Bone Marrow Aspirate Samples Research Articles

Bone Marrow Morphology: A Key Diagnostic Tool in Various Hematological and Non Hematological Disorders

Bone marrow morphology means microscopic examination of bone marrow cells and tissue samples obtained by aspirate and trephine biopsy. An invaluable tool for diagnosis of many hematological and non-hematological disorders, various types of cancer staging and metastases detection. Different kinds of anemia and leukemia can be accurately diagnosed and monitored. Objective: To check the spectrum of diseases which could be identified and diagnosed with the help of bone marrow morphology examination thus demonstrating its diagnostic utility and to check diagnostic concordance between aspirate and core biopsy. Methods: Cross sectional study with consecutive sampling technique conducted over period of one year. Samples of both Bone Marrow Aspiration (BMA) and trephine biopsy (BMB) were collected from all patients included in study after consent using standard protocols. Air dried smears were prepared from bone marrow aspiration samples while trephine biopsy tissues were processed by histopathological techniques. Routine staining such as Wright Giemsa stain, H and E stain and special cytochemistry were used to visualize the cellular architecture of bone marrow. Results: Out of 471 samples, males were predominant with the frequency of 63.9% and maximum patients (31.8%) were from the age group of 35-40 years. Most common clinical indication to conduct bone marrow examination was unexplained Cytopenia which accounted for 35.2% cases, followed by suspicion of Leukemia (28.5%). Malignant hematological disorders were more common as compared to benign disorders (64% vs 17.5%). Acute leukemia was the most commonly identified cancer with frequency of 27.6%. Conclusions: Bone marrow morphology till date remains low risk, economical, crucial diagnostic tool in especially in under resource country like Pakistan. It can guide physicians to plan proper and timely management of patients.

CiRS-7 circular RNA overexpression in plasma cells is a promising molecular biomarker of unfavorable prognosis in multiple myeloma.

Several non-coding RNAs are known to be associated with the pathobiology and progression of multiple myeloma (MM). ciRS-7 (also known as CDR1-AS), a key oncogenic circular RNA (circRNA) that sponges miR-7-5p and other cancer-related microRNAs, was recently found to be downregulated in malignant plasma cells resistant to immunomodulatory drugs. Considering that various circRNAs have a strong potential as molecular biomarkers, we aimed to investigate the expression of ciRS-7 in plasma cell disorders, assess its prognostic importance in MM, and compare these findings with those of individuals with smoldering MM (SMM) and monoclonal gammopathy of unknown significance (MGUS). This study included 171 patients (110 newly diagnosed MM, 34 SMM, and 27 MGUS cases), from which bone marrow aspirate samples were collected for CD138+ plasma cell selection. Total RNA was reversely transcribed using random hexamer primers, and the expression levels of ciRS-7 were quantified using an in-house-developed protocol that includes pre-amplification and real-time quantitative polymerase chain reaction. ciRS-7 levels were found to significantly differ among CD138+ plasma cells of MM, SMM, and MGUS patients. ROC analysis indicated that ciRS-7 expression effectively distinguishes between MM and SMM patients. Moreover, high levels of ciRS-7 were associated with unfavorable prognosis in MM, independently of MM patients' age and Revised International Staging System stage. Additionally, in silico analysis predicted the binding of 85 microRNAs to ciRS-7. In conclusion, this study provides novel insights into the role of ciRS-7 as a promising molecular marker able to distinguish MM from SMM and predict prognosis in MM.

Computing cell state discriminates the aberrant hematopoiesis and activated microenvironment in Myelodysplastic syndrome (MDS) through a single cell genomic study

