scholarly journals Topographic Analysis of Low-Grade Myeloid Neoplasms By Multiparametric in Situ Imaging of Human Bone Marrow Core Biopsy Tissues

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2593-2593
Author(s):  
Sanjay Patel ◽  
Viktor Svekolkin ◽  
Arina Varlamova ◽  
Ilia Galkin ◽  
Itzel Valencia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Core Biopsy ◽  
Stock Options ◽  
Cell Types ◽  
Low Grade ◽  
Topographic Analysis ◽  
Myeloid Neoplasms ◽  
Tissue Area ◽  
Privately Held

Abstract Diagnosis of low-grade myelodysplastic syndromes (LG-MDS) is one of the most challenging in hematopathology as it relies predominantly on morphologic assessment of dysplasia. Prior studies have demonstrated poor interobserver agreement among pathologists. Histomorphological evaluation of bone marrow core biopsy samples remains the gold standard for diagnostic workup of LG-MDS, including myelodysplastic syndromes (MDS) and other myeloid neoplasms. However, this approach may be subjective, and cannot quantitatively assess subtle differences in marrow topography and the cellular microenvironment. Multiparametric in situ imaging (MISI) through various techniques enables multiple biomarker detection in a single tissue. BostonGene has developed an AI-based image analysis platform to reveal spatial information and subtle histomorphologic features in an objective, quantitative fashion. Here, we demonstrate the potential for automated AI-based imaging analysis of MISI to assist in the differentiation of LG-MDS samples from normal marrow tissues (NBM). Decalcified human bone marrow core biopsy tissues from LG-MDS (n=6) and uninvolved staging marrows (NBM, n=4) were first prepared by immunofluorescence-based MISI via staining with DAPI and CD34, CD38, CD117, CD71, CD15, and CD61 antibodies. BostonGene analyzed the resulting images (fig.1) using a proprietary AI-based imaging platform to identify cells by segmentation performed with the pre-trained instance segmentation neural network. Cell types were identified with mean marker expression values using an accelerated version of BostonGene's phenograph clustering algorithm. Pathologists manually masked fat and bone trabeculae. Using a combination of cell size/shape parameters and antigen expression levels, the following unique cell types were identified: myeloblasts, proerythroblasts, erythroid normoblasts, maturing granulocytes, megakaryocytes, mast cells, plasma cells, and B-cell precursors (hematogones). Data revealed differences in the cellular content of NBM and LG-MDS samples, and separation of LG-MDS samples with the del(5q) subtype (n=2). While linear slender islet-like small clusters of erythroid normoblasts were detected in NBM, we observed a chaotic arrangement of them in LG-MDS samples. In LG-MDS samples, we found an increase in the total number of erythroid normoblasts from 17% to 31%, LG-MDS-del(5q) had 14%. The ratio of maturing granulocytes to erythroid normoblasts (M:E ratio) was significantly lower in LG-MDS (0.63) than in NBM (1.95). The M:E ratio generated by MISI strongly correlated to the M:E ratio produced by manual differential count of bone marrow aspirate samples (R=0.83, p < 0.003). Additionally, fewer hematogones were identified in LG-MDS marrows compared to NBM samples, as reported by others using orthogonal methods. Topographic analysis showed the fat to cellular tissue area ratio was higher in NBM (0.73) than LG-MDS (0.41), but the ratio of trabecular area to total tissue area was higher in LG-MDS (1.67) than NMB (0.74). Spatially, myeloblasts and megakaryocytes were found closer to trabeculae in NBM than LG-MDS;12 different cell communities were identified;2 of them (cluster 3 - erythroid normoblasts enriched, cluster 5 - erythroid normoblasts contacting proerythroblasts) were distributed statistically significantly differently in NBM and LG-MDS samples, indicating the use of MISI with AI-based imaging to distinguish LG-MDS from NBM. Patients of MDS-del(5q) subtype differ significantly from other MDS samples and are more similar to NBM. AI-based image analysis applied to MISI of bone marrow tissue revealed multiple cell types in single tissue sections, along with histologically subtle differences in topography between NBM and LG-MDS samples. These results highlight the importance of integrating in situ tissue analysis with techniques that examine single cell characteristics for a more comprehensive picture of the differences between normal tissue and tumor samples. Coupling sophisticated imaging analytics with this imaging method may provide a more powerful tool for novel biomarker discovery of prognostic and therapeutic significance in the management of MDS and other marrow-based disorders. Figure 1 Figure 1. Disclosures Svekolkin: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Varlamova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Galkin: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Akaeva: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Smirnova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Ovcharov: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Polyakova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Tabakov: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company. Postovalova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Koltakova: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company. Gunn: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company. Bagaev: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene.

