Bone Marrow Aspirates Of Patients Research Articles

Bone Marrow Aspiration and Biopsy Findings in Patients with Hematological Disorders in a Tertiary Care Center from Eastern Nepal: An Institutional Experience

Background: Bone marrow study is an important study which is performed when the peripheral blood examination and other laboratory tests and clinical signs and symptoms are suggestive and sometimes inconclusive of both hematological neoplastic and non-neoplastic conditions. The aim of this study was to study about the indications of bone marrow to assess the diagnostic value and comparison of microscopic findings with immunophenotyping and molecular studies. Methods: A total of 102cases were analyzed retrospectively and prospectively from January 2022 to October 2023. All the bone marrow aspiration and trephine biopsy received at the laboratory during this period were included in the study and other cases were analyzed form medical record of the department. Results: Among the 102 cases, mean age of the patient was 48 years and majority were male patients. The age range was between 17-80 years. The male: female ratio was 1.25:1. Weakness was most common presenting complain. Acute leukemia was the most frequent diagnosis (24.5%) followed by myeloproliferative neoplasm (7.8%), primary aplastic anemia (6.9%), myelodysplastic syndrome (5.8%), megaloblastic anemia (4.9%) and plasma cell dyscrasia (3.9%), CLPD (2.9%), ITP (1.9%). Reactive marrow constituted 3.9% and few of the rare diagnosis included hypereosinophilia with PDGFRA mutation, Leishmaniasis etc. Conclusions: The study concludes the bone marrow aspirate and biopsy examination is still the gold standard technique for screening and definite diagnosis of hematological conditions. Immunophenotyping and molecular studies are important for treatment purpose and in case of cytomorphological dilemma.

A Comparative Study of B Cell Blast Isolation Methods from Bone Marrow Aspirates of Pediatric Leukemia Patients.

Density gradient centrifugation is a conventional technique widely utilized to isolate bone marrow mononuclear cells (BM-MNC) from bone marrow (BM) aspirates obtained from pediatric B-cell acute lymphoblastic leukemia (B-ALL) patients. Nevertheless, this technique achieves incomplete recovery of mononuclear cells and is relatively time-consuming and expensive. Given that B-ALL is the most common childhood malignancy, alternative methods for processing B-ALL samples may be more cost-effective. In this pilot study, we use several readouts, including immune phenotype, cell viability, and leukemia-initiating capacity in immune-deficient mice, to directly compare the density gradient centrifugation and buffy coat processing methods. Our findings indicate that buffy coat isolation yields comparable BM-MNC product in terms of both immune and leukemia cell content and could provide a viable, lower cost alternative for biobanks processing pediatric leukemia samples.

Newly identified roles for PIEZO1 mechanosensor in controlling normal megakaryocyte development and in primary myelofibrosis.

Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in mouse and human Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Yet, a role for PIEZO1 in megakaryopoiesis and proplatelet formation has never been investigated. Here, we show that activation of PIEZO1 increases the number of immature Mks in mice, while the number of mature Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its activation reduces Mk size, ploidy, maturation, and proplatelet extension. Resulting effects of PIEZO1 activation on Mks resemble the profile in Primary Myelofibrosis (PMF). Intriguingly, Mks derived from Jak2V617F PMF mice show significantly elevated PIEZO1 expression, compared to wild-type controls. Accordingly, Mks isolated from bone marrow aspirates of JAK2V617F PMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2V617F PMF patients are rescued upon PIEZO1 inhibition. Together, our data suggest that PIEZO1 places a brake on Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease.

Use of extracellular vesicle microRNA profiles in patients with acute myeloid leukemia for the identification of novel biomarkers.

This study aimed to establish clinically significant microRNA (miRNA) sets using extracellular vesicles (EVs) from bone marrow (BM) aspirates of patients with acute myelogenous leukemia (AML), and to identify the genes that interact with these EV-derived miRNAs in AML. BM aspirates were collected from 32 patients with AML at the time of AML diagnosis. EVs were isolated using size-exclusion chromatography. A total of 965 EV-derived miRNAs were identified in all the samples. We analyzed the expression levels of these EV-derived miRNAs of the favorable (n = 10) and non-favorable (n = 22) risk groups; we identified 32 differentially expressed EV-derived miRNAs in the non-favorable risk group. The correlation of these miRNAs with risk stratification and patient survival was analyzed using the information of patients with AML from The Cancer Genome Atlas (TCGA) database. Of the miRNAs with downregulated expression in the non-favorable risk group, hsa-miR-181b and hsa-miR-143 were correlated with non-favorable risk and short overall survival. Regarding the miRNAs with upregulated expression in the non-favorable risk group, hsa-miR-188 and hsa-miR-501 were correlated with non-favorable risk and could predict poor survival. Through EV-derived miRNAs-mRNA network analysis using TCGA database, we identified 21 mRNAs that could be potential poor prognosis biomarkers. Overall, our findings revealed that EV-derived miRNAs can serve as biomarkers for risk stratification and prognosis in AML. In addition, these EV-derived miRNA-based bioinformatic analyses could help efficiently identify mRNAs with biomarker potential, similar to the previous cell-based approach.

Utility of bone marrow examination (BME) in the diagnosis of pyrexia of unknown origin (PUO).

Bone marrow examination (BME) is a useful tool in the diagnosis of haematological and non-haematological diseases. It plays an important role in early diagnosis of the underlying cause of pyrexia of unknown origin (PUO) and can influence the management of patients. Bone marrow aspiration (BMA) plays a very important role in establishing a definitive diagnosis in cases of PUO. The aim of this study was to review the indications and usefulness of bone marrow aspirates sent for microbiological evaluation as a diagnostic tool with histopathological correlation. A prospective study was conducted from 1 January 2017 to 30 September 2019 in the Department of Microbiology and Pathology on the bone marrow aspirates of patients of all groups. A total of 148 bone marrow aspirates were included. The cases were categorized as classical PUO (n = 81/148, 54.7%), nosocomial PUO (n = 4 /148, 2.7%), neutropenic PUO (n = 18/148, 12.1%), and immunocompromised PUO (n = 45/148, 30.4%), among which were systemic lupus erythematosus cases n = 8/45 (22.2%), human immunodeficiency virus positive cases n = 10/45 (17.7%), and renal transplant cases n = 27/45 (60%). A total of 28 BMAs were positive for microorganisms, out of which bacterial pathogens were n = 12 (42.8%), mycobacterial n = 12, 42.8%, fungal (n = 3, 10.7 %), and viruses (n = 1, 3.5%). This study helped in highlighting the role of bone marrow examination as an important diagnostic method in the diagnosis of infectious diseases.

