Bone Marrow Collection Research Articles

Identifying ADGRG1 as a specific marker for tumor-reactive T cells in acute myeloid leukemia

Besides chemotherapy and hematopoietic stem cell transplantation (HSCT), autologous T cells can also serve as a new treatment approach for AML patients. However, the features of tumor-reactive T cells and their distinctive markers still lack full description. To evaluate the characteristics of tumor-reactive T cells, we collected bone marrow (BM) T cells from newly diagnosed AML patients with RUNX1::RUNX1T1 as examples for paired single-cell RNA sequencing and single-cell V(D)J sequencing. Based on the STARTRAC-like algorithm, we defined bystander T cells and tumor-reactive T cells. Compared with bystander T cells, tumor-reactive T cells presented as senescent-like cytotoxic terminally differentiated T cells (Temra) with upregulated NK-related markers. Additionally, we found ADGRG1 could serve as the specific marker of CD8+ T tumor-reactive T cell and validated it through the Runx1Runx1t1/+; Mx1-Cre mouse model. In chimeric antigen receptor (CAR)-T and target cell system, ADGRG1 was selectively upregulated upon antigen-TCR encounter. Moreover, ADGRG1+CD8+ T cells released a higher level of IFN-γ and showed higher cell-killing ability when exposed to matched AML blasts. Together, our findings depict the single-cell profile of tumor-reactive T cells in AML BM and propose that ADGRG1 can act as an indicator of T cell tumor reactivity in AML, which may be further harnessed for adoptive cell therapy and tumor-reactive TCR enrichment.

A Comprehensive Protocol for the Collection, Differentiation, Cryopreservation, and Resuscitation of Primary Murine Bone Marrow Derived Macrophages (BMDM)

ABSTRACT Background The field of immunology has undoubtedly benefited from the in vitro use of cell lines for immunological studies; however, due to the “immortal” nature of many cell lines, they are not always the best model. Thus, direct collection and culture of primary cells from model organisms is a solution that many researchers utilize. To the best of our knowledge, there is not a singular protocol which encompasses the entire process of bone marrow cell collection through cryopreservation and resuscitation of cells from a murine model. Methods Bone marrow cells were collected from mice with a C57BL6 genetic background. Cells were differentiated using L929 conditioned media. Cells were assessed using a combination of microscopy, differential staining, immunocytochemistry, and trypan blue. Results: Primary murine BMDMs that underwent cryopreservation followed by resuscitation retained a high degree of viability. Furthermore, these BMDMs retained on overall ability to clear S. aureus. Results Primary murine BMDMs that underwent cryopreservation followed by resuscitation retained a high degree of viability. Furthermore, these BMDMs retained on overall ability to clear S. aureus. Conclusion Crypopreserved and resuscitated primary murine BMDMs were viable and retained their pverall S. aureus clearance ability.

CRIP1 regulates osteogenic differentiation of bone marrow stromal cells and pre-osteoblasts via the Wnt signaling pathway

With the aging of the global demographic, the prevention and treatment of osteoporosis are becoming crucial issues. The gradual loss of self-renewal and osteogenic differentiation capabilities in bone marrow stromal cells (BMSCs) is one of the key factors contributing to osteoporosis. To explore the regulatory mechanisms of BMSCs differentiation, we collected bone marrow cells of femoral heads from patients undergoing total hip arthroplasty for single-cell RNA sequencing analysis. Single-cell RNA sequencing revealed significantly reduced CRIP1 (Cysteine-Rich Intestinal Protein 1) expression and osteogenic capacity in the BMSCs of osteoporosis patients compared to non-osteoporosis group. CRIP1 is a gene that encodes a member of the LIM/double zinc finger protein family, which is involved in the regulation of various cellular processes including cell growth, development, and differentiation. CRIP1 knockdown resulted in decreased alkaline phosphatase activity, mineralization and expression of osteogenic markers, indicating impaired osteogenic differentiation. Conversely, CRIP1 overexpression, both in vitro and in vivo, enhanced osteogenic differentiation and rescued bone mass reduction in ovariectomy-induced osteoporosis mice model. The study further established CRIP1's modulation of osteogenesis through the Wnt signaling pathway, suggesting that targeting CRIP1 could offer a novel approach for osteoporosis treatment by promoting bone formation and preventing bone loss.

Turning Trash Into Treasure: A Simple Protocol for Human Mesenchymal Stromal Cell Isolation From Used Bone Marrow Collection Kits.

The therapeutic potential of mesenchymal stromal cells (MSCs) has been extensively investigated in both preclinical and clinical settings. Recent years have witnessed the emergence of numerous isolation protocols and culture techniques, ranging from the selection of subpopulations to preserve stemness to preconditioning strategies aimed at enhancing therapeutic efficacy, tailored to the specific tissue source. In this protocol, we present a straightforward and cost-effective method for isolating human MSCs (hMSCs) from discarded bone marrow collection kits (comprising bag and filter systems) originally intended for removing impurities and unwanted cellular debris from the collected bone marrow aspirate, ensuring the purity of the stem cell population during stem cell transplantation. Utilizing basic laboratory equipment, we demonstrate the isolation of hMSCs, highlighting the expression of specific surface antigens, and multilineage differentiation into adipogenic, osteogenic, and chondrogenic lineages in vitro. This sustainable and resource-efficient approach not only contributes to reducing medical waste but also holds promise for advancing regenerative medicine applications. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of human mesenchymal stromal cells from bone marrow collection kits Basic Protocol 2: Culture of human mesenchymal stromal cells Basic Protocol 3: Characterization of human mesenchymal stromal cells with flow cytometry analysis Basic Protocol 4: Characterization of human mesenchymal stromal cells with multilineage differentiation under in vitro conditions.

