Bone Marrow Samples Research Articles

Increased co-expression of ICOS and PD-1 predicts poor overall survival in patients with acute myeloid leukemia

BackgroundInducible co-stimulatory factor (ICOS) has a dual role: activating cytotoxic T cells against tumors or exacerbating immunosuppression of regulatory T cells (Tregs) to participate in immune evasion. However, the correlation between ICOS and its co-expression with inhibitory immune checkpoints (IICs) and prognosis in acute myeloid leukemia (AML) is little known. MethodsThe prognostic importance of ICOS and IICs in 62 bone marrow (BM) samples of de novo AML patients from our clinical center (GZFPH) was explored and then the RNA sequencing data of 155 AML patients from the Cancer Genome Atlas (TCGA) database was used for validation. ResultsIn both GZFPH and TCGA cohorts, high expression of ICOS was significantly associated with poor overall survival (OS) in patients with AML (P < 0.05). Importantly, co-expression of ICOS and PD-1, PD-L1, PD-L2, CTLA-4, and LAG-3 predicted poor OS in AML; among them, ICOS/PD-1 was the optimal combination of immune checkpoints (ICs). The co-expression of ICOS and PD-1 was correlated with poor OS in non-acute promyelocytic leukemia (non-APL) patients following chemotherapy. Additionally, ICOS/PD-1 was an independent OS-predicting factor (P < 0.05). Notably, a nomogram model was constructed by combining ICOS/PD-1, age, European Leukemia Net (ELN) risk stratification, and therapy to visually and personalized predict the 1-, 3-, and 5-year OS of patients with non-APL. ConclusionIncreased expression of ICOS predicted poor outcomes, and ICOS/PD-1 was the optimal combination of ICs to predict outcomes in patients with AML, which might be a potential immune biomarker for designing novel AML therapy.

Utility of immature platelet fraction in the Sysmex XN-1000V for the differential diagnosis of central and peripheral thrombocytopenia in dogs and cats.

The immature platelet fraction (IPF), a parameter obtained by the Sysmex XN-1000V analyzer, is used in humans to differentiate between central (CEN) and peripheral (PER) thrombocytopenia (TP) but has not been evaluated in small animals. Compare IPF between healthy, clinical non-TP and TP dogs and cats, study IPF in different causes of TP in dogs and cats and, establish IPF reference intervals (RIs), and study the effect of age and sex on IPF in healthy dogs and cats. A total of 3281 dogs and 726 cats. Retrospective review of medical records. Animals were classified as nonthrombocytopenic (healthy group and group of clinical patients without TP [NTP]) or TP. These latter animals were subclassified as pseudothrombocytopenia (PSE), CEN and PER, based on evaluation of platelet clumps, estimated platelet count in blood smears and final diagnosis. Blood samples were evaluated using a Sysmex XN-1000V with a specific platelet channel (PLT-F). The IPF was significantly different between each subtype of TP in both species. Immature platelet fractions <6.9% in dogs or 13.6% in cats, once PSE has been eliminated by review of blood smears, are indicative of CEN. Reference intervals for IPF were 0.5%-8% in healthy dogs and 1%-40.3% in healthy cats. We determined that IPF can differentiate between CEN and PER in dogs and cats, guiding additional testing and avoiding more invasive procedures (bone marrow sampling). A blood smear always should be evaluated to rule out platelet clumping.

Abstract P07: Extracellular Vesicles Secreted from Daratumumab Resistant Cells Promote Resistance and Proliferation of Daratumumab Sensitive cells, possibly Through the Transfer of miRNA Cargo