Relationship between Mitochondrial Oxphos Kinetics and Response to Induction Chemotherapy in Patients with Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy carrying a poor prognosis. Relative to non-leukemic myeloid progenitors, AML cells present with elevations in mitochondrial metabolism driven by higher mitochondrial content. Despite having more mitochondria per cell, AML cells are inefficient at coupling respiration to ATP synthesis, indicative of intrinsic deficiencies in oxidative phosphorylation (OXPHOS) flux. The present study aimed to determine the relationship between mitochondrial OXPHOS kinetics and chemotherapy response in a clinical cohort of AML patients by comparing mitochondrial bioenergetic profiles between responders to induction chemotherapy with those who have persistent disease, and between adverse risk and intermediate risk AML. Methods: In this prospective study from January 2020 to February 2023, we collected bone marrow aspirate samples from patients with new diagnosis of AML on day 1 to evaluate the bioenergetic phenotype of the AML cells. We collected data on primary induction therapy, response to induction therapy, relapse, and other clinical features. We compared the bioenergetic phenotypes between the responders to primary induction chemotherapy and non-responders who had persistent disease post induction chemotherapy. We did the same comparison between patients with adverse risk and intermediate risk AML per European LeukemiaNet (ELN) 2022 risk stratification. Mitochondrial bioenergetic profiles were generated via high resolution respirometry using freshly isolated mononuclear cells from each patient's bone marrow aspirate. The Fisher exact test was used for categorical variables and the Wilcoxon Rank Sum for continuous variables. Results: In 18 AML patients included in the study, median age was 64 years (range 30-85 years), 67% (N=12) were male, 33% female, 61%(n=11) were Caucasian and 39%(n=7) were African Americans. Day 1 median blast count was 57% (range 20-92%). Initial induction therapy with cytarabine/daunorubicin (standard 7+3) with or without midostaurin (in patients with FLT3 mutations) was done in 12 (67%) patients and hypomethylating agent/venetoclax was used in 5 (28%) patients. One patient was managed with azacitidine only. Seven (39%) patients had persistent disease after induction and others responded to the initial induction. Relapsed disease after achieving remission with initial induction was seen in 8 (44%) patients. No patients had favorable risk cytogenetics/molecular findings, 10 had adverse risk AML and 9 had intermediate risk AML. TP53 mutation was observed in 4 patients, FLT3 mutation in 5 patients, IDH1 mutation in 4, and IDH2 mutation in 3 patients. The fraction of the respiratory network capable of performing OXPHOS (i.e., Fractional OXPHOS) was 0.426 in responders and 0.629 in non-responders (p=0.93). In responders, basal respiration in intact cells was non-significantly (p=0.54) lower ( JO2 9.838 pmol/s/million cells) compared to non-responders ( JO2 11.52 pmol/s/million cells). Adverse risk patients had lower fractional OXPHOS, and higher basal and maximal respiration rates as compared to intermediate risk AML patients (Table 1). Conclusion: While Fractional OXPHOS and basal respiration were lower in the patients who had remission after induction therapy, this difference was not statistically significant. We did not find a clinically significant correlation in the bioenergetic profile of AML cells; however, our study is limited by small sample size. Further research is warranted to investigate the role of OXPHOS kinetics in patients of AML that can translate into clinically meaningful outcomes.

Newly Identified Roles for PIEZO1 Mechanosensor in Controlling Normal Megakaryocyte Development and in Primary Myelofibrosis

Introduction: Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix (ECM) to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Primary Myelofibrosis (PMF) is a Chromosome Philadelphia-negative Myeloproliferative Neoplasm (MPN), characterized by altered stiffer ECM and megakaryocytosis, often driven by the gain-of-function mutation in JAK2 V617F. Here, we study a role for PIEZO1 in megakaryopoiesis and proplatelet formation under normal physiological conditions and in the context of PMF. Methods: Bone marrow Mks from C57BL/6J control and myelofibrotic mice carrying the human JAK2 V617F+ mutation, or Mks derived from stem cells of patients carrying the same mutation were analyzed at mRNA and protein levels for Piezo1 expression. Effects of Piezo1 activation or inhibition on Mk maturation and ploidy were assessed by flow cytometry, and proplatelet formation by fluorescent microscopy. Human peripheral blood samples from PMF patients, diagnosed according to established criteria, or healthy individuals were used to compare the two categories for Piezo1 relative expression on mRNA and protein levels and platelet biogenesis. Mk-specific double knockout mice of Piezo1/2 were generated to compare platelet counts under normal conditions or bone marrow ablation with 5-FU challenge. Culture of mouse and human Mks in different 3D microenvironments was used to demonstrate in vitro association of Piezo1 expression levels with different ECM stiffness substrates. Results: Mouse Mks express PIEZO1 and negligible levels of PIEZO2. Pharmacological activation of mouse PIEZO1 increases the number of immature CD41 + Mks, while the number of mature CD41 +CD42 + Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its pharmacological activation reduces Mk size, ploidy, maturation, and proplatelet development. Resulting effects of PIEZO1 activation on Mks resemble the profile in PMF. Intriguingly, Mks derived from PMF mice bearing the Jak2 V617F mutation show significantly elevated PIEZO1 expression, compared to wild-type controls. Pharmacological activation of PIEZO1 in these cells significantly augments the number of immature CD41 + Mks, and sharply reduces the number of mature CD41 +CD42 + cells and ploidy level. Accordingly, Mks isolated from bone marrow aspirates of JAK2 V617FPMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Additionally, PIEZO1 was overexpressed in 3D culture of human and mouse Mks in stiffer microenvironments as compared to softer ones and liquid medium. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2 V617FPMF patients are rescued upon PIEZO1 inhibition with GsMTx4. Discussion: Our data show that PIEZO1 might serve as a break of Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease. Our finding that inhibition of PIEZO1 activity partially rescues the aberrant Mk phenotype associated with PMF suggests that GsMTx4 holds potential as a therapeutic strategy to mitigate the pathological effects observed in Mk development in PMF patients.