Abstract 2743: Molecular characterization and prognostic evaluation of disseminated cancer cell subpopulations detected in bone marrow of non-small cell lung cancer patients

Abstract Introduction: Disseminated cancer cells (DCC) can be detected years before metastatic manifestation in bone marrow of lung cancer patients. Although a consensus protocol has been established, the impact of markers and methods for DCC detection are still not settled. We recently found that EpCAM+ DCC detected by immunofluorescence (IF) outperform the classical immunocytologic (IC) detection by cytokeratin (CK) antibodies regarding prognostic impact. However, it remained unclear whether this difference resulted from biological traits associated with EpCAM+ vs CK+ DCC or from methodological reasons. We therefore performed a side-by-side comparison of IC vs IF for CK and EpCAM. Methods: We prospectively collected bone marrow (BM) aspirates from 56 non-metastasized NSCLC patients that underwent tumor resection with curative intention. After density gradient centrifugation, we analyzed the mononucleated cells by (i) IC using the anti-CK antibody A45-B/B3 and (ii) by IF using the anti-CK antibody A45-B/B3 and the anti-EpCAM antibody HEA-125. IF signals were captured and objectively quantified using ImageJ, whereas IC was evaluated by two independent observers. Marker-positive cells were isolated and subjected to Whole Genome Amplification (WGA) and whole genome sequencing for assessment of copy number alterations (CNA). Survival analysis was performed for progression-free survival (PFS), tumor-specific survival (TSS) and overall-survival (OS) after a median follow-up time of 72.5, 78.7, 78.7 months, respectively. Results: IC and IF revealed the presence of CK+ DCCs in 37.5% and 41.1% of cases, respectively (P=0.34). IF identified EpCAM+ DCCs in 55.4% of patients, resulting in a 63.8% DCC-positivity rate found by IF, when combined with CK. The average number of DCCs per million MNC, the DCC density (DCCD), was 0.92, 0.93 and 1.44 for CKIC and CKIF and for EpCAMIF, respectively. No association was found between CKIC + DCCs and PFS, TSS or OS. In contrast, detection of EpCAMIF + and/or CKIF + DCCs showed a trend towards reduced PFS (P=0.055), TSS (P=0.225) and OS (P=0.155). Subgroup analysis revealed that both EpCAMIF + and CKIF + cells were correlated with worse PFS (P=0.016 and P=0.040, respectively) and TSS (P=0.033 and P=0.008, respectively). CKIF-positivity was also associated with worse OS (P=0.014), whereas EpCAM was not. CKIF + DCCs remained an independent prognostic variable for TSS and OS upon multivariate testing (hazard ratio: 6.45 and 3.31, respectively), while EpCAMIF + was not. The IF-marker-defined cell phenotypes exhibited comparable frequencies and distributions of CNAs across the entire genome (~50% of cells). Conclusions: Detection of CK+ DCC by quantified IF analysis apparently outperforms CK detection by IC in survival analysis. The impact of CK and EpCAM expression levels on outcome are under investigation. Citation Format: Daniel Spitzl, Tobias Mederer, Julius Zank, Severin Gütter, Michael Ried, Hans-Stefan Hofmann, Christoph A. Klein. Molecular characterization and prognostic evaluation of disseminated cancer cell subpopulations detected in bone marrow of non-small cell lung cancer patients [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 2743.

Addressing a Pre-Clinical Pipeline Gap: Development of the Pediatric Acute Myeloid Leukemia Patient-Derived Xenograft Program at Texas Children's Hospital at Baylor College of Medicine.

The survival rate of pediatric acute myeloid leukemia (pAML) is currently around 60%. While survival has slowly increased over the past few decades, the development of novel agents likely to further improve survival for this heterogeneous patient population has been limited by gaps in the pAML pre-clinical pipeline. One of the major hurdles in evaluating new agents for pAML is the lack of pAML patient-derived xenograft (PDX) models. Unlike solid tumors and other types of leukemias, AML is notoriously hard to establish in mouse models, likely due in part to the need for specific human microenvironment elements. Our laboratory at TCH/BCM addressed this gap by establishing a systematic PDX workflow, leveraging advanced immunodeficient hosts and capitalizing on our high volume of pAML patients and close coordination between labs and clinical sections. Patients treated at TCH are offered the chance to participate in specimen banking protocols that allow blood and bone marrow collection as well as the collection of relevant clinical data. All patients who consent and have samples available are trialed for PDX development. In addition, samples from the Children's Oncology Group (COG) are also trialed for PDX generation. Serially transplanting PDX models are validated using short tandem repeat (STR) and characterized using both targeted DNA/RNA next generation sequencing and RNAseq. As of March 2023, this systematic approach has resulted in 26 serially transplanting models. Models have been shared with requesting labs to facilitate external pAML pre-clinical studies. Available PDX models can be located through the BCM PDX Portal. We expect our growing PDX resource to make a significant contribution to expediting the testing of promising novel therapeutics for pAML.