Abstract Daratumumab (dara) is an anti-CD38 monoclonal antibody used for Multiple Myeloma (MM) treatment, but responses are heterogeneous, and resistance is inevitable. We established three dara resistant (dara R) cell lines in Natural Killer T-cell Lymphoma (NKTL), T-cell acute lymphoblastic leukaemia (T-ALL) and MM via long-term exposure to increasing concentrations of dara. We have previously demonstrated that the exosome biogenesis pathway plays a significant role in the survival of dara R cell lines. Coculture of dara sensitive (dara S) cell lines with dara R cell lines separated by a cell-impermeable transwell insert promoted cell proliferation and reduced response to dara treatment in dara S cell lines thereby suggesting a potential role for extracellular vesicles (EVs) in mediating these responses. To assess the specific role of EVs, we isolated dara R EVs and added it to the culture of dara S cells. We observed that uptake of dara R EVs directly promote proliferation and reduced cytotoxic response to dara treatment in dara S cells. In this study, we seek to investigate the role of EVs secreted by dara R cells, in particular, that of EV-derived miRNAs. Exosomal miRNAs are known to play a role in regulating cellular pathways driving cancer cell resistance and survival. To evaluate the potential role of EV-derived miRNA cargo, we performed miRNA sequencing of EVs isolated from the plasma fraction of bone marrow samples obtained from MM patients. We found that the EVs from patients with a progression free survival (PFS) of less than 2 years after dara based therapy had an enrichment of mir-155 and mir-181a as compared to those with a PFS longer than 2 years. These data align with our findings in the cell lines and suggest that these miRNAs may be utilised as potential biomarkers for daratumumab resistance. Citation Format: Muhamad Irfan Bin Azaman, Wee Joo Chng, Sanjay de Mel, Nurulhuda Mustafa. Extracellular Vesicles Secreted from Daratumumab Resistant Cells Promote Resistance and Proliferation of Daratumumab Sensitive cells, possibly Through the Transfer of miRNA Cargo [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P07.

Prevalence of massively diluted bone marrow cell samples aspirated from patients with myelodysplastic syndromes (MDS) or suspected of MDS: A retrospective analysis of nationwide samples in Japan.

Bone marrow (BM) examination is a key element in the diagnosis and prognostic grading of myelodysplastic syndromes (MDSs), and obtaining adequate BM cell samples is critical for accurate test results. Massive haemodilution of aspirated BM samples is a well-known problem; however, its incidence in patients with MDS has not been well studied. We report the first study to examine the incidence of massive haemodilution in nationwide BM samples aspirated from patients diagnosed with or suspected of MDS in Japan. Among 283 cases available for analysis, BM smears from 92 cases (32.5%) were hypospicular (massively haemodiluted) and, particularly, no BM particles were observed in 52 cases (18.4%). Regarding hypospicular cases, we examined how the doctors in charge interpreted the BM smears of their patients. In only 19 of 92 cases (20.7%), doctors realised that the BM smears were haemodiluted. Furthermore, the BM biopsy, which can help diagnose hypospicular cases, was oftentimes not performed when the haemodilution was overlooked by doctors (not performed in 50 of 73 such cases). These real-world data highlight that not only researchers who are working to improve diagnostic tests but also clinicians who perform and use diagnostic tests must realise this common and potentially critical problem.

Evaluation of the toxicogenetic potential of visceral leishmaniasis in naturally infected dogs and salivary samples for definitive disease diagnosis