The Genomic and Transcriptomic Landscape of Myeloid Sarcoma and Associated Acute Myeloid Leukemia

Background: Myeloid sarcoma (MS) is a distinct form of acute myeloid leukemia (AML) found in ~10% of patients, in which mass-forming myeloid blasts proliferate in extramedullary sites. MS may present as an isolated lesion as the primary manifestation of disease, co-occur with AML, or, more commonly, arise in the setting of AML relapse. It is associated with poor outcomes, and treatment options are sparse. While increased knowledge of AML and its biology have improved the care and survival of AML patients, those with MS continue to have limited successful treatment options, as MS has typically not been included in AML genetic and genomic profiling efforts, and patients are excluded from clinical trials. Leukemia cutis (LC) is a form of extramedullary AML characterized by cutaneous involvement, often with an infiltrative and non-mass forming pattern of growth. Still, historically the literature has not made a clear distinction between MS and LC. Although recent small pilot studies have suggested differences in AML-associated mutations between medullary AML and MS, knowledge of the complete molecular and cellular composition of MS and comprehensive comparison with AML is lacking. While molecular characterization of the extramedullary disease site is encouraged in the updated clinical recommendations, no definitive guidance exists for changes in the management and care of AML patients with MS, given the substantial gap in our current knowledge and understanding of extramedullary AML. Methods: Here, we performed RNA-Seq (n=96), single-cell (sc) RNA-Seq (n=33), and mutation profiling using either whole exome sequencing (WES, n=6) or a 409-gene targeted sequencing panel (n=48) of MS or LC patient samples and bone marrow (BM) aspirates from AML patients with and without extramedullary disease to present the first comprehensive characterization of MS and LC. Results: Our WES data showed thatMS and LC evolve independently in the extramedullary site from concurrent medullary disease with distinct dominant clones, characterized by higher median tumor mutation burden (1.66 vs. 064 mutations per megabase, respectively; p=0.0059) and increased copy number aberrations, which both validated in an MS Tet2 HR mouse model. Notably, we observed location-specific molecular evolution in patients with multiple concurrent MS sites (Figure 1A) and patients with recurring MS in WES and targeted panel data. While targetable mutations (i.e., IDH1/2 mutations and FLT3-ITD/TKD) occur, the inter-site variability of molecular aberrations may explain the short-lived responses of AML patients with MS to IDH/FLT3 inhibitors. Transcriptional profile differences between MS and associated medullary disease included up-regulation of extracellular matrix organization and cell-substrate adhesion and down-regulation of inflammatory response pathways in both bulk and scRNA-seq data (Figure 1B). Further, involved BM malignant cells of MS patients compared to patients without MS up-regulated specific pathways, including interferon-gamma response observed in both bulk and scRNA-seq, as well as chromatin organization and splicing. We further revealed that MS patients without overt BM involvement show remodeling of the BM immune microenvironment, with increased fractions of hematopoietic stem and progenitor cells that down-regulate HLA class II genes. Comparing LC and MS in the skin revealed distinct differences in their expression profiles, including high expression of genes involved in cell migration and immune response in LC, suggesting LC and MS represent distinct disease entities. Lastly, we demonstrated high expression of BCL2 in extramedullary disease (p=0.0074) and presented initial evidence that treatment with BH3-mimetics may be beneficial for the treatment of isolated MS. Conclusions: Myeloid sarcoma shows distinct molecular evolution and microenvironmental composition compared to medullary disease, including location-specific evolution necessitating personalized management consideration. Leukemia cutis is distinct from MS tumorous lesions and should be considered a separate entity. High BCL2 expression in extramedullary disease might represent a promising targetable vulnerability in patients with isolated MS.

STMN1 promotes cell malignancy and bortezomib resistance of multiple myeloma cell lines via PI3K/AKT signaling

ABSTRACT Background This study investigates the biological functions of Stathmin1 (STMN1) involving drug resistance and cell proliferation in multiple myeloma (MM) and its related mechanisms. Methods Bone marrow aspirates were collected from 20 MM patients, and the bone marrow mononuclear cells (BMMCs) were separated by Ficoll-Hypaque density gradient centrifugation. Blood samples of 20 patients with monoclonal gammopathy of undetermined significance (MGUS) and 20 healthy donors were collected. Normal plasma cells sorted from the peripheral blood of MGUS patients and healthy subject as controls. Two bortezomib (BTZ)-resistant MM cell lines were established, namely NCI-H929/BTZ and KM3/BTZ cells, and then transfected with lentiviruses packaging sh-STMN1 to knock down STMN1 level in BTZ-resistant cells. Expression of STMN1 was assessed by RT-qPCR and western blotting. CCK-8 assays were performed to assess 50% growth inhibition (IC50) values. Green fluorescent protein in BTZ-resistant cells infected with lentiviruses was observed by fluorescence microscopy. Cell viability, proliferation, cell cycle, and apoptosis were evaluated through MTT assays, colony formation assays, flow cytometry analyses, and TUNEL staining. Results STMN1 was upregulated in MM cells and bone marrow aspirates of MM patients. Additionally, STMN1 depletion attenuated BTZ resistance in MM cells. Moreover, downregulation of STMN1 limited the malignant phenotypes of BTZ-resistant cells. Mechanistically, the PI3K/Akt signaling was inactivated by STMN1 downregulation in BTZ-resistant cells. Conclusion STMN1 silencing inhibits cell proliferation and BTZ resistance in MM by inactivating the PI3K/Akt signaling.

High-Dimensional Mass Cytometry Analysis of Embryonic Antigens and Their Signaling Pathways in Myeloid Cells from Bone Marrow Aspirates in AML Patients at Diagnosis.