Clinical Study on the Efficacy and Safety of Peg Rhg CSF for Autologous Hematopoietic Stem Cell Mobilization in Lymphoma and Multiple Myeloma：a Randomized Controlled, Multicenter Clinical Study

Introduction: PEG rhG CSF has been widely used in the prevention and treatment of bone marrow suppression after chemotherapy for a variety of tumors, but whether it can be used for lymphoma and Multiple myeloma autologous hematopoietic stem cell mobilization has no relevant clinical evidence. Therefore we carried out this randomized controlled, multicenter clinical study to evaluate the efficiency and safety of PEG-rhG-CSF in the mobilization of autologous hematopoietic stem cells for lymphoma and Multiple myeloma. Methods:This experiment selected an appropriate mobilization based on the patient's willingness and physical condition of the subjects. Chemotherapy mobilized patients underwent bone marrow mobilization when white blood cells reached their lowest point and began to rebound. The experimental group received a fixed dose of 12mg of polyethylene glycol recombinant human granulocyte stimulating factor injection (PEG-rhG-CSF) for mobilization; the control group received recombinant human granulocyte stimulating factor (rhG-CSF) 5-10μg/kg daily. Collect blood and bone marrow samples from patients daily and test white blood cells and CD34+cells every day. Record CD34+cells proportion until its qantity reached 2×10 6 proportion. The interval from mobilization to collection, the time for hematopoietic reconstruction after transplantation, and complications were also collected. Results : A total of 60 patients were included in this study, including 31 in the experimental group and 29 in the control group. There were no statistically significant differences in gender, age, disease classification, and pre harvest CD34+levels between the two groups of patients. The CD34+cells collected from both the experimental and control groups reached ≥ 2.0×10 6/kg and there was no statistically significant difference (Figure 1). There was no statistical difference between the experimental group and the control group in terms of CD34+cell collection frequency, CD34+cell count, interval from mobilization to collection time, neutrophil reconstruction time, and platelet reconstruction time. In terms of related side effects there was no statistically significant difference in the incidence of rash, fever, infection, and diarrhea between the two groups of patients. The incidence of bone pain in all patients was 15% (9/60), with 8 cases being mild and only 1 case being moderate. There was no significant statistical difference in the incidence of bone pain between the experimental group and the control group (Table 1). Conclusion : PEG-rhG-CSF is safe and effective for autologous hematopoietic stem cell mobilization in lymphoma and Multiple myeloma, and its efficacy is consistent with that of rhG CSF. However, the subcutaneous injection of rhG CSF is less frequent, which is easier for patients to accept. [Key words] PEG rhG CSF; matopoietic stem cell mobilization;ymphoma and Multiple myeloma; inical study

Measuring Minimal Residual Disease in Plasma of Multiple Myeloma Patients By Intact Mass Spectrometry

Multiple Myeloma (MM) is a hematological cancer that is currently treatable but incurable, and is characterized by the accumulation of malignant plasma cells in bone marrow. Clinical studies have shown that post-cancer treatment, the minimal residual disease (MRD) status is a prognostic factor indicative of the extent of disease remission. MRD assessment has been primarily assessed in bone marrow (BM) using flow cytometry or next generation sequencing. Not only these measurements may lead to false-negative results due to spatial heterogeneity in the BM, hemodilution and/or extramedullary disease as indicated by Derman et al. in Blood Cancer Journal ((2021), 11:19) but the procedure for BM collection is invasive and painful for the patient. These authors present a peripheral blood (PB)-based method that potentially can overcome the barriers in assessing for residual disease in MM patients. We hereby present a non-invasive blood-test for measurement of MRD using mass spectrometry (intact mass approach) for the detection of Ig product in MM patients. We will describe the assay development, analytical characterization of assay performance, and data from the analysis of plasma/serum samples from MM patients enrolled in a clinical trial. A very low volume of patient plasma/serum sample (10 μL) is required to perform this assay. The plasma/serum sample is diluted in purification buffer, applied to Melon gel resin and incubated for 5 min, followed by centrifugation and reduction with DTT. The supernatant is then analyzed by LC-MS on a Q-Exactive mass spectrometer. Deconvolution of the intact protein m/z spectra is performed to yield intact mass of the specific protein target species of interest. Overall, the method can process up to 96 samples in parallel within 3 hrs, making this procedure highly scalable for clinical applications. Data obtained from the assay characterization indicate that the assay is sensitive with an LOD <500 ng/mL and differentiates MM patient from a healthy donor as well allows for the monitoring of MM patient before and after treatment. In conclusion, although still an exploratory analysis, the blood-based mass spectrometry method can be used to screen for MRD in patients who have undetectable disease by flow cytometry or next generation sequencing.

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

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

Myelodysplasia-Related Gene Mutations for NGS-Based MRD Assessment in Acute Myeloid Leukemia