scholarly journals Hematopoiesis in 3 dimensions: human and murine bone marrow architecture visualized by confocal microscopy

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. e41-e55 ◽  
Author(s):  
Tomoiku Takaku ◽  
Daniela Malide ◽  
Jichun Chen ◽  
Rodrigo T. Calado ◽  
Sachiko Kajigaya ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Three Dimensional ◽  
Cell Types ◽  
Bone Marrow Tissue ◽  
Murine Bone Marrow ◽  
3 Dimensional ◽  
Vascular Structures ◽  
Bone Structures ◽  
Surrounding Bone

AbstractIn many animals, blood cell production occurs in the bone marrow. Hematopoiesis is complex, requiring self-renewing and pluripotent stem cells, differentiated progenitor and precursor cells, and supportive stroma, adipose tissue, vascular structures, and extracellular matrix. Although imaging is a vital tool in hematology research, the 3-dimensional architecture of the bone marrow tissue in situ remains largely uncharacterized. The major hindrance to imaging the intact marrow is the surrounding bone structures are almost impossible to cut/image through. We have overcome these obstacles and describe a method whereby whole-mounts of bone marrow tissue were immunostained and imaged in 3 dimensions by confocal fluorescence and reflection microscopy. We have successfully mapped by multicolor immunofluorescence the localization pattern of as many as 4 cell features simultaneously over large tiled views and to depths of approximately 150 μm. Three-dimensional images can be assessed qualitatively and quantitatively to appreciate the distribution of cell types and their interrelationships, with minimal perturbations of the tissue. We demonstrate its application to normal mouse and human marrow, to murine models of marrow failure, and to patients with aplastic anemia, myeloid, and lymphoid cell malignancies. The technique should be generally adaptable for basic laboratory investigation and for clinical diagnosis of hematologic diseases.

scholarly journals Heterogeneity of Minimal/Measurable Residual Disease (MRD) Practices in Adult B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) in the United States

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4478-4478
Author(s):  
Juliana E. Hidalgo-Lopez ◽  
Gail J. Roboz ◽  
Brent Wood ◽  
Michael Borowitz ◽  
Elias J. Jabbour ◽  
...  
Keyword(s):  
United States ◽  
Bone Marrow ◽  
Best Practices ◽  
Stock Options ◽  
Research Funding ◽  
The United States ◽  
Bone Marrow Sample ◽  
Sample Collection ◽  
Nccn Guidelines ◽  
Privately Held

