Neoplastic Bone Marrow Research Articles

Optimized multiplex immunofluorescence for the characterization of tumor immune microenvironment in neoplastic paraffin-preserved tissues.

The study of neoplastic cells enabled the discovery of important tumor-related biomarkers which resulted in new forms of early diagnosis, therapeutic options, and prognostic markers. Thus, immunofluorescence (IF), a high throughput imaging technology, represents a valuable method that enables the virtual characterization and localization of diverse cell types and targets, preserving tissue architecture and spatial surroundings. IF staining and analysis of formalin-fixed paraffin-embedded (FFPE) tissues are considered a challenge due to several difficulties, such as tissue autofluorescence, non-specific antibody binding, and image acquisition and quality. This study aimed to develop a multiplex-fluorescence staining technique with high-contrast and high-quality multiple-color images to enrich the investigation of important biomarkers. We present a robust optimized multiple-immunofluorescence procedure that reduced sample autofluorescence, enabled the use of simultaneous antibodies on the same sample, and showed super-resolution imaging through precise antigen localization. We illustrated the utility of this powerful method in FFPE neoplastic appendix, lymph node and bone marrow biopsies, and a 3D-coculture system, in which cells are enabled to grow and interact with their surroundings in all 3D dimensions. Our optimized multiple-immunofluorescence method represents a powerful tool for better understanding the complexity of tumor cells, characterizing cell populations and spatial localization, revealing predictive and prognostic biomarkers, and identifying immunologic phenotypes in a single and limited sample. Thisvaluable IF protocol successfully enables tumor microenvironment profiling that could contribute to the study of cellular crosstalk and the niche, and to the identification of predictive biomarkers for neoplasms.

Acute cerebral ischemic stroke as a presentation of polycythemia vera: a case report and a review of literature.

In the presenting study, we report a 60-year-old male patient who presented with a right-side weakness for 3 days. After laboratory and brain imaging, he was diagnosed with an acute cerebral infarct affecting the left basal ganglion secondary to PV. PV as the cause of ischemic stroke is a rare condition but can be encountered in clinical practice, and clinicians should be familiar with this combination.

EE11 Burden of Myelodysplastic Syndromes Part II: Systematic Literature Review of Economic Burden

Myelodysplastic syndromes (MDS) are malignant clonal neoplastic bone marrow disorders characterized by ineffective hematopoiesis, leading to abnormal and/or dysfunctional blood cells that express as different forms of cytopenia. A comprehensive assessment of the disease burden is lacking.

Neoplastic bone marrow invasion:rapid exclusion of hematological disease by flow cytometric routine panels

Multiparametric flow cytometry is an extensively used technique to assess the presence of different cellular populations in immunology and hematology. During routine immunophenotyping analysis, it is not uncommon to face cells of non-hemopoietic origin, negative for CD45 and other myeloid, megakaryocytic, B and T lineage antigens and positive for at least one antibody among CD56, CD117 and CD138. If cytology cannot identify cell origin, especially in cases of unclear interpretation, the contribution of multiparametric flow cytometry analysis can be crucial. We report 6 patients with a clinical suspicion of hematological disease in which multiparametric flow cytometry was extremely useful to quickly exclude blood disorders in order to initiate patients to the most appropriate diagnostic process.

Immune Biomarkers of Survival and Severe Infection in Newly Diagnosed Multiple Myeloma (NDMM) Patients (pts) Treated with the Backbone Regimen Lenalidomide and Dexamethasone (Rd)