Introduction: Measurable residual disease (MRD) plays an increasing role for treatment management of AML patients. While next-generation-sequencing (NGS) based MRD assessment can be well standardized, there is uncertainty about the type of gene mutations that associate with prognosis and are useful for MRD assessment. The 2022 ELN recommendations assign patients with at least one mutation in the genes ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1 or ZRSR2 to the subgroup of myelodysplasia-related gene mutations (MRGM), which is associated with poor prognosis. We aimed to define the prognostic effect of MRD after induction chemotherapy in AML patients with MRGM using MRG mutations for MRD assessment by NGS. Methods: We retrospectively collected bone marrow and peripheral blood samples from AML patients in complete remission (CR) or CR with incomplete hematologic recovery (CRi) after one to two cycles of 7+3-based standard induction chemotherapy (IC) with at least one MRGM detected at diagnosis by myeloid panel sequencing covering 48 AML associated genes. Patients treated with one course of IC were included if they proceeded to allogeneic hematopoietic cell transplantation (alloHCT) already after one cycle. Molecular MRD status was assessed using targeted NGS and bioinformatics error-correction with 0.01% sensitivity as described before using only MRGM as MRD markers. Results: MRD was assessed on 104 adult newly-diagnosed AML patients (median age 58.6) with a total of 166 MRGM, resulting in a median of one MRGM per patient (range 1-4). 43 (41.3%) patients had secondary or therapy-related AML and 20 (19.2%) had myelodysplasia-related cytogenetic abnormalities. 68 (65.4%) patients were MRD positive for at least one MRGM after one (18 patients (17.3%)) or two (86 patients (82.7%)) cycles of intensive chemotherapy, while 36 (34.6%) became MRD negative. Measured variant allele frequencies (VAF) of positive samples ranged from 0.02-43.1% (median VAF 1.34%).MRD positive patients were older and more likely to be male compared to MRD negative patients, while cytogenetic risk, WBC count, Hgb, platelet count and ECOG status at diagnosis were similar to MRD negative patients. 53 (77.9%) MRD positive and 23 (63.9%) MRD negative patients underwent alloHCT in first CR/CRi.Median follow-up was 3.35 years. OS (P = 0.92), RFS (P = 0.99) and CIR (P = 0.98) were similar in MRD positive (at least one MRGM detectable) and negative (no MRGM detectable) patients (Figure 1). OS, RFS and CIR were also similar between MRD positive and negative patients, when MRD negativity was defined by conversion of at least one MRGM to negativity (55 (52.9%) MRD positive, 49 (47.1%) MRD negative), and when ASXL1 was excluded from the analysis. As variants with VAF >5% in CR/CRi samples may indicate clonal hematopoiesis, we excluded all variants with VAF >5% and repeated the analysis with the 88 (84.6%) remaining patients. Again, OS, RFS and CIR were similar for MRD positive and negative patients. We next reasoned that reduction of VAF correlates with the sensitivity of leukemic cells and possibly with depth of remission and long-term outcome. The fold-reduction of VAFs from diagnosis to the time of MRD analysis was calculated for all variants. For patients with several MRGMs, the variant with the largest fold-reduction was considered including undetectable variants. Patients with more or less than a 2-log, 3-log or 4-log reduction (log 10) had similar OS, RFS and CIR, respectively. Evaluating each MRGM individually, only RUNX1-MRD associated with RFS and by trend CIR (comparing MRD positive to negative patients for OS, HR 3.41, 95% CI 0.43-26.82, P = 0.24; RFS, HR 4.37, 95% CI 1.07-17.79, P = 0.04; CIR, HR 3.30, 95% CI 0.79-13.82, P = 0.1). Conclusion: 65.4% of AML-MRGM patients in CR/CRi remained MRD positive when MRGMs were used for MRD assessment by NGS. MRD positivity was associated with age above 60 years and male sex.Using all MRG mutations for MRD assessment at a threshold of 0.01% or by various degrees of log-reduction was not predictive of outcome after intensive induction chemotherapy. This limits the number of potential MRD markers for NGS analysis before alloHCT, although individual markers like RUNX1 may be of prognostic value.

Monitoring of Systemic Light-Chain Amyloidosis Patients Using Mass Spectrometry (M-inSight) in Serum

Introduction: Systemic light-chain amyloidosis (AL) is a rare disorder characterized by extracellular deposition of monoclonal light-chain immunoglobulins forming insoluble aggregates in different organs. Kidney, heart and liver are principal organs affected by the disease inducing dysfunctions and failures. In this disease, even a low level of circulating light chain can cause significant damages, making challenging the measure of disease burden via peripheral blood. Although a bone marrow aspiration is required to confirm the diagnosis, the evolution of the disease is usually monitored using free light chains dosage, serum and urine electrophoresis, as well as immunofixation. However, these techniques have demonstrated some limitations in terms of sensitivity. Sebia had recently introduced M-inSight TM, a novel, ultra-sensitive and non-invasive assay for monitoring low level disease on blood using targeted mass spectrometry. In this pilot study, M-inSight TM technology is employed to assess the use of mass spectrometry to monitor AL patients. Methods: 14 AL patients were selected to assess the use of M-InSight to detect and quantify AL light chain. 8/14 patients were selected from the AMYDARA study (NCT02816476), consisting in refractory patients treated with Daratumumab. Serum free light-chain assay (Freelite TM), immunofixation on serum/urine as well as bone marrow collection for DNA (ClonoSeq) and RNA (RACE-RepSeq) immunoglobulin next generation sequencing were used to monitor the disease at baseline and end of treatment (EOT) and compared to M-inSight TM. Additionally, 6/14 multicenter patients with normal free light chain level at diagnosis were selected to evaluate the sensitivity of M-InSight. Total mRNA was extracted from BM samples, retrotranscribed to cDNA and then used for immunoglobulin repertoire high-throughput sequencing assay (RACE-RepSeq). cDNA assembly pipeline using IMGT/HighV-QUEST and Vidjil software were used to construct clonotypes and to identify clonal molecular fingerprints and finally their clonotypic peptides for M-InSight analysis. This technology consists of sequence the AL light chain from serum sample and choose clonotypic peptides from the hypervariable regions which then tracked down in the follow-up sample. Results Clonotypic peptides were identified in all eight patients from the baseline sample, which were then confirmed by mass spectrometry. Out of the 8 AMYDARA patients, 5/8 patients achieved very good partial response (VGPR), 2/8 achieved partial response (PR) and 1/8 resulted in complete response (CR). 6 patients had a decrease of the dFLC ratio measure with Freelite TM of 80% or more. 6 out of these 8 patients had MS data highly concordant with that of the serum free light-chain assay. 2 patients had a strong decrease in dFLC while MS measurements were stable or increased. However, detection of the clonotype using RACE-RepSeqand Clonoseq remained elevated, which was more in line with MS than Freelite TM. All patients with normalization of their dFLC were still detected using M-inSight, even those reaching MRD negativity (1 patient) or low number of cells (~10-5, 2 patients) by ClonoSeq. These results were consistent with the RACE-RepSeq which detected the light chain clonotypes in all patients at EOT. Clonotypic peptides using M-InSight were also detected in all 7 patients with normal free light chain at diagnosis. However, in one bi-clonal patient, peptides from only one of both chains were detected, suggesting that only one clone was secreting light chain. Conclusion The goal of this study was to evaluate the M-InSight technique to monitor the evolution of systemic AL amyloidosis in blood samples. The results showed a high concordance with bone marrow MRD measurement and a higher sensitivity than dFLC to monitor the disease. This study shows that MRD monitoring on blood is feasible, using an ultra-sensitive MS technique, avoiding invasive bone-marrow aspirate. M-inSight can track low level AL disease.