Visceral leishmaniasis is an infectious and zoonotic disease caused by the protozoan Leishmania infantum chagasi, transmitted through the bite of the sandfly Lutzomyia longipalpis, with dogs serving as the primary reservoir. Given the increasing spread of this disease in dogs and its epidemiological and public health significance, this study aimed to investigate whether salivary samples from naturally infected dogs could serve as a diagnostic tool, attempting to replace invasive conventional methods. Additionally, the study analyzed the potential toxicogenetic effects of the disease. Ten adult dogs, both males and females, intact and neutered, of mixed breed, from Rifaina (SP) and attended at the Veterinary Hospital of the University of Franca, were included. These dogs exhibited various clinical signs suggestive of visceral leishmaniasis, with five of them confirmed through serological and parasitological examinations. Salivary samples were collected using sterile swabs from the five seropositive dogs, placed on glass slides, and stained with hematoxylin-eosin for subsequent analysis of amastigote forms of the protozoan using bright-field microscopy; the results were descriptive. Furthermore, bone marrow samples from the sternum were collected from the five seropositive dogs and compared with those from the five seronegative dogs to investigate the toxicogenetic potential of the disease through the micronucleus test. To assess possible genotoxicity, each slide made from bone marrow was analyzed for 2000 immature erythrocytes (IE), totaling 4000 IE/animal, and those containing micronuclei (MN) were identified (IE-MN). Cytotoxicity was determined by analyzing 1000 erythrocytes/slide, totaling 2000 erythrocytes/dog, and subsequently calculating the ratio of IE/IE+NCE (normochromatic erythrocytes). Toxicogenetic results of seropositive dogs were compared with seronegative dogs and statistically verified by simple analysis of variance (ANOVA). The protozoan Leishmania was not detected in any of the salivary samples from seropositive dogs. The mean frequency of IE-MN in seropositive dogs was statistically higher compared to seronegative dogs (p&lt;0.0001), indicating the genotoxicity of the disease. In two of the five seropositive dogs, a decrease in erythrocyte production suggested cytotoxicity of the disease. Given the established methodology, it can be inferred that simple saliva analysis, without more specific assays, does not detect amastigote forms of the protozoan in dogs seropositive for visceral leishmaniasis and should not replace established diagnostic methods. Additionally, the micronucleus test suggested the genotoxicity and cytotoxicity of the disease.

Gene expression patterns of Sirtuin family members (SIRT1 TO SIRT7): Insights into pathogenesis and prognostic of Myelodysplastic neoplasm

To assess and validate the gene expression profile of SIRTs (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7) in relation to the pathogenesis and prognostic progression of Myelodysplastic neoplasm (MDS). Eighty bone marrow samples of patients with de novo MDS were diagnosed according to WHO 2022 and IPSS-R criteria. Ten bone marrow samples were obtained from elderly healthy volunteers and used as control samples. Gene expression levels of all SIRTs were assessed using RT-qPCR assays. Downregulation of SIRT2 (p = 0.009), SIRT3 (p = 0.048), SIRT4 (p = 0.049), SIRT5 (p = 0.046), SIRT6 (p = 0.043), and SIRT7 (p = 0.047) was identified in MDS patients compared to control individuals. Also, we identified that while SIRT2-7 genes are typically down-regulated in MDS patients compared to normal controls, there are relative expression variations among MDS patient subgroups. Specifically, SIRT4 (p = 0.029) showed increased expression in patients aged 60 or above, and both SIRT2 (p = 0.016) and SIRT3 (p = 0.036) were upregulated in patients with hemoglobin levels below 8 g/dL. SIRT2 (p = 0.045) and SIRT3 (p = 0.033) were highly expressed in patients with chromosomal abnormalities. Different SIRTs exhibited altered expression patterns concerning specific MDS clinical and prognostic characteristics. The downregulation in SIRTs genes (e.g., SIRT2 to SIRT7) expression in Brazilian MDS patients highlights their role in the disease's development. The upregulation of SIRT2 and SIRT3 in severe anemia patients suggests a potential link to manage iron overload-related complications in transfusion-dependent patients. Moreover, the association of SIRT2/SIRT3 with genomic instability and their role in MDS progression signify promising areas for future research and therapeutic targets. These findings underscore the importance of SIRT family in understanding and addressing MDS, offering novel clinical, prognostic, and therapeutic insights for patients with this condition.

Development of a Multivariable Model to Predict the Need for Bone Marrow Sampling in Persons With Monoclonal Gammopathy of Undetermined Significance : A Cohort Study Nested in a Clinical Trial.

Monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) are asymptomatic precursor conditions to multiple myeloma and related disorders. Smoldering multiple myeloma is distinguished from MGUS by 10% or greater bone marrow plasma cells (BMPC) on sampling, has a higher risk for progression, and requires specialist management. To develop a multivariable prediction model that predicts the probability that a person with presumed MGUS has 10% or greater BMPC (SMM or worse by bone marrow criteria) to inform the decision to obtain a bone marrow sample and compare its performance to the Mayo Clinic risk stratification model. iStopMM (Iceland Screens, Treats or Prevents Multiple Myeloma), a prospective population-based screening study of MGUS. (ClinicalTrials.gov: NCT03327597). Icelandic population of adults aged 40 years or older. 1043 persons with IgG, IgA, light-chain, and biclonal MGUS detected by screening and an interpretable bone marrow sample. Monoclonal gammopathy of undetermined significance isotype; monoclonal protein concentration; free light-chain ratio; and total IgG, IgM, and IgA concentrations were used as predictors. Bone marrow plasma cells were categorized as 0% to 4%, 5% to 9%, 10% to 14%, or 15% or greater. The c-statistic for SMM or worse was 0.85 (95% CI, 0.82 to 0.88), and calibration was excellent (intercept, -0.07; slope, 0.95). At a threshold of 10% predicted risk for SMM or worse, sensitivity was 86%, specificity was 67%, positive predictive value was 32%, and negative predictive value was 96%. Compared with the Mayo Clinic model, the net benefit for the decision to refer for sampling was between 0.13 and 0.30 higher over a range of plausible low-risk thresholds. The prediction model will require external validation. This accurate prediction model for SMM or worse was developed in a population-based cohort of persons with presumed MGUS and may be used to defer bone marrow sampling and referral to hematology. International Myeloma Foundation and the European Research Council.

Xenotransplantation of Human Hematopoietic Stem Cells into NBSGW Mice: A Basic Model for Preclinical Development of Gene Therapy Approaches

Background. The gene therapy based on hematopoietic cell xenotransplantation is becoming a powerful and universally applied therapeutic strategy in an ever-expanding range of human diseases. One of the current issues in implementing the techniques of genome modification in hematopoietic stem cells (HSCs) into clinical practice is to assure the quality and safety of gene and cell therapy products for human use. This is achieved by animal model testing at the stage of preclinical studies. With this purpose in view, NBSGW mice seem to be a unique and promising model for human HSC engraftment without pre-conditioning.&#x0D; Aim. To test the NBSGW mouse model for human HSC engraftment, to optimize the methods of assessing the state of the animals and monitoring the chimerism level for translational preclinical development of HSC-based products for gene and cell therapy.&#x0D; Materials &amp; Methods. The xenograft models of NBSGW mice were generated using the samples of the selected peripheral blood CD34+ HSCs from a healthy donor. Serial transplantation was performed by intravenous injection of bone marrow cells from primary recipients with a high chimerism level. Engraftment efficiency was evaluated by flow cytofluorometry (FCF) and droplet digital PCR (ddPCR). Subpopulation pattern of human cell engraftment was assessed by FCF.&#x0D; Results. The tested HSC transplantation regimen is characterized by favorable toxicity profile. In the entire study sample of mice, the FCF analysis showed a long-term engraftment of human cells with a high chimerism level (23.5–93.6 %) in the bone marrow of the animals, also after serial transplantation, which was confirmed by ddPCR. The B-lineage differentiation cells predominated in all tested samples (of peripheral blood, bone marrow, and spleen) from mice after primary and serial transplantation. The ddPCR assay can be used as an additional tool for validating the level of human cell engraftment determined by FCF.&#x0D; Conclusion. NBSGW mice present a promising reference model for preclinical development of gene and cell therapy products based on human primary HSCs with a modified genome.

Pursuing dynamics of minimal residual leukemic subclones in relapsed and refractory acute myeloid leukemia during conventional therapy.