Embryonic antigens (EA) regulate pluripotency, self-renewal, and differentiation in embryonic stem (ES) cells during their development. In adult somatic cells, EA expression is normally inhibited; however, EAs can be re-expressed by cancer cells and are involved in the deregulation of different signaling pathways (SPs). In the context of AML, data concerning the expression of EAs are scarce and contradictory. We used mass cytometry to explore the expression of EAs and three SPs in myeloid cells from AML patients and normal bone marrow (NBM). Imaging flow cytometry was used for morphological assessment of cells in association with their OCT3/4 expression status (positive vs. negative). An overall reduction in or absence of EA expression was observed in immature myeloid cells from AML patients compared to their normal counterparts. Stage-specific embryonic antigen-3 (SSEA-3) was consistently expressed at low levels in immature myeloid cells, whereas SSEA-1 was overexpressed in hematopoietic stem cells (HSCs) and myeloblasts from AML with monocytic differentiation (AML M4/M5). Therefore, these markers are valuable for distinguishing between normal and abnormal myeloid cells. These preliminary results show that the exploration of myeloid cell intracellular SPs in the setting of AML is very informative. Deregulation of three important leukemogenic SPs was also observed in myeloid cells from AML. Exploring EAs and SPs in myeloid cells from AML patients by mass cytometry may help identify characteristic phenotypes and facilitate AML follow-up.

Abstract A14: Ex vivo immune activation with the macrophage-targeting immunotherapy, anti-Clever-1 antibody bexmarilimab, in acute myeloid leukemia and myelodysplastic syndrome

Abstract The potential of immunotherapies in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) remains still under investigation. Clever-1 (also known as Stabilin-1) is a multifunctional scavenger and adhesion receptor expressed by monocytes and immunosuppressive macrophages. Bexmarilimab, a Clever-1 targeting antibody, demonstrates many immunomodulatory effects1 along with promising anti-tumor activity against solid tumors in patients with multiple lines of previous treatment2. In AML, high STAB1 (mRNA) levels associate with poor survival and resistance to therapy3. The aim of this study was to profile Clever-1 expression in AML and MDS and test in preclinical models the growth inhibitory and immunomodulatory potential of bexmarilimab, as a single agent and in combination with azaciditine and venetoclax. AML cell lines (n=11) and frozen mononuclear cells, extracted from AML (n=42) and very high risk MDS (n=4) patient bone marrow (BM) aspirates provided by the Finnish Hematology Registry and Biobank, were used. Samples were treated for 48h with bexmarilimab alone, or in combination with azacytidine and/or venetoclax. Flow cytometry was used to detect different myeloid and TBNK cell populations along with Clever-1, HLA-DR, PD-(L)1 and additional T cell activation markers. Our results confirmed Clever-1 protein expression in AML cell lines and bexmarilimab treatment induced metabolic and growth inhibition of KG1 blasts (Clever-1high). In patients with AML, FAB M4/M5 subtypes exhibited highest Clever-1 levels, along with FAB M2 patients with consequent rapid relapse. Clever-1 expression correlated negatively with monocyte MHC class II molecule, HLA-DR, expression, and BM T-cell frequency, in line with the immunosuppressed state associated with high Clever-1. Ex vivo treatment of the primary AML BM cells with bexmarilimab resulted in a notable, 5-10x fold increase in monocyte HLA-DR in samples with low basal HLA-DR and high Clever-1. The combination of azacitidine with bexmarilimab augmented HLA-DR induction by 20-110% (mean 44%). FAB M1/M2 AML showed increase in activation markers, such as Ki67, CXCR3 and Granzyme B after ex vivo bexmarilimab treatment in CD8+ T-cells. Furthermore, bexmarilimab reduced PD-1 expression in NK- and CD8+CXCR3+ T-cell populations of FAB M0-M2 AML and MDS-EB2. This project is the first comprehensive investigation of Clever-1 expression in AML and MDS patient BM blasts and monocytes. Ex vivo treatment with bexmarilimab, alone or in combination with azacytidine or venetoclax, indicate enhanced antigen presentation capability and immunological activation. These results validate the therapeutic potential of bexmarilimab in myeloid malignancies. The safety, tolerability and preliminary efficacy of bexmarilimab is now further investigated in combination with venetoclax and/or azacytidine in a phase I/II clinical trial BEXMAB (NCT05428969).1Viitala et al., Clin Cancer Res 2019;25:3289-303; 2 Bono et al., Annals of Oncology; 2021. p 32, 5: S1283-S346; 3 Lin et al., Mol Ther Nucleic Acids 2019;18:476-84 Citation Format: Arno Ylitalo, Sofia Aakko, Heikki Kuusanmäki, Mari Björkman, Juho Jalkanen, Marie-Louise Fjällskog, Caroline Heckman, Maija Hollmén, Mika Kontro. Ex vivo immune activation with the macrophage-targeting immunotherapy, anti-Clever-1 antibody bexmarilimab, in acute myeloid leukemia and myelodysplastic syndrome [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A14.

Patient Demographic Factors Are Not Associated With Mesenchymal Stromal Cell Concentration in Bone Marrow Aspirate Concentrate

To describe the capacity for concentration of a single processing machine for bone marrow aspirate concentrate (BMAC) production and investigate the effects of demographic factors on the number of mesenchymal stromal cells (MSCs) in BMAC. Patients enrolled in our institution's randomized control trials involving BMAC who had complete BMAC flow cytometry data were included. Multipotent MSC phenotype, defined as cell-surface coexpression of specific-identifying antigens (≥95% positive) and the absence of hematopoietic lineage markers (≤2% positive), was determined for both patient bone marrow aspirate (BMA) and BMAC samples. The ratio of cells in BMA:BMAC samples was calculated and Spearman correlations (i.e., body mass index [BMI]) and Kruskall-Wallis (i.e., age: <40, 40-60, >60 years) or Mann-Whitney (i.e., sex) tests were used to determine the relationship of cell concentration to demographic factors. Eighty patients were included in analysis (49% male, mean age: 49.9 ± 12.2 years). Mean concentration of BMA and BMAC was 2,048.13 ± 2,004.14 MSCs/mL and 5,618.87 ± 7,568.54 MSC/mL, respectively, with a mean BMAC:BMA ratio of 4.35 ± 2.09. A significantly greater MSC concentration was observed in the BMAC samples when compared with BMA (P= .005). No patient demographic factors (age, sex, height, weight, BMI) were found to predict MSC concentration in the BMAC samples (P ≥ .01). Demographic factors, including age, sex, and BMI do not impact the final concentration of MSCs in BMAC when using a single harvest technique (anterior iliac crest) and a single processing system. As the role of BMAC therapy expands in clinical application, it becomes increasingly important to understand the determinants of BMAC composition and how it is affected by different harvesting techniques, concentrating processes, and patient demographics.