Characterization of Rabbit Mesenchymal Stem/Stromal Cells after Cryopreservation.

Adipose tissues (ADPs) are an alternative source for mesenchymal stem/stromal cells (MSCs), given that conventional bone marrow (BM) collection is painful and yields limited cell numbers. As the need for easily accessible MSCs grows, cryopreservation's role in regenerative medicine is becoming increasingly vital. However, limited research exists on the characteristics and functional properties of rabbit-derived MSCs from various anatomical sources before and after cryopreservation. We examined the effects of cryopreservation using Bambanker. We found that cryopreservation did not adversely affect the morphology, viability, and adipogenic or chondrogenic differentiation abilities of ADP MSCs or BM MSCs. However, there was a notable drop in the proliferation rate and osteogenic differentiation capability of BM MSCs post-cryopreservation. Additionally, after cryopreservation, the surface marker gene expression of CD90 was not evident in ADP MSCs. As for markers, ADIPOQ can serve as an adipogenic marker for ADP MSCs. ACAN and CNMD can act as chondrogenic markers, but these two markers are not as effective post-cryopreservation on ADP MSCs, and osteogenic markers could not be validated. The study highlights that compared to BM MSCs, ADP MSCs retained a higher viability, proliferation rate, and differentiation potential after cryopreservation. As such, in clinical MSC use, we must consider changes in post-cryopreservation cell functions.

An in vivo study to investigate an original intramedullary bone graft harvesting technology

BackgroundHarvesting bone graft (BG) from the intramedullary canal to treat bone defects is largely conducted using the Reamer–Irrigator–Aspirator (RIA) system. The RIA system uses irrigation fluid during harvesting, which may result in washout of osteoinductive factors. Here, we propose a new harvesting technology dedicated to improving BG collection without the potential washout effect of osteoinductive factors associated with irrigation fluid. This novel technology involves the conceptual approach of first aspirating the bone marrow (BM) with a novel aspirator prototype, followed by reaming with standard reamers and collecting the bone chips with the aspirator (reaming–aspiration method, R–A method). The aim of this study was to assess the harvesting efficacy and osteoinductive profile of the BG harvested with RIA 2 system (RIA 2 group) compared to the novel harvesting concept (aspirator + R–A method, ARA group).MethodsPre-planning computed tomography (CT) imaging was conducted on 16 sheep to determine the femoral isthmus canal diameter. In this non-recovery study, sheep were divided into two groups: RIA 2 group (n = 8) and ARA group (n = 8). We measured BG weight collected from left femur and determined femoral cortical bone volume reduction in postoperative CT imaging. Growth factor and inflammatory cytokine amounts of the BGs were quantified using enzyme-linked immunosorbent assay (ELISA) methods.ResultsThe use of the stand-alone novel aspirator in BM collection, and in harvesting BG when the aspirator is used in conjunction with sequential reaming (R–A method) was proven feasible. ELISA results showed that the collected BG contained relevant amounts of growth factors and inflammatory cytokines in both the RIA 2 and the ARA group.ConclusionsHere, we present the first results of an innovative concept for harvesting intramedullary BG. It is a prototype of a novel aspirator technology that enables the stepwise harvesting of first BM and subsequent bone chips from the intramedullary canal of long bones. Both the BG collected with the RIA 2 system and the aspirator prototype had the capacity to preserve the BG’s osteoinductive microenvironment. Future in vivo studies are required to confirm the bone regenerative capacity of BG harvested with the innovative harvesting technology.

Compressed Sensing SEMAC MRI of Hip, Knee, and Ankle Arthroplasty Implants: A 1.5-T and 3-T Intrapatient Performance Comparison for Diagnosing Periprosthetic Abnormalities.