Acute myeloid leukemia (AML) is characterized by clonal heterogeneity, leading to frequent relapses and drug resistance despite intensive clinical therapy. Although AML's clonal architecture has been addressed in many studies, practical monitoring of dynamic changes in those subclones during relapse and treatment is still understudied. Fifteen longitudinal bone marrow (BM) samples were collected from three relapsed and refractory (R/R) AML patients. Using droplet digital polymerase chain reaction (ddPCR), the frequencies of patient's leukemic variants were assessed in seven cell populations that were isolated from each BM sample based on cellular phenotypes. By quantifying mutant clones at the diagnosis, remission, and relapse stages, the distribution of AML subclones was sequentially monitored. Minimal residual (MR) leukemic subclones exhibit heterogeneous distribution among BM cell populations, including mature leukocyte populations. During AML progression, these subclones undergo active phenotypic transitions and repopulate into distinct cell population regardless of normal hematopoiesis hierarchic order. Of these, MR subclones in progenitor populations of patient BM predominantly carry MR leukemic properties, leading to more robust expansion and stubborn persistence than those in mature populations. Moreover, a minor subset of MR leukemic subclones could be sustained at an extremely low frequency without clonal expansion during relapse. In this study, we observed treatment persistent MR leukemic subclones and their phenotypic changes during the treatment process of R/R AML patients. This underscores the importance of preemptive inhibition of clonal promiscuity in R/R AML, proposing a practical method for monitoring AML MR subclones.

450 Developing Methods for High-Resolution Characterization of Plasma Cells

OBJECTIVES/GOALS: Antibodies play an important role in the pathogenesis of a wide range of diseases, including cancer, autoimmune diseases, and infections. There are currently no reliable methods to isolate and study specific plasma cell subpopulations as antibody production sources. We aim to develop methods to study plasma cells in high resolution. METHODS/STUDY POPULATION: We will use molecular cloning to engineer fusion proteins that would bind plasma cell proteins to study these cells based on their surface features. The first phase of our study consists of assessing the efficacy of this plasma cell isolation method in established cell lines (e.g., RPMI 8226) and also antibody-secreting cell lines that we are establishing as a part of this study. In the second phase of the study, we will assess the efficacy of this method by studying antigen-specific plasma cell populations in the bone marrow aspiration samples of 20 healthy volunteers using various assays, including ELISPOT, flow cytometry, and fluorescent microscopy. RESULTS/ANTICIPATED RESULTS: We have designed the constructs and have completed the cloning. The final plasmids have been verified using various restriction enzymes and Sanger sequencing. Following the transfection of Freestyle HEK 293F cells and isolation of respective proteins, we expect to be able to utilize these engineered proteins to differentiate various antibody-secreting plasma cells. We will use cell lines for proof-of-concept experiments and will subsequently move this method to human bone marrow samples. We expect to be able to visualize multiple specific antibody-secreting plasma cell populations using fluorescent microscopy and utilize this method to isolate them by cell sorting via flow cytometry. DISCUSSION/SIGNIFICANCE: We expect to be able to use this method to target specific plasma cell clones in the advancement of precision medicine regarding the treatment of plasma cell disorders (e.g., multiple myeloma) and also expand its use in other areas, such as antibody discovery and the assessment of the humoral immune responses in infectious diseases.

Identification of cells of leukemic stem cell origin with non-canonical regenerative properties

Despite most acute myeloid leukemia (AML) patients entering remission following chemotherapy, outcomes remain poor due to surviving leukemic cells that contribute to relapse. The nature of these enduring cells ispoorly understood. Here, through temporal single-cell transcriptomic characterization of AML hierarchical regeneration in response to chemotherapy, we reveal a cell population: AML regeneration enriched cells(RECs). RECs are defined by CD74/CD68 expression, and although derived from leukemic stem cells (LSCs), are devoid of stem/progenitor capacity. Based on REC in situ proximity to CD34-expressing cells identified using spatial transcriptomics on AML patient bone marrow samples, RECs demonstrate the ability to augment or reduce leukemic regeneration invivo based on transfusion or depletion, respectively. Furthermore, RECs are prognostic for patient survival as well as predictive of treatment failure in AML cohorts. Our study reveals RECs as a previously unknown functional catalyst of LSC-driven regeneration contributing to the non-canonical framework of AML regeneration.

Decreased neutrophil function in newly diagnosed multiple myeloma patients is restored with lenalidomide therapy.