BackgroundMyelodysplastic syndrome (MDS) is a complicated hematopoietic malignancy characterized by bone marrow (BM) dysplasia with symptoms like anemia, neutropenia, or thrombocytopenia. MDS exhibits considerable heterogeneity in prognosis, with approximately 30% of patients progressing to acute myeloid leukemia (AML). Single cell RNA-sequencing (scRNA-seq) is a new and powerful technique to profile disease landscapes. However, the current available scRNA-seq datasets for MDS are only focused on CD34+ hematopoietic progenitor cells. We argue that using entire BM cell for MDS studies probably will be more informative for understanding the pathophysiology of MDS.MethodsFive MDS patients and four healthy donors were enrolled in the study. Unsorted cells from BM aspiration were collected for scRNA-seq analysis to profile overall alteration in hematopoiesis.ResultsStandard scRNA-seq analysis of unsorted BM cells successfully profiles deficient hematopoiesis in all five MDS patients, with three classified as high-risk and two as low-risk. While no significant increase in mutation burden was observed, high-risk MDS patients exhibited T-cell activation and abnormal myelogenesis at the stages between hematopoietic stem and progenitor cells (HSPC) and granulocyte–macrophage progenitors (GMP). Transcriptional factor analysis on the aberrant myelogenesis suggests that the epigenetic regulator chromatin structural protein-encoding gene HMGA1 is highly activated in the high-risk MDS group and moderately activated in the low-risk MDS group. Perturbation of HMGA1 by CellOracle simulated deficient hematopoiesis in mouse Lineage-negative (Lin-) BM cells. Projecting MDS and AML cells on a BM cell reference by our newly developed MarcoPolo pipeline intuitively visualizes a connection for myeloid leukemia development and abnormalities of hematopoietic hierarchy, indicating that it is technically feasible to integrate all diseased bone marrow cells on a common reference map even when the size of the cohort reaches to 1,000 patients or more.ConclusionThrough scRNA-seq analysis on unsorted cells from BM aspiration samples of MDS patients, this study systematically profiled the development abnormalities in hematopoiesis, heterogeneity of risk, and T-cell microenvironment at the single cell level.

Haematological Profile in Patients With Acute Falciparum Malaria: A Hospital-Based Study.

Introduction Malaria is the most common parasitic disease affecting humans. Haematological alterations in malaria are expected, and these changes play a significant role in fatal complications. The present study aims to assess the clinical and haematological profile in patients with acute falciparum malaria and the significance of various haematological and coagulation alterations with the clinical severity of malaria. Methods The prospective cross-sectional study included 68 acute falciparum malaria cases. Thick and thin blood film microscopy and a rapid diagnostic kit were used to diagnose malaria. The cases were subjected to various haematological and biochemical investigations. Bone marrow aspiration samples were also collected. Using appropriate statistical methods, the findings were compared between severe and uncomplicated malaria cases. A p-value below 0.05 was considered significant. Results The participants' ages ranged from 14 to 78. Most participants (n = 51, 75%) were male and belonged to the lower income group (33, 48.5%). Significant variations in mean parasite count between severe and uncomplicated malaria cases (p-value < 0.01) were observed. The severe and uncomplicated groups showed significant differences in haemoglobin (gm/dL), haematocrit, red blood cell count, reticulocyte, serum iron, and ESR levels (p-value < 0.05). The severe malaria group had considerably reduced mean platelet counts (p-value < 0.01). Only five instances (7.3%) had an appropriate erythropoietic response after day 28. Erythroid hyperplasia with dyserythropoietic alterations was most common in patients with severe anaemia and low-grade parasitaemia. Conclusion Acute falciparum malaria is often associated with haematological alterations. Anaemia and thrombocytopenia were the most expected alterations associated with disease prognosis and mortality.

Hematological cytomorphology: Where we are.

The manuscript discusses the historical evolution of observing blood cell morphology under an optical microscope, from the earliest microscopes in the 17th century to the modern digital era, highlighting key advancements and contributions in the field. Blood has historically held symbolic importance in various cultures, with early medical observations dating back to Hippocrates and Galeno. The discovery of cells and subsequent advancements in microscopy by scientists like Hooke and van Leeuwenhoek paved the way for understanding blood cell morphology. Influential figures such as Hewson, Donné, and Ehrlich followed. Diagnostic cytology evolved from manual cell counting to the development of automated hematological systems. Automated complete blood counting came to support microscopic examination in diagnosing hematological disorders. Morphology is crucial in predicting disease outcomes and guiding treatment decisions, particularly hematological neoplasms. The introduction of flow cytometry and its integration with traditional morphological analysis and the new cytogenetic and molecular techniques revolutionized the classification and prognostication of hematologic disorders. Digital microscopy has emerged as a powerful tool in recent years, offering rapid acquisition and sharing of blood cell images. Integrating Artificial Intelligence with digital microscopy has further enhanced morphological analysis, improving diagnostic efficiency. We also discuss the prospects of AI in pre-classifying blood cells in bone marrow aspirate samples, potentially revolutionizing diagnostic pathways for hematologic diseases. Overall, the manuscript provides a comprehensive overview of the historical development, clinical significance and technological advancements in observing blood cell morphology, underscoring its continued relevance in modern hematology practice.