Background. The utility of 3-T MRI for diagnosing joint disorders is established, but its performance for diagnosing abnormalities around arthroplasty implants is unclear. Objective. This study compared 1.5-T and 3-T compressed sensing slice encoding for metal artifact correction (CS-SEMAC) MRI for diagnosing periprosthetic abnormalities around hip, knee, and ankle arthroplasty implants. Methods. Forty-five participants (mean age ± SD, 71 ± 14 years; 26 women, 19 men) with symptomatic lower extremity arthroplasty (hip, knee, and ankle, 15 each) prospectively underwent consecutive 1.5- and 3-T MRI with intermediate-weighted (IW) and STIR CS-SEMAC sequences. Using a Likert scale, three radiologists evaluated presence or absence of periprosthetic abnormalities, including bone marrow edema-like signal, osteolysis, stress reaction/fracture, synovitis, tendon abnormalities and collections, image quality, and visibility of anatomic structures. Statistical analysis included nonparametric comparison and interchangeability testing. Results. For diagnosing periprosthetic abnormalities, 1.5-T and 3-T CS-SEMAC MRI were interchangeable. Across all three joints, 3 T produced lower noise than 1.5 T (median IW/STIR score at 3 T vs 1.5 T, 4/4 [range, 2-5/3-5] vs 3/3 [2-5/2-4]; p < .01 for both), sharper edges (4/4 [both 2-5] vs 3/3 [2-4/2-5]; p < .02/p < .05), and more effective metal artifact reduction (4/4 [3-5/2-5] vs 4/4 [both 3-5]; p < .02/p = .72). Agreement was moderate to substantial for image contrast (IW/STIR, 0.66/0.54 [95% CI, 0.41-0.91/0.29-0.80]; p = .58/p = .16) and joint capsule visualization (0.57/0.70 [0.32-0.81/0.51-0.89]; p = .16/p = .19). The bone-implant interface was more visible at 1.5 T (median IW/STIR score, 4/4 [range, both 2-5] at 1.5 T vs 3/3 [both 2-5] at 3 T; p = .08/p = .58), but periprosthetic tissues had superior visibility at 3 T (4/4 [both 3-5] at 3 T vs 4/4 [2-5/3-5] at 1.5 T; p = .07/p = .19). Conclusion. Optimized 1.5-T and 3-T CS-SEMAC MRI are interchangeable for diagnosing periprosthetic abnormalities, although metallic artifacts are larger at 3 T. Clinical Impact. With CS-SEMAC MRI, lower extremity arthroplasty implants can be scanned at 3 T rather than 1.5 T.

The incidence of translocation t(11;14) among patients with multiple myeloma in a single clinic.

Data regarding bone marrow (BM) sampling and cytogenetic testing rates for identification of translocation (11q13;14q32) and their changes over time in a multiple myeloma (MM) population are limited. We analyzed these metrics at a clinic specializing in the treatment of MM. A total of 760 BM aspirate samples from 351 patients were collected between August 2004 and October 2021. We analyzed BM sampling statistics, cytogenetic testing frequency, and the incidence rates for the t(11;14) translocation in a single clinic specializing in the treatment of MM. We report that most (54.4%) patients had only 1 aspirate collected; the main reason (64.6%) for BM collection was to confirm disease progression. Less than half (47.5%) of BM samples collected for evaluation of MM disease had cytogenetic testing, but the rates have markedly increased in recent years. Our data demonstrated an incidence rate of 19.3% for t(11;14). This report suggests that some patients may need to retest for this genetic aberration due to the possibility of false negatives and the potential benefit of identifying the t(11;14) marker for patients who may be candidates for a highly effective targeted therapy consisting of the BCL-2 inhibitor venetoclax.

Differential expression analysis of miRNAs in macrophage-derived exosomes in the tuberculosis-infected bone microenvironment.

Macrophages play an important role in regulating the course of spinal tuberculosis within the bone microenvironment. This study aimed to investigate the differential expression of miRNA in macrophage-derived exosomes within the tuberculosis-infected bone microenvironment, to identify miRNAs that hold potential as diagnostic markers and therapeutic targets. We established study cohorts for spinal tuberculosis, collected bone marrow blood samples, isolated macrophage exosomes, and performed exosome miRNA sequencing. A miRNA-mRNA co-expression network was constructed using WGCNA analysis. Gene GO analysis and KEGG pathway enrichment analysis were performed using KOBAS software. Target miRNAs were selected based on fold change, P-value, and false discovery rate, and their validation was carried out using qRT-PCR and ROC curve studies. Subsequently, we constructed a target gene network for these miRNAs and performed KEGG pathway enrichment analysis to explore the potential signaling mechanisms involved in regulating the disease course of spinal tuberculosis. Our findings revealed that macrophages from the tuberculosis-infected bone microenvironment exhibited an M1 phenotype. The successful extraction of exosomes from macrophage supernatants was confirmed through electron microscopy, particle size analysis, and protein blot analysis. Exosome miRNA-seq demonstrated that 28 miRNAs were up-regulated, while 34 miRNAs were down-regulated in individuals with spinal tuberculosis. GO analysis and KEGG pathway enrichment analysis indicated that the differentially expressed miRNAs were involved in various biological processes, cell components, molecular functions, and signaling pathways, which collectively contribute to the regulation of the disease course of spinal tuberculosis. Notably, miRNA-125b-5p was successfully selected based on fold change, p-value, and false discovery rate. qRT-PCR validation further confirmed the significant up-regulation of miRNA-125b-5p in spinal tuberculosis. The ROC curve revealed that miR-125b-5p is a potential diagnostic biomarker for spinal tuberculosis. Moreover, construction of the miRNA-125b-5p target gene network and subsequent KEGG enrichment analysis highlighted the importance of MAPK, TNF, Ras, Rap1, and the PI3K-Akt signaling pathways in the regulation of the disease course of spinal tuberculosis. Our study demonstrates differential expression of miRNAs in macrophage-derived exosomes in the tuberculosis-infected bone microenvironment. Specifically, MiRNA-125b-5p is significantly up-regulated in spinal tuberculosis and shows potential as a diagnostic biomarker for spinal tuberculosis.

Development of an in-house bone marrow collection kit: The Catalan bone marrow transplantation group experience.

Bone marrow (BM) harvesting is one of the essential sources of stem cells for haematopoietic stem cell transplantation. In 2019, commercial BM collection kits became unavailable in Europe. Consequently, we created an in-house BM collection kit as an alternative. We compared two groups of BM collections. The first collections were taken using an in-house kit from June 2022 through February 2023 and the second with a commercial kit from February 2021 through May 2022. These all took place at seven collection centres (CC). We analysed the harvest quality (cell blood count, CD34+ cells, viability, potency and sterility), the incidents occurring with each kit and the time to neutrophil and platelet engraftment in recipients. A total of 23 donors underwent BM harvesting with the in-house kit and 23 with the commercial one. Both cohorts were comparable regarding donor characteristics, CC and time to procedure. No statistical differences were found in harvest quality between the in-house and commercial kits. A new transfusion set was required in three BM harvests (13%) with the in-house kit because of filter clogging. The median time to neutrophil and platelet engraftment was 21 days for both cohorts and 29 days (in-house) and 33 days (commercial), p = 0.284, respectively. The in-house BM collection kit offers a real approach to solve the diminished supply of commercial kits. A higher risk of filter clogging was observed compared with commercial kits due to the lack of 850 and 500 μm filters.