Abstract Background: MRD testing in BCP-ALL is critical for appropriate patient management, but little is known regarding sample acquisition and testing heterogeneity across clinical practice settings. These factors may impact the quality and reliability of MRD assessment. Methods: Thirty-minute online surveys were conducted in May 2021 with hematologists/oncologists (HEME/ONCs) in the United States in both academic (acad) and community (comm) settings. Respondents were licensed physicians board certified in oncology and/or hematology who treated ≥2 BCP-ALL patients/year or ≥10 patients in the past 5 years, with over 25% of time spent in the clinical setting; pediatric HEME/ONCs were excluded. Survey enrollment is ongoing, with interim results presented here; a related survey for pathologists (PATHs) is underway. Results: HEME/ONC respondents (acad n=40, comm n=57, from 29 states) had been practicing as specialists for a median of between 11-15 years (choices were ranges, eg 6-10, 11-15, min-max was 1-34 years), and typically spent over 75% of their time in the clinic; 94% of respondents had ≥5 BCP-ALL patients/year and 92% ordered MRD tests for ≥5 patients/year. Typical timepoints for MRD testing included the end of induction/suspected complete remission, the end of consolidation, and at suspected disease progression; testing after the end of consolidation was infrequent in both groups (Table). Testing for MRD at the end of consolidation was notably more frequent in the academic setting. In both settings, the HEME/ONC ordering the MRD test generally also performed the bone marrow collection procedure (acad: 78%, comm: 56%). Resources consulted on bone marrow collection best practices included UpToDate (21%), ASH and ASCO (13%), NCCN guidelines (13%), and hematology/oncology journals. About half of practices had defined institutional protocols for bone marrow collection (acad: 55%, comm: 47%), nearly all of which were developed internally. The amount of bone marrow sample collected showed high variability, ranging from 1-10 draws (median=3) and 1-30 mL sample per draw (median=5 mL). While 49% of HEME/ONCs performed <5 draws and extracted ≤6 mL per draw, 22% collected 10 mL/draw, and 10% collected 20 mL/draw; the remaining 18% reported >5 draws and/or >6 mL per draw. In both settings, the first pull was identified and labeled in 35% of procedures; in those cases, the first-pull samples were used primarily for MRD testing in 60% of cases as recommended by NCCN guidelines (vs for morphology assessment and cytogenetic studies). HEME/ONCs typically relied on the expertise of pathologists to choose MRD testing methodology.Survey results indicate that external labs (both national clinical reference labs and commercial labs) were most commonly used for MRD assessments (63%); comm HEME/ONCs were more likely to use external reference labs and acad HEME/ONCs were more likely to use in-house labs. When asked to estimate the frequency with which different MRD methods were used, mean responses were 54% flow cytometry and 40% next-generation sequencing. While all HEME/ONCs indicated that MRD results were presented clearly in lab reports, there was a desire to include more guideline information about MRD interpretation and BCP-ALL treatment. Conclusion: Interim results identified broad heterogeneity in clinical practices affecting sample collection for MRD assessment in Ph- BCP-ALL in the US, indicating several opportunities for harmonization of routine MRD assessment in BCP-ALL. These opportunities include optimization of bone marrow sample collection techniques (volume/draw and identification/use of first pull for MRD), timing/frequency of specimen collection, serial MRD surveillance after consolidation, MRD method chosen, and standardizing reports to include guideline information. There were gaps in awareness of FDA-approved methods of MRD testing for BCP-ALL. Initiatives supporting provider education and harmonization of best practices from professional guideline committees/organizations are needed to optimize outcomes of BCP-ALL patients. Figure 1 Figure 1. Disclosures Hidalgo-Lopez: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Roboz: Janssen: Research Funding; Daiichi Sankyo: Consultancy; MEI Pharma - IDMC Chair: Consultancy; Actinium: Consultancy; AbbVie: Consultancy; Mesoblast: Consultancy; Bayer: Consultancy; Blueprint Medicines: Consultancy; Jazz: Consultancy; Janssen: Consultancy; Astex: Consultancy; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Agios: Consultancy; Astellas: Consultancy; Jasper Therapeutics: Consultancy; Helsinn: Consultancy; Glaxo SmithKline: Consultancy; Novartis: Consultancy; Amgen: Consultancy; AstraZeneca: Consultancy; Otsuka: Consultancy; Pfizer: Consultancy; Roche/Genentech: Consultancy. Wood: Pfizer, Amgen, Seattle Genetics: Honoraria; Juno, Pfizer, Amgen, Seattle Genetics: Other: Laboratory Services Agreement. Borowitz: Amgen, Blueprint Medicines: Honoraria. Jabbour: Amgen, AbbVie, Spectrum, BMS, Takeda, Pfizer, Adaptive, Genentech: Research Funding. Velasco: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Elkhouly: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Adedokun: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Zaman: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Iskander: Amgen Inc.: Current Employment, Current holder of stock options in a privately-held company. Logan: Amgen, Pfizer, AbbVie: Consultancy; Pharmacyclics, Astellas, Jazz, Kite, Kadmon, Autolus, Amphivena: Research Funding.

scholarly journals Tumor-Confirmed Follicular Lymphoma Mutations Are Detectable in Peripheral Blood Years Prior to Clinical Diagnosis

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 709-709
Author(s):  
Joseph G Schroers-Martin ◽  
Joanne Soo ◽  
Gabriel Brisou ◽  
Florian Scherer ◽  
David M. Kurtz ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
Clinical Diagnosis ◽  
Peripheral Blood ◽  
Stock Options ◽  
Research Funding ◽  
Low Grade ◽  
Pet Ct ◽  
Significant Difference ◽  
Tumor Biopsies ◽  
Privately Held