Flow Cytometry-Based Detection of Minimal/Measurable Residual Disease Predicts Survival Outcomes in Pediatrics, Adolescents, and Young Adults With T-acute Lymphoblastic Leukemia.

Measurable/minimal residual disease (MRD) is considered the single most powerful high-risk factor in acute leukemia, including T-cell acute lymphoblastic leukemia (T-ALL). In this study, we evaluated the impact of flow cytometry (FC)-based detection of MRD on survival outcomes in pediatrics, adolescents, and young adults (AYA) with T-ALL. We included 139 patients, 88 pediatric patients between the ages of one and 14 years, and 51 AYA patients between 15 and 39 years of age, over a period of three years and were treated with the Indian Collaborative Childhood Leukemia Group (ICiCLe) protocol. MRD assessment was performed on post-induction (PI) bone marrow aspirate samples using a 10-color 11-antibody MRD panel on a Gallios instrument (Beckman Coulter, Miami, FL, USA). MRD value > 0.01% was considered positive. PI-MRD status was available in 131 patients. The five-year event-free survival (5-year EFS) in PI-MRD positive patients was inferior to those of negative patients (13.56% vs 79.06%), which was statistically significant (P < 0.001). However, the five-year overall survival (5-year OS) did not show any statistically significant difference between PI-MRD positive and negative T-ALL patients (92.93% vs 94.28%). The hazard ratio (HR) for 5-year EFS and MRD positivity was 8.03 (p-value < 0.0001). HR for 5-year EFS and early T-cell precursor ALL (ETP-ALL) was 2.63 (p = -0.02). PI-MRD detected using FC is a strong predictive factor of inferior survival outcomes in pediatrics, AYA patients with T-ALL. PI-MRD positivity can be used to modify the treatment of T-ALL patients, especially in resource-constrained developing countries where molecular tests are not widely available.

Genomic evolution of patients with myeloid neoplasms and known antecedent clonal hematopoiesis.

6584 Background: Clonal hematopoiesis (CH) refers to the clonal expansion of hematopoietic stem cells (HSCs) with one or more somatic mutations in the absence of a hematologic malignancy. Although it is associated with normal aging, CH confers an increased risk of several comorbidities and hematologic malignancies, including myeloid neoplasms (MNs). However, the outcomes of patients with CH that transformed to MN are unknown. Here, we aim to evaluate the clinicopathologic characteristics and survival outcomes of patients with MN with known antecedent CH. Methods: We retrospectively evaluated patients seen at a tertiary cancer center from May 2016 to July 2023 with myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), or acute myeloid leukemia (AML) and identified those who had CH prior to MN diagnosis. At the time of MN transformation, genomic data was extracted from whole bone marrow aspirate samples and subject to 81-gene target PCR-based sequencing using a next-generation sequencing platform. Results: A total of 36 patients were identified; at the time of CH diagnosis, 79% had known cardiovascular comorbidities and 81% had a prior diagnosis of non-myeloid malignancies for which 72% received prior chemotherapy and 25% radiation. According to the Clonal Hematopoiesis Risk Score (CHRS), 22% were low, 56% deemed intermediate, and 22% at high risk of transformation to MNs. The most frequently observed CH-associated mutations were TET2 (41%), TP53 (38%), IDH2 (16%), EZH2 (16%), ASXL1 (13%), and DNMT3A (13%); 69% had more than one somatic mutation, and 61% had normal cytogenetics. The median time to transformation to MN was 15 months, 26 months, and 24 months for MDS (n=28), CMML (n=2), and AML (n=6) patients, respectively (p=0.72). The median overall survival from MN diagnosis was 54 months, not reached, and 2 months for MDS, CMML, and AML, respectively (p=0.0015). We further delved into the genomic evolution from CH to MN. In the 6 pts who transformed to AML, 4 had a TP53 mutation at CH with expansion of the clone at MN transformation, 2 acquired NPM1 mutations and 1 acquired FLT3 mutations. Both pts who progressed to CMML had expansion of their underlying CH clones (KRAS and ZRSR2). In those who developed MDS, 22 pts had expansion of their CH-mutated clone at the time of MN, most frequently in TET2 (36%), IDH2 (18%), and TP53 (14%); 5 pts acquired new driver mutations outside of their CH clone; and 1 pt lost their CH clone with no new somatic mutations detected. Conclusions: Individuals with CH are at risk of progressing to MN, especially with external selective pressures like antineoplastic therapy that are not accounted for in prognostic systems like CHRS. Patients often have underlying high-risk clones at the time of CH diagnosis that expand at the time of MN, though some patients acquired new driver mutations.