P-800 Autologous stem cell ovarian transplant induced long-lasting effects on the plasma proteome of women with premature ovarian insufficiency

Abstract Study question Does autologous stem cell ovarian transplant (ASCOT) induce any long-term modifications in the plasma proteomic profile of women with premature ovarian insufficiency (POI)? Summary answer Stem cell mobilization and infusion into the ovary elicit long-term proteomic changes in peripheral blood plasma composition in women with POI, reverting age-related proteomic changes. What is known already Patients with POI are challenging to treat, with oocyte donation remaining as the only feasible option to achieve pregnancy. Previous attempts to overcome the fertility problems of these patients have mainly been based on ovarian stimulation, and were likely unsuccessful due to the lack of stimulable antral follicles remaining in the ovaries. Our group described that ASCOT in patients with impaired ovarian function improves ovarian reserve biomarkers, follicle and oocyte quantity enabling pregnancy and delivery of healthy babies. Beneficial effects of ASCOT were associated with the presence of different stem-cell secreted factors in apheresis plasma. Study design, size, duration Experimental cohort study with plasma samples of POI women undergoing stem cell mobilization with granulocyte-colony stimulating factor (mobilization arm; N = 3) or stem cell mobilization and infusion into the ovarian artery (ASCOT arm; N = 3). Peripheral plasma samples were collected from all patients at recruitment (Pre), during bone marrow stem cell mobilization and collection by apheresis (Apheresis), and three months after mobilization or ASCOT (Post). Participants/materials, setting, methods Proteins were isolated from plasma samples and quantified by SWATHTM to compare the plasma proteomic profile of the different time points (Pre, Apheresis, Post), using an ElasticNet penalized linear regression method. To determine the biological processes affected by stem cell mobilization and infusion into the ovary, a Gene Ontology Enrichment Analysis for the differentially expressed proteins (DEPs) was performed, considering significantly enriched those GO Biological Processes with a False Discovery Rate &amp;lt;0.05. Main results and the role of chance Discriminant analysis highlighted clear distinctions between the plasma proteome at the three evaluated time points. Both the stem cell mobilization and ASCOT technique induced statistically significant modifications in the plasma composition, reversing some age-related protein expression changes. Specifically, differences between Apheresis and Pre samples were explained by fourteen DEPs, revealing the functional analysis an enrichment in processes related to the complement cascade, immune system, and platelet degranulation. When the link of these proteomic changes with aging was evaluated, we found three proteins that were upregulated in Apheresis [C1QC, LYSC, LYAM1] whose plasma levels decrease with aging, and one downregulated protein [CRP] whose expression rises with age. Regarding Post samples, twenty-four DEPs with respect to Pre condition were found within the mobilization arm, being two of them linked with aging [MMP2 and ALDOC]. These DEPs were related with immune response and platelet degranulation but also with processes associated with responses to oxygen-containing compounds and growth hormones, and blood vessel maturation. Within the ASCOT arm, Pre and Post samples were clearly differentiated by eleven DEPs, although any GO Biological Process were found. Nevertheless, among these DEPs, we found DIAC which increases in plasma with aging and was downregulated after ASCOT. Limitations, reasons for caution Considering the small sample size used in this study, further experimental studies will be needed to validate the results. Moreover, further research will be necessary to determine the extent of the regenerative effects of the identified DEPs within the ovaries, and their direct implications in ovarian aging. Wider implications of the findings Our findings highlight the potential proteins and biological processes that may promote the follicle activation and growth observed after ASCOT. Identifying plasma proteins that regenerate aged or damaged ovaries could lead to more effective, targeted and/or preventive therapies for affected patients. Funding: CIPROM/2021/058, PI21/00170, CP19/00141 and CD20/00116. Trial registration number NCT03535480

CLONAL ARCHITECTURE OF RELAPSED OR REFRACTORY FOLLICULAR HELPER T‐CELL LYMPHOMA: AN ANCILLARY STUDY OF THE ORACLE TRIAL, A LYSA STUDY