Abstract Background: Mutations in chromatin modifying genes (CMGs) including KMT2D, CREBBP, EZH2, and EP300 have been inferred as early events in follicular lymphoma (FL) by truncal status in mature tumors and persistence between diagnosis and relapse. We previously reported frequent detection of CREBBP lysine acetyltransferase (KAT) domain mutations in pre-diagnostic blood and tissue specimens from individuals later developing FL (Schroers-Martin et al, ASH Annual Meeting 2017). However, the limited availability of paired tumor biopsies has precluded confirmation of concordance between precursor lesions and subsequent clinical malignancy. Methods: The American Cancer Society (ACS) Cancer Prevention Study-II (CPS-II) LifeLink cohort collected screening blood or saliva samples from over 100,000 cancer-free American participants between 1998 and 2002. To evaluate detection of tumor-confirmed variants in pre-diagnostic specimens, we identified 29 FL patients with available FFPE tumor biopsy and screening sample (Fig A). The median age at screening was 71 years (range 56-83) with a median time to FL diagnosis of 56 months (TTD, range 6-139). Tumor biopsies were sequenced utilizing hybrid capture sequencing for commonly mutated lymphoma genes. DNA extracted from pre-diagnostic blood or saliva cell pellet specimens was sequenced utilizing error-corrected CAPP-Seq (Newman et al Nat Biotech 2016) to a median depth of 5204x. We sequenced to similar depths peripheral blood DNA from control cohorts of individuals with detectable t(14;18) but no subsequent lymphoma diagnosis (n=14) and healthy individuals without detectable t(14;18) by PCR (n=20). Results: Coding mutations were identified from all tumors with a mutational distribution similar to prior FL sequencing studies. Tumor-derived variants were detected in 7 of 29 paired pre-diagnostic specimens (24%) at a median TTD of 44 months (range 11-112 months, Fig B). The statistical significance of detection was assessed using a previously described approach based on Monte Carlo sampling (Newman et al Nat Biotech 2016) and the error distribution of affected loci in control cohorts. While an outlier case contained concordant TNFRSF14, FOXO1, and STAT6 mutations 90 months pre-diagnosis at an elevated allelic fraction (AF) of 1.8%, the mean AF of other detected precursor variants was 0.091%. Individuals with detected variants were not older (Fig C) nor significantly closer to clinical diagnosis (Fig D). The most frequently detected lesions were CREBBP (6/15 cases, 40%) and BCL2 (3/13, 23%) with one case demonstrating a fuller mutational profile including FOXO1 and ARID1A at 44 months before diagnosis. All detected precursor CREBBP variants localized to the KAT domain, reflecting prior observations in pre-diagnostic samples without confirmatory biopsy (Fig E). Of note, saliva cell pellets may contain 30% or more hematopoietic DNA (Kaur et al Chimerism 2012) and we detected tumor-confirmed variants in both saliva and blood screening specimens (Fig F) with no significant difference in AF (Fig G). In an illustrative independent case with available imaging, a patient undergoing radical prostatectomy was found to have involvement of a pelvic lymph node with in situ follicular neoplasia (ISFN). Staging PET/CT showed no evidence of FL (Fig H) and he was followed expectantly for 4 years without emergent disease. Eight years after surgery he presented with inguinal swelling and bilateral FDG-avid adenopathy on PET/CT. Excisional biopsy confirmed low grade FL and sequencing for M7-FLIPI revealed a CREBBP KAT domain variant. Retrospective sequencing of serial peripheral blood specimens from his initial surveillance showed detectable CREBBP R1446C at the earliest collected time point (AF range 0.019-0.046%) rising to AF 0.082% after clinical diagnosis. Conclusions: Precursor FL mutations are detectable in peripheral blood and saliva years prior to clinical diagnosis with a spectrum of variants enriched in CREBBP and BCL2 and concordant with subsequent FL tumors. Such lesions may assist in stratifying individuals at elevated risk of clinical malignancy, including after identification of pathologic precursors such as ISFN. Figure 1 Figure 1. Disclosures Kurtz: Foresight Diagnostics: Consultancy, Current holder of stock options in a privately-held company; Roche: Consultancy; Genentech: Consultancy. Khodadoust: Alexion, AstraZeneca Rare Disease: Other: Study investigator; CRISPR Therapeutics, Nutcracker Therapeutics: Research Funding; Myeloid Therapeutics: Membership on an entity's Board of Directors or advisory committees. Diehn: Roche: Consultancy; AstraZeneca: Consultancy; RefleXion: Consultancy; BioNTech: Consultancy; Varian Medical Systems: Research Funding; Illumina: Research Funding; CiberMed: Current holder of stock options in a privately-held company, Patents & Royalties; Foresight Diagnostics: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Roulland: BMS: Research Funding. Alizadeh: Bristol Myers Squibb: Research Funding; Gilead: Consultancy; CAPP Medical: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Celgene: Consultancy, Research Funding; Roche: Consultancy, Honoraria; Janssen Oncology: Honoraria; Foresight Diagnostics: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Cibermed: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Forty Seven: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

scholarly journals Machine Learning Algorithms Accelerate Throughput of a Flow - Sequencing Cell Based Assay for an Acute Myeloid Leukemia (AML) Therapeutic Discovery Platform

