%, mean 76%). Conclusion: These studies support further investigation of whether simultaneous blockade of CXCR4 and ADBR2 may potentiate chemotherapy response in AML by limiting microenvironment mediated chemotherapy protection and reveal that patients with new diagnosis may be more susceptible to this approach. Citation Format: Brenda Loera, Chunxiao Zhang, Pina M. Cardarelli, Niña G. Caculitan, Sylvia Chien, Jin Dai, Vivian G. Oehler, Jianying Zhang, Guido Marcucci, Pamela S. Becker. Toward optimizing CXCR4 inhibition with beta adrenergic blockade to enhance chemotherapy response in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 732.

Abstract 2305: Label-free high dimensional single cell morphological profiling of different hematological malignancies by ghost cytometry

Abstract Characterizing the heterogeneity in human leukemias is critical for understanding mechanisms of disease onset, uncovering biomarkers for disease diagnosis, and guiding research into novel treatment approaches. While extensive research focus has been placed on characterizing the molecular heterogeneity in leukemia by modern omics technologies, comparatively less emphasis has been placed on studying the diversity in holistic morphological changes that occur in single cells in the context of disease. Here, we report on a novel approach to understanding and characterizing different hematological malignancies using ghost cytometry, a recently developed high-content flow cytometric method that leverages high-speed, artificial intelligence (AI) driven morphological characterization and analysis of single cells. We performed morphometric profiling on human bone marrow samples from patients with T and B-cell acute lymphoblastic leukemia (ALL, n=5), acute myeloid leukemia (AML, n=5), multiple myeloma (MM, n=5) and healthy bone marrow mononuclear cell (BM-MNC) controls (n=6). Morphological profiles from individual cells were analyzed by unsupervised machine learning (universal manifold approximation and projection, UMAP) and compared within disease subsets and against controls. We identified disease-specific cell populations from a mixture of healthy control and disease samples without any labeling, based on completely label-free information. For validation of the specific cell subsets, we overlaid known cell surface markers (e.g. CD45 for blast cells, CD10/CD19 for B-ALL, CD33/CD34 for AML, CD38/CD138 for MM). The intensity of CD45 was dimmer and disease-specific markers were highly expressed in the disease-specific cell population. We also identified a unique cell subpopulation in the disease-specific cell population that could not be distinguished by existing markers. We also analyzed three human myeloma cell lines (AMO1, KMS, and L363) for comparison to the primary MM samples. In the UMAP analysis using LF-GC data, in all three types of cell lines, Bortezomib-resistant cell lines exhibited different distributions from the control parental cell lines. In addition, we observed distinct morphological profiles between primary myeloma samples and myeloma cell lines. Here we present a novel cell characterization approach for human hematological diseases that leverages AI-based, label-free, high-speed morphological characterization of single cells. We demonstrate that the approach can be used to identify subtle morphological differences in blast cells from a range of blood cancer subtypes. Application of morphological profiling and AI can be used to identify measurable, disease-related changes in these diseases that have diagnostic, drug screening, and therapeutic monitoring potential. Citation Format: Asako Tsubouchi, Juho J. Miettinen, Keisuke Wagatsuma, Satoru Akai, Yuri An, Mika Uematsu, Philipp Sergeev, Dimitrios Tsallos, Markus J. Vähä-Koskela, Sadao Ota, Caroline A. Heckman. Label-free high dimensional single cell morphological profiling of different hematological malignancies by ghost cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2305.

Abstract 3788: EpCAM+ DCCs isolated from the bone marrow of breast cancer patients display high stemness and potency scores