Bacterial infections are common and a major cause of morbidity and mortality in multiple myeloma (MM). We have investigated the function of polymorphonuclear leukocyte (PMN), the immune system's first line of defense against bacteria, in peripheral blood (PB) and bone marrow (BM) samples from patients with newly diagnosed MM (NDMM), smoldering MM (SMM), monoclonal gammopathy of undetermined significance (MGUS) and healthy controls. Phagocytosis and oxidative burst in PMN cells from patients and healthy donors were investigated using PhagoTest and PhagoBurst assay. PMN from NDMM, SMM, and MGUS patients had reduced phagocytosis and oxidative burst ability compared with healthy controls. The dysfunction was most prominent in BM samples from MM, SMM, and MGUS patients. Importantly the reduced phagocytosis in MM patients was restored in patients on lenalidomide therapy. Consistently the ability of Escherichia coli stimulated oxidative burst in BM was reduced for the MM, SMM, and MGUS cohort in contrast to the healthy controls and the patients on lenalidomide treatment. Our results show that MM patients have neutrophil dysfunction that could contribute to susceptibility for bacterial infections and that lenalidomide therapy was associated with restored PMN function.

Using Targeted Transcriptome and Machine Learning of Pre- and Post-Transplant Bone Marrow Samples to Predict Acute Graft-versus-Host Disease and Overall Survival after Allogeneic Stem Cell Transplantation.

Acute graft-versus-host disease (aGvHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). We performed RNA analysis of 1408 candidate genes in bone marrow samples obtained from 167 patients undergoing HSCT. RNA expression data were used in a machine learning algorithm to predict the presence or absence of aGvHD using either random forest or extreme gradient boosting algorithms. Patients were randomly divided into training (2/3 of patients) and validation (1/3 of patients) sets. Using post-HSCT RNA data, the machine learning algorithm selected 92 genes for predicting aGvHD that appear to play a role in PI3/AKT, MAPK, and FOXO signaling, as well as microRNA. The algorithm selected 20 genes for predicting survival included genes involved in MAPK and chemokine signaling. Using pre-HSCT RNA data, the machine learning algorithm selected 400 genes and 700 genes predicting aGvHD and overall survival, but candidate signaling pathways could not be specified in this analysis. These data show that NGS analyses of RNA expression using machine learning algorithms may be useful biomarkers of aGvHD and overall survival for patients undergoing HSCT, allowing for the identification of major signaling pathways associated with HSCT outcomes and helping to dissect the complex steps involved in the development of aGvHD. The analysis of pre-HSCT bone marrow samples may lead to pre-HSCT interventions including choice of remission induction regimens and modifications in patient health before HSCT.

Performance of a novel eight-color flow cytometry panel for measurable residual disease assessment of chronic lymphocytic leukemia.

Measurable residual disease (MRD) is an important prognostic indicator of chronic lymphocytic leukemia (CLL). Different flow cytometric panels have been developed for the MRD assessment of CLL in Western countries; however, the application of these panels in China remains largely unexplored. Owing to the requirements for high accuracy, reproducibility, and comparability of MRD assessment in China, we investigated the performance of a flow cytometric approach (CD45-ROR1 panel) to assess MRD in patients with CLL. The European Research Initiative on CLL (ERIC) eight-color panel was used as the "gold standard." The sensitivity, specificity, and concordance rate of the CD45-ROR1 panel in the MRD assessment of CLL were 100% (87/87), 88.5% (23/26), and 97.3% (110/113), respectively. Two of the three inconsistent samples were further verified using next-generation sequencing. In addition, the MRD results obtained from the CD45-ROR1 panel were positively associated with the ERIC eight-color panel results for MRD assessment (R = 0.98, p < 0.0001). MRD detection at low levels (≤1.0%) demonstrated a smaller difference between the two methods (bias, -0.11; 95% CI, -0.90 to 0.68) than that at high levels (>1%). In the reproducibility assessment, the bias was smaller at three data points (within 24, 48, and 72 h) in the CD45-ROR1 panel than in the ERIC eight-color panel. Moreover, MRD levels detected using the CD45-ROR1 panel for the same samples from different laboratories showed a strong statistical correlation (R = 0.99, p < 0.0001) with trivial interlaboratory variation (bias, 0.135; 95% CI, -0.439 to 0.709). In addition, the positivity rate of MRD in the bone marrow samples was higher than that in the peripheral blood samples. Collectively, this study demonstrated that the CD45-ROR1 panel is a reliable method for MRD assessment of CLL with high sensitivity, reproducibility, and reliability.