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4930-4930
Author(s):  
Bradley A Patay ◽  
Alyssa M Zlotnicki ◽  
Swati Shah ◽  
Robert Apilado ◽  
Robert Andrews
Keyword(s):  
Neural Network ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Quality Control ◽  
Acute Myeloid Leukemia ◽  
Stock Options ◽  
Myeloid Leukemia ◽  
Cell Populations ◽  
Acute Myeloid ◽  
Privately Held

Abstract Introduction Optimal therapeutic cancer discovery pipelines rely on high throughput, reproducibility and minimal bias for candidate discovery and clinical trials. Acute Myeloid Leukemia (AML) cell-based assays, combining flow cytometry (FCM) immunophenotyping with genomic sequencing, provide comprehensive characterization. However, sequential manual gating by expert flow analysts has been shown to have interlaboratory variation, weaknesses with higher dimensional feature spaces due to increased fluorescent channels, and limited scalability for throughput due to the necessary expert interpretation. Automated analysis of Flow Cytometry Standard (FCS) data using a combination of unsupervised and supervised algorithms that combine quality control, gating and clustering strategies offers a more scalable and sustainable option. Given these established breakthroughs, we investigated the use of these informatic tools, combined with sequencing data, to create a therapeutic discovery platform. Methods Peripheral blood and bone marrow samples with abnormal myeloid population and normal controls were exposed to multiple chemotherapeutic small molecules at varying concentrations. Samples were flow cytometrically evaluated with an 8-color tube of antibodies targeting various antigens allowing for apoptosis, viability and blast characterization using standardized lab protocols with a flow cytometer, followed by hematopathologist review. Furthermore, the samples underwent molecular sequencing and annotation with the MyAML® assay to interpret results in the context of AML molecular classification. The computational pipeline utilized Bioconductor version 3.11 open-source packages for quality control, Logicle transformation, apoptosis and viability analyses utilizing a supervised gating strategy. Once viability was established, a Self-Organizing Map (unsupervised dimensionality reduction) algorithm was used to cluster cells into different cell populations. Moreover, a neural network utilizing TensorFlow combined with the API, Keras, was used to detect cell populations from these clusters. The neural network classifier was trained, validated and tested on these clusters compared to expert interpretation. Results Six bone marrow and three peripheral blood samples with the appropriate controls were evaluated via the discovery pipeline. Each sample was exposed to various chemotherapeutic molecules for a total of 203 samples for the pipeline with 50,000 cells per sample. Sequencing demonstrated these samples reflected various European LeukemiaNet (ELN) molecular risk categories. The correlation between the total viable cells in 59 samples as determined by manual and automated gating processes was 98% (r 2=0.98) (Figure 1a). Heatmaps were generated for data visualization of therapeutic efficacy using this viable cell data as demonstrated in one example, Figure 1b, which was consistent with biological hypotheses. Beyond total cell viability detection, cell populations were determined. The validation and test accuracy of our lymphocyte classification model were both 99%, and F1 statistic for model's testing performance was 0.98. Figure 2a displays the ROC curve. The classification of lymphocyte cell population using our combined unsupervised learning/neural network approach for 59 samples had a correlation of 97% (r 2=0.97) (Figure 2b). Conclusions Based on a preliminary comparison with manual gating by an expert flow analyst, we show an automated scalable protocol gives comparable results; therefore, it would be an appropriate alternative to use to screen multiple cells for downstream therapeutic analysis with increased throughput for both cell viability and cell population identification. Figure 1 Figure 1. Disclosures Patay: Invivoscribe: Current Employment, Current holder of stock options in a privately-held company. Zlotnicki: Invivoscribe: Current Employment. Shah: Invivoscribe: Current Employment, Current holder of stock options in a privately-held company. Apilado: Invivoscribe: Current Employment. Andrews: Invivoscribe: Current Employment, Current holder of stock options in a privately-held company.