Evaluating the association between radiomic features on breast MRI and dormant tumor cell presence in the SURMOUNT trial.

561 Background: While breast cancer treatment is effective at eradicating primary disease, approximately 20% of patients will ultimately recur at a distant site. Recurrence has been linked to the presence of minimal residual disease (MRD) after treatment, which presents as circulating micrometastases and dormant tumor cells (DTCs) located primarily in the bone marrow. Radiomic features on MRI are imaging biomarkers that have been linked with lesion heterogeneity, which in turn has been associated with disease aggressiveness. Here, we examine the association between radiomic features from the primary lesion on MRI and the presence of post-treatment DTCs. Noninvasively identifying patients at high risk of MRD would facilitate optimized treatment strategies to reduce recurrence risk. Methods: Dynamic contrast-enhanced MRI data from breast cancer survivors enrolled in the SURMOUNT trial (N=104) were retrospectively analyzed. Participants with MRIs collected within 90 days of diagnosis who consented to provide a bone marrow aspirate (BMA) sample were eligible. Working with a board-certified radiologist, we successfully identified and segmented the primary lesion for N=70 patients with BMA outcomes. Using the publicly available CaPTk software, we computed 33 radiomic features for each segmented lesion based on pixel intensity, morphology, and grey level textures. To reduce the dimensionality of the feature space, we used these feature values to group the participants into clusters—termed radiomic phenotypes — via agglomerative hierarchical clustering with significance testing. Fisher’s exact test was applied to test the statistical significance of the association between radiomic phenotype and BMA outcome (± DTC). For all statistical tests, p&lt;0.05 was considered significant. Results: Of the N=70 participants analyzed, 58 were negative and 12 were positive for DTCs present in the bone marrow. Two statistically significant radiomic phenotypes ( p&lt;0.05) were identified that were strongly associated with BMA outcome ( p=0.0251). 11 out of 12 positive outcomes were grouped into the second radiomic phenotype (Table). Conclusions: We found a statistically significant association between DTC presence and radiomic phenotypes derived from MRI data. Given the large percentage of positive outcomes assigned to the second radiomic phenotype, the radiomic phenotypes may indicate low and high risk of MRD, respectively. Importantly, this analysis shows promise for the ability to predict MRD from noninvasive imaging features. These results motivate continued data collection to increase sample size and identify a robust set of radiomic features, which may ultimately be used as predictors in potential models of MRD risk. [Table: see text]

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.