Abstract 184: Multiple Myeloma 3D model: A platform for testing drug effects on myeloma in conjunction with the bone marrow niche

Abstract Background: Multiple myeloma is a cancer that remains mostly incurable despite of the wide array of treatment options. In part, this may be due to its ability to develop resistance to therapy with time. To evaluate key aspects of the disease such as the protective effects of the tumor niche, the immune repertoire that could influence immunotherapies and the ability to study primary tumor cells, developing a new platform is necessary. In order to overcome the obstacles of growing primary myeloma cells and having a model that better replicates the tumor microenvironment, we used a 3D organoid model with patient-derived cells to evaluate different conditions to observe how current therapies affects cancer cells and the tumor niche in which they live. Methods: We used a 3D organoid model using patient bone marrow aspirates containing myeloma cells and stroma in a Matrigel scaffold and incubated over 21 days with partial media change containing key growth factors found in the bone marrow space to maintain cells alive. Established agents used to treat myeloma were used to expose the cultures and then monitored at different timepoints to study its immediate and delayed effect in the tumor cells, immune repertoire, and stroma. Bortezomib, Lenalidomide, Selinexor, and Dexamethasone, which are commonly used to treat myeloma patients and influence the immune compartment, were used at different concentrations. ATP, alamar blue, histology, and flow cytometry were performed at key timepoints of the experiment. Results: H&amp;E sections demonstrated hematopoietic elements with good cellular preservation suggesting that the model may be a functional way to recapitulate the tumor environment. Patient-derived Multiple Myeloma organoids treated with Bortezomib 10nM and Selinexor 10uM and 30uM showed significant inhibition in viability compared to control. Lenalidomide 100 uM had variable effect in viability between organoids. Flow Cytometry analysis captured the immunomodulatory effect of Lenalidomide on the immune cells. Conclusions: The 3D organoid model was successful at maintaining viability for 21 days and having a heterogeneous representation of the tumor niche when using bone marrow aspirates of myeloma patients. The experiments performed allowed us to explore not just the effect of drugs on myeloma cells, but we can see the impact they have on the other cells in the bone marrow niche. A unique interest in the ability to evaluate changes in the immune compartment may help better understand immunotherapy and optimize personalized medicine by helping select the most effective treatments for a particular patient. Citation Format: Hanadi M. Rashad, Giovanni Insuasti, Graca Almeida-Porada, Cesar Rodriguez. Multiple Myeloma 3D model: A platform for testing drug effects on myeloma in conjunction with the bone marrow niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 184.

EVALUATION OF PANCYTOPENIA WITH BONE MARROW INTERPRETATION

Background: Pancytopenia is one of the most common clinico-haematological entity observed in our day to day clinical practice. It is a disorder in which all the three major elements of blood (i.e. red blood cells, white blood cells and platelets) are decreased in number. The causes of pancytopenia may be due to decrease in hematopoietic cell production in the marrow resulting from infections, toxins, malignant cell inltration, post- chemotherapy or post-radiation. Aims and Objectives: 1) To study the etiology and clinical presentation of pancytopenia in all age groups. 2) To correlate with bone marrow interpretation Materials &amp; Methods: This is a prospective study which was conducted among 50 patients of pancytopenia in the Clinical Pathology, Government General Hospital,Kurnool from January 2021 to October 2022 Bone marrow aspiration was done by using Salah's bone marrow puncture needle. Smears were made from bone marrow aspirate (BMA) and stained by Leishman stain and special stains like Perl`s wherever necessary. The smears were assessed for cellularity, differentiation and maturation of erythroid, myeloid and megakaryocytic lineage, M:E ratio, Plasma cells, Lymphocytes and parasites/ abnormal cells. In the present study the commonest cause of Pancytopenia was Megaloblastic anemia (70%) follo Results: wed by Dimorphic anemia (20%). The less common conditions were Multiple Myeloma (6%),Chronic Myeloid Leukemia(2%),Acute Leukemia(2%). The present study concludes that com Interpretation and Conclusion: plete primary hematological investigations along with bone marrow aspiration in pancytopenic patients are helpful for understanding disease process and to diagnose or to the rule out causes of pancytopenia. These are also helpful in planning for further investigations and management.

SegPC-2021: A challenge &amp; dataset on segmentation of Multiple Myeloma plasma cells from microscopic images

Multiple Myeloma (MM) is an emerging ailment of global concern. Its diagnosis at the early stages is critical for recovery. Therefore, efforts are underway to produce digital pathology tools with human-level intelligence that are efficient, scalable, accessible, and cost-effective. Following the trend, a medical imaging challenge on "Segmentation of Multiple Myeloma Plasma Cells in Microscopic Images (SegPC-2021)" was organized at the IEEE International Symposium on Biomedical Imaging (ISBI), 2021, France. The challenge addressed the problem of cell segmentation in microscopic images captured from the slides prepared from the bone marrow aspirate of patients diagnosed with Multiple Myeloma. The challenge released a total of 775 images with 690 and 85 images of sizes 2040×1536 and 1920×2560 pixels, respectively, captured from two different (microscope and camera) setups. The participants had to segment the plasma cells with a separate label on each cell's nucleus and cytoplasm. This problem comprises many challenges, including a reduced color contrast between the cytoplasm and the background, and the clustering of cells with a feeble boundary separation of individual cells. To our knowledge, the SegPC-2021 challenge dataset is the largest publicly available annotated data on plasma cell segmentation in MM so far. The challenge targets a semi-automated tool to ensure the supervision of medical experts. It was conducted for a span of five months, from November 2020 to April 2021. Initially, the data was shared with 696 people from 52 teams, of which 41 teams submitted the results of their models on the evaluation portal in the validation phase. Similarly, 20 teams qualified for the last round, of which 16 teams submitted the results in the final test phase. All the top-5 teams employed DL-based approaches, and the best mIoU obtained on the final test set of 277 microscopic images was 0.9389. All these five models have been analyzed and discussed in detail. This challenge task is a step towards the target of creating an automated MM diagnostic tool.