Abstract Metastatic dissemination of cancer cells from primary to distant sites often occurs early and it has been shown that their detection is linked to poor outcomes. Eradication of disseminated cancer cells (DCC) has therefore become the primary goal of adjuvant therapies. Since the phenotype of DCC is largely unknown, we set out to search, isolate, and molecularly characterize these candidate metastasis founder cells from bone marrow of breast cancer patients, years before manifestation of metastasis. We screened more than 200 bone marrow samples of breast cancer patients with no evident metastasis (UICC stage M0) for EpCAM-positive cells. EpCAM positive cells in the bone marrow of healthy donors were used as a negative control. In addition, we isolated DCC from breast cancer patients with manifest metastasis (UICC stage M1). We then performed single cell RNA sequencing after whole transcriptome amplification of the collected DCC and the healthy donor (HD) control cells. To confirm the malignant origin of picked DCCs, we projected the transcriptome data into the bone marrow atlas, inferred copy number alterations, and searched for mutations shared with the matched primary tumor. EpCAM-positive cells from control patients mostly comprised plasma cells. In contrast, DCCs derived from M0-stage breast cancer patient formed a unique, non-overlapping cluster in the bone marrow atlas. UMAP-clustering placed M1-stage DCC clearly separate from M0 stage DCC. Among the collected M0-stage DCCs, we identified at least three separate subclusters. Strikingly, M0-stage DCC displayed much higher stemness and potency scores than M1-stage DCCs, reminiscent of embryonic cells. DCCs, identified by EpCAM, display very high transcriptional stemness scores when isolated before metastatic manifestation. Upon expansion and proliferation, stemness scores are reduced indicating epithelial re-differentiation. The embryonic phenotype of M0-stage DCCs may reflect an early adaptive mechanism that enables DCC to survive in an ectopic environment and may impact on the metastatic potential of the DCC. Citation Format: Elia Raya, Huiqin Koerkel-Qu, Laura Rudhart, Lisa-Marie Köhler, Anna Damboeck, Christoph Irlbeck, Catherine Botteron, Melanie Werner-Klein, Stephan Seitz, Christoph Klein. EpCAM+ DCCs isolated from the bone marrow of breast cancer patients display high stemness and potency scores [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3788.

Abstract 6169: High throughput in-situ spatial sequencing of proteins and RNA in FFPE tissue

Abstract We have developed an high throughput high resolution in situ multi-omic platform to enable studies in the rapidly evolving landscape of oncology and immunology research. Despite the potential of existing tools, the speed and throughput of current methodologies pose significant limitations. Here, we introduce a novel spatial sequencing platform employing a rapid 4-color SBS chemistry with sub-micron resolution imaging and an ultra-high throughput capacity. The platform simultaneously profiles RNA transcripts and proteins within formalin-fixed paraffin-embedded (FFPE) tissues. With up to 40 cm^2 of tissue area imaging within a 24-hour timeframe, the platform can power extensive, multi-omic studies in cancer research. To evaluate the performance of the platform, we prepared 5 um serial sections from healthy and tumor kidney FFPE block and used a novel tool to precisely position multiple tissue sections on a slide. Following probe binding and amplification, we used SBS readout to profile 105 transcripts and 6 proteins (via DNA-conjugated antibodies). Images were analyzed using a custom pipeline for feature detection and decoding, and cell segmentation using Cellpose. Results showed strong correlation to single-cell transcription profiles and cell type signatures defined by scRNA-Seq. Additionally, we see good agreement between single cell transcript and protein profiles. Existing spatial biology platforms are known to struggle with degraded and challenging clinical sample types (e.g. decalcified bone marrow), due to RNA fragmentation. We successfully profiled immune cells in FFPE-embedded bone marrow biopsies, measuring 143 transcript targets and 8 proteins. We believe this represents a significant step forward for in-situ analysis of bone marrow samples, and could be valuable for studying the pathology and response to treatment in blood cancers such as AML and CML. In-situ sequencing of antibody genes and T-cell receptors provides a deeper view of cellular immune response. As a step towards this goal, we demonstrated in-situ sequencing of antibody heavy and light chains within a B cell line, resulting in accurate readout of 28 bases within the CDR3 regions. In conclusion, we present an SBS-based approach for in-situ detection of targeted gene transcripts and proteins at an unprecedented throughput, and its application to the analysis of human kidney and bone marrow FFPE tissue samples. The capability to deeply profile tissues at high throughput could enable larger translational studies, and assembly of 3D maps of gene and protein expression at sub-cellular resolution. Citation Format: Michael Lawson, Yuji Ishitsuka, Andrew Pawlowski, Zhenmin Hong, Jake Koh, Kenneth Gouin, Nathan Ing, Richard Que, Yeoan Youn, Sara Ding, Tony Facchini, Ryan Costello, Katelyn Nelson, Jamie Stover, Howon Lee, Sarthak Duggal, Taylor Plaziak, Kaitlin Cameron, Mimi Abdu, Daan Witters, Martin Fabani, Eli Glezer. High throughput in-situ spatial sequencing of proteins and RNA in FFPE tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6169.