Introduction: Rd is a backbone of treatment in MM. The strong immunomodulatory effects of this regimen would suggest that immune profiling might complement conventional prognostic factors. However, immune signatures predictive of survival were not well defined in the past, and nowadays it is challenging to detach the effect of Rd from other backbone drugs in triplets or quadruplets, as well from high dose therapy in transplant eligible patients. Aim: Identify immune biomarkers in elderly NDMM pts treated with Rd. Methods: The GEM-CLARIDEX trial randomized transplant ineligible NDMM pts to Rd +/- clarithromycin until disease progression. Because clarithromycin had no effect in survival, the trial design yielded a unique opportunity to identify immune biomarkers in elderly MM pts treated with Rd. Immune profiling was performed in bone marrow (BM) at baseline in 186 pts, using multidimensional and computational flow cytometry together with lasso penalized Cox regression and 10-fold cross validation, for a holistic and unbiased definition of prognostic immune signatures. 38 cell populations were identified, including seven myeloid/erythroid and 29 lymphoid subsets plus normal and clonal plasma cells (PC). Single cell RNA sequencing (scRNAseq, 10x Genomics) was performed on BM immune cells sorted from healthy adults, MGUS, smoldering and active MM pts (n = 17). Results: An immune signature associated with inferior survival was defined based on increased frequency of cytotoxic CD314+ NK and CD4+ central memory (CM) CD127+ PD1+ T cells, and decreased percentages of CD4+ CM CD127+ PD1- and CD8+ CD127low PD1+ terminally differentiated T cells, as well as of B cell precursors. Median progression-free survival (PFS) of NDMM pts with high risk immune score (106/186 [57%]) was 18 months vs not reached in those with low risk (80/186 [43%]) (p=.0008, Fig 1A). The respective median overall survival (OS) rates at three years were 50% and 80% (p = .0021, Fig 1B). In multivariate analyses including age, ISS, LDH and cytogenetic risk, immune profiling showed independent prognostic value for PFS (HR: 2.6, p = .001) and OS (HR: 2.9, p = .003). Increased frequency of cytotoxic CD314+ NK cells was the immune biomarker with the highest negative impact in the survival predictive model. In an attempt to better understand this finding, we analyzed scRNAseq data of 61,637 NK cells from normal and tumor BM. Up to 47 NK-cell clusters were identified, and the one showing the highest expression of CD314 also displayed upregulation of granzymes K, H and B, perforin, CD16, CD38, CD69 and IFNγ, and therefore appeared to be associated with cytolytic activity and inflammation. Interestingly, there was a progressive expansion of this cluster within the NK cell compartment of normal vs pre-malignant vs neoplastic BM samples. Similarly, in genetically engineered BIcγ1 mice that develop active MM preceded by precursor states, there was a progressive expansion of CD314+ NK cells in normal vs benign vs malignant BM (p = .001). Being infection an important cause of morbidity and mortality in elderly MM pts as those included in this study, we next investigated if immune profiling at diagnosis could help predicting severe infection (≥ grade 3 CTCAE v4.0, n=43/186). The combination of five clinical features - age ≥ 75y, low hemoglobin and albumin, high β2m and LDH - plus seven immune biomarkers - increased percentages of eosinophils, together with decreased frequencies of CD4+ CM CD127+ PD1-, CD8+ naïve and CD127low PD1+ and PD1- terminally differentiated T cells, B cell precursors and normal PC - resulted in the most effective model to predict severe infection. B cell precursors and eosinophils, which are easily identified using flow cytometry at diagnosis, showed the strongest association. The area under the curve (AUC) of the model was 0.90 with a 4-fold cross validation showing a mean AUC = 0.86 ± 0.06. Conclusions: This study shows the clinical value of immune profiling at diagnosis and defined immune cell types, including a previously unidentified subset of CD314+ cytotoxic NK cells, which were predictive of survival in transplant ineligible NDMM pts treated with the backbone regimen Rd. Furthermore, this study uncovered immune biomarkers complementary to clinical features for predicting severe infection in elderly MM pts, which could be used to intensify prophylactic measures in specific subgroups that are more susceptible to severe infection. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

An Automated Pipeline for Cell Differentials on Whole-Slide Bone Marrow Aspirate Smears

