Bone Marrow Samples Of Patients Research Articles

Neuroblastoma plasticity during metastatic progression stems from the dynamics of an early sympathetic transcriptomic trajectory.

Despite their indisputable importance in neuroblastoma (NB) pathology, knowledge of the bases of NB plasticity and heterogeneity remains incomplete. They may be rooted in developmental trajectories of their lineage of origin, the sympatho-adrenal neural crest. We find that implanting human NB cells in the neural crest of the avian embryo allows recapitulating the metastatic sequence until bone marrow involvement. Using deep single cell RNA sequencing, we characterize transcriptome states of NB cells and their dynamics over time and space, and compare them to those of fetal sympatho-adrenal tissues and patient tumors and bone marrow samples. Here we report remarkable transcriptomic proximities restricted to an early sympathetic neuroblast branch that co-exist with phenotypical adaptations over disease progression and recapitulate intratumor and interpatient heterogeneity. Combining avian and patient datasets, we identify a list of genes upregulated during bone marrow involvement and associated with growth dependency, validating the relevance of our multimodal approach.

Beyond myeloid neoplasms germline guidelines: Validation of the thresholds criteria in the search of germline predisposition variants

AbstractIntroductionGermline predisposition to myeloid neoplasms can be suspected in patients younger than 50 years or when harboring mutations with a variant allele frequency (VAF) higher than 30% for point mutations in specific genes. To investigate the VAF thresholds’ accuracy we have explored the prevalence of germline variants below the 30% VAF threshold.MethodsA total of 40 variants with VAF lower than 30% in bone marrow samples of myeloid neoplasm patients were selected and studied in CD3+ cells.ResultsAll the selected variants were not found in CD3+ cells except one variant in the SF3B1 gene. However, the whole series was found somatic. Selected variants were also evaluated with our previously studied series of 52 variants with VAF higher than 30%.ConclusionOur study suggests that variants with VAF below 30% are strong somatic candidates but the variants with VAF higher than 30% cannot be considered of germline origin.

Novel method for the detection of circulating tumor plasma cells in multiple myeloma.

e19520 Background: Multiple Myeloma (MM) is characterized by the proliferation of abnormal plasma cells within the bone marrow. A bone marrow examination is essential for the diagnosis of multiple myeloma. This is a test that causes great pain to patients because it requires collecting and analyzing the bone marrow fluid inside the bone. In addition, there is a disadvantage that it cannot be carried out frequently due to cost issues. Therefore, the development of new diagnostic methods is absolutely necessary. The aim of this study is to access the potential of circulating tumor plasma cells (CTPC) for the diagnosis of MM using CytoGen's Smart Biopsy platform. Methods: Spiking tests using MM cell line (RPMI8226) was conducted with the purpose of making an environment similar to the MM patient's blood. RPMI8226 cells (200 cells) stained with CellTracker were spiked into human blood sample (5mL, healthy donor). Then, spiked cells were isolated by CytoGen’s Smart Biopsy isolator, subsequently recovery rate was accessed by counting of isolated RPMI8226 or immunofluorescence (IF) staining with multiple myeloma-specific marker, CD138. Then, clinical evaluation was performed with bone marrow (3mL) and peripheral blood (5mL) samples of MM patients (n=20). The presence of CTPC in patient samples was tested by IF staining with anti-CD138 antibody. Results: In in vitro experiments to test recovery rate of CTPC using CytoGen's Smart Biopsy isolator, MM cell line’s recovery rate was validated to over 80%. CD138 expression was clearly detected by IF staining of spiking samples of multiple myeloma cell line and was not detected at all in the negative controls. In clinical evaluation study, CTPC was successfully observed after the isolation from bone marrow and peripheral blood using CytoGen's Smart Biopsy isolator by IF staining with CD138. Conclusions: This study shows a novel method for the detection of MM with liquid biopsy. CTPC from liquid biopsy of MM could be isolated using CytoGen's Smart Biopsy isolator with high recovery rate and can clearly be detected with specific marker of MM, CD138 by IF staining. These results suggested that CytoGen's Smart Biopsy platform might be an effective diagnostic tool for the detection of MM. Expanded clinical study will be performed with more MM samples for clinical validation. Clinical trial information: 2013-09-009 .

Long non-coding RNA PXN-AS1 promotes glutamine synthetase-mediated chronic myeloid leukemia BCR::ABL1-independent resistance to Imatinib via cell cycle signaling pathway