Transcriptome free energy can serve as a dynamic patient-specific biomarker in acute myeloid leukemia

Acute myeloid leukemia (AML) is prevalent in both adult and pediatric patients. Despite advances in patient categorization, the heterogeneity of AML remains a challenge. Recent studies have explored the use of gene expression data to enhance AML diagnosis and prognosis, however, alternative approaches rooted in physics and chemistry may provide another level of insight into AML transformation. Utilizing publicly available databases, we analyze 884 human and mouse blood and bone marrow samples. We employ a personalized medicine strategy, combining state-transition theory and surprisal analysis, to assess the RNA transcriptome of individual patients. The transcriptome is transformed into physical parameters that represent each sample’s steady state and the free energy change (FEC) from that steady state, which is the state with the lowest free energy.We found the transcriptome steady state was invariant across normal and AML samples. FEC, representing active molecular processes, varied significantly between samples and was used to create patient-specific barcodes to characterize the biology of the disease. We discovered that AML samples that were in a transition state had the highest FEC. This disease state may be characterized as the most unstable and hence the most therapeutically targetable since a change in free energy is a thermodynamic requirement for disease progression. We also found that distinct sets of ongoing processes may be at the root of otherwise similar clinical phenotypes, implying that our integrated analysis of transcriptome profiles may facilitate a personalized medicine approach to cure AML and restore a steady state in each patient.

Abstract 4215: Single-cell proteomic characterization of the tumor microenvironment in humanized NOG-EXL patient-derived xenograft (PDX) models

Abstract Introduction: CD34+ hematopoietic stem cell (HSC) derived humanized mice engineered to express human cytokine transgenes are promising models for the evaluation of novel immunotherapeutic treatments. While flow cytometry-based analyses have confirmed the presence of many human immune cell populations within humanized mice bearing patient-derived tumor xenografts (PDX), comprehensive high-dimensional immunophenotyping of the tumor microenvironment (TME) of these models remains limited. Beyond this, the relative influence of PDX vs. donor CD34+ HSC populations on TME composition is poorly understood. Here, we characterize the quantity and diversity of immune cell subpopulations in the TME, bone marrow, and spleen of humanized NOG-EXL (huNOG-EXL) mice using Cytometry by Time-Of-Flight (CyTOF) data with &amp;gt; 60 features. Methods: We performed CyTOF analysis on 250 dissociated samples from primary tumors (n = 15; n = 6 head and neck cancer, n = 6 ovarian cancer, n = 3 renal cell carcinoma), as well as xenograft (n = 59), spleen (n = 88), and bone marrow (n = 88) samples from huNOG-EXL mice (Taconic). Each sample was profiled with two partially overlapping antibody panels; an innate immunity-focused panel (40 features), and an adaptive immunity-focused panel (40 features). Results: CyTOF data were collected from over 10 million live immune cells for each of the adaptive and innate antibody panels. Unbiased clustering of single-cell marker expression revealed clusters corresponding to all major lymphoid and myeloid cell types. Therapeutically relevant cell states were identified including exhausted CD8+ T cells marked by PD-1 and Tim-3 expression, and PD-L1+ macrophages. Hierarchical clustering of bone marrow and spleen samples suggested that systemically, immune composition was not driven by tumor-intrinsic factors alone with many samples clustering on the basis of CD34+ stem cell donor ID. Conversely, clustering PDX samples revealed strong consistency in xenografts originating from the same primary tumor sample regardless of the chosen stem cell donor. The immune composition of primary tumor samples showed greater similarity with matched PDX samples versus unmatched PDX samples, further supporting that tumor-intrinsic factors drive immune composition in xenografts. Conclusions: We provide a high-resolution characterization of the immune phenotypes present in huNOG-EXL PDX models across three cancer types. These data captured substantial heterogeneity in the immune composition of humanized xenograft samples, and we demonstrate that immune composition in humanized xenografts is strongly driven by tumor-intrinsic features. Overall, this resource data supports the use of these models for immuno-oncology studies with future work focused on functionally validating specific immune cell populations of interest. Citation Format: Daniel Stueckmann, Jalna Meens, Sally Zhang, Shirley Hui, Sujana Sivapatham, Sephane Chevrier, Jennifer Pfeil, Lisa Martin, Maria Komisarenko, Philip Dube, Susan Prendeville, Hartland Jackson, Antonio Finelli, Gary Bader, Bernd Bodenmiller, Keith A. Lawson, Laurie Ailles. Single-cell proteomic characterization of the tumor microenvironment in humanized NOG-EXL patient-derived xenograft (PDX) models [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 4215.