Abstract Current pathologic diagnosis of benign and neoplastic bone marrow disorders relies in part on the microscopic analysis of bone marrow aspirate (BMA) smears and manual counting of nucleated cell populations to obtain a cell differential. This manual process has significant limitations, including the limited sample of cells analyzed by a conventional 500-cell differential compared to the thousands of nucleated cells present, as well as the inter-observer variability seen between differentials on single samples due to differences in cell selection and classification. To address these shortcomings, we developed an automated computational platform for obtaining cell differentials from scanned whole-slide BMAs at 40x magnification. This pipeline utilizes a sequential process of identifying BMA regions with high proportions of marrow nucleated cells that are ideal for cell counting, detecting individual cells within these optimal regions, and classifying cells into one of 11 types within the differential. Training of convolutional neural network models for region and cell classification, as well as a region-based convolutional neural network for cell detection, involved the generation of an annotated training data set containing 10,948 BMA regions, 28,914 cell boundaries, and 23,609 cell classifications from 73 BMA slides. Among 44 testing BMA slides, an average of 19,209 viable cells per slide were identified and used in automated cell differentials, with a range of 237 to 126,483 cells. In comparing these automated cell differential percentages with corresponding manual differentials, cell type-specific correlation coefficients ranged from 0.913 for blast cells to 0.365 for myelocytes, with an average coefficient of 0.654 among all 11 cell types. A statistically significant concordance was observed among slides with blast percentages less or greater than 20% (p=1.0x10-5) and with plasma cell percentages less or greater than 10% (p=5.9x10-6) between automated and manual differentials, suggesting potential diagnostic utility of this automated pipeline for malignancies such as acute myeloid leukemia and multiple myeloma. Additionally, by simulating the manual counting of 500 cells within localized areas of a BMA slide and iterating over all optimal slide locations, we quantified the inter-observer variability associated with limited sample size in traditional BMA cell counting. Localized differentials exemplify an average variance ranging from 24.1% for erythroid precursors to 1.8% for basophils. Variance in localized differentials of up to 44.8% for blast cells and 36.9% for plasma cells was observed, demonstrating that sample classification based on diagnostic thresholds of cell populations is variable even between different areas within a single slide. Finally, pipeline outputs of region classification, cell detection, cell classification, and localized cell differentials can be visualized using whole-slide image analysis software. By improving cell sampling and reducing inter-observer variability, this automated pipeline has potential to improve the current standard of practice for utilizing BMA smears in the diagnosis of hematologic disorders.

Pathogenic and Prognostic Roles of Paraneoplastic Leukocytosis in Cervical Cancer: Can Genomic-Based Targeted Therapies Have a Role? A Literature Review and an Emblematic Case Report.

Tumor-associated leukocytosis has been associated with poor prognosis in cervical cancer. Leukemoid reaction (i.e., white blood cell count > 40,000/μL) is defined paraneoplastic (PLR) when it occurs in the presence of a cytokine-secreting tumor (CST) without neoplastic bone marrow infiltration. Cervical cancers displaying PLR represent a peculiar entity characterized by a rapidly progressive behavior typically associated with chemo-radioresistance. The present paper aims to review the literature about the pathogenetic mechanisms of PLR and its prognostic role in cervical cancer. Moreover, it reports the emblematic case of a patient with an advanced cervical cancer associated with PLR that was chemotherapy resistant. The patient underwent a palliative cytoreductive surgery of high complexity, obtaining a temporary regression of PLR. The tumor sample stained positive for G-CSF and IL-6, thus indicating a CST. Notably, the tumor genomic analysis revealed a PI3CKA mutation. Therefore, at the instrumental evidence of a rapidly progressive disease relapse, which was accompanied by reappearance of PLR, we started a targeted treatment with a selective PIK3 inhibitor alpesilib combined with the JAK1-2 inhibitor ruxolitinib. We achieved a relief of symptoms and leukocytosis; however, severe side effects necessitated the treatment suspension. In conclusion, as therapeutic strategies for cancer with PLR are scarcely reported in literature, our study could contribute to expand our understanding of the topic and provide a basis for further research.

P1027: RESPONSES TO AVAPRITINIB IN PATIENTS WITH ADVANCED SYSTEMIC MASTOCYTOSIS: HISTOPATHOLOGIC ANALYSES FROM EXPLORER AND PATHFINDER CLINICAL STUDIES