BackgroundChronic myeloid leukemia (CML) is a common hematological malignancy, and tyrosine kinase inhibitors (TKIs) represent the primary therapeutic approach for CML. Activation of metabolism signaling pathway has been connected with BCR::ABL1-independent TKIs resistance in CML cells. However, the specific mechanism by which metabolism signaling mediates this drug resistance remains unclear. Here, we identified one relationship between glutamine synthetase (GS) and BCR::ABL1-independent Imatinib resistance in CML cells.MethodsGS and PXN-AS1 in bone marrow samples of CML patients with Imatinib resistance (IR) were screened and detected by whole transcriptome sequencing. GS expression was upregulated using LVs and blocked using shRNAs respectively, then GS expression, Gln content, and cell cycle progression were respectively tested. The CML IR mice model were established by tail vein injection, prognosis of CML IR mice model were evaluated by Kaplan–Meier analysis, the ratio of spleen/body weight, HE staining, and IHC. PXN-AS1 level was blocked using shRNAs, and the effects of PXN-AS1 on CML IR cells in vitro and in vivo were tested the same as GS. Several RNA-RNA tools were used to predict the potential target microRNAs binding to both GS and PXN-AS1. RNA mimics and RNA inhibitors were used to explore the mechanism through which PXN-AS1 regulates miR-635 or miR-635 regulates GS.ResultsGS was highly expressed in the bone marrow samples of CML patients with Imatinib resistance. In addition, the lncRNA PXN-AS1 was found to mediate GS expression and disorder cell cycle in CML IR cells via mTOR signaling pathway. PXN-AS1 regulated GS expression by binding to miR-635. Additionally, knockdown of PXN-AS1 attenuated BCR::ABL1-independent Imatinib resistance in CML cells via PXN-AS1/miR-635/GS/Gln/mTOR signaling pathway.ConclusionsThus, PXN-AS1 promotes GS-mediated BCR::ABL1-independent Imatinib resistance in CML cells via cell cycle signaling pathway.Graphic

The unnecessary use of short tandem repeat testing on bone marrow samples in patients after 1 year following allogeneic hematopoietic stem cell transplant.

To determine whether the information provided by short tandem repeat (STR) testing and bone marrow (BM) biopsy specimens following hematopoietic stem cell transplant (HSCT) provides redundant information, leading to test overutilization, without additional clinical benefit. Cases with synchronous STR and flow cytometric immunophenotyping (FCI) testing, as part of the BM evaluation, were assessed for STR/FCI concordance. Of 1199 cases (410 patients), we found the overall concordance between STR and FCI was 93%, with most cases (1063) classified as STR-/FCI-. Of all discordant cases, 75 (6%) were STR+/FCI-, with only 5 (6.7%) cases best explained as identification of disease relapse. Eight cases were STR-/FCI+, representing relapsed/residual disease. Analysis of cases 1 year or more from transplant (54% of all cases) indicated only 9 (1.5%) were STR+/FCI-, and none uniquely identified relapse. These data suggest that STR analysis performed 1 year or more post-HSCT does not identify unknown cases of relapse. Furthermore, while STR testing is critical for identifying graft failure/rejection within the first year posttransplant, FCI appears superior to STR at detecting late relapses with low-level disease. Therefore, STR testing from patients 1 year or more post-HSCT may be unnecessary, as BM biopsy evaluation is sufficient to identify disease relapse.

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

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

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

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

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

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

Morinda officinalis polysaccharide promotes the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells via microRNA-210-3p/scavenger receptor class A member 3.

Morinda officinalis polysaccharide (MOP) is the bioactive ingredient extracted from the root of Morinda officinalis, and Morinda officinalis is applied to treat osteoporosis (OP). The purpose of this study was to determine the role of MOP on human bone marrow mesenchymal stem cells (hBMSCs) and the underlying mechanism. HBMSCs were isolated from bone marrow samples of patients with OP and treated with MOP. Quantitative real-time polymerase chain reaction was adopted to quantify the expression of microRNA-210-3p (miR-210-3p) and scavenger receptor class A member 3 (SCARA3) mRNA. Cell Counting Kit-8 assay was employed to detect cell viability; Terminal-deoxynucleotidyl Transferase Mediated Nick End Labeling assay and flow cytometry were adopted to detect apoptosis; Alkaline Phosphatase (ALP) activity assay kit was applied to detect ALP activity; Western blot was executed to quantify the expression levels of SCARA3, osteogenic and adipogenic differentiation markers. Ovariectomized rats were treated with MOP. Bone mineral density (BMD), serum tartrate-resistant acid phosphatase 5b (TRACP 5b), and N-telopeptide of type I collagen (NTx) levels were assessed by BMD detector and Enzyme-linked immunosorbent assay kits. It was revealed that MOP could promote hBMSCs' viability and osteogenic differentiation and inhibit apoptosis and adipogenic differentiation. MOP could also upregulate SCARA3 expression through repressing miR-210-3p expression. Treatment with MOP increased the BMD and decreased the TRACP 5b and NTx levels in ovariectomized rats. MOP may boost the osteogenic differentiation and inhibit adipogenic differentiation of hBMSCs by miR-210-3p/SCARA3 axis.

Imaging flow cytometric detection of del(17p) in bone marrow and circulating plasma cells in multiple myeloma.

Detection of del(17p) in myeloma is generally performed by fluorescence in situ hybridization (FISH) on a slide with analysis of up to 200 nuclei. The small cell sample analyzed makes this a low precision test. We report the utility of an automated FISH method, called "immuno-flowFISH", to detect plasma cells with adverse prognostic risk del(17p) in bone marrow and blood samples of patients with myeloma. Bone marrow (n = 31) and blood (n = 19) samples from 35 patients with myeloma were analyzed using immuno-flowFISH. Plasma cells were identified by CD38/CD138-immunophenotypic gating and assessed for the 17p locus and centromere of chromosome 17. Cells were acquired on an AMNIS ImageStreamX MkII imaging flow cytometer using INSPIRE software. Chromosome 17 abnormalities were identified in CD38/CD138-positive cells in bone marrow (6/31) and blood (4/19) samples when the percent plasma cell burden ranged from 0.03% to 100% of cells. Abnormalities could be identified in 14.5%-100% of plasma cells. The "immuno-flowFISH" imaging flow cytometric method could detect del(17p) in plasma cells in both bone marrow and blood samples of myeloma patients. This method was also able to detect gains and losses of chromosome 17, which are also of prognostic significance. The lowest levels of 0.009% (bone marrow) and 0.001% (blood) for chromosome 17 abnormalities was below the detection limit of current FISH method. This method offers potential as a new means of identifying these prognostically important chromosomal defects, even when only rare cells are present and for serial disease monitoring.