Abstract 2277: Understanding the impact of clonal hematopoiesis on multiple myeloma: Insights from exosomal RNA analysis and survival analysis

Abstract Clonal Hematopoiesis of Indeterminate Potential (CHIP) is a trait characterized by the accumulation of somatic mutations in hematopoietic stem cells in certain adults. It has been observed that the presence of CHIP can have significant implications for the development and prognosis of various blood cancers. Multiple myeloma (MM), a cancer involving the abnormal proliferation of malignant plasma cells in the bone marrow, is one such condition. Exosomes, a type of extracellular vesicles, play a role in cell communication by carrying information between cells. Analysis of UK Biobank data confirmed the poor prognosis of multiple myeloma patients with CHIP and identified a potential of paracrine effect on plasma cancer cells. To gain deeper insights into this paracrine effect on plasma cells, we attempted to analyze cell-to-cell interaction, such as the expression of miRNAs in exosomes, through exosome RNA sequencing. In our research, exosomes were extracted from bone marrow samples of 30 multiple myeloma patients, and exosome RNA sequencing was performed. Differential expression of miRNAs was identified. A total of 14 down-regulated miRNAs were identified based on adjusted p value < 0.05, log2 fold change < -1. We found that hsa-let-7f-5p, hsa-let-7a-5p, and hsa-miR-148a-3p, which are mainly miRNAs targeting MAP kinase, were significantly down-regulated in the CHIP cases. A computational tool was used to predict and integrate the target genes and pathways regulated by these DE miRNAs. The results suggested that certain inflammatory and oncogenic pathways including the MAPK signaling pathways, were not effectively suppressed in cases with CHIP (KEGG, p < 0.005). In addition, the chemokine signaling pathway and cytokine-cytokine receptor interaction (KEGG, p < 0.005) related to the paracrine effect, integrins in angiogenesis (PID, p < 0.005) related to poor prognosis and metastasis of cancer were also not adequately inhibited in the CHIP cases. Using UK Biobank data, we called somatic CHIP variants excluding those with VAF < 0.02 and analyzed them against ICD-10 codes. Hazard ratios (HR) were computed through Cox proportional regression with covariates. CHIP presence significantly correlated with MM diagnosis (HR: 1.64, CI: 1.35-2, p < 0.001), notably in TET2 mutation carriers (HR: 2.35, CI: 1.63-3.4, p < 0.001). TET2 mutations intensified MGUS to MM progression (HR: 5.0, CI: 2.27-11.0, p < 0.001). CHIP in MM impacted EFS (HR: 1.61, CI: 1.25-2.1, p < 0.001), with TET2 mutation carriers facing higher hazards (HR: 1.66, CI: 1.05-2.6, p = 0.031). CHIP, especially TET2 mutations, significantly contribute to adverse MM outcomes and MGUS to MM evolution. Our findings provide insight into how CHIP influences the tumorigenesis and cancer progression of MM. Citation Format: Jeongmin Park, GangPyo Ryu, Daniel Nachun, Maggie Maurer, Siddhartha Jaiswal, Dong-Yeop Shin, Ja Min Byun, Junshik Hong, Youngil Koh, Sung-Soo Yoon. Understanding the impact of clonal hematopoiesis on multiple myeloma: Insights from exosomal RNA analysis and survival analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2277.

Abstract 3333: Targeting the MDM2-p53 interaction: Time- and concentration-dependent studies in tumor and normal human bone marrow cells reveal strategies for an enhanced therapeutic index