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 6869: Paired single-cell B-cell transcriptomics and receptor sequencing reveal activation states and clonal signatures that characterize B cell immunity in acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is the second most common adult leukemia with a five-year survival rate around 30%. For decades, AML treatment has centered around intensive chemotherapy frequently followed by allogeneic stem cell transplant (Allo-SCT). The power of Allo-SCT to induce lasting remission has been attributed to a potent graft-versus-leukemia effect and has inspired efforts to stimulate host immunity against AML via immune checkpoint blockade (ICB). Thus far, these studies have yielded mixed results. While tumor-infiltrating T-cells have long been the focus of immunotherapy investigations, a growing body of data points to a central role of B-cells in mediating anti-tumor immunity, and a comprehensive assessment of B lymphocytes within the AML immune microenvironment has never been carried out with single cell clonal resolution. We performed paired single-cell transcriptomic and B cell receptor (BCR) analysis on 52 bone marrow aspirate samples. These samples included 6 from healthy bone marrow donors (normal), 24 from newly diagnosed AML patients (NewlyDx), and 22 from 8 relapsed or refractory AML patients (RelRef), who underwent assessment both before and after ICB with azacitidine/nivolumab. We first used transcriptomic data to delineate canonical B-cell subpopulations and observed a reduction in nascent B cells and an expansion of differentiated B cells in AML patients (RelRef&amp;gt;NewlyDx). Overlaying BCR sequencing and clonotypic analysis revealed clonal expansion, low clonotypic diversity, and extensive somatic hypermutation in the RelRef samples, suggesting antigen-driven affinity maturation within the tumor microenvironment. Furthermore, we identified robust AP-1 expression (a marker of activation) in B-cells from NewlyDx patients and a marked loss of AP-1 in RelRef patients, suggesting that B-cell exhaustion may be a key immunologic feature of AML relapse. Importantly, we observed that AP-1 activity is restored and that specific B-cell clones expand after ICB, but only among patients who clinically respond to ICB. In line with these observations, we also noted the clonal expansion of AML-associated plasma cells and loss of clonotypic diversity among ICB clinical responders, pointing to an antitumor role for these clones. Finally, we characterized atypical memory B cells which demonstrate high antigen presentation activity, close interaction with AML cells and are associated with unfavorable clinical outcomes, suggesting that they play a direct role in aiding AML survival and immune escape. Collectively, our findings establish the dynamic landscape of AML-associated B-cells while identifying specific B-cell subpopulations and B-cell-AML interactions that may be targeted by the next generation of AML-directed immunotherapies. Citation Format: Shengnan Guo, Gopi S. Mohan, Bofei Wang, Tianhao Li, Naval Daver, Yuting Zhao, Patrick K. Reville, Dapeng Hao, Hussein A. Abbas. Paired single-cell B-cell transcriptomics and receptor sequencing reveal activation states and clonal signatures that characterize B cell immunity in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6869.

Immunophenotypic characterization of leukemic stem cells in acute myeloid leukemia using single tube 10-colour panel by multiparametric flow cytometry: Deciphering the spectrum, complexity and immunophenotypic heterogeneity.

Despite extensive research, comprehensive characterization of leukaemic stem cells (LSC) and information on their immunophenotypic differences from normal haematopoietic stem cells (HSC) is lacking. Herein, we attempted to unravel the immunophenotypic (IPT) characteristics and heterogeneity of LSC using multiparametric flow cytometry (MFC) and single-cell sequencing. Bone marrow aspirate samples from patients with acute myeloid leukaemia (AML) were evaluated using MFC at diagnostic and post induction time points using a single tube-10-colour-panel containing LSC-associated antibodies CD123, CD45RA, CD44, CD33 and COMPOSITE (CLL-1, TIM-3, CD25, CD11b, CD22, CD7, CD56) with backbone markers that is, CD45, CD34, CD38, CD117, sCD3. Single-cell sequencing of the whole transcriptome was also done in a bone marrow sample. LSCs and HSCs were identified in 225/255 (88.2%) and 183/255 (71.6%) samples, respectively. Significantly higher expression was noted for COMPOSITE, CD45RA, CD123, CD33, and CD44 in LSCs than HSCs (p < 0.0001). On comparing the LSC specific antigen expressions between CD34+ (n = 184) and CD34- LSCs (n = 41), no difference was observed between the groups. More than one sub-population of LSC was demonstrated in 4.4% of cases, which further revealed high concordance between MFC and single cell transcriptomic analysis in one of the cases displaying three LSC subpopulations by both methods. A single tube-10-colour MFC panel is proposed as an easy and reproducible tool to identify and discriminate LSCs from HSCs. LSCs display both inter- and intra-sample heterogeneity in terms of antigen expressions, which opens the facets for single cell molecular analysis to elucidate the role of subpopulations of LSCs in AML progression.