ULK2 Is a Key Pro-Autophagy Protein That Contributes to the High Chemoresistance and Disease Relapse in FLT3-Mutated Acute Myeloid Leukemia.

We recently demonstrated that a small subset of cells in FLT3-mutated acute myeloid leukemia (AML) cell lines exhibit SORE6 reporter activity and cancer stem-like features including chemoresistance. To study why SORE6+ cells are more chemoresistant than SORE6- cells, we hypothesized that these cells carry higher autophagy, a mechanism linked to chemoresistance. We found that cytarabine (Ara-C) induced a substantially higher protein level of LC3B-II in SORE6+ compared to SORE6- cells. Similar observations were made using a fluorescence signal-based autophagy assay. Furthermore, chloroquine (an autophagy inhibitor) sensitized SORE6+ but not SORE6- cells to Ara-C. To decipher the molecular mechanisms underlying the high autophagic flux in SORE6+ cells, we employed an autophagy oligonucleotide array comparing gene expression between SORE6+ and SORE6- cells before and after Ara-C treatment. ULK2 was the most differentially expressed gene between the two cell subsets. To demonstrate the role of ULK2 in conferring higher chemoresistance in SORE6+ cells, we treated the two cell subsets with a ULK1/2 inhibitor, MRT68921. MRT68921 significantly sensitized SORE6+ but not SORE6- cells to Ara-C. Using our in vitro model for AML relapse, we found that regenerated AML cells contained higher ULK2 expression compared to pretreated cells. Importantly, inhibition of ULK2 using MRT68921 prevented in vitro AML relapse. Lastly, using pretreatment and relapsed AML patient bone marrow samples, we found that ULK2 expression was higher in relapsed AML. To conclude, our results supported the importance of autophagy in the relapse of FLT3-mutated AML and highlighted ULK2 in this context.

No evidence on infectious DNA-based agents in pediatric acute lymphoblastic leukemia using whole metagenome shotgun sequencing.

The etiology of pediatric acute lymphatic leukemia (ALL) is still unclear. Whole-metagenome shotgun sequencing of bone marrow samples in patients with treatment-naïve ALL (n=6) was performed for untargeted investigation of bacterial and viral DNA. The control group consisted of healthy children (n=4) and children with non-oncologic diseases (n=2) undergoing bone marrow sampling. Peripheral blood of all participants was investigated at the same time. After bioinformatical elimination of potential contaminants by comparison with the employed controls, no significant amounts of microbial or viral DNA were identified.

Effects of circ-SFMBT2 on Proliferation, Migration and Invasion of Acute Myeloid Leukemia Cells by Regulating miR-491-5p/HOXA9 Axis

To explore the effects and molecular mechanism of circ-SFMBT2 on the proliferation, migration and invasion of acute myeloid leukemia (AML) cells. Bone marrow samples from 35 pediatric AML patients and 35 healthy controls in Henan Provincial Children's Hospital from April 2015 to April 2017 and human bone marrow stromal cell lines (HS-5) and AML cell lines (HL-60, THP-1, U-937 and Kasumi-1) were collected. The expressions of circ-SFMBT2, miR-491-5p and homeobox A9 (HOXA9) in bone marrow samples and cells were detected by RT-qPCR and Western blot. The Pearson method was used to analyze the correlation of circ-SFMBT2, miR-491-5p and HOXA9 mRNA expression levels in bone marrow samples of AML patients. HL-60 cells were cultured in vitro and divided into 5 groups: Control, si-NC, si-circ-SFMBT2, si-circ-SFMBT2+anti-NC and si-circ-SFMBT2+anti-miR-491-5p, HL-60 cells were transfected with si-NC, si-circ-SFMBT2, anti-NC, and miR-491-5p inhibitor with Lipofectamine™ 3000. RT-qPCR and Western blot were performed to detect the expression levels of circ-SFMBT2, miR-491-5p and HOXA9 in cells of each group. The proliferation activity of HL-60 cells in each group was detected by CCK-8 assay at 24, 48 and 72 h after transfection, respectively. The apoptosis rate was detected by flow cytometry. The migration and invasion abilities of cells were detected by Transwell assay. The regulatory roles of circ-SFMBT2, miR-491-5p and HOXA9 in AML cells were verified by dual-luciferase reporter gene assay, RNA pull-down and RNA-binding protein immunoprecipitation (RIP) experiments. The expression levels of circ-SFMBT2 and HOXA9 mRNA were increased in bone marrow samples and cell lines (HL-60, THP-1, U-937 and Kasumi-1) of children with AML (P <0.001), while the expression level of miR-491-5p was significantly decreased (P <0.001). Pearson correlation analysis showed that the expression levels of circ-SFMBT2 and miR-491-5p in bone marrow samples of AML children were negatively correlated (r =-0.905), miR-491-5p was also negatively correlated with HOXA9 mRNA (r =-0.930), while the expression levels of HOXA9 mRNA and circ-SFMBT2 was positively correlated (r =0.911). The overall survival rate of AML children with high expression of circ-SFMBT2 was significantly decreased than those with low expression of circ-SFMBT2 (P <0.05). Silencing of circ-SFMBT2 could greatly up-regulate the expression of miR-491-5p, decrease the expression of HOXA9, inhibit the proliferation, migration and invasion of AML cells, and promote cell apoptosis (P <0.05). Down-regulation of miR-491-5p expression greatly attenuated the inhibitory effects of circ-SFMBT2 silencing on cell proliferation, migration and invasion (P <0.05). Dual-luciferase reporter gene assay, RNA pull-down and RIP experiments confirmed that circ-SFMBT2 could target miR-491-5p and negatively regulate the expression of miR-491-5p in AML, and HOXA9 was the target of miR-491-5p. Silencing of circ-SFMBT2 may inhibit the proliferation, migration and invasion of AML cells by regulating the miR-491-5p/HOXA9 axis.