Abstract Aim We aimed to design an MDM2-p53 antagonist with a differentiated tolerability profile that could be used to treat patients with wild-type TP53 malignancies. As part of an alliance between Newcastle University, Astex Pharmaceuticals, and Cancer Research Horizons, we discovered ASTX295, a potent inhibitor of the MDM2-p53 interaction that is currently under clinical investigation in patients with solid tumors (NCT03975387). We selected ASTX295 as a compound with a predicted short plasma half-life, which we hypothesised would help to mitigate the dose-limiting neutropenia and thrombocytopenia observed with earlier MDM2-p53 antagonists in clinical studies. To examine this hypothesis in vitro, we determined time- and concentration-dependent responses to ASTX295 treatment in healthy volunteer-derived human bone marrow cells, megakaryocytes, and in a panel of human tumor cell lines. Methods Samples containing bone marrow cells from healthy patients undergoing hip surgery were obtained under the ethical approval of the Newcastle Biobank (REC 12/NE/0395). Following Lymphoprep™ separation, cells were treated ex vivo with ASTX295 and seeded for Granulocyte-macrophage (GM) colony-forming assays in methylcellulose. Megakaryocytes were obtained from in vitro differentiation of CD34+ stem/progenitor cells. Human tumor cell lines (including MDM2-amplified SJSA-1) were treated with ASTX295 in vitro and seeded at low density for colony-forming assays. Exposures of 6, 12, or 24h were examined, and the data plotted to calculate LC50 values. Results The clonogenic survival of tumor cells was time-dependent, with LC50 values (mean ± SEM) in SJSA1 cells being 238 ± 46nM and 75 ± 7nM respectively (n = 3-4), following a 12h or 24h exposure to ASTX295. Time-dependent effects were also evident in five human bone marrow samples but with LC50 values of 1.9, >3, >10, >10, and >10uM being achieved at 12h, and 860 ± 268nM at 24h. Megakaryocytes showed similar time-dependent sensitivities in which daily treatment of 2 or 6h over three days did not induce apoptosis while significant cell death was observed when the treatment time was extended to 16-24h daily. In contrast, short, daily pulse treatment of 2-6h in cell lines (MV4-11, MOLM-13, SJSA-1) over three days was sufficient to induce cell death. Conclusions ASTX295 is a potent antagonist of the MDM2-p53 interaction. Collectively, our in vitro data suggest that a shorter exposure to ASTX295 (up to 12h), may help to spare healthy bone marrow cells whilst killing tumor cells. Hence, intermittent exposure to an MDM2-p53 antagonist could favourably modulate its therapeutic index. The predicted short plasma half-life of ASTX295 should provide flexibility in controlling the duration of exposure in vivo, potentially enabling a more bone-marrow sparing approach to MDM2-p53 antagonism to be utilised. Citation Format: Elaine Willmore, Maria Ahn, Suzanne Kyle, Yan Zhao, Huw Thomas, Kenneth S. Rankin, Luke Bevan, Lynsey Fazal, Keisha Hearn, Nicola Wilsher, Justyna Kucia-Tran, Nicola Ferrari, Nicola Wallis, Neil Thompson, John Lyons, Duncan Miller, Celine Cano, Martin E. Noble, Ian R. Hardcastle, Steven Howard, Gianni Chessari, John Lunec, David R. Newell, Steve R. Wedge. Targeting the MDM2-p53 interaction: Time- and concentration-dependent studies in tumor and normal human bone marrow cells reveal strategies for an enhanced therapeutic index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3333.

Abstract 3400: Single-cell proteogenomics analysis of AML samples treated with decitabine and venetoclax reveals divergent treatment escape mechanisms