The Role of Serum Lactate Dehydrogenase in Etiological Diagnosis of Macrocytic Anemia

Background: Macrocytic anemia is common and is characterized by decreased hemoglobin levels with elevated Mean Corpuscular Volume (MCV), due to a range of diseases and divided into Megaloblastic and Non-megaloblastic anemia. Serum vitamin B12 and folic acid tests are usually performed but they are limited by their low sensitivity and specificity. To confirm diagnosis of macrocytic anemia bone marrow examination is required but it is invasive procedure. Vitamin B12 and/or B9 deficiency leads to a defect in DNA synthesis leading to ineffective erythropoiesis and intramedullary hemolysis in patients of megaloblastic anemia, this leads to increased serum LDH (Lactate Dehydrogenase) and unconjugated bilirubin. Aims of study: This study was carried out to evaluate the role of serum LDH in the distinction between megaloblastic and non-megaloblastic anemia, and to study the correlation between serum LDH and MCV Patient and Methods: The study included 60 patients with non-regenerative macrocytic anemia (We exclude patients with regenerative macrocytic anemia because elevated reticulocytes leads us to hymolysis anemia or bleeding and it’s not a diagnostic problem(. Complete blood count, biochemical investigation, peripheral blood examination, reticulocyte count, bone marrow examination was performed in all cases Results: The most common cause of macrocytic anemia was Megaloblastic anemia (65%). The other causes were primary bone marrow disorders (35%). There was a significant difference in the mean values of serum LDH and MCV between two groups ( megaloblastic and non-megaloblastic anemia). When LDH&gt;2076.5 IU/L, there is more probability of having megaloblastic anemia than non-megaloblastic anemia but when LDH&gt;2076.5 and MCV&gt;109.45 with bilirubin&gt; 1.2mg/dl will must more probably not have non-megaloblastic anemia (the specificity was 100%), all three parameters combined together can be used as screening test to distinguish between the 2 groups of macrocytic anemia (megaloblastic and non-megaloblastic) without necessity of bone marrow aspiration in patients of non- regenerative macrocytic anemia. Present study had also shown that there were a positive relationship between serum LDH and MCV (r=+0.613).

Single Cell Transcriptomic Analysis of the Bone Marrow Microenvironment Uncovers a Role for Antigen Presentation Processes in MDS/AML Relapse after Allogeneic Hematopoietic Stem Cell Transplantation

Background: Bone marrow (BM) diseases such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are characterized by a poor overall survival with allogeneic hematopoietic stem cell transplantation (allo-HSCT) often representing the only curative treatment option. However, patient mortality even after allo-HSCT remains high, mainly due to relapse. Previous studies showed involvement of immune escape processes in relapsed patients via downregulation of human leukocyte antigen class II (HLA-II) genes on leukemic blasts, or T cell exhaustion. Nevertheless, other components of the BM microenvironment (BMME) including mesenchymal stromal cells (MSC), endothelial cells and immune cells from the innate and adaptive immune system might play a role in relapse after allo-HSCT. This role remains unclear and needs further characterization. Therefore, we performed single cell transcriptomic analysis of different subpopulations of the BMME in MDS/AML patients who either achieved a long-term complete remission (CR) or relapsed after allo-HSCT. Methods: Patient BM aspirates were obtained before and after 3-6 months post allo-HSCT after written informed consent, and mononuclear cells were stored in liquid nitrogen. To reduce confounding factors (i.e., pre-conditioning treatment, age, sex, diagnosis, graft versus host disease), "best-matched" patient pairs in CR or relapse were established. Subsequently, 2 pairs of matched CR/relapse patients were chosen for further evaluation (Table 1). From each patient, 6 different BM subpopulations were FACS-sorted to enrich for rare cell populations: hematopoietic stem and progenitor cells (HSPC, CD45dim/CD34+), MSC (CD45-/CD271+), endothelial cells (CD45-/CD31+), monocytes (CD45dim/CD14+), granulocytes (CD45dim/CD11b+), T cells (SSClow/CD45hi/CD3+) and NK cells (SSClow/CD45hi/CD3-/CD56+/CD16+). A maximum number of 5x103 cells from each of these 6 cell populations from a single patient and time point were pooled back together in one lane of a 10X Genomics chip for single cell RNA sequencing library preparation. Data analysis was performed in R. Results: After UMAP clustering, 15 groups of cells could be identified by their conserved expression patterns, recapitulating the different input sorted subpopulations. Gene set enrichment analysis comparing cell types from patients in CR and relapse revealed enrichment in immune related pathways in many clusters from relapsing patients both before and after allo-HSCT. In contrast, this pathway enrichment was only observed in classical monocytes from patients in CR after allo-HSCT. Further gene expression analysis showed that within these pathways, the most significantly differentially expressed genes were HLA class I (HLA-I) and HLA-II genes, as well as the transcription factor STAT1. Of note, STAT1 expression was increased 1.2 to 2.3 fold in several cell types from patients in relapse compared to patients in CR. Similarly, the known STAT1 target genes HLA-A and HLA-C showed a 1.2 to 7.5 fold higher expression in several cell types from relapsed patients. Importantly, upregulations were observed in progenitor cells as well as in lymphocytes both before and after allo-HSCT, suggesting that STAT1 upregulation might trigger HLA-I genes expression and play a role in and/or serve as predictive marker of post-transplant relapse. Interestingly, HLA-II gene enrichment patterns were more heterogeneous. Indeed, before transplantation, genes such as HLA-DRA, HLA-DPB1 or CD74 were upregulated 1.4 to 3.0 fold in most cell types of patients in relapse, while after allo-HSCT a downregulation of 1.5 to 3.5 fold was observed in progenitor cells from patients in relapse, and was even more prominent in classical monocytes showing a 2 to 8 fold downregulation (Figure 1). This observation suggests that by preventing T cell activation via HLA-II antigen presentation, monocytes might also play a role in relapse mechanisms after allo-HSCT. Conclusions: Altogether, our results suggest that antigen presentation processes in the BMME via HLA-I and HLA-II could be involved in relapse mechanisms. Their expression level even before transplantation might be used as a predictor of relapse after allo-HSCT. Further analyses are ongoing to confirm our preliminary findings including functional analyses linking relapse with those observed differences in gene expression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Monosomy 7 and Del(7q) Cause Selective Sensitivity to Inhibitors of Nicotinamide Phosphoribosyltransferase in Acute Myeloid Leukemia