Abstract B002: Pharmacodynamics and antitumor mechanism of the BRG1/BRM (SMARCA4/2) inhibitor FHD-286 in a Phase 1 study in subjects with AML or MDS

Abstract BRG/Brahma-associated factors (BAF) chromatin remodeling complexes are critical to the regulation of cellular differentiation and are implicated in cancer and 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 extended exposure of acute myeloid leukemia (AML) cells to FHD-286 led to upregulation of the myeloid maturation marker CD11b, downregulation of BRG1, and decreased cell proliferation. 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 overcoming this differentiation block. Biomarkers identified from these nonclinical studies were analyzed in a Phase 1 dose escalation study of FHD-286 monotherapy in patients with relapsed or refractory AML or myelodysplastic syndrome (MDS) (Study FHD-286-C-002). Flow cytometric analysis was performed on serially collected peripheral blood mononuclear cells (PBMCs) from a total of 31 subjects across 4 dose cohorts (2.5, 5, 7.5 and 10 mg once-daily). Peripheral blasts showed time- and dose-dependent upregulation of myeloid maturation markers, including CD11b and CD64, and concomitant decreases in BCL2, CD34, and BRG1. At the highest dose, substantial biomarker modulation was evident beginning at Cycle 1 Day 15 (C1D15), whereas significant modulation was not observed before C2D1 at the lower dose levels. Single cell RNA sequencing of bone marrow aspirates revealed distinct populations of blasts expressing leukemic stem cell (LSC) markers versus those expressing myeloid differentiation markers. In the majority of on-treatment bone marrow samples, blast populations expressing LSC markers were reduced, while populations expressing myeloid differentiation markers were expanded in a generally dose-dependent manner. Evidence of differentiation was also observed by morphological assessment of patient bone marrow samples. Importantly, these effects were observed in subjects with a wide range of mutation profiles, including those considered high risk. In summary, analysis of patient samples from Study FHD-286-C-002 revealed dose-dependent impacts on stemness and maturation markers in peripheral and bone marrow blasts. These results suggest that FHD-286 forces stem-like blasts to mature, possibly reducing their capacity for self-renewal, and leading to a gradual reduction in circulating blasts. Consistent with this, decreases in bone marrow and peripheral blast counts were observed across cohorts. These findings suggest combining FHD-286 with cytoreductive agents may be a promising strategy to target both LSC-like and proliferative blasts, which is supported by nonclinical data showing enhanced antitumor activity with various combination partners. Citation Format: Mike Collins, Astrid Thomsen, GiNell Elliott, Jessica Wan, Kim Horrigan, Laure Delestre, Virginie Penard-Lacronique, Stephane De Botton, Warren Fiskus, Kapil N. Bhalla, Courtney DiNardo, Samuel Agresta, Jessica Piel, Martin Hentemann. Pharmacodynamics and antitumor mechanism of the BRG1/BRM (SMARCA4/2) inhibitor FHD-286 in a Phase 1 study in subjects with AML or MDS [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 B002.

High Dimensional Clustering and Identification of Immune Cell Hybrid Populations in B-ALL across the Disease Continuum

Introduction: Nearly half of adults with acute lymphoblastic leukemia (ALL) will experience relapse of their disease, and those with relapsed ALL carry a worse prognosis than those who remain in remission. The influence of the tumor immune microenvironment (TME) on relapse and survival outcomes in ALL is not well understood. Mass cytometry (CyTOF) enables immune profiling based on single cell protein expression and can identify phenotypic diversity of cells in the TME that cannot reliably be detected by standard flow cytometry. Recently, T-cell/monocyte hybrid cells have been discovered in patients with uveal melanoma and latent tuberculosis infections. However, cells expressing CD19, a surface antigen expressed on B-ALL cells, and markers of other cells in the TME including T cells and tumor-associated macrophages (TAMs), have not been described in ALL or other hematologic malignancies. The presence of CD3/CD163 hybrid cells in the TME may have predictive and prognostic implications for adults with ALL, as TAMs play a role in suppressing antitumor immunity which can lead to T-cell exhaustion. In this study, we identify the presence of multilineage hybrid cells in peripheral blood (PB) and bone marrow (BM) samples of patients with B-ALL alongside accompanying evolution of the TME at each disease stage. Methods: PB (N=16) and BM aspirate (N=16) isolates were collected from patients with B-ALL at diagnosis, relapse, or remission, as well as from healthy donors (HDs, N=7) for comparison of cellular profiles. A 28-marker CYTOF panel was designed to evaluate cell surface and intracellular markers of interest in the myeloid compartment. Antibodies were purchased pre-conjugated or conjugated in house based on manufacturer guidelines (Standard BioTools). Samples were barcoded with palladium isotopes prior to staining to reduce the impact of batch effect, and were then analyzed on a first generation CyTOF system (Standard BioTools). Following background attenuation and singlet gating in FlowJo (BD), the CATALYST R package enabled deconvolution and visualization of single cell data, including dimensional reduction techniques (tSNE) and clustering of all cell populations. An iterative approach was taken to determine a stable configuration of clusters across sample populations to protect from over-fragmentation of data, further informed by K-means clustering using the elbow method. Heatmaps were then generated based on median scaled expression of markers contained in each cluster. Results: The CyTOF panel identified several different myeloid cell populations in newly diagnosed, relapsed, and remission samples in both PB and BM compared to HDs. Most interestingly, quantifiable levels of a cluster of cells co-expressing CD19, CD3 and CD163 as well as a second cluster of cells expressing CD19 and CD163 were detected in newly diagnosed and relapsed patients, but not in HDs. A single patient with the largest proportion of these cell populations in their PB at the time of relapse presented with an aggressive leukemia variant and relapsed multiple times prior to succumbing to illness. On investigation of BM aspirates, the triple positive hybrid population was found in a similar distribution amongst the newly diagnosed and relapsed patients, as well as affecting one patient who achieved remission. This patient also had a highly aggressive leukemia that was refractory to chemotherapy and immunotherapy, and only achieved this remission following CAR-T cell therapy. She subsequently underwent stem cell transplant and is in remission. Conclusions: Our findings add to the literature highlighting the utility of high parameter single cell capture techniques in detecting rare cell populations in the leukemic TME. Further, we identified two unique hybrid cell populations that are reliably detected across different disease stages in patients with B-ALL. Importantly, these populations persisted following stringent gating protocols to rule out doublets and debris. Although our sample size was too small to demonstrate a prognostic impact of these hybrid cell populations in patients with B-ALL, the identification of these populations alone is a significant finding. Further investigation and patient sample accrual are warranted to better understand the role of these cell types in tumor biology, which may pave the way towards new prognostic models that can leverage well-established immune monitoring techniques.

Single Cell Whole Genome Measurement of Genomic Heterogeneity and Clonal Evolution in Multiple Myeloma

Background: Sequencing studies have advanced our understanding of the genomic underpinnings of Multiple Myeloma (MM). While whole genome sequencing (WGS) has emerged as a comprehensive approach for profiling the genomic landscape of MM, WGS has limited sensitivity in defining the intrapatient genomic heterogeneity. Single cell RNA approaches can capture phenotypic heterogeneity but fail to recover most of the key genomic drivers. To overcome these historical limitations, we utilized a newer single cell whole genome direct library preparation (scWGS DLP+; Lacks et al. Cell 2019) sequencing technology able to detect early changes and study clonal population dynamics, providing a more precise understanding of the emergence and clinical impact of genomic events during MM evolution. Methods: scWGS DLP+ was applied to CD138 + Plasma Cells (PC) isolated from bone marrow samples of MM patients. Using newly developed computational approaches, we characterized the Single Nucleotide Variants (SNVs), copy number changes and complex Structural Variants (SVs) at a single cell level and use this information to reconstruct the evolutionary histories of each patient's myeloma. Results: We applied DLP to 7 MM patients obtaining 800-1600 single cell whole genomes per patient. All canonical IgH translocations identified by FISH were recovered in the scWGS data. Single cell copy number heterogeneity was observed in all but 1 patient, including heterogeneity affecting known drivers. The number of subclones ranged from 1 to 15, with an average of 6 subclones detected per patient (≥10 cells, ≥5MB distinct segmental difference). This is in striking contrast with bulk WGS where we are usually able to define no more than one or two subclones. Gain of 1q (3 copies) was observed in all patients but was either rare (1-3%, 3 patients) or clonal (&amp;gt;99%, 4 patients), with two clonal 1q patients exhibiting convergent subclonal 1q amplification (&amp;gt;3 copies). Loss of previously gained chromosomes was observed including loss of ancestrally gained chromosome 15 in hypodiploid Patient 2, and sporadic loss of 1q in patients with ubiquitous 1q gain. For 3 of 7 patients, hypodiploidy (HRD) had arisen before the most recent clonal expansion. HRD clones continued to diversify, accruing multiple additional events on HRD chromosomes including large or focal gains, or deletions subsequent to a gain. Interestingly, one case had 1 small hypodiploid clone co-existing with a completely different major clone with 1q and t(14;20) IgH translocation, suggesting the existence of two distinct clonal plasma cell populations (one malignant and one likely bening). Two of the 7 patients harbored both small (&amp;lt;20 cells) whole genome doubled (WGD) subclones, and small numbers of highly aneuploid likely produced by multi-polar cell division post WGD. We explored two patient's evolutionary histories in detail, highlighting the unique resolution of scWGS DLP+ to detect CNV but also SNV, mutational signatures and SVs. Patient 3 exhibited 4 distinct subclones with distant evolutionary origin as evidenced by the relatively small number of shared SNV. APOBEC signature mutations were detected in all 4 subclones suggesting APOBEC mutagenesis was still ongoing across the cell populations in this patient. In Patient 1, a complex SV on chromosome 17 resulted in loss of most of 17q and an amplification of 17q segments. The heterogeneous structure of chromosome 17 across clones suggests a late onset of this complex event and ongoing instability. Chromosome 16 exhibited a pattern of cell specific arm loss likely resulting from telomere loss. Interestingly, a minor clone showed complex amplification of 16p affecting TNFRSF17, suggesting that chromosome 16 is at an earlier stage of a process of instability that starts with cell specific arm loss, and ends with clonal expansion of a complex amplification SV targeting a gene with fitness benefit. Discussion: Applied to Multiple Myeloma, scWGS shows higher clonal heterogeneity than previously described with multiple clones exhibiting diverse genetic alterations. The findings underscore the genomic complexity and dynamic nature of MM, highlighting the need for sensitive techniques like scWGS to capture the full spectrum and chronological order of genomic events.

Mesenchymal Stromal Cell-Derived MMP14 Facilitates Leukemia Progression and Chemotherapy Resistance

Abstract i ntroduction:Acute Myeloid Leukemia (AML) stands as a menacing hematological malignancy, characterized by the aberrant proliferation and development of myeloid cells, entailing formidable therapeutic complexities. Matrix metalloproteinases (MMP) represent pivotal enzymes that dismantle the extracellular matrix by disintegrating tissue barriers. Among these, MMP14 assumes significance as it governs the invasion and metastasis of cancer cells, exhibiting heightened expression in solid tumors, including prostate cancer, colon cancer, pancreatic cancer, breast cancer, and skin cancer, thereby endorsing the invasiveness and metastatic potential of angiogenic inflammatory cancer cells. Nonetheless, the effect of abnormal expression of MMP14 derived from mesenchymal stem cells (MSCs) in AML patients upon the bone marrow microenvironment and AML pathogenesis, along with its underlying mechanism, remains unexplored. Methods: AML-MSCs were extracted from bone marrow samples of AML patients through whole bone marrow adherent culture. Lentiviral vectors carrying cDNA of the MMP14 gene and empty lentiviral vectors were meticulously constructed and subsequently introduced into AML-MSCs. Brdu and CFU-F assayed the proliferative capacity of knockdown and control MSCs, and osteogenic induced differentiation was performed in vitro on both groups of MSCs.AML cell lines Kasumi-1 and Molm-13 were subjected to co-culture with AML-MSCs to evaluate the migratory and invasive capabilities of AML cell lines , while the proliferative activity of AML cell lines was assessed via BrdU. Following co-cultivation, AML cell lines were exposed to 1μM cytarabine for 48 hours, and cell viability was gauged using flow cytometry. Moreover, CD34+ cells were isolated from leukemia patients' bone marrow using magnetic beads and co-cultivated with the aforementioned MSC groups to explore the impact of MMP14 derived from MSCs on the resistance of leukemia stem cells to cytarabine. Results: Knockdown of MMP14 reduces MSC proliferation and colony-forming ability while also impeding MSC osteogenic differentiation. AML cell lines subjected to co-culture with the MSC knockdown group exhibited a remarkable reduction in both invasion and migration abilities. The BrdU assay demonstrated a significant decrease in the proliferation rate of AML cell lines within the MMP14 knockdown group. Moreover, following 48 hours of cytarabine administration, flow cytometric analysis revealed a lower proportion of viable cells in the knockdown group when compared to the control group. The same trend was observed in primary cells, as evidenced by a decreased percentage of proliferating CD34+ cells within the knockdown group, along with a reduced proportion of viable cells following cytarabine treatment. Conclusion: The MMP14 derived from MSCs fosters AML progression and confers chemoresistance. Our study provides valuable insights into the significance of MMP14 in AML drug resistance and underscores its potential as a promising preclinical target for AML treatment. DisclosuresNo relevant conflicts of interest to declare.

CD28 CAR T Cells for the Treatment of T Cell Malignancies

CONCLUSION While synthetic immunotherapy has been introduced into the standard of care treatment of patients with B-lineage malignancies, patients with T-lineage malignancies have not gained benefit from this novel approach, yet. The main reason is the challenging choice of target antigen in T-lineage malignancies such as pediatric T cell acute lymphoblastic leukemia (T-ALL). As activating mutations of CD28 have already been described in T cell malignancies, we set out to quantify surface expression of CD28 and other costimulatory molecules on primary pediatric T-ALL samples and explore the utility of this functionally relevant molecule as a novel target antigen. Bone marrow (BM) samples of pediatric T-ALL patients (n=54) at time of diagnosis and healthy control individuals (n=14) were collected. We quantified surface expression of CD28 and 25 additional co-stimulatory or co-inhibitory molecules by flow cytometry after gating on T cell precursors (living singlets, CD45 dim/SSC-A low and CD7 +). Subsequently, we generated a set of 20 CD28 directed second-generation chimeric antigen receptors (CARs) based on five different monoclonal antibodies with variation in hinge domain and light chain / heavy chain chronology. We analyzed specific CAR activity against CD28 expressing T-ALL cell lines after retroviral transduction into primary human T cells. Additional CD28 knockout (KO) by CRISPR/Cas9 could prevent T cell fratricide. CAR constructs were subjected to in vivo testing in a T-ALL NSG mouse model with transplantation of 7.5e4 CCRF-CEM cells transduced with firefly luciferase on day 0 and transfer of 2.5e6 CD28 KO T cells on day 3 harboring either of both CD28 CAR molecules. We used CD7 CAR T cells as positive control, CD19 CAR T cells and CD28 KO T cells without CAR as negative controls. We observed significant upregulation of CD28 on T-ALL leukemia when compared to healthy BM donors (Figure 1A, mean 68.8% vs. 3.7%, p=0.0002). We confirmed upregulation of CD28 in published T-ALL RNA-expression data sets. Interestingly, other co-stimulatory molecules such as CD127 were upregulated, too (mean 50.0% vs. 14.6%, p=0.0001), while co-inhibitory molecules such as CD160, TIGIT and TIM-3 were strongly downregulated (&amp;gt;10fold and p&amp;lt;0.0001 each). We hypothesized that this could imply a functional relevance of CD28 in T-ALL. Therefore, we performed co-culture assays of monocyte derived dendritic cells (CDs) expressing CD28 ligands CD80 and CD86 with different T-lineage leukemia cell lines. The presence of DCs significantly increased proliferation of CD28 + T-ALL cell lines under stress conditions. In order to test the feasibility of CD28 CAR T cells, we generated CD28 KO primary T cells without CAR. When tested in co-culture assays with leukemia cells and T cell engagers, CD28 KO T cells showed unchanged cytotoxic capacity and target-dependent activation illustrating that CD28 KO T cells retain short-term effector functions. Based on cytotoxic capacity and CAR T cell expansion, we identified two lead CD28 CAR candidates out of a pool of 20 CD28 CARs. After CD28 KO, CD28 CAR-T cells showed in vitro expansion comparable to CD19 CAR T cells. Next, we asked how CD28 CAR T cells compare to CD7 CAR T cells, which are currently evaluated clinical trials for T-ALL therapy. Therefore, we generated CD7 KO T cells that were subsequently transduced with a functional CD7 CAR. In vitro, both CD28 CAR T cells and CD7 CAR T cells showed killing of CD28 +CD7 + CCRF-CEM cells of &amp;gt;90% at an effector:target (E:T) ratio of 0.2:1, with CD7 being outperformed by one of the CD28 CARs while outperforming the other CD28 CAR at an E:T ratio of 0.04:1 (Figure 1B). Finally, we went on to validate the functionality of CD28 CAR T cells in vivo. We observed significantly prolonged survival of mice treated with CD28 CAR T cells and CD7 CAR T cells when compared to CD19 CAR T cells or control T cells. No difference in survival between CD7 and CD28 CAR T cell treated mice could be observed in two independent experiments. We identify CD28 as novel target antigen for pediatric T-ALL and provide evidence that CD28 is an immunotarget with functional relevance for T-ALL. We demonstrate the feasibility of CD28 CAR T cell generation and that these novel CAR T cells perform equally well as CD7 CAR T cells both in vitro and in vivo. Novel and functionally relevant CAR targets will facilitate clinical development of immunotherapy for T-lineage malignancies.

Clonal Hematopoiesis in Cartilage-Hair Hypoplasia

Background: Cartilage-hair hypoplasia (CHH) is an autosomal recessive disorder characterized by short stature, hypotrichosis, combined immunodeficiency, and macrocytic anemia. CHH is caused by germline mutations in the RMRP gene encoding the RNA component of the mitochondrial RNA processing endoribonuclease. CHH is heavily enriched in the Finnish (1 in 23000 births) and the Amish (1 in 1340 births) populations. Transient macrocytic anemia is common in children with CHH, whereas severe hypoplastic macrocytic anemia is only present in 10% of pediatric CHH patients and can be treated with allogeneic hematopoietic stem cell transplantation (HSCT). The mechanism behind hypoplastic anemia in CHH is unknown: while erythroid differentiation from hematopoietic stem cells is impaired in vitro in CHH, immune dysregulation may also play a role in this phenotype. Clonal hematopoiesis is a common phenomenon of aging. Patients with inherited and immune-mediated bone marrow failure (BMF) show distinct spectra of clonal hematopoiesis mutations, reflecting context-specific selection of hematopoietic stem cell clones. However, the spectrum of clonal hematopoiesis is yet to be characterized in CHH. Methods: To assess the landscape of somatic alterations in hematopoietic cells in CHH, we performed whole exome sequencing (WES) on twelve bone marrow samples of four pediatric (age 4 to 13 years) and eight adult (age 20 to 52 years) unrelated subjects with CHH. Three children with CHH had severe hypoplastic anemia at the time of DNA sampling and three patients underwent HSCT during follow-up. Seven adults with CHH had normal blood counts and no severe immune phenotypes despite other clinical manifestations of CHH. Matched skin fibroblast samples were available for five CHH adults in the cohort for whom we performed tumor-normal variant calling. For the remaining cases, variant calling was based on tumor-only samples and stringent filtering to exclude germline variants. Additionally, we queried the FINRISK dataset, a Finnish cohort comprising WES and SNP microarray data from 10 129 participants, for the presence of CHH associated RMRP variants and evaluated their association with clonal hematopoiesis of indeterminate potential (CHIP) and mosaic chromosomal alterations (mCAs). Results: The median WES coverage of the bone marrow samples was 445x (347-505). Eleven out of 12 patients were homozygous for the RMRP founder variant n.71A&amp;gt;G and one patient was compound heterozygous for two pathogenic RMRP variants. We observed no somatic reversion of the germline RMRP variants. Two pediatric patients had variants in the CHIP genes: CHH1 (age 13 years) with TP53 hotspot variant R175C (variant allele frequency, VAF 0.006) and CHH2 (age 4 years) with BCORL1 P1681Qfs*20 (VAF 0.025). One fifty-year-old patient had a DNMT3A splicing variant c.1555-1G&amp;gt;C (VAF 0.022), likely consistent with CHIP observed in older general population. To identify clonal hematopoiesis outside the classical CHIP genes, we performed conservative filtering of the remaining somatic variant calls, resulting in a list of 36 high-confidence coding variants (VAF &amp;gt;0.02). These variants were present in 10 out of 12 patients; no recurrent genetic alterations were observed. Finally, the analysis of the FINRISK dataset revealed eleven participants with RMRP disease-associated variants; each participant had one heterozygous RMRP variant. No enrichment of CHIP variants or mCAs were observed in carriers of RMRP variants. Conclusion: We observed TP53 and BCORL1 mutations in the bone marrow samples of pediatric patients with CHH. In comparison to other CHIP-associated conditions, BCORL1 variants are disproportionately represented in immune-mediated aplastic anemia, potentially suggestive of similar immune-mediated origin of hypoplastic anemia in CHH. TP53 mutations are abundant in inherited BMF syndromes; however and in contrast to classical inherited BMF syndromes, CHH is not associated with increased risk of myeloid malignancies. No enrichment of classical or non-classical clonal hematopoiesis mutations were observed in adults with clinical CHH diagnosis or in RMRP variant carriers in the population-level FINRISK cohort. This is consistent with transient selection pressure on hematopoietic stem cells in childhood CHH. To our knowledge, our study is the first attempt to characterize the spectrum of clonal hematopoiesis in CHH.

Optical Genome Mapping for Genome-Wide Structural Variation Analysis in Hematologic Malignancies: A Prospective Study Demonstrates Additional Findings Compared to Standard-of-Care (SOC) Cytogenomic Methods

Genomic structural variants (SVs) are key in understanding the pathogenesis of hematologic malignancies. The current standard-of-care (SOC) cytogenomic methods, including chromosome karyotyping (KT) and fluorescence in situ hybridization (FISH), have inherent limitations in identifying SVs, while next-generation sequencing technologies have limited ability to detect most SVs. Optical Genome Mapping (OGM) is a high-resolution cytogenomic technique that overcomes the limitations of SOC cytogenomic methods and detects all classes of SVs. Recent studies, mostly retrospective, have demonstrated OGM's analytic validity and clinical utility for detecting SVs in both hematologic malignancies and constitutional genetic disorders. This prospective study, using OGM as a validated laboratory developed test (LDT) for detection of SVs, included 112 patients with a wide variety of hematologic malignancies undergoing clinical evaluation and management and demonstrates OGM as superior to SOC cytogenomic methods. Patient blood or bone marrow samples were subject to the Bionano OGM-Dx™ HemeOne assay. OGM analysis was successful for 105/112 (94%) samples with 7/112 (6%) samples failing analysis. Of the 105 analyzed samples, 74 (70%) had abnormal and 31 (30%) had normal findings by OGM analysis, with 49% of abnormal OGM findings classified as complex. At initial diagnosis, 49 cases had guideline and SOC result-based disease risk stratification with 18/49 (37%) graded as high risk, 15/49 (31%) as intermediate risk, and 16/49 (32%) as low risk. Additionally, 60 cases had SOC cytogenomic analysis (KT or FISH) results available; 37% with KT and FISH, 27% with only KT, and 35% with only FISH. OGM cases with normal findings were concordant with KT in 9/10 (90%) of cases and FISH in 8/12 (67%) of cases. For those cases that had abnormal findings by KT and/or FISH, OGM detected additional cytogenomic findings in 79% of cases. A change of risk stratification was made in 12/42 (29%) of cases with additional findings by OGM analysis compared to KT and/or FISH; 9/12 (75%) with upgraded risk and 3/12 (25%) with downgraded risk stratification. In 10/42 (24%) of cases, OGM findings resulted in a change in therapeutic management recommendations. This is exemplified by an acute lymphoblastic leukemia case: KT detected a sole t(9;22) abnormality however, OGM detected t(9;22) and additional findings of gains at chromosomes 1q, 17q, and 19p and losses at chromosomes 5q, 7p, and 11q (see figure 1), changing the risk stratification from intermediate to adverse and directly leading to a change in therapeutic management. The impact of OGM on disease risk stratification and case management is in alignment with previously published studies with OGM analysis demonstrating its power to detect additional findings not seen by SOC cytogenomic analysis. The prospective study outcomes suggest a significant role for OGM in delivering comprehensive results for improved diagnostic assessment and significant prognostic information for better case management and care compared to SOC cytogenomic methods.