Abstract The combination therapy of hypomethylating agents and venetoclax (HMA+Ven) has emerged as a standard of care for acute myeloid leukemia (AML) patients unfit for intensive chemotherapy. Nonetheless, significant clinical challenges persist including relapse and treatment resistance, to which our understanding remains incomplete. Previous studies have revealed genetic (RAS/RTK pathway mutations and TP53 mutations) and phenotypic (monocytic differentiation) resistance mechanisms. In this context, we hypothesized that integrative assessment of both genetic and phenotypic factors could further unveil the contributors of HMA+Ven resistance. To determine the predictors of treatment response, we first analyzed the baseline characteristics of 208 newly diagnosed, secondary, and relapse/refractory AML patients treated with Decitabine and Venetoclax (Dec+Ven). While complex cytogenetics, KRAS and TP53 mutations were significantly associated with treatment resistance, phenotypic factors such as bone marrow cell compositions or French-American-British (FAB) classifications were not. To identify the factors of resistance at the cellular level, we performed a single-cell targeted DNA-antibody sequencing (DAb-seq) on a subset of 33 patients. In total, 60 multi-timepoint bone marrow samples were analyzed, which included five patients with paired baseline-relapse timepoints. From all five patients, we identified that stem-like cells were largely eradicated and replaced by more differentiated cells such as monocyte-like or erythroid-like cells. At relapse, we identified three patterns of phenotypic shifts: monocytic shift, erythroid shift, and mixed monocytic/erythroid shift. The phenotypic shifts were not always associated with the expansion of new genetic mutations, except for in two cases: one patient had monocytic shift accompanied by the expansion of KRAS mutation, and another patient had erythroid shift accompanied by the expansion of NRAS mutation. To examine the mechanisms behind the phenotypic shifts, we performed a single-cell RNA sequencing on three of the relapsed patients. As a result, we confirmed the upregulation of BCL2L1 in the patient with erythroid shift (N=1), and MCL1 in the patients with monocytic shifts (N=2), but not BCL2 in both. These findings are consistent with known dependency of these cells on non-BCL-2 anti-apoptotic proteins such as MCL-1 and BCL-xL. To summarize, we conclude both genetic and phenotypic factors play a role in Dec+Ven resistance. Most importantly, we report that divergent phenotypic transitions, from stem-like toward more differentiated phenotypes (monocytic or erythroid), were frequently observed in AML relapse following Dec+Ven therapy. Close monitoring of phenotypic alterations during HMA+Ven treatment may facilitate the precise identification of AML patients predisposed to relapse. Citation Format: Yi June Kim, Zongrui Li, Abhishek Maiti, Tomoyuki Tanaka, Hidetaka Uryu, Ken Furudate, Kala Hayes, Naval Daver, Naveen Pemmaraju, Tapan M. Kadia, Farhad Ravandi, Nicholas J. Short, Maro Ohanian, Gautam Borthakur, Nitin Jain, Ghayas C. Issa, Lucia Masarova, Steven M. Kornblau, William G. Wierda, Sanam Loghavi, Sa A. Wang, Guillermo Garcia-Manero, Hagop Kantarjian, Courtney D. DiNardo, Marina Y. Konopleva, Koichi Takahashi. Single-cell proteogenomics analysis of AML samples treated with decitabine and venetoclax reveals divergent treatment escape mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3400.

Novel Angiogenesis Role of GLP-1(32-36) to Rescue Diabetic Ischemic Lower Limbs via GLP-1R-Dependent Glycolysis in Mice.

Restoring the capacity of endothelial progenitor cells (EPCs) to promote angiogenesis is the major therapeutic strategy of diabetic peripheral artery disease. The aim of this study was to investigate the effects of GLP-1 (glucagon-like peptide 1; 32-36)-an end product of GLP-1-on angiogenesis of EPCs and T1DM (type 1 diabetes) mice, as well as its interaction with the classical GLP-1R (GLP-1 receptor) pathway and its effect on mitochondrial metabolism. In in vivo experiments, we conducted streptozocin-induced type 1 diabetic mice as a murine model of unilateral hind limb ischemia to examine the therapeutic potential of GLP-1(32-36) on angiogenesis. We also generated Glp1r-/- mice to detect whether GLP-1R is required for angiogenic function of GLP-1(32-36). In in vitro experiments, EPCs isolated from the mouse bone marrow and human umbilical cord blood samples were used to detect GLP-1(32-36)-mediated angiogenic capability under high glucose treatment. We demonstrated that GLP-1(32-36) did not affect insulin secretion but could significantly rescue angiogenic function and blood perfusion in ischemic limb of streptozocin-induced T1DM mice, a function similar to its parental GLP-1. We also found that GLP-1(32-36) promotes angiogenesis in EPCs exposed to high glucose. Specifically, GLP-1(32-36) has a causal role in improving fragile mitochondrial function and metabolism via the GLP-1R-mediated pathway. We further demonstrated that GLP-1(32-36) rescued diabetic ischemic lower limbs by activating the GLP-1R-dependent eNOS (endothelial NO synthase)/cGMP/PKG (protein kinase G) pathway. Our study provides a novel mechanism with which GLP-1(32-36) acts in modulating metabolic reprogramming toward glycolytic flux in partnership with GLP-1R for improved angiogenesis in high glucose-exposed EPCs and T1DM murine models. We propose that GLP-1(32-36) could be used as a monotherapy or add-on therapy with existing treatments for peripheral artery disease. URL: www.ebi.ac.uk/metabolights/; Unique identifier: MTBLS9543.