Follicular helper T-cell lymphoma (TFHL) results from the oncogenic transformation of a TFH cell, driven by mutations in genes involved in epigenetic regulation (TET2, DNMT3A, IDH2) and T-cell signaling (RHOA). Demonstration of TET2 and DNMT3A mutations in B cells, myeloid cells, or hematopoietic progenitor cells have suggested that TFHL can emerge from clonal hematopoiesis (CH) in a multi-step process. However, the frequency, the extension, and the relevance of such CH in TFHL oncogenesis are unclear. To describe and characterize the clonal architecture of refractory/relapsed (R/R) TFHL, we collected bone marrow samples of patients included in the ORACLE trial (NCT03593018), a phase 3 trial evaluating the oral 5-azacitidine (CC-486) compared to single-agent chemotherapy in patients with R/R TFHL. Bone marrow (BM) cells from 32 patients at inclusion were cultured in methylcellulose supplemented with standard growth factors. BFU-E, CFU-GM, and CFU-GEMM were counted and individually harvested. A total of 517 colonies derived from the 32 patients were then individually genotyped by next-generation sequencing (NGS) using a capture-based panel (limit of detection: 1%) covering 43 genes recurrently mutated in CH and/or TFHL, including TET2, DNMT3A, IDH2, and RHOA. Meanwhile, bulk BM cells from 29 patients were sequenced with the same NGS panel and then compared to the mutations found in cfDNA (31 patients) and tumor biopsies (28 patients). Overall, the frequency of CH and the phylogenetic link between the myeloid progenitors and the TFHL was evaluable in 30 TFHL patients. We successfully genotyped 471/517 (91%) colonies. CH defined by at least one somatic mutation detected in at least one myeloid colony or total BM cells was found in 29/30 (97%) patients. Among the 26/29 patients with mutated myeloid colonies, the median percentage of mutated colonies was 21% (IQR 7%–53%) and 7 had subclonal architecture with a median of 3 different clones. The 2 most frequent mutations were DNMT3A and TET2, detected in 24/30 (80%) and 18/30 (60%) patients' BM, respectively. We confirmed the absence of RHOAG17V and IDH2R172 mutations in BM cells of TFHL patients. While 17/18 patients with a detectable TET2 mutation in CH had a TET2 mutated TFHL, only 13/23 patients with a DNMT3A mutated CH had a DNMT3A mutated TFHL. We thus identified 3 groups of patients according to the distribution of mutations found in the BM and the LN lymph nodes: 16/30 (53.3%) patients had a TFHL clonally derived from CH (CH-related TFHL), 13 (43.3%) had TFHL, and clonally distinct CH (CH-unrelated TFHL), and one patient (3.3%) had undetectable CH. No significant difference in patient outcome was observed regarding the presence of CH-related or unrelated TFHL, but our results are limited by the small number of patients and treatment heterogeneity. Encore Abstract—previously submitted to EHA 2023 Keywords: aggressive T-cell non-Hodgkin lymphoma, tumor biology and heterogeneity No conflicts of interests pertinent to the abstract.

P52 RISK STRATIFICATION COMBINING SKY92 GENE EXPRESSION PROFILING AND TRADITIONAL FISH IN MULTIPLE MYELOMA: THE FIRST PROSPECTIVE EVIDENCE IN THE R2-ISS ERA

High-risk (HR) multiple myeloma (MM) patients still have a very poor prognosis. However, the definition of HR MM remains controversial. Currently, FISH is the most commonly used method for risk stratification in MM. According to the Second Revised International Staging System (R2-ISS), HR cytogenetics is defined as presence of at least one of the following: del(17p), t(4;14), and 1q CNA. In recent years, gene expression profiling (GEP) approaches, e.g. SKY92, have been developed for detection of HR patients. In the R2-ISS era, however, data on MM risk stratification applying SKY92 in combination with traditional FISH is still lacking. Therefore, we performed the first prospective study evaluating the prognostic value of the combined SKY92 and FISH HR detection in newly diagnosed (ND) and relapsed/refractory (RR) MM. We prospectively collected bone marrow (BM) samples and clinical data of 147 MM patients. Cytogenetics were analyzed on purified CD138 positive MM cells by FISH, and HR cytogenetics was defined according to the R2-ISS classification. SKY92 risk status was determined with MMprofiler gene expression assay. Whole genome sequencing (WGS) was performed to compare the both risk stratification systems SKY92 and FISH. We included 147 patients in our study (NDMM: n=51, RRMM: n=96). SKY92 classification was available for 121 (82.3%) patients. HR SKY92 was significantly enriched in RRMM (40/76) compared with NDMM (6/45) (P<0.0001). RRMM patients with HR SKY92 showed significantly shorter progression free survival (PFS) (P<0.0001) and overall survival (OS) (P=0.0004) than standard-risk (SR). In NDMM, HR SKY92 also indicated a significantly inferior PFS (P=0.001) in comparison with SR. Of note, samples of 26 (17.7%) patients, who showed significantly lower bone marrow infiltration than the remaining patients (median: 25% vs 55%, P=0.038), did not meet the SKY92 quality control criteria. We then combined the SKY92 classification with cytogenetic analyses by FISH, which were available in 99 patients (NDMM: n=33; RRMM: n=66). We noticed a discrepancy between both risk stratification systems, with 44 (44.4%) and 58 (58.6%) patients being classified as HR by SKY92 and FISH, respectively. In total, 28 (42.4%) RRMM patients and 6 (18.2%) NDMM patients displayed HR in both SKY92 and FISH (double-HR). Regarding survival outcome in RRMM, double-HR patients showed the worst PFS (P<0.0001) and OS (P=0.0007). In NDMM, double-HR presented a negative prognostic factor for PFS (P=0.01). To compare the FISH and SKY92 classifications, we performed WGS in 16 patients who exhibited either only HR SKY92 (n=7) or only HR FISH (n=9). Interestingly, 1 patient with bi-allelic TP53 inactivation (deletion + mutation) and 6 patients harbouring 1q CNA were determined as SR by SKY92 but as HR by FISH. The median PFS was not reached after a median follow up of 371 days in these 9 patients. Moreover, 4 out of 7 patients with only HR SKY92 but SR FISH displayed 1q CNA, which was detected only by WGS, and del1p32 was found in 1 patients. Interestingly, we found CRBN mutation in 3 out of 7 patients with only HR SKY92 but SR FISH. The remaining 2 patients did not show any known HR genomic alterations, suggesting that HR MM may be associated with other factors, e.g. epigenetic modifications. Here, we provide the first prospective evidence in the R2-ISS era that advanced risk stratification combining SKY92 and FISH may help to identify the highest-risk MM.