Utility of Next-Generation Sequencing-Based Chimerism Analysis for Early Relapse Prediction following Allogenic Hematopoietic Cell Transplantation.

Chimerism monitoring following allogeneic hematopoietic cell transplantation (HCT) plays a pivotal role in evaluating engraftment status and identifying early indicators of relapse. Recent advancements in next-generation sequencing (NGS) technology have introduced AlloSeq HCT as a more sensitive alternative to short tandem repeat (STR) analysis. This study aimed to compare AlloSeq HCT with STR, focusing on the prediction of early relapse post-allogeneic HCT. Chimerism levels in 29 HCT recipients were assessed using both STR and NGS, employing a total of 125 whole blood or bone marrow aspirate samples (68 post-HCT and 57 pre-HCT samples from recipients or donors). AlloSeq HCT exhibited high concordance with STR and demonstrated the potential for early detection of chimeric changes, particularly at extremely low levels. The combined advantages of high sensitivity and automated data analysis offered by AlloSeq HCT substantiate its clinical adoption for effective chimerism monitoring.

Deficient Generation of Spike-Specific Long-Lived Plasma Cells in the Bone Marrow After Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Generation of a stable long-lived plasma cell (LLPC) population is the sine qua non of durable antibody responses after vaccination or infection. We studied 20 individuals with a prior coronavirus disease 2019 infection and characterized the antibody response using bone marrow aspiration and plasma samples. We noted deficient generation of spike-specific LLPCs in the bone marrow after severe acute respiratory syndrome coronavirus 2 infection. Furthermore, while the regression model explained 98% of the observed variance in anti-tetanus immunoglobulin G levels based on LLPC enzyme-linked immunospot assay, we were unable to fit the same model with anti-spike antibodies, again pointing to the lack of LLPC contribution to circulating anti-spike antibodies.

Paired single-B-cell transcriptomics and receptor sequencing reveal activation states and clonal signatures that characterize B cells in acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is associated with a dismal prognosis. Immune checkpoint blockade (ICB) to induce antitumor activity in AML patients has yielded mixed results. Despite the pivotal role of...

Novel Four-Way Variant Translocation, t(1;9;22;16)(q21;q34;q11.2;q24), in a Patient with Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is characterized by the Philadelphia (Ph) chromosome resulting from the translocation of t(9;22)(q34;q11), producing the BCR::ABL1 fusion gene. Variant Ph chromosome translocations, involving rearrangements in chromosomes other than 9 and 22, occur in 5–10% of CML cases. Herein, we report a unique case of a 36-year-old male with a four-way variant Ph chromosome. Conventional chromosomal analysis performed on bone marrow aspirate samples showed 46, XY, t(1;9;22;16)(q21;q34;q11.2;q24). Nested RT-PCR of the BCR::ABL1 gene revealed a major BCR::ABL rearrangement. The treatment with nilotinib achieved a complete hematologic, cytogenetic, and molecular response after 12 months.

Prognostic Impact of Bone Marrow Fibrosis and Effects of Tyrosine Kinase Inhibitors on Bone Marrow Fibrosis in Chronic Myeloid Leukemia

BackgroundMyelofibrosis is reported in around 40% of newly diagnosed chronic myeloid leukemia (CML) patients and have an important role in the pathobiology and prognosis of CML. This retrospective study aimed to evaluate the effects of bone marrow (BM) fibrosis on disease prognosis and the effects of specific tyrosine-kinase inhibitors (TKIs) on BM fibrosis in CML patients. MethodsThe study included 96 patients (>18 years) diagnosed with chronic phase (CP) CML. The clinical and demographic information were collected from the medical files. Post-treatment BM aspirate and core biopsy samples were analyzed for the presence of fibrosis and dysplasia. ResultsThe mean age of the study patients was 52.69 years; 47.9% of the patients were female. At the onset, 53 (63.1%) patients had BM fibrosis. The difference in the overall survival of the patients with respect to BM fibrosis grades was significant (p = .001). Within the BM fibrosis grade groups, there were significant differences between grade 0 vs. grade 2, grade 0 vs. grade 3, and grade 1 vs. grade 3 (p = .005, p = .002, and p = .003 respectively) There was no significant association between the presence of BM fibrosis at the onset and not responding to first-line therapy (p = .724). Moreover, no significant association was found between the presence of BM fibrosis at the onset and molecular (p = .623) or cytogenetic response (p = .535) to first-line therapy. Additionally, the association between the type of second-line and third-line therapy and molecular response (p = .773 and p = .424, respectively) or cytogenetic response (p = .298 and p = .641) was not significant. ConclusionAlthough BM fibrosis seems to be a crucial complication of CML with a poor prognosis, it can be reversed via TKI treatment which may result in improved survival. It might be considered to check the BM for this complication on a regular basis during therapies to test its prognostic influence in CML patients in prospective controlled trials. Further studies focused on this issue are required to utilize BM fibrosis as a candidate prognostic factor.