scholarly journals Single Agent CD117-Targeted Antibody Drug Conjugate in Combination with Lymphodepleting Antibodies Enables Allogenic Hematopoietic Stem Cell Transplantation in Mice without Chemotherapy or Radiation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1682-1682
Author(s):  
Leanne Lanieri ◽  
Tahirih Lamothe ◽  
Prashant Bhattarai ◽  
Rahul Palchaudhuri ◽  
Lisa Olson
Keyword(s):  
Bone Marrow ◽  
Single Dose ◽  
Peripheral Blood ◽  
Stock Options ◽  
Positive Control ◽  
Antibody Drug Conjugate ◽  
Hematopoietic Stem ◽  
Donor Chimerism ◽  
Conditioning Regimens ◽  
Privately Held

Abstract Hematopoietic stem cell transplant (HSCT) is a highly effective and potentially curative treatment for malignant and non-malignant blood disorders. However, patient eligibility for this procedure can be limited due to the mortality and morbidity risks associated with current conditioning regimens, including organ toxicity, infertility, and secondary malignancies. We have developed a novel anti-CD117 antibody drug conjugate (ADC) that, in combination with lymphodepleting antibodies, can effectively condition mice to support a full allogeneic (allo) transplant. Specifically, we used a tool anti-mouse (anti-m) CD117-PBD ADC in combination with anti-mCD4 and CD8 depleting antibodies and assessed the ability of the combination to successfully condition in a murine model of allo-HSCT. Our tool ADC, anti-mCD117-PBD, was engineered for rapid clearance to enable a timely HSCT following conditioning. A single dose of 1 mg/kg robustly depleted long-term hematopoietic stem cells (LT-HSC) by 97% compared to PBS controls in C57BL/6 mice. We first evaluated the ability of single doses of 1 and 3 mg/kg anti-mCD117-PBD to condition for transplant in a congenic mouse model (C57BL/6 hosts [CD45.2+] with B6.SJL-Ptprca Pepcb/boyJ donors [CD45.1+]). We then evaluated conditioning with a single dose of 3 mg/kg anti-mCD117-PBD, in combination with 250 μg/mouse anti-mCD4 (GK1.5) and anti-mCD8 (YTS 169.4) antibody, in a fully mismatched allo transplant model (C57BL/6 hosts [CD45.2+] with CByJ.SJL(B6)-ptprca/J donors [CD45.1+]). In both studies, a dose matched non-targeted isotype-PBD (iso-PBD) was used as a negative control, while 9 Gy total body irradiation (TBI) was used as a fully myeloablative positive control. Anti-rat (anti-r) IgG isotype (LTF-2) was used as a negative lymphodepletion control antibody in the allo-HSCT study. Conditioned mice were transplanted with 2e7 whole BM cells. Lymphodepleting antibodies were dosed daily for three consecutive days before transplant. Peripheral blood chimerism was assessed over 16 weeks (congenic model) to 24 weeks (allo model), at which time donor HSC chimerism was evaluated in the terminal bone marrow. In the congenic HSCT model, conditioning recipient mice with a single dose of 3 mg/kg anti-mCD117-PBD enabled robust donor chimerism in the peripheral blood and bone marrow, as well as reconstitution of the T-, B- and myeloid cell compartments, that was comparable to the 9 Gy TBI positive control for myeloablative conditioning. Treatment with the iso-PBD control at 3 mg/kg was not effective at enabling HSC engraftment. In the fully mismatched allo-HSCT model, recipient mice conditioned with 3 mg/kg anti-mCD117-PBD in combination with 250 μg/mouse lymphodepleting anti-mCD4 + anti-mCD8 antibodies enabled full donor chimerism, achieving >90% engraftment by week 12 in the peripheral blood which was sustained through the end of the study at week 24 (Figure 1A). Multilineage reconstitution of immune cell subsets was also observed in this study, with >90% donor chimerism seen in the B cell and myeloid compartments and T cell reconstitution above 75% (Figure 1B-D). There was 99% donor HSC engraftment in the bone marrow at study termination (Figures 1E & F). These results were comparable to the chimerism observed in the 9 Gy TBI positive control mice. Groups conditioned with the non-targeting iso-PBD or anti-rIgG isotype antibody controls did not support donor engraftment in the model. In conclusion, conditioning with 3 mg/kg anti-mCD117-PBD, in combination with lymphodepleting antibodies anti-mCD4 and anti-mCD8, enables complete donor chimerism in a fully mismatched allo-HSCT murine model. This targeted conditioning approach could offer a more favorable risk-benefit profile over currently available conditioning regimens and could extend the curative potential of allo-HSCT to more patients with malignant and non-malignant diseases who otherwise would not be eligible for HSCT. Figure 1 Figure 1. Disclosures Lanieri: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Lamothe: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bhattarai: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Palchaudhuri: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Olson: Magenta Therapeutics: Current Employment, Current holder of stock options in a privately-held company.