PHF19 before and post induction treatment possess favorable potency of reflecting treatment response to protease inhibitors, event-free survival, and overall survival in multiple myeloma patients

ABSTRACT Objective Plant homeodomain finger protein 19 (PHF19) regulates hematopoietic stem cell differentiation and promotes multiple myeloma (MM) progression. This study intended to explore the potency of PHF19 at baseline and post induction treatment in estimating treatment response to protease inhibitors and survival in MM patients. Methods This retrospective study screened 69 MM patients who received protease inhibitors with bone marrow (BM) samples available at both baseline and post induction treatment. Twenty healthy BM donors were included as healthy controls (HCs). PHF19 in plasma cells from BM was quantified by reverse transcription-quantitative polymerase chain reaction. Results PHF19 at baseline and post induction treatment in MM patients were increased than in HCs. In MM patients, PHF19 was declined post induction treatment. Elevated PHF19 at baseline and post induction treatment were correlated with renal impairment, beta-2-microglobulin ≥5.5 mg/L, t (4; 14), higher international staging system (ISS) stage, and higher revised ISS (R-ISS) stage. Concerning treatment response, PHF19 at baseline and post induction treatment were negatively associated with complete response and overall response rate. Notably, abnormal PHF19 (above 95% quantile value of PHF19 in HCs) at baseline and post induction treatment were linked with shortened event-free survival (EFS) and overall survival (OS). After adjustment, abnormal PHF19 post induction treatment was independently related to shortened EFS (hazard ratio = 2.474) and OS (hazard ratio = 3.124). Conclusion PHF19 is aberrantly high and declines post induction therapy, which simultaneously reflects unfavorable treatment response to protease inhibitors as well as shorter EFS and OS in MM patients.

Monophosphoryl lipid A and poly I:C combination enhances immune responses of equine influenza virus vaccine

Equine influenza is a contagious respiratory disease caused by H3N8 type A influenza virus. Vaccination against equine influenza is conducted regularly; however, infection still occurs globally because of the short immunity duration and suboptimal efficacy of current vaccines. Hence the objective of this study was to investigate whether an adjuvant combination can improve immune responses to equine influenza virus (EIV) vaccines. Seventy-two mice were immunized with an EIV vaccine only or with monophosphoryl lipid A (MPL), polyinosinic-polycytidylic acid (Poly I:C), or MPL + Poly I:C. Prime immunization was followed by boost immunization after 2 weeks. Mice were euthanized at 4, 8, and 32 weeks post-prime immunization, respectively. Sera were collected to determine humoral response. Bone marrow, spleen, and lung samples were harvested to determine memory cell responses, antigen-specific T-cell proliferation, and lung viral titers. MPL + Poly I:C resulted in the highest IgG, IgG1, and IgG2a antibodies and hemagglutination inhibition titers among the groups and sustained their levels until 32 weeks post-prime immunization. The combination enhanced memory B cell responses in the bone marrow and spleen. At 8 weeks post-prime immunization, the combination induced higher CD8+ central memory T cell frequencies in the lungs and CD8+ central memory T cells in the spleen. In addition, the combination group exhibited enhanced antigen-specific T cell proliferation, except for CD4+ T cells in the lungs. Our results demonstrated improved immune responses when using MPL + Poly I:C in EIV vaccines by inducing enhanced humoral responses, memory cell responses, and antigen-specific T cell proliferation.

Gene expression prognostic of early relapse risk in low-risk B-cell acute lymphoblastic leukaemia in children.

ETV6::RUNX1 is the most common fusion gene in childhood acute lymphoblastic leukaemia (ALL) and is associated with favorable outcomes, especially in low-risk children. However, as many as 10% of children relapse within 3 years, and such early relapses have poor survival. Identifying children at risk for early relapse is an important challenge. We interrogated data from 87 children with low-risk ETV6::RUNX1-positive B-cell ALL and with available preserved bone marrow samples (discovery cohort). We profiled somatic point mutations in a panel of 559 genes and genome-wide transcriptome and single-nucleotide variants. We found high TIMD4 expression(>85th-percentile value) at diagnosis was the most important independent prognostic factor of early relapse (hazard ratio [HR]=5.07 [1.76, 14.62]; p=0.03). In an independent validation cohort of low-risk ETV6::RUNX1-positive B-cell ALL (N=68) high TIMD4 expression at diagnosis had an HR=4.78 [1.07, 21.36] (p=0.04) for early relapse. In another validation cohort including 78 children with low-risk ETV6::RUNX1-negative B-cell ALL, high TIMD4 expression at diagnosis had an HR=3.93 [1.31, 11.79] (p=0.01). Our results suggest high TIMD4 expression at diagnosis in low-risk B-cell ALL in children might be associated with high risk for early relapse.