Abstract 112: The NSG-Flt3KO; Tg(hFLT3LG); hIL15 mouse strain improves engraftment of innate immune cells post-CD34+ humanization

Abstract Human immune system mouse models play critical roles in numerous preclinical drug development pipelines, including immunology, infectious diseases, and immuno-oncology. These models are constantly evolving, with addition of human cytokines to sustain engraftment of human immune cells. To date, most humanized models have primarily focused on T lymphocyte cell biology, leaving room for improvement in developing models that effectively represent NK cells and the myeloid lineage. Previously, we demonstrated that the introduction of human IL-15 (NSG-IL-15) or FLT3L (NSG-FLT3L) to NOD-scid IL2rγnull (NSG) mice enhanced engraftment of innate immune cells, including human CD56+ NK cells, CD141+ and CD1c+ DC subsets, CD123+ pDCs, and CD14+ monocytes. In this study, we describe a new NSG mouse variant (NSG-Flt3KO; Tg(hFLT3LG); hIL15) that expresses physiological levels of both human IL-15 and FLT3L and detail the immune cell diversity following HSC engraftment. NSG-Flt3KO; Tg(hFLT3LG); hIL15 mice, along with control strains NSG, NSG-IL15, and NSG-FLT3L, were engrafted with human umbilical cord blood derived CD34+ hematopoietic stem cells from three different donors, and blood samples were collected until Week 20 post-transplantation. Blood samples were analyzed via flow cytometry to assess the engraftment of innate and adaptive immune cells. Spleen and bone marrow samples were collected at Week 12 post-transplantation and analyzed for lymphoid and myeloid immune cell populations via flow cytometry. To compare the differences among the strains in terms of immune cell sub-populations across weeks, a Two-Way ANOVA (mixed-model) was conducted, with p&amp;lt;0.05 considered statistically significant. Overall, the results demonstrate that NSG-Flt3KO; Tg(hFLT3LG); hIL15 mice have enhanced CD56+/dim CD16+ NK cells compared to NSG-FTL3L strain (20.5±12.8% vs 7.0±3.5% at Week 12), but comparable to NSG-IL15. Furthermore, myeloid sub-lineage cells in NSG-IL15-FLT3L mice, such as monocytes, macrophages (M1), granulocytes, dendritic cells, and neutrophils, were greater than in NSG or NSG-IL15 strains, but comparable to NSG-FLT3L mice. Overall levels of hCD45+ and T cell sub-populations (CD3+ or CD4+ or CD8+) were comparable across all strains tested. In sum, our NSG-Flt3KO; Tg(hFLT3LG); hIL15 engraftment data suggest an improved model that supports drug development targeting human myeloid and NK cell populations. Citation Format: Srinivas Reddy Boreddy, Shuang Hu, Brian Soper. The NSG-Flt3KO; Tg(hFLT3LG); hIL15 mouse strain improves engraftment of innate immune cells post-CD34+ humanization [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 112.

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.