Monosomy 7 and deletion of chromosome arm 7q (-7/-7q) occur in up to 10% of patients with acute myeloid leukemia (AML). In AML, -7/-7q are associated with resistance to chemotherapy and adverse prognosis. As such, treatment outcomes for this subgroup of patients remain particularly dismal, and targeted therapies are urgently needed. To identify targeted therapies for AML with -7/-7q we analyzed ex vivo drug sensitivities in 158 AML samples. The presence of -7/-7q (n=18) was determined using karyotyping and exome-sequencing of bone marrow (BM) mononuclear cells and a matched skin biopsy control. Mononuclear cells from fresh bone marrow aspirates of AML patients were incubated with a library of up to 525 oncology drugs for 72h, with each drug pre-plated in 5 concentrations. Cell viability was measured using the CellTiter Glo (CTG) assay, and the response of each sample was summarized as a drug sensitivity score (DSS), which is a modified integration of the dose-response inhibition curve, directly proportional to the sensitivity of a sample. We found that AML samples with -7/-7q were highly sensitive to the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor daporinad (p-value ~ 4.2e-06) (Figure A). NAMPT is the rate-limiting enzyme in the NAD+ salvage pathway. The NAMPT gene is located at 7q22.3. Monosomy 7 and del(7q) lead to the deletion of one copy (i.e., haploinsufficiency) of NAMPT. Notably, a rare AML with del(7q) in which the deletion did not include the NAMPT gene (and which was thus diploid for NAMPT) was not sensitized to NAMPT inhibition. Taken together, this data demonstrates a significant association between the NAMPT haploinsufficiency in AML with -7/-7q and increased sensitivity to NAMPT inhibition. To further support our findings, we analyzed the sensitivity of AML cells without or with -7/-7q to four NAMPT inhibitors (daporinad, GMX1778, KPT-9274, and LSN3154567) using multiparametric flow cytometry. Viably frozen BM mononuclear cells from AML patients with (n=11) and without (n=51) -7/-7q were thawed and incubated with drugs in 8 concentrations for 72 hours. After incubation, cell viability was analyzed using the iQue Screener Plus flow cytometer with a panel consisting of CD45, CD3, CD34, CD117, CD14, and CD38 antibodies, to identify myeloid and lymphoid cell populations, as well as Annexin V and 7-AAD to discriminate live from dead cells. We found that CD34+ myeloblasts from AML with -7/-7q were selectively killed by all four NAMPT inhibitors, validating our initial results (p-value ~ 1.4e-02). An in-depth analysis of cellular subpopulations revealed that while CD34+CD38+ cells (representing more differentiated myeloblasts) were significantly more sensitive to NAMPT inhibition in -7/-7q samples compared to wild-type samples (p-value ~ 4.5e-04), the CD34+CD38- compartment (anticipated to contain the immature leukemic stem and progenitor cells) was not sensitized in this group (Figure B). Interestingly, the analysis of ex vivo drug sensitivities also revealed that while the majority of AML with -7/-7q are resistant to the FDA-approved BCL2 inhibitor venetoclax, they are highly sensitive to daporinad (Figure A). This finding proposes NAMPT inhibitors as a suitable alternative to venetoclax for -7/-7q patients. Additionally, since venetoclax has previously shown significant efficacy against the highly BCL2-dependent undifferentiated (CD34+CD38-) myeloblasts, its combination with NAMPT inhibitors represents a promising strategy for achieving total leukemic cell death in these patients. In conclusion, we have found that AML with NAMPT haploinsufficiency caused by -7/-7q shows selective sensitivity to NAMPT inhibition. This suggests that NAMPT inhibitors have the potential to be a potent targeted therapy for AML with -7/-7q, a disease with a particularly adverse prognosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ISB 2001, a First-in-Class Trispecific BCMA and CD38 T Cell Engager Designed to Overcome Mechanisms of Escape from Treatments for Multiple Myeloma By Targeting Two Antigens