Exploring the molecular biomarker utility of circCCT3 in multiple myeloma: A favorable prognostic indicator, particularly for R-ISS II patients.

Circular RNAs (circRNAs) are associated with the pathobiology of multiple myeloma (MM). Recent findings regarding circCCT3 support its involvement in the development and progression of MM, through microRNA sponging. Thus, we aimed to examine the expression of circCCT3 in smoldering and symptomatic MM and to assess its clinical importance. Three cell lines from plasma cell neoplasms were cultured and bone marrow aspirate (BMA) samples were collected from 145 patients with MM or smoldering MM. Next, CD138+ enrichment was performed in BMA samples, followed by total RNA extraction and reverse transcription. Preamplification of circCCT3 and GAPDH cDNA was performed. Finally, a sensitive assay for the relative quantification of circCCT3 using nested real-time quantitative polymerase chain reaction was developed, optimized, and implemented in the patients' samples and cell lines. MM patients exhibited significantly higher intracellular circCCT3 expression in their CD138+ plasma cells, compared to those from SMM patients. In addition, MM patients overexpressing circCCT3 had longer progression-free and overall survival intervals. The favorable prognostic significance of high circCCT3 expression in MM was independent of disease stage (either International Staging System [ISS] or revised ISS [R-ISS]) and age of MM patients. Interestingly, circCCT3 expression could serve as a surrogate molecular biomarker of prognosis in MM patients, especially those of R-ISS stage II. In conclusion, our study sheds new light on the significance of circCCT3 as a promising molecular marker for predicting MM patients' prognosis.

Diagnostic Utility of Bone Marrow Aspiration, Trephine Biopsy, and Flow Cytometry in the Evaluation of Various Haematological and Non Haematological Disorders: A Cross-sectional Study from Northern India

Introduction: The spectrum of haematological and non haematological disorders is vast in various age groups. Complete blood counts and other routine laboratory tests are not always sufficient to diagnose these diseases. Bone marrow examination plays an important role in diagnosing their underlying causes. Aim: To analyse the spectrum of various haematological and non haematological disorders reported in Bone Marrow Aspiration (BMA) and compare them with Bone Marrow Trephine Biopsy (BMB) and Flow Cytometry (FCM) findings where applicable. Materials and Methods: A one-year cross-sectional study was conducted in the Department of Clinical Pathology, PGIMS, Rohtak, Haryana, India from April 2022 to March 2023. A total of 518 consecutive BMA samples were morphologically analysed. Comparative evaluations were performed among BMA, BMB, and FCM where applicable. Diagnostic accuracy was calculated, and the findings of discordant cases were tabulated. Definitive diagnosis of lymphoma/leukaemia and Paroxysmal Nocturnal Haemoglobinuria (PNH) cases were made using FCM. Results: The patients’ ages ranged from three months to 86 years, with a mean age of 38.4 years. The male to female ratio was 1.3:1, with a slight male predominance. The highest number of cases were of anaemia {183 (35.3%) and 164 (31.7%), respectively} and leukaemia {128 (24.7%) and 134 (25.9%), respectively}, followed by normal marrow studies {39 (7.5%) and 32 (6.2%), respectively} and megakaryocytic thrombocytopenia {24 (4.6% in each} in both BMA and biopsy. Among anaemia cases {183 and 164 cases in BMA and BMB}, the majority were of the megaloblastic type {62 (33.8%) and 54 (32.9%), respectively}, followed by hypoplastic/aplastic type {40 (21.8%) and 50 (30.5%), respectively}. In cases of leukaemia (128 and 134 cases in BMA and BMB), acute leukaemia cases (76 (59.4%) and 82 (61.2%), respectively} outnumbered chronic leukaemia cases {52 (40.6%) and 52 (38.8%), respectively} in both BMA and BMB. The concordance and discordance rate between BMA and BMB were 87.6% (419 cases) and 12.4% (59 cases), respectively. Diagnosis was exclusively made by BMB in cases of myelofibrosis, granulomatous disease, and Hodgkin’s lymphoma. Conclusion: BMA cytology is a relatively safe and mildly invasive technique for evaluating various haematological and non haematological disorders with better preservation of cellular morphology. However, in cases with dry/blood taps and focal marrow involvement, BMB should be performed, as it shows well-preserved marrow architecture with all its cellular and stromal components. FCM is a definitive diagnostic modality for further categorisation of acute leukaemia and Chronic Lymphoproliferative Disorders (CLPD).