scholarly journals Use of repetitive DNA sequences to distinguish Mus musculus and Mus caroli cells by in situ hybridization

Development ◽  
1983 ◽  
Vol 73 (1) ◽  
pp. 163-178
Author(s):  
L. D. Siracusa ◽  
V. M. Chapman ◽  
K. L. Bennett ◽  
N. D. Hastie ◽  
D. F. Pietras ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Situ Hybridization ◽  
Dna Sequences ◽  
Mus Musculus ◽  
Bone Marrow Cells ◽  
Cell Types ◽  
Parental Cell ◽  
F1 Hybrid ◽  
Mus Caroli

Mammalian chimaeras have proved useful for investigating early steps in embryonic development. However, a complete clonal analysis of cell lineages has been limited by the lack of a marker which is ubiquitous and can distinguish parental cell types in situ. We have developed a cell marker system which fulfils these criteria. Chimaeric mice were successfully producedfrom two mouse species which possess sufficient genetic differences to allow unequivocal identification of parental cell types. DNA-DNA in situ hybridization with cloned, species-specific sequences was performed to distinguish the parental cell types. We have identified a cloned, Mus musculus satellite DNA sequence which shows hybridization differencesbetween Mus musculus and Mus caroli DNA. This clone was used asa probe in in situ hybridizations to bone marrow chromosomes from Mus musculus, Mus caroli, and an interspecific F1 hybrid. The clone could qualitatively distinguish Mus musculus from Mus caroli chromosomes after in situ hybridization, even when they were derived from the same F1 hybrid cell. Quantitation of this hybridization to interphase nuclei from bone marrow spreads indicates that the probe can successfully distinguish Mus musculus from Mus caroli cells and can determine the percentage contribution of Mus musculus in mixtures of bone marrow cells of these species and in chimaeric bone marrow cell preparations.

scholarly journals A Novel Lair-1 Antibody Selectively Targets Acute Myeloid Leukemia (AML) Stem Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3413-3413
Author(s):  
Linjie Tian ◽  
Ana Paucarmayta ◽  
Rustin Lovewell ◽  
Karla Maloveste ◽  
Junshik Hong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Death ◽  
Stock Options ◽  
Hematopoietic Stem ◽  
Isotype Control ◽  
Cd34 Cells ◽  
Survival Signals ◽  
Privately Held