In recent years, multiple myeloma (MM) patients have benefited from new treatments targeting MM associated antigens such as CD38 and BCMA. However, despite exceptional overall response rates observed using CAR T cell therapies or bispecific antibodies, durable responses beyond 2 years are still limited (PFS below 40% for treatment with idecabtagene vicleucel1 or teclistamab2). It has been proposed that one of the potential reasons for patient relapse may be downregulation of the target or expansion of clones lacking sufficient expression of targets3. This low expression of targets in the MM population has been observed after treatment with CD38-targeted daratumumab4, and BCMA specific CAR T cells5. Simultaneously targeting two antigens has been proposed as promising approach to prevent escape by antigen loss6. Here, we propose to simultaneously target BCMA and CD38 using ISB 2001, a first-in-class trispecific molecule based on the TREAT™ (Trispecific Engagement by Antibodies based on the TCR) technology. ISB 2001 targets CD3 on T cells and co-targets BCMA and CD38 on MM cells. ISB 2001 binding arms were derived from a synthetic phage display library with common light chain (Vκ3-15 + IgκJ1) and a silenced Fc (L234A, L235A, P329A mutations) to suppress FcγR-mediated interactions. In order to mimic antigen escape due to target downregulation post monotargeted therapies, ISB 2001 was evaluated in vitro comparing MM cell lines expressing either low levels of both CD38 and BCMA (KMS-12-BM), or high levels of CD38 and very low levels of BCMA (MOLP-8). Compared to monospecific T cell engagers, the binding of ISB 2001 to these cells was strongly increased due to avidity, while retaining reduced affinity to cells devoid of either CD38 or BCMA. ISB 2001 exhibited potent killing with EC50 ranging from 0.4 to 1.2 pM in different cell lines, which was 3 - 41-fold superior to that of EM801, a BCMA-targeted T cell engager7. The potency of ISB 2001 was superior to the combination of the CD38-specific and BCMA-specific T cell engagers constructed with the same binding domains, demonstrating the advantage of avidity augmentation when binding to CD38 and BCMA simultaneously. ISB 2001 was only modestly affected by soluble BCMA or APRIL when compared to EM801, demonstrating killing at 3 pM (EC50) in the presence of these soluble factors. Given the wide expression of CD38 on healthy immune cells, we evaluated whether the avidity induction enabled by the dual targeting could preferentially target MM cells while sparing normal immune cells. ISB 2001 induced low T cell activation and cytokine secretion in the absence of tumor cells, similar to EM801. This was in line with low binding to T cells (apparent cell-based affinity approx. 150 nM) and minimal binding to circulating CD38+ BCMA- healthy cells, indicative of low on-target off-tumour activity of ISB 2001. ISB 2001 showed potent anti-tumor activity in bone marrow aspirates of MM patients and in blood samples from plasma cell leukemia patients, indicating the ability of ISB 2001 to leverage its cytotoxic properties with available immune cells. The half-life of 7 days for ISB 2001 was determined in a preclinical study in human FcRn receptor-expressing TG32 mice. When evaluated in a therapeutic PBMC-humanized mouse model subcutaneously engrafted with KMS-12-BM cells, ISB 2001 was able to induce tumor regression at doses of 0.1 mg/kg. Even at doses as low as 0.02 mg/kg, ISB 2001 demonstrated significant growth inhibition. The in vivo efficacy was associated with T cell infiltration, T cell activation and cytokine release at the tumour site but not systemically. As reported here, ISB 2001 data support future clinical development as a potent treatment of MM through co-targeting CD38 and BCMA. Based on the specific dual targeting of MM cells, significant benefit is anticipated for relapsed/refractory patients who may experience tumor escape through target downregulation mechanisms. Preparations for the initiation of a Phase 1 clinical trial are ongoing. References Munshi, N. C. et al.384, 705-716 (2021).Moreau, P. et al. NEJMoa2203478 (2022) doi:10.1056/NEJMoa2203478.Rodríguez-Lobato, L. G., et al. Hemato2, 1-42 (2020).Nijhof, I. S. et al. Blood128, 12 (2016).Cohen, A. D. et al.J. Clin. Invest.129, 2210-2221 (2019).Fernández de Larrea, C. et al. D. Blood Cancer Discov.1, 146-154 (2020).Seckinger, A. et al.Cancer Cell31, 396-410 (2017).

Utilizing CLL-1 Positive Leukemia Stem Cells As a Marker of Early Treatment Response in Acute Myeloid Leukemia

Background: Current methods to evaluate therapeutic response in Acute Myeloid leukemia (AML) rely primarily on morphological evaluation of leukemia blasts in bone marrow at end of treatment cycles, with complete remission (CR) defined as less than 5% bone marrow blast counts without leukemic phenotype. Measurable residual disease (MRD) has been shown highly prognostic for outcome. MRD by molecular methods is limited to subgroup of patients with defined genetic mutations. MRD detection by Multicolor Flow Cytometry (MFC) is still being standardized. Leukemia stem cells (LSC) has been established as source of treatment resistance and disease recurrence in AML. In this study, we aim to analyze change in LSC during treatment using immunophenotypically defined markers and examine whether information gained from LSC analysis could provide additional insights into disease course and assist management of AML under routine clinical practice setting. Methods: We analyzed flow cytometry data from bone marrow aspirate (BMA) and/or peripheral blood (PB) in patients with AML (excluding acute promyelocytic leukemia) before and after treatment. "Leukemic blasts” were identified by morphology and Leukemia-associated Immunophenotype by MFC. Hematopoietic stem cells (HSCs) gating was based on CD45dim/SSC and CD34+CD38low/- expression. Leukemic stem cells (LSC) fractional change was analyzed using CD371 (CLL-1), CD366 (TIM3), and CD45RA percent of positive over HSCs. A chart review was conducted to determine correlation between LSC fractional change and clinical outcome. Statistical analysis was performed using GraphPad Prism version 9. Results: Fifty AML cases with both pre- and post-treatment data from a single institution in the past 3 years were included in this study. Consistent with published literature, patients with significant reduction in LSC subsets was associated with longer remission, while patients with no significant decrease in the LSC subsets demonstrated no or only a temporary remission. Intriguingly, several patients with drastic reduction in LSC after induction chemotherapy achieved complete remission, despite initial post-treatment bone marrow showing persistence of ">5% leukemia blasts” indicating possibly failed induction therapy. In contrast, in patients with persistently high or increased LSC fraction post-treatment, overt clinical relapse occurred quickly despite "absence” or <1% of leukemia blast differential counts in initial bone marrow. In a series of patients who received allogeneic stem cell transplant (Allo-SCT), rise of LSC fraction post Allo-SCT was associated with early AML relapse (within 6 months), while the decrease of LSC fraction was associated with prolonged remission (Representative patient cases shown in Figure 1). Lastly, among the three LSC-specific markers analyzed, the changes in LSC characterized by CLL-1 expression showed the best correlation with clinical course. In addition, the LSC fraction measured by CLL-1 in the peripheral blood is highly correlated with that of the bone marrow (R2=0.93, P<0.0001). Conclusion: This retrospective study explored the value and applicability of LSC detection in routine clinical practice. Importantly, conventional methods relying on evaluating leukemic blast counts in post-treatment bone marrow can sometimes over- or under- estimate the benefit of certain treatment regimen, leading to inappropriate continuation of the same treatment or switch of a potentially effective treatment regimen. In those groups of patients, the change in LSC fraction provides additional information for early treatment response evaluation, refines outcome prediction, and helps with treatment regimen selection. Trending LSC fractional change identifies impending relapse in post Allo-SCT. LSC fractional change determined by CLL-1 showed the best clinical correlation and should be incorporated into routine evaluation for AML patient in clinical practice, and possibly for those participating in investigational trials. Future studies will focus on collecting more data to validate above findings, determining the thresholds of LSC fractional change which constitute potential treatment response vs failure and testing the feasibility of tracking LSC subsets non-invasively in peripheral blood during treatment to predict therapeutic outcome and potentially guide treatment decisions. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal