Cells In Bone Marrow Aspirates Research Articles

FLAER Revealed Normally Expected Non-PNH FLAER-Dim Immature Myeloid Cells (CD117+/CD34-) In Bone Marrow Aspirates and Could Be Utilized as a Marker of Hierarchical Hematopoiesis.

Fluorescently labeled aerolysin (FLAER) is widely used for the identification of paroxysmal nocturnal hemoglobinuria (PNH) clones in peripheral blood (PB) samples. However, there are only a few reports on the differential fluorescent intensity of FLAER in normal bone marrow (BM) cell subpopulations. The purpose of this study was to evaluate FLAER expression during normal and pathological hematopoiesis, to map the critical existence of non-PNH FLAER-dim cells. A total of 54 BM aspirates were prospectively analyzed with FLAER-based flow cytometric (FC) protocols, during their routine work-up. These were obtained from patients with the following diagnoses: PNH (3), infections/reactive (5), myelodysplastic syndromes (MDS) (7), myelodysplastic/myeloproliferative neoplasms (MDS/MPN) (4), chronic myelogenous leukemia (CML) (3), acute myelogenous leukemia (AML) at diagnosis (20), AML in measurable residual disease (MRD) assessment (7), and B-cell acute lymphoblastic leukemia (B-ALL) in MRD assessment (5). The applied protocols consisted of FLAER, HLA-DR, CD14, CD33, CD34, CD66b, CD38, CD117, CD64, CD45, and FLAER, CD66c, CD14, CD33, CD34, CD66b, CD123, CD16, CD64, and CD45, respectively. FLAER expression was assessed in CD34++/CD38- and CD34+/CD38+ stem cells, CD34-/CD117+/HLA-DR+/CD33+ myeloid precursors, and CD64+/CD14-/HLA-DR+ monocyte precursors but also in mature myeloid cells. All patients revealed an intermediate FLAER intensity in CD34++/CD38- stem cells, with a discrete FLAER-negative subpopulation observed only in maturing CD34+/CD38+ stem cells of patients with PNH. The lowest FLAER intensity was noticed in CD34-/CD117+/HLA-DR+/CD33+ myeloid precursors, not only in patients with PNH but also in PNH-negative BM aspirates. An ascending FLAER intensity was further observed during monocytic and granulocytic maturation, with a discrete FLAER-negative population in CD64+/CD14-/HLA-DR+ monocyte precursors and maturing neutrophils and monocytes of patients with PNH only. The maturation pattern of FLAER expression was further confirmed in a patient with acute promyelocytic leukemia treated with all-trans retinoic acid (ATRA), where FLAER was concurrently upregulated with CD66b in a consecutive series of PB samples tested over a 20-day-period after diagnosis. The application of FLAER in PNH-positive and PNH-negative reactive or malignant BM aspirates identified normally expected non-PNH FLAER-dim CD34-/CD117+/HLA-DR+/CD33+ myeloid precursors in all samples. A specific FLAER-associated maturation pattern was observed, which is proposed for further study within MRD and diagnostic protocols.

Comparison of cytomorphology and histomorphology in myelodysplastic syndromes.

Gold standard for the establishment of the diagnosis of myelodysplastic syndromes (MDS) are cytomorphological features of hematopoietic cells in peripheral blood and bone marrow aspirates. There is increasing evidence that bone marrow histomorphology not only aids in the diagnosis of MDS but can provide additional prognostic information, particularly through assessment of fibrosis and cellularity. However, there is only sparse data on direct comparison between histological and cytomorphological findings within the same MDS patient cohort. Therefore, we performed such an analysis under exceptionally well-standardized conditions. We reexamined biopsy material of 128 patients from the Düsseldorf MDS registry who underwent bone marrow trephine biopsy (in addition to bone marrow aspiration) at the time of diagnosis, addressing the following items: a. Analysis of concordance of diagnoses made by histology and cytomorphology b. Analysis of additional information by histology with regard to the diagnosis and prognosis. The respective biomaterials were available at our institution and had been processed according to unchanged protocols between 1992 and 2010. Fresh histopathological sections were obtained from the tissue blocks, stained under identical conditions and re-assessed by a designated expert pathologist (C.B.) without knowledge of the previous histopathological report or the respective cytomorphological diagnosis. The latter, likewise, was uniformly made by the same expert cytomorphologist (U.G.). Histopathology of bone marrow trephine biopsies reliably captured the diagnosis of MDS. Assignment to the diagnostic WHO subgroup was not entirely concordant with cytomorphology, mainly due to incongruences between the proportion of CD34-positive cells on histopathology and the cytomorphological blast count. Histopathology provided additional diagnostic and prognostic information with high diagnostic and prognostic significance, such as fibrosis. Likewise, histopathology allowed more reliable estimation of bone marrow cellularity.

Deep Learning for Morphology-Based, Bone Marrow Cell Classification

The morphological classification of cells in bone marrow aspirate (BMA) is central to the diagnosis of hematologic diseases, including leukemias. Despite being a critical task, its monotonous, time-consuming nature and dependency on highly skilled clinical experts makes it prone to human error. Such errors can lead to delays and misdiagnoses that negatively impact patient care. To counter these challenges, we curated an expansive dataset of more than 40,000 hematopathologist consensus-annotated single-cell images, extracted from BMA whole slide images (WSIs), each annotated into one of 23 distinct morphologic classes. We then utilized this data to develop DeepHeme, a convolutional neural network classifier designed for bone marrow cell typing tasks. DeepHeme achieves state-of-the-art performance in both the breadth of differentiable classes and accuracy across these classes. By comparing its performance to that of individual hematopathologists from three premier academic medical centers, using our gold standard consensus-labelled images, we found our AI algorithm either matched or surpassed the average performance across all classes. In addition, we integrated DeepHeme with internally developed region classifier and cell detection algorithms, culminating in a comprehensive diagnostic pipeline for whole slide cell differential. We next tested DeepHeme on slides from an external hospital system at a major cancer center to evaluate the generalizability of our model, a necessary precondition to widespread application. DeepHeme demonstrated a high level of generalizability, evidenced by a decrease of only 4% in the mean F-1 score, from 0.89 to 0.85, across all 23 cell classes. Lastly, to improve access to the DeepHeme algorithm results and encourage further real-world generalizability testing, we developed a web application that allows scientists and clinicians to test the DeepHeme algorithm on either test images from our study or their own user-uploaded aspirates.

Undetectable MRD Status in Patients with R/R CLL/SLL with Stable Disease after Lisocabtagene Maraleucel Treatment: Exploratory Analysis of the TRANSCEND CLL 004 Study

Background:For patients (pts) with R/R CLL/SLL after failure of Bruton tyrosine kinase inhibitor (BTKi) and B-cell lymphoma 2 inhibitor (BCL2i)-based treatment, there is no established standard of care and therefore there is a high unmet therapeutic need. The phase 1/2 TRANSCEND CLL 004 study (NCT03331198) investigated the efficacy and safety of lisocabtagene maraleucel(liso-cel) monotherapy, an autologous CD19-directed CAR T cell therapy, in pts with heavily pretreated R/R CLL/SLL and ≥ 2 previous lines of therapy, including a BTKi. The primary analysis demonstrated durable CR/CR with incomplete marrow recovery (CRi) per 2018 International Workshop on CLL (iwCLL 2018) criteria, high undetectable MRD (uMRD; 10 −4 sensitivity) rates, and a manageable safety profile in pts who received liso-cel (Siddiqi T, et al. Lancet 2023). All evaluable responders (CR/CRi/PR) achieved blood uMRD status. Among evaluable pts with stable disease (SD), uMRD status was associated with longer median PFS (6.4 months) versus MRD-positive (MRD+) status (PFS, 2.8 months). This exploratory analysis aimed to better understand potential variables associated with achieving uMRD in pts who had SD with liso-cel. Methods: Among 96 pts in the full efficacy set who were treated with liso-cel at dose level 1 (50 × 10⁶ CAR + T cells) or 2 (100 × 10⁶ CAR + T cells), 80 pts were evaluable for peripheral blood MRD. Peripheral blood MRD status was assessed by next-generation sequencing (NGS; clonoSEQ) assay; uMRD was defined as < 10 −4 (< 1 CLL cell per 10,000) at 1 or more postinfusion time points. Additional exploratory analysis leveraged sample-level MRD results defined by total clonal cells/total nucleated cells assessed by NGS. Cellular kinetics were analyzed in peripheral blood samples by qPCR to detect the liso-cel transgene. Correlative analysis studies of baseline markers of tumor burden (sum of the product of diameters; CD19 + cells/μL; percent CLL cells in bone marrow biopsies or aspirates) with liso-cel expansion were evaluated by MRD status. Statistical analyses were performed using the Wilcoxon signed-rank test. Results:Of the 80 pts who were evaluable for peripheral blood MRD, 34 had a best overall response of SD, 19 of whom achieved uMRD and 15 who were MRD+. Greater liso-cel expansion parameters (median maximum expansion and area under the curve from 0-28 days postinfusion) were observed in all pts, including those with SD, who achieved uMRD versus MRD+ status ( P < 0.0001), suggesting that higher liso-cel expansion after infusion was associated with achievement of uMRD independent of iwCLL 2018 response. Regardless of baseline tumor burden, pts with higher liso-cel expansion achieved uMRD after liso-cel treatment. A positive association between tumor burden and liso-cel expansion in pts with SD and uMRD was also observed. In contrast, pts with SD who were MRD+ had lower liso-cel expansion and trends of higher baseline disease burden. Liso-cel expansion in pts with SD and uMRD clustered with the responders, who all achieved uMRD status (in those evaluable for MRD). Notably, 4 pts with SD and uMRD had a durable disease control of > 22 months. We wanted to further understand the relationship between uMRD and durable disease control. Given that the median PFS of pts with SD and uMRD was 6 months, we evaluated the association between tumor burden and liso-cel expansion in pts who had a PFS of ≤ 6 months or > 6 months. Although no clear association was observed with liso-cel expansion and tumor burden within these 2 groups, 4 out of 5 pts with SD and uMRD with longer disease control (PFS > 6 months) had a > 98% decrease in sample-level MRD from Month 1 to Month 3. In contrast, in pts with SD and uMRD with PFS ≤ 6 months, 5 out of 7 pts had a > 70% increase in sample-level MRD from Month 1 to Month 3. Conclusions:In this exploratory analysis of pts with R/R CLL who had SD by iwCLL 2018 criteria after liso-cel treatment, greater liso-cel expansion, regardless of baseline tumor burden, correlated with blood uMRD status. Additionally, those with a PFS > 6 months achieved deeper clearance of blood sample-level MRD within 3 months of treatment versus those with a PFS ≤ 6 months, suggesting that MRD kinetics may identify pts with SD who can achieve durable disease control despite not achieving a response per iwCLL 2018 criteria. Further investigation is needed to better understand which subsets of pts with CLL who had SD may derive clinical benefit from liso-cel.

Ultrasensitive chimerism enhances measurable residual disease testing after allogeneic hematopoietic cell transplantation

AbstractIncreasing mixed chimerism (reemerging recipient cells) after allogeneic hematopoietic cell transplant (allo-HCT) can indicate relapse, the leading factor determining mortality in blood malignancies. Most clinical chimerism tests have limited sensitivity and are primarily designed to monitor engraftment. We developed a panel of quantitative polymerase chain reaction assays using TaqMan chemistry capable of quantifying chimerism in the order of 1 in a million. At such analytic sensitivity, we hypothesized that it could inform on relapse risk. As a proof-of-concept, we applied our panel to a retrospective cohort of patients with acute leukemia who underwent allo-HCT with known outcomes. Recipient cells in bone marrow aspirates (BMAs) remained detectable in 97.8% of tested samples. Absolute recipient chimerism proportions and rates at which these proportions increased in BMAs in the first 540 days after allo-HCT were associated with relapse. Detectable measurable residual disease (MRD) via flow cytometry in BMAs after allo-HCT showed limited correlation with relapse. This correlation noticeably strengthened when combined with increased recipient chimerism in BMAs, demonstrating the ability of our ultrasensitive chimerism assay to augment MRD data. Our technology reveals an underappreciated usefulness of clinical chimerism. Used side by side with MRD assays, it promises to improve identification of patients with the highest risk of disease reoccurrence for a chance of early intervention.

Genomic analysis to identify determinants of inherent response and resistance to elranatamab in MagnetisMM-3 cohort A.

8045 Background: MagnetisMM-3 (NCT04649359) is an open-label, multicenter, non-randomized phase 2 study of elranatamab monotherapy in patients (pts) with multiple myeloma refractory to at least 1 proteasome inhibitor, 1 immunomodulatory drug and 1 anti-CD38 antibody. This analysis examined molecular correlates of elranatamab response and resistance in pts naïve to B-cell maturation antigen (BCMA)-directed therapy (Cohort A). Methods: Bone marrow aspirate (BMA) samples collected at screening were analyzed by whole exome and whole transcriptome sequencing. To investigate the contribution of the tumor microenvironment (TME) in elranatamab response, the abundance of cell types in the BMA samples collected at screening was estimated using single sample gene set enrichment analysis (ssGSEA) of LM22 cell type signatures. Response was defined as a best overall response of very good partial response or better (partial response was considered non-response for this analysis). Results: TNFRSF17 (BCMA encoding gene) expression correlated with markers of disease burden: levels increased with disease stage (with progressively higher levels in R-ISS stages I, II, and III; p= 0.014), were higher in pts with high-risk cytogenetics ( p= 0.002) and correlated with plasma cell content in BMA samples ( ρ= 0.80; p< 10-10). TNFRSF17 expression trended higher in non-responders ( p= 0.08), but this trend was diminished when adjusting for disease burden. These findings suggest TNFRSF17 expression in bulk BM samples is associated with higher disease burden and likely poorer response. According to ssGSEA of LM22 cell types, plasma cells in BMA were associated with non-responders ( p= 0.03). Further multivariable modeling (controlling for plasma cell content) revealed additional cell types associated with response, including macrophages and monocytes, which were associated with poor outcome. Pts with both low plasma and low myeloid cells were most likely to respond. Genome wide copy number analysis showed that TNFRSF17 locus amplification was associated with non-response ( p= 0.008). Chromosomal alterations associated with non-response were identified, including genomic loci known to define high-risk MM (eg, 1q21+) and loci not known to be associated with high-risk MM (eg, 17q21+ and 6p21+). Conclusions: Genomic analysis of BMA samples from MagnetisMM-3 identified an association between higher TNFRSF17 expression in the TME and unfavorable outcomes, likely due to its surrogacy with increased tumor burden. Features of high-risk disease were also associated with lack of response to elranatamab. Adjusting for tumor cell content revealed additional aspects of the TME associated with poor response, including increased myeloid cell populations. Lastly, alterations in specific genomic loci were also associated with response, consistent with tumor intrinsic features influencing elranatamab response. Clinical trial information: NCT04649359 .

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

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

Preclinical Evaluation of ISB 1442, a First-in-Class CD38 and CD47 Bispecific Antibody Innate Cell Modulator for the Treatment of AML and T-ALL

ISB 1442 is a bispecific antibody (BsAb) using Ichnos' proprietary Bispecific Engagement by Antibodies based on the TCR (BEAT®) platform. A fully human BsAb with anti-CD38 and CD47 binding arms, ISB 1442 was developed for the treatment of hematologic malignancies. The CD38 binding arm consists of two bi-paratopic Fabs that strongly bind to CD38 through avidity-induced interactions. The anti-CD47 arm comprises a single Fab designed to block the interaction between CD47 and the signal-regulatory protein alpha (SIRPα) receptor present on phagocytes. With this design, the CD38 Fab arm preferentially drives binding to tumor cells and enables blocking of proximal CD47 receptors on the same cell via avidity-induced binding. Hence, ISB 1442 is anticipated to induce minimal effects on red blood cells (RBC) compared to benchmark anti-CD47 monoclonal antibodies (mAb), such as magrolimab. The Fc portion of ISB 1442 is engineered to enhance antibody dependent cell phagocytosis (ADCP), antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Preclinical activity of ISB 1442 for the treatment of relapsed/refractory multiple myeloma (rrMM), including increased killing potency relative to daratumumab and magrolimab benchmarks as well as more favorable off tumor/on target specificity, was reported previously (Sammicheli et al. ASH 2021). ISB 1442 is currently in a Phase 1 clinical trial for rrMM. Beyond MM, CD38 and CD47 are expressed in other hematologic cancers, including acute myeloid leukemia (AML) and T cell acute lymphoblastic leukemia (T-ALL). In AML, unleashing tumor cell phagocytosis using magrolimab shows clinical benefit in combination with demethylating agents and BCL-2 inhibitors. T-ALL is characterized with high CD38 expression, and daratumumab in combination with chemotherapy is under investigation (NCT03384654) for this indication. ISB 1442 has the potential to treat AML and T-ALL patients with its first-in-class molecular attributes of co-targeting CD38 and CD47 by a single antibody with enhanced Fc effector functions. ISB 1442 induced potent phagocytosis of both AML and T-ALL cell lines in vitro. The induction of phagocytosis was more prominent in cell lines expressing high level of CD38. ISB 1442 also induced higher phagocytosis of both AML and T-ALL tumor cells as compared to daratumumab and anti-CD47 (5F9) in cell lines expressing low level of CD38 and CD47. In addition to phagocytosis, ISB 1442 induced potent ADCC of both AML and T-ALL cell lines. To characterize the complex mechanisms of action of ISB 1442 in a single assay, a multiple mode of action of killing (MMoAK) in vitro assay was established where macrophages and PBMCs are incubated with AML or T-ALL tumor cells and human serum. With this approach, tumor cells can be targeted simultaneously by NK cells from PBMCs (ADCC Mechanism of Action (MOA)), macrophages (ADCP MOA), and complement from human serum (CDC MOA). ISB 1442 induced higher killing of AML and T-ALL cell lines in MMoAK assays as compared to daratumumab or anti-CD47 (5F9). ISB 1442 induced potent killing of primary tumor samples from both AML and T-ALL patients in a heterologous assay using macrophages from healthy donors as effector cells, suggesting that target cancer cells do not have intrinsic mechanisms protecting them from the immune cell-mediated MOA of ISB 1442 killing . In patients' samples having detectable levels of myeloid or NK cells in bone marrow aspirates, ISB 1442 induced killing of tumor cells in the autologous setting in a direct killing assay ex vivo. In summary, these data support the clinical development of 1442 in AML and T-ALL. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to have antitumor activity in AML and T-ALL patients in a single antibody relative to anti-CD38 or anti-CD47 monoclonal antibodies as well as their combination.

Poster 104: The Role of Patient Demographic Factors in the Composition of Bone Marrow Aspirate Concentrate: A Flow Cytometry Analysis

Objectives:Bone marrow aspirate concentrate (BMAC) is one type of orthobiologic that has gained increased popularity over the last decade for a variety of orthopedic procedures. Multiple factors have been demonstrated to influence the MSCs concentration and chondrogenic potential in BMA and thus BMAC, ranging from aspiration location to amount extracted to peripheral blood platelet count. Some studies have suggested that age may affect chondrogenic potential of BMAC derived MSCs but may not affect initial MSC concentration. Therefore, the purpose of this study was to 1) investigate the concentration value and variance BMAC content and 2) investigate the effects of donor age, sex, and other demographic factors on the amount of MSCs as measured by flow cytometry in bone marrow aspirate concentrate (BMAC).Methods:A review of patients who underwent BMAC treatment as part of a clinical trial were enrolled and complete BMAC flow cytometry data were included in analysis. Chart review was performed to identify patient demographic factors to determine their relationship to flow cytometry cell count. Flow cytometry was performed on both patient bone marrow aspirate (BMA) and BMAC samples to identify multipotent MSC phenotype defined as cell-surface co-expression of the antigens CD105, CD73, and CD90 (≥95% positive) and the absence of hematopoietic lineage markers CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR (≤2% positive). The ratio of cells in BMA: BMAC samples was calculated, and Spearman correlations and Kruskall-Wallis tests were used to determine the relationship of cell concentration to demographic factors.Results:A total of 80 patients were included in analysis (49% male, mean age: 49.93±12.17 years). Mean concentration of BMA and BMAC was 2,048.13±2,004.14 MSCs/mL and 5618.87±7568.54 MSC/mL, respectively, with a mean BMAC:BMA ratio of 4.35 ± 2.09 (Figure 1). A significantly higher MSC concentration was observed in the BMAC samples (P=0.005). No patient demographic factors (age, sex, height, weight, BMI, smoking status) significantly impacted MSC concentration in the BMAC samples.Conclusions:This study validates that BMAC concentrates and increases MSCs from BMA approximately four-fold. In addition, in opposition to our hypothesis, no demographic factors affected the final number of MSCs observed with flow cytometry analysis. These preliminary results suggest that BMAC successfully concentrates BMA and provides a higher number of MSCs. Baseline patient characteristics likely have minimal effects on the number and concentration of MSCs in BMAC, and therefore may not limit patient selection for BMAC for clinical use.Figure 1.Distribution of the ratio of BMAC to BMA

PB2000: LESSONS LEARNED FROM CERBA RESEARCH CHIMERIC ANTIGEN RECEPTOR T-CELL (CAR T) MULTIPLE MYELOMA RUNNING TRIALS

Background: The pipeline has 359 liquid tumor trials that are CAR T driven where Cerba Research (CR) understands that there is a demand to provide innovative specialty testing, such as flow cytometry, sequencing and downstream biomarker analysis. Herein, CR examined 38 of our active multiple myeloma (MM) trials since 2016 with a focus on CAR T therapy. We identified 15 of those trials where CR participated as a central laboratory. Aims: The aim of this evaluation is to identify gaps within current CAR T MM trial endpoints and to improve for the future of our liquid tumor trials. Methods: We analyzed internal trial matrix database and examined other documents such as laboratory specification documents, trial design, protocol, and schedule of activities. We used descriptive statistics to illustrate our specialty testing involvement and to identify gaps in our offerings. Results: CR currently has 38 running MM trials, in which 15 (39%) have a CAR T therapy. Sixteen (42%) of our MM trials are phase I/first-in-human (FIH), whereas 14 (37%) and 7 (18%) are phase II and III, respectively. Within our MM CAR T trials, we perform flow cytometry (FCM) in 87% of cases. Our involvement in FCM is exploratory in nature, and the design of the panels is performed with continued scientific discussions with sponsors. Some of the flow cytometry panels performed in our CAR T trials are: - Enumeration of CAR T cells in whole blood and bone marrow aspirates, including PK evaluation - Immunophenotyping of CAR T cells (memory, regulatory, activation status, exhaustion status) - Minimal residual disease (MRD) CR is often involved across full CAR T programs starting at FIH up to registration trials. We have robust upstream capabilities where we perform peripheral blood mononuclear cell (PBMC) isolation (60%), bone marrow mononuclear cell (BMMC) isolation (53%), DNA extraction (47%), cell sorting (53%), serum and urine protein electrophoresis/immunofixation (47%), and histopathology (decalcification, embedding, sectioning) (53%). We are also involved in routine testing such as hematology (27%), biochemistry (33%) and coagulation (27%). However, except for FCM (87%), immunohistochemistry (IHC 47%) and FISH (47%), we identified a gap in requests for sequencing within MM CAR T trials (0%.) Next generation sequencing (NGS), which was mostly for MRD assessment, was performed by referral labs. We also identified up to 11 referral labs participating in MM trials which significantly adds to a trial operation complexity. Summary/Conclusion: CR currently has strong FCM fit-for-purpose design and execution capabilities as demonstrated by our participation in CAR T programs as a central laboratory. Using FCM in CAR T trials is important as it can aid in determining effectiveness of therapy, any potential side effects, and general health of the immune system by performing longitudinal monitoring of the immune system. We also frequently isolate PBMCs/BMMCs, extract DNA and perform histopathology on a global scale for clinical trial purposes. Herein, we determined that we have good downstream efforts with FCM, IHC and FISH and plan on growing NGS offerings by creating a new MRD NGS oncopanel and compare it with FCM for validation and concordance in multiple myeloma.

Abstract 410: Development of novel, non-toxic rifamycins that reverse drug resistance

Abstract Introduction: We have discovered novel non-toxic rifamycins that are extremely potent at sensitizing many drug-resistant cancers, including DLBCL, to standard-of-care chemotherapeutics. Methods: CHOP-resistant DLBCL cell lines were derived from CHOP-sensitive cells by “on-off” cycles of CHOP treatment, analogous to clinical therapy. The CHOP-resistant cells were used in high-throughput screening of a highly diverse collection of approximately 50,000 drug-like molecules to identify small molecules that reverse CHOP-resistance. The FDA-approved drug, Rifabutin, was identified as a non-cytotoxic compound that potently reversed resistance to CHOP. Structure-Activity-Relationships (SAR) on Rifabutin were conducted, which led to the generation of a new more potent CHOP-chemosensitizing agent, designated RTI. Results: RTI was highly synergistic with variety of chemotherapeutics, including doxorubicin (DOX), epirubicin, vinblastine, etoposide in drug-resistant NHL cells. Combination therapy of DOX+RTI in mouse xenograft models of DLBCL was much more effective at repressing tumor growth than with DOX alone. RTI lowered the IC90 in a dose-dependent manner in CD20-positive B cells in bone marrow aspirates from both CHOP-naïve and CHOP-relapsed patients, and in a metastatic lymph node. RTI’s PK characteristics are similar to rifabutin, and it exhibited no overt toxicity in mice or pigs at high doses. RTI rapidly induced mitochondrial superoxide, membrane potential, and fission. The superoxide dismutase, FeTCP, antagonized RTI-induced ROS and potentiation of DOX cytotoxicity. RTI reduced the activity of the Nrf-2 antioxidant protein, upregulated proteins involved in the unfolded protein response (UPR), and induced metabolic reprogramming as indicated by Seahorse assays that showed increased glycolysis and decreased oxygen consumption from 3 to 24 hrs. Conclusions: RTI has a broad spectrum of action in both double- and triple-hit DLBCL and synergizes with many different chemotherapeutics to restore drug sensitivity. RTI-79 works by dramatically increasing intracellular superoxide through redox cycling, triggers UPR, and downregulates Nrf-2 activity. Thus, RTI-79 increases oxidative stress through the squelching of Nrf-2’s ability to respond to chemotherapeutic stress. Since the parent compound, rifabutin, binds to the aryl hydrocarbon receptor (AhR) and modulates its activity, we hypothesize that RTI mediates its unique pleiotropic chemosensitizing mechanism through targeting of AhR, and provides for a broad, safe, and novel approach to treating drug resistant cancers. Ongoing experiments are investigating the effect of RTI on the AhR-signaling pathway and the role it plays in RTI-mediated chemosensitization and potentiation of chemotherapeutics. A clinical trial of companion dogs with CHOP-relapsed DLBCL treated with combination CHOP+RTI is currently in progress. Citation Format: Steve A. Maxwell, Deeann Wallis, Nian Zhou, Dwight Baker, Seyed H. Mousavi-Fard, Kimberly Loesch, Stacy Galaviz, Liam Guthrie, Thomas Snavely, Qingan Sun, Carolina M. Rojas, David W. Threadgill, Thomas Ioerger, Wen Dong, Gwen Seemann, Theresa W. Fossum, James C. Sacchettini. Development of novel, non-toxic rifamycins that reverse drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 410.

Enrichment-Free Single-Cell Detection and Morphogenomic Profiling of Myeloma Patient Samples to Delineate Circulating Rare Plasma Cell Clones.

Multiple myeloma is an incurable malignancy that initiates from a bone marrow resident clonal plasma cell and acquires successive mutational changes and genomic alterations, eventually resulting in tumor burden accumulation and end-organ damage. It has been recently recognized that myeloma secondary genomic events result in extensive sub-clonal heterogeneity both in localized bone marrow areas and circulating peripheral blood plasma cells. Rare genomic subclones, including myeloma initiating cells, could be the drivers of disease progression and recurrence. Additionally, evaluation of rare myeloma cells in blood for disease monitoring has numerous advantages over invasive bone marrow biopsies. To this end, an unbiased method for detecting rare cells and delineating their genomic makeup enables disease detection and monitoring in conditions with low abundant cancer cells. In this study, we applied an enrichment-free four-plex (CD138, CD56, CD45, DAPI) immunofluorescence assay and single-cell DNA sequencing for morphogenomic characterization of plasma cells to detect and delineate common and rare plasma cells and discriminate between normal and malignant plasma cells in paired blood and bone marrow aspirates from five patients with newly diagnosed myeloma (N = 4) and monoclonal gammopathy of undetermined significance (n = 1). Morphological analysis confirms CD138+CD56+ cells in the peripheral blood carry genomic alterations that are clonally identical to those in the bone marrow. A subset of altered CD138+CD56- cells are also found in the peripheral blood consistent with the known variability in CD56 expression as a marker of plasma cell malignancy. Bone marrow tumor clinical cytogenetics is highly correlated with the single-cell copy number alterations of the liquid biopsy rare cells. A subset of rare cells harbors genetic alterations not detected by standard clinical diagnostic methods of random localized bone marrow biopsies. This enrichment-free morphogenomic approach detects and characterizes rare cell populations derived from the liquid biopsies that are consistent with clinical diagnosis and have the potential to extend our understanding of subclonality at the single-cell level in this disease. Assay validation in larger patient cohorts has the potential to offer liquid biopsy for disease monitoring with similar or improved disease detection as traditional blind bone marrow biopsies.

EFFECT OF BONE MARROW ASPIRATE IN DENERVATION-INDUCED SKELETAL MUSCLE ATROPHY

The aim: To evaluate muscle changes after sciatic nerve damage with the injection of bone marrow aspirate cells. Materials and methods: 36 rabbits underwent sciatic nerve cross-section and neuroraphy, bone marrow aspirate cells were injected directly or 7 weeks after neuroraphy. Changes in skeletal muscle morphology (photomicrographs of histological sections were analyzed for morphometric analysis of collagen region, quantitative analysis of conducted collagen density and measurement of muscle fibers diameter) and biochemical parameters (catalase activity, superoxide dismutase and glutathione peroxidase measurements and level of TBARS was determined) at 8, 12, and 16 weeks were examined. Results: There is atrophy of muscle fibers in denervated muscles, and it has a negative tendency between 8 and 12 weeks. Delayed bone marrow aspirate cells injection into the muscles at 7 week – delayed atrophy and formation of TBA reactive substances. But bone marrow aspirate cells injection into the muscles directly after neuroraphy increased collagen formation, and development of fibrosis in areas of atrophy. Conclusions: Sciatic nerve injury results in atrophy of muscle tissue, which is partially delayed after delayed bone marrow aspirate cells injection at week 7. Muscle atrophy was characterized by a sharp increase in TBARS levels at 12 and 16 weeks and catalase activity at 12 weeks, and changes in biochemical parameters were partially normalized after the use of cell aspirates, to a greater extent with delayed injection.

Dysplasia and PNH-type cells in bone marrow aspirates of myelodysplastic syndromes.

Flow cytometry is increasingly applied in cytopenic patients suspected for myelodysplastic syndromes (MDS). Analysis includes evaluation of antigen expression patterns in granulocytes of which, for example, partial lack of CD16 may indicate dysplasia, but presence of paroxysmal nocturnal hemoglobinuria (PNH)-type cells should be considered. However, diagnostic bone marrow (BM) samples hamper PNH analysis because immature stages in the granulo-/monocytic compartment lack expression of certain glycophosphatidyl-inositol-anchored proteins. In this prospective study, we evaluated the presence of PNH-type cells in BM next to aberrancies from routine MDS immunophenotyping. We combined antibodies defining maturation trajectories with FLAER. Validation of the designed method against routine PNH analysis and parallel analysis of BM and blood samples revealed similar results (granulocytes: Wilcoxon p= 0.25 and p= 0.82, respectively). We analyzed BM samples from 134 MDS, 17 chronic myelomonocytic leukemia, 15 aplastic anemia (AA), 1 PNH, 51 non-clonal cytopenic controls, and 12 normal controls. Most AA/PNH-BM samples showed clear PNH clones: median 1.1% (0%-35%); CD16 loss on mature neutrophils paralleled PNH-clone sizes. In MDS-BM, only 3.7% of cases showed ≥0.1% PNH-type cells, whereas partial CD16 loss was more frequent and abundant. Our findings confirm that dysplastic features in MDS-BM may point to presence of PNH-type cells, though only few cases displayed FLAER-negative cells. We showed that identification of these cells in the granulocyte compartment of BM specimen is feasible, but-according to international guidelines-results need to be confirmed in peripheral blood.

High Grade of Amplification of Six Regions on Chromosome 2p in a Neuroblastoma Patient with Very Poor Outcome: The Putative New Oncogene TSSC1.

Simple SummaryHere, a case of neuroblastoma (NB) carrying a high-grade amplification of six loci besides MYCN is described. Since the patient had a very poor outcome, we postulated that these DNA co-amplifications might have a synergistic effect in increasing NB cell proliferation. In order to verify this hypothesis, we analyzed in silico the impact of high expression of the genes located within the amplifications on the NB patients’ outcome using a large dataset integrating three different platforms. These analyses disclosed that high expression of the TSSC1 gene was the most significantly associated with reduced overall survival of NB patients, suggesting that it may have a potential prognostic role in NB in both MYCN amplified and MYCN not amplified tumors. Further studies on TSSC1 interactions and functioning could lead to possible focused therapies for high-risk NB patients.We observed a case of high-risk neuroblastoma (NB) carried by a 28-month-old girl, displaying metastatic disease and a rapid decline of clinical conditions. By array-CGH analysis of the tumor tissue and of the metastatic bone marrow aspirate cells, we found a high-grade amplification of six regions besides MYCN on bands 2p25.3–p24.3. The genes involved in these amplifications were MYT1L, TSSC1, CMPK2, RSAD2, RNF144A, GREB1, NTSR2, LPIN1, NBAS, and the two intergenic non-protein coding RNAs LOC730811 and LOC339788. We investigated if these DNA co-amplifications may have an effect on enhancing tumor aggressiveness. We evaluated the association between the high expression of the amplified genes and NB patient’s outcome using the integration of gene expression data of 786 NB samples profiled with different public platforms from patients with at least five-year follow-up. NB patients with high expression of the TSSC1 gene were associated with a reduced survival rate. Immunofluorescence staining on primary tumor tissues confirmed that the TSSC1 protein expression was high in the relapsed or dead stage 4 cases, but it was generally low in NB patients in complete remission. TSSC1 appears as a putative new oncogene in NB.

Pharmacodynamics of SEA-BCMA, a Nonfucosylated Antibody Targeting BCMA, in Patients with Relapsed/Refractory Multiple Myeloma

Patients with relapsed/triple class refractory (refractory to a proteasome inhibitor, immunomodulatory drug or anti-CD38 antibody) multiple myeloma (MM) have limited treatment options. Versatile therapies, such as unconjugated antibodies (Abs), that are well tolerated and can be combined with multiple modalities are critical for the MM treatment landscape. SEA-BCMA is an investigational, humanized, nonfucosylated IgG1 monoclonal Ab targeting B-cell maturation antigen (BCMA) on malignant plasma cells. Preclinical data show that SEA-BCMA blocks BCMA-mediated pro-survival and proliferative cell signaling and mediates antibody-dependent cellular phagocytosis and enhanced antibody-dependent cellular cytotoxicity via increased binding to activating Fc receptor, FcγRIIIa (Van Epps 2018). A phase 1, open-label, multicenter study to evaluate the safety, tolerability, and antitumor activity of SEA-BCMA in adults with relapsed or refractory MM (SGNBCMA-001; NCT03582033) is ongoing. To support maximal ligand blocking and immune effector engagement by SEA-BCMA, we investigated its binding and saturation pharmacodynamics (PD) in patients enrolled in dose escalation and currently recruiting dose expansion cohorts.Novel quantitative assays were developed to assess the BCMA target and the availability of SEA-BCMA in patients enrolled in the study. These assessments include: a liquid chromatography-mass spectrometry (LC-MS) soluble BCMA (sBCMA) assay, a BCMA receptor occupancy assay on malignant plasma cells in bone marrow aspirates, and a receptor binding assay to assess the direct binding and saturation capacity of SEA-BCMA in patients' serum samples and bone marrow aspirates to a BCMA-expressing cell in vitro. Results reported as median (min-max) values unless specified.At baseline, the median sBCMA level from patients enrolled in SGNBCMA-001 was 8.5(0.5-217.0) ng/mL. After the first dose of SEA-BCMA, a rapid and sustained increase in sBCMA was observed [32.0 (3.0-139.0) fold]. Mechanistically, because SEA-BCMA can bind to sBCMA, accumulation of sBCMA may occur due to formation of the sBCMA:SEA-BCMA complex, resulting in reduced clearance. Importantly, for patients at the dose selected for expansion (1600mg), the molar ratio of SEA-BCMA to sBCMA remained in excess (10:1-400:1), thereby overcoming the liabilities of this complex formation and supporting malignant plasma cell drug exposure. Results of a receptor binding assay using patients' serum samples also demonstrated a dose-dependent ability to saturate BCMA-expressing cells. Evaluation of receptor occupancy in patient bone marrow aspirates showed that at baseline, median unoccupied membrane BCMA (unbound by ligand) was 3,500(1,500-30,000) copies per cell with one excluded outlier that exhibited 270,000 copies per cell. On-treatment, unoccupied membrane BCMA reductions were observed ranging from 50% to 100% from baseline in majority of evaluable patients at the 1600mg dose. An on-treatment increase in total BCMA expression was also observed, which is being further investigated.Maximal ligand blocking and immune effector engagement is best achieved through saturation of a target receptor. Overall, these PD results provide insight into the saturation potential of plasma cell membrane BCMA by SEA-BCMA and informed the dose selection and schedule in expansion cohorts. Further evaluation of these relationships to patient response is ongoing as the Phase 1 study continues to enroll. DisclosuresTaft: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. Henderson: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. O'Day: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. Yu: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company. Li: Seagen Inc.: Current Employment, Current equity holder in publicly-traded company. Ho: Seagen Inc.: Current Employment, Current equity holder in publicly-traded company. Van Epps: Seagen, Inc: Current Employment, Current equity holder in publicly-traded company.

Damages of Neuromuscular System After Mechanical-Induced Limb Ischemia (Experimental Study)

Relevance. Traumatic and ischemic injury of the limbs is accompanied by damage of the skeletal muscles and peripheral nerves of the limbs. The dynamics and consequences of ischemic lesions remain poorly understood and need to be corrected.&#x0D; Objective: using quantitative morphological and sonographic methods, to study the dynamics of skeletal muscle damage of the limb after traumatically induced ischemia with and without the injection of platelet-rich plasma, bone marrow aspirate, and adipose tissue fraction.&#x0D; Materials and Methods. In 3 experiments, rabbits were modeled with 6-hour limb ischemia by applying an elastic tourniquet. After compartment syndrome detection, based on the assessment of subfascial pressure, cell suspensions were injected into the leg muscles. Sonographic and histological examination of the muscles was performed on days 5, 15, and 30. The results of sonography and morphometry were evaluated by statistical methods.&#x0D; Results. The developed model of ischemia consists of 6-hour immobilization of the limb, on тwhich medical elastic tourniquets were imposing. The action of the tourniquets causes high subfascial pressure and necrosis of the superficial muscle groups of the lower third of the thigh and lower leg. According to sonography, the δ-entropy of damaged tissues on day 5 is reduced relative to the intact limb, as in the case of administration of bone marrow aspirate cells. On days 15 and 30, sonography showed no difference between the comparison groups. The dynamics of morphological features of limb tissue damage consist of necrosis of superficial muscle groups, atrophy in the middle layers, and almost intact deep muscle groups. Necrosis was replaced by scar tissue, the density of which increases 11-14 times, and does not differ in the period 5-30 days. The administration of platelet plasma, bone marrow aspirate, and adipose tissue fraction did not change the dynamics of fibrotic changes in ischemic damaged muscles. Muscle atrophy is accompanied by activation of endogenous repair of single muscle fibers, which tended to intensify after injection of bone marrow aspirate. The sciatic nerve of the injured limb was not structurally damaged according to the deep topography, while the nerves of the tibia develop degenerative changes from the 15th day.

OAB-014: Newly diagnosed Multiple Myeloma patients with high levels of circulating tumor cells are distinguished by increased bone marrow plasma cell proliferation

<h3>Background</h3> Circulating tumor cells (CTCs) are present in the blood of all patients with (precursor)stages of Multiple Myeloma (MM). The levels of CTCs vary widely between patients, yet the biology behind this variability is unknown. Importantly, MM patients with a higher percentage of CTCs have an inferior prognosis independent of high-risk cytogenetics, suggesting that CTC numbers are a relevant reflection of tumor cell biology. Here, we set out to identify differences in CTCs and paired bone marrow plasma cells (BM PCs). Additionally, we compared BM PCs from patients with high and low levels of CTCs with the goal of identifying mechanistic drivers of high-risk disease. <h3>Methods</h3> We isolated PCs from viably frozen mononuclear cells of peripheral blood and bone marrow aspirates of newly diagnosed MM patients. Common high-risk mutations were present in both groups. We performed single cell RNA sequencing of paired CTCs and BM PCs from five patients with a high percentage of CTCs (0.5%-8%). In addition, we generated single cell transcriptomes of BM PCs of eight patients with a high percentage of CTCs (2-22%) and 13 patients with low percentage of CTCs (0.004%-0.08%) <h3>Results</h3> Single cell transcriptomes were generated from 44,779 CTCs and 35,697 bone marrow PCs. When paired CTCs and BM PCs were integrated, we identified 9 common clusters, no cell specific cluster for either source was detected. Moreover, only 25 genes were significantly differentially expressed between CTCs and BM PCs. The absence of unique clusters in either CTCs or BM PCs, and the transcriptional overlap between these two sources indicate that CTC levels are not driven by the emergence of a transcriptionally different migratory clone, but are likely a reflection of altered BM PC biology. To identify such alterations, we compared bone marrow PCs from patients with high and low percentages of CTCs. Single cell transcriptomes were generated from 74,830 bone marrow PCs. Integration of all patients lead to the identification of 8 distinct PC clusters, one of which was characterized by active proliferation as defined by transcription of STMN1 and MKI67. Interestingly, this proliferative cluster was larger in patients with a high percentage of CTCs. Furthermore, cell cycle analyses based on canonical G2M and S phase markers revealed that actively cycling PCs were more frequent in the BM of patients with a high percentage of CTCs (64% versus 30%, p<0.001), irrespective of the cluster in which these cells were contained. <h3>Conclusions</h3> Through single cell transcriptomic analyses we reveal that CTCs and MM cells from BM are transcriptionally similar. Importantly, we identify increased BM PC proliferation as a significant difference between patients with high and low levels of CTCs, implicating an increased entry into the cell cycle as one of the mechanisms driving CTC levels and MM disease pathobiology.

Photobiomodulation of fresh bone marrow aspirate for regenerative therapy

Use of mesenchymal stem cells and low-level laser therapy (LLLT) have been widely studied to promote bone healing. evaluate effect of photobiomodulation on total number of cells (TNC) and cell viability (CV) of fresh bone marrow aspirate (BMA). Femur BMA from 10 adult rats was collected and a cell concentration of 1x107 cell/mL was obtained. Cell suspension was deposited on 96 well cell culture plates and distributed in groups: 1) RPMI, positive control; 2) Distilled Water, negative control; 3) Red Laser (RL); 4) Infrared Laser (IRL). Groups RL and IRL received LLLT application right after incubation. Cells were incubated for 24 h. TNC and CV were assessed through trypan blue assay after 1, 3, 6, 10 and 24 h of incubation. Data distribution was verified by Shapiro-Wilk test. Kruskal-Wallis test was used for intergroup and intragroup comparisons (p&lt;0.05). TNC: after 1 and 3 h, groups RL and IRL presented significantly higher TNC than Group Water; after 6 and 10 h, groups RPMI, RL and IRL presented significantly higher TNC than Group Water. CV: after 1 h, groups RL and IRL showed significantly higher percentage of VC than Group Water; after 3, 6 and 10 h, all groups presented significantly higher percentage of VC than Group Water. It can be concluded that LLLT enhanced number and viability of fresh bone marrow aspirate cells.

Abstract 408: Quantification of cell surface HLA-A2 and intracellular Survivin protein levels for tumor blasts and non-blast immune cells in multiple myeloma bone marrow aspirates using a rapid sample preservation methodology followed by 10-color FACS assay

Abstract Introduction: Multiple myeloma (MM) is a plasma cell disorder characterized by clonal expansion and accumulation of immature undifferentiated neoplastic cells in the bone marrow (BM). Recent advances in fluorochrome chemistries, flow cytometry (FACS) instrumentation and rapid tissue preservation methodologies have created opportunities for broader adoption of FACS in the clinical management of multiple myeloma patients. Methods: We have developed a 10 color, 2 tube FACS assay based on recommendations made by the European Myeloma Network for phenotyping plasma cell gammopathies to identify abnormal blasts in multiple myeloma patient bone marrow aspirates. The assay includes essential plasma cell markers CD138, CD38, CD56 and CD19 to differentiate abnormal blasts from reactive plasma cells. Use of a multi epitope fluorochrome conjugated anti-CD38 antibody enables CD38 detection in patients on anti-CD38 therapy. Results: Quantification of cell surface HLA-A2 and intracellular survivin protein was made possible using fluorochrome conjugated antibodies in combination with bead standards. The SmartTube™ proteomic stabilizer system for rapid preservation was used to freeze BM aspirate specimens for later analysis at locations with appropriate FACS equipment and trained staff. Consistent with previously published literature, patient survivin levels were elevated on abnormal MM blasts in comparison to survivin levels on non-blast immune cells. Conclusion: We have developed a robust FACS methodology that would be easy to implement in the clinical trial and development setting with minimal training. The assay can consistently identify abnormal blasts in BM samples of multiple myeloma patients and quantify intracellular and cell surface biomarkers related to MM disease or disease treatment responses. Citation Format: Tejaswini Hardas, Hyun-Jeong Ra, James Sheridan, Maria Kovalenko, Catherine Tribouley. Quantification of cell surface HLA-A2 and intracellular Survivin protein levels for tumor blasts and non-blast immune cells in multiple myeloma bone marrow aspirates using a rapid sample preservation methodology followed by 10-color FACS assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 408.