Abstract Extensive research has led to recent approval of novel therapies such as mylotarg, venetoclax, glasdegib and CC486, and small molecule inhibitors against actionable mutations such as ivosidenib (IDH1), enasidenib (IDH2), gliteritinib and midostaurin (FLT3) in AML. However, the mainstay of treatment in AML remains unchanged since the 1970s. There is a significant unmet need for AML patients that fail to respond to or relapse after standard-of-care (SOC) treatments including allogeneic stem cell transplantation and targeting actionable mutations. In addition, a large fraction of SOC patients invariably relapse due to persistence of chemotherapy-resistant leukemia stem cells (LSCs) or immune evasion. Therefore, identification of unique therapies that preferentially target elusive LSCs and promote immune responses to AML to prevent relapse are highly sought after. Unlike, targeting acute lymphoblastic leukemia (ALL) with CD19 or CD22 with various modalities, when developing AML therapies, it is of paramount importance to differentiate LSCs from hematopoietic stem cells (HSCs) to lessen or abolish unavoidable cytopenias. Leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) is an immune checkpoint receptor on T cells and myeloid cells that delimits immune cell activation through binding to endogenous collagen ligands. In addition, LAIR-1 is universally expressed on AML blasts and may sustain AML survival signals. We demonstrated using multi-color flow cytometry that LAIR-1 is highly expressed in AML blasts (n=9 of 9) and that LAIR-1 expression in LSCs (markers: CD34 +CD38 -CD90 -CD45RA +/- or CD34 -CD117 +CD244 +/-) is high compared with negligible expression of LAIR-1 in HSCs (markers: CD34 +CD38 -CD90 +CD99 -) (n=3) (Figure 1). Based on these findings, we hypothesized that a LAIR-1 monoclonal antibody (mAb) would disrupt LAIR-1 mediated survival signaling and preferentially target LAIR-1 expressing AML LSCs and blast cells but not HSCs. To test this, we developed a novel LAIR-1 targeting mAb with a functional human IgG 1 isotype that blocks LAIR-1 binding to its ligands (including collagens, complement component C1q, MBL and SP-D) To characterize the anti-leukemic effect of the LAIR-1 mAb we performed an in vitro antibody dependent cell cytotoxicity (ADCC) assay with LAIR-1 expressing AML cells (MOLT4 and MV-4-11). Compared with isotype control, the LAIR-1 mAb significantly increased leukemia cell death (MV411 = 17% above isotype, and MOLT4 = 29.24% above isotype at 15 µg/ml), suggesting that the LAIR-1 mAb confers ADCC activity against LAIR1 + AML cells (Figure 2). To elucidate if the LAIR-1 mAb has a direct signaling effect on LAIR-1 + AML cells, a colony forming unit assay using primary AML cells was carried out. Interestingly, the LAIR-1 mAb inhibited colony formation by AML CD34 + cells (40-60% decreased compared with isotype control, N=4), but not normal CD34 + cells. These data suggests that our LAIR-1 mAb stimulated LAIR-1 signaling that inhibits LSC self-renewal. We then tested the in vivo anti-leukemia effect of the mAb in cell line derived xenograft (CDX) models (immune deficient mice transplanted with MV-4-11 expressing luciferase). In vivo bioluminescence imaging indicated that the LAIR-1 mAb significantly inhibited in vivo AML growth (91% reduction of total flux)(Figure 3). A significant increase in cell death was observed in the presence of the mAb in the blood (47%), spleen (89.4%) and bone marrow (27.6%). Similar to the anti-leukemic effect in CDX AML models, the LAIR-1 mAb significantly suppressed in vivo growth of AML patient derived xenografts (5 different primary AML donors) (10-90% human CD33 + AML cells in isotype control treatment vs 0.5-5% CD33 + AML cells in anti-LAIR-1 treatment, N=3) (Figure 4), while minimally impacting normal immune cells. Taken together, our studies suggest that the LAIR-1 mAb we generated is a novel AML immunomedicine that preferentially eradicates AML LSCs and blasts while preserving healthy HSCs through disruption of AML survival signals and clearance of AML through ADCP and ADCC. Additional studies are currently evaluating if this novel LAIR-1 mAb has other mechanisms of action that contribute to overall in vivo activity, including reduction of AML niche implantation, regulation of bone marrow homing and regulation of anti-tumor immunity. Figure 1 Figure 1. Disclosures Tian: NextCure: Ended employment in the past 24 months. Paucarmayta: NextCure: Current Employment. Lovewell: NextCure: Current Employment. Maloveste: NextCure: Current Employment. Copeland: NextCure: Current Employment. O'Neill: NextCure: Current Employment. Patel: NextCure: Current Employment. Liu: NextCure: Current Employment, Current holder of stock options in a privately-held company. Myint: NextCure: Current Employment, Current holder of stock options in a privately-held company. Langermann: NextCure: Current Employment, Current holder of stock options in a privately-held company. Flies: NextCure: Current Employment, Current holder of stock options in a privately-held company. Kim: Nextcure: Research Funding.

scholarly journals Cell types associated with fibronectin in long-term mouse bone marrow cultures.

1982 ◽  
Vol 30 (3) ◽  
pp. 235-244 ◽  
Author(s):  
U Reincke ◽  
P Hsieh ◽  
P Mauch ◽  
S Hellman ◽  
L B Chen
Keyword(s):  
Bone Marrow ◽  
Hematopoietic Cells ◽  
Cell Types ◽  
Mouse Bone Marrow ◽  
Cell Adherence ◽  
Fibronectin Matrix ◽  
Bone Marrow Cultures ◽  
Marrow Cultures

The formation of fibronectin matrix was studied in long-term mouse bone marrow cultures. Stromal and hematopoietic cells were observed in situ under phase contrast optics and quantified according to their staining characteristics on smear preparations. Surface fibronectin was demonstrated by indirect immunofluorescence. While only stromal and no hematopoietic cells participated, various stromal cell types differed in their expression of cell surface fibronectin: Reticulum cells contributed the major portion of fibronectin matrix. Elongated, meshwork-forming histiocytes expressed some surface fibronectin, while the flattened, macrophagic histiocytes remained fibronectin negative. These findings were recapitulated during regeneration of scrape wounds in the adherent layers. Isolated fibronectin matrix did not support hematopoietic cell adherence or maintenance, although it had marked effects on stromal cells.

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