Abstract A013: Leukemic stem cell differentiation visible at single-cell resolution in AML patients treated with BRG1/BRM inhibitor FHD-286

Abstract BAF chromatin remodeling complexes are critical to the regulation of cellular differentiation and are implicated in cancer as well as other diseases. FHD-286 is a first-in-class dual inhibitor of the BAF catalytic subunits BRM and BRG1 (SMARCA2/4) that is being developed for the treatment of advanced hematologic malignancies. We previously reported that exposure of acute myeloid leukemia (AML) cells to pharmacologically relevant concentrations of FHD-286 led to downregulation of leukemic stem cell (LSC) marker CD34, and upregulation of the myeloid maturation marker CD11b. Morphologic features of differentiation were also observed, suggesting that BAF functions to maintain AML cells in an undifferentiated state, and that FHD-286 may inhibit AML cell growth by reducing their capacity for stem cell renewal. Using single-cell transcriptomic data, we further assessed the impact of FHD-286 on differentiation markers of tumor cells from clinical AML patients. scRNA-seq data was obtained for 26 bone marrow aspirate samples from 11 AML and 2 myelodysplastic syndrome (MDS) patients from the Phase 1 clinical study evaluating FHD-286. Non-tumor cell types (T-cells, erythroid, B cells, megakaryocytes) were identified using published gene markers and removed from the dataset, leaving only tumor cells enriched for either LSC markers (CD34, CD123) or CD11b. We next identified differentially expressed genes between patient LSCs and CD11b+ cells. The top 50 genes associated with each cell group were taken as signature markers of stemness or differentiation, and their mean expression used as phenotypic scores for all patient tumor cells. Clustering analysis of the patient single-cell data showed that cells grouped according to cell type, treatment timepoint, and patient identity. Tumor cells expressing LSC markers CD34 and CD123 clustered separately from those expressing differentiation marker CD11b, and the relative percentage of CD11b+ tumor cells increased on treatment for 8 out of 12 patients dosed at &amp;gt;=5mg, consistent with preclinical AML models. Differential expression analysis revealed additional genes upregulated in either LSCs or CD11b+ cells, which were identified in literature as markers of stemness or myeloid differentiation, respectively. Single-cell LSC and differentiation signature scores showed a strongly statistically significant shift away from LSC identity and toward differentiation for 10 out of 12 patients. Single-cell RNA data from AML patients validates the preclinical observation that FHD-286 treatment induces changes in LSCs resembling differentiation towards a terminal myeloid cell state. This dataset further indicates that nearly all patients undergo some degree of tumor cell differentiation after treatment, suggesting broad on-target activity of FHD-286. Citation Format: GiNell Elliott, Jessica Wan, Kim Horrigan, Mike Collins, Astrid Thomsen, Ashley K Gartin, Ammar Adam, David L Lahr, Gabriel Sandoval, Sameul Agresta, Jessica Piel, Martin Hentemann. Leukemic stem cell differentiation visible at single-cell resolution in AML patients treated with BRG1/BRM inhibitor FHD-286 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A013.