Background: Systemic mastocytosis (SM) is a mast cell (MC) neoplasm driven by the KIT D816V mutation in ~95% of cases. Diagnosis includes evaluation of MC aggregates in extracutaneous organs, atypical MC morphology, and MC immunophenotype. Avapritinib, a potent and selective KIT D816V inhibitor, has been approved in the US for patients with advanced SM (AdvSM) based on the phase 1 EXPLORER and phase 2 PATHFINDER studies. Aims: Here, we describe a comprehensive evaluation of the effect of avapritinib on bone marrow (BM) pathology in patients treated in these studies. Methods: Patients aged ≥18 years with diagnoses of AdvSM per local investigator were treated with once-daily avapritinib <200 mg (n=17), 200 mg (n=126), and ≥300 mg (n=50). Bone marrow biopsies (BMBs), aspirates (BMAs), and peripheral blood (PB) smears were obtained at screening, and after 8 and 24 weeks of treatment with avapritinib. Evaluations were performed using standard Wright-Giemsa and H&E staining, and IHC was performed on formalin-fixed EDTA-decalcified BMBs using standard techniques for CD25 and CD30. Changes in fibrosis were assessed by the European Consensus on grading of BM fibrosis (MF score). Results: As of April 2021, 193 patients enrolled in EXPLORER (n=86) and PATHFINDER (n=107) were included in analyses with centrally confirmed diagnoses of aggressive SM (n=29), SM with an associated hematologic neoplasm (n=119), mast cell leukemia (n=28), indolent SM (n=14), smoldering SM (n = 2) or CMML (n=1). Median (range) age was 67 years (31–88), 56% were male and 88% had a KIT D816V mutation. Following treatment with avapritinib, a decrease in the proportion of patients with multifocal dense aggregates of MCs in BMBs was observed from 93% at screening to 50% by Week 8 (Table). Avapritinib treatment reduced the proportion of aberrant CD25+ and CD30+ MCs in BMBs by Week 8 and Week 24 (Table). In BMAs, avapritinib reduced the mean MC burden from 11% of total nucleated cells at screening to 2% at Week 8 and by Week 24, with reduced proportions of immature and spindle-shaped MCs (Table). Of 9 patients with circulating MCs at screening and post-screening sample measurements (6 with SM with associated hematologic neoplasm diagnoses and 3 with MCL), 8 had no detectable MCs in PB by Week 8. The proportion of patients with fibrosis in BMBs decreased from 96% (178/186) at screening to 90% (142/158) at Week 8 to 84% (103/123) through week 24, and MF scores were reduced during avapritinib treatment in those patients presenting with increased fibrosis at screening (Table). Image:Summary/Conclusion: Patients treated with avapritinib were observed to have rapid (Week 8) and marked (Week 24) reductions in neoplastic BM MCs with a return to a normal morphologic appearance and immunophenotype. This was accompanied by an improvement of fibrosis and a decrease in circulating MCs. These improvements in disease histopathology provide further support that treatment with this highly selective and potent KIT D816V inhibitor is disease modifying in AdvSM.

GLUT1 Immunohistochemistry Is a Highly Sensitive and Relatively Specific Marker for Erythroid Lineage in Benign and Malignant Hematopoietic Tissues.

Glucose transporter 1 (GLUT1), a glucose transporter, is an abundant protein in erythrocytes with expression beginning early in erythropoiesis. We sought to evaluate the utility of GLUT1 immunohistochemistry (IHC) as a diagnostic marker for identifying erythroid differentiation in hematopoietic tissues, including neoplastic erythroid proliferations. A variety of benign and neoplastic bone marrow biopsy specimens containing variable proportions of erythroid precursors were selected (n = 46, including 36 cases of leukemia). GLUT1 IHC was performed using a commercially available polyclonal antibody. Each case was evaluated for staining of erythroid precursors, nonerythroid hematopoietic cells, and blasts. A GATA1/GLUT1 double stain was performed on one case to confirm coexpression of GLUT1 on early erythroid precursors. Staining was compared with other erythroid markers, including glycophorin C. GLUT1 demonstrated strong membranous staining in erythroid precursors of all cases, which was restricted largely to the erythroid lineage. Of the 36 leukemia cases, all 6 cases of pure erythroid leukemia and both cases of therapy-related acute myeloid leukemia with erythroid differentiation showed positive GLUT1 staining in blasts. Otherwise, only lymphoblasts in B-lymphoblastic leukemia showed weak to moderate granular cytoplasmic staining (four of five cases). GLUT1 IHC is a highly sensitive and relatively specific marker for erythroid lineage in benign and neoplastic bone marrow biopsy specimens.

Machine Learning-Based Automated Selection of Regions for Analysis on Bone Marrow Aspirate Smears

Abstract Manual microscopic examination of bone marrow aspirate (BMA) smears and counting of cell populations remains the standard of practice for accurate assessment of benign and neoplastic bone marrow disorders. While automated cell classification software using machine learning models has been developed and applied to BMAs, current systems nonetheless require manual identification of optimal regions within the slide that are rich in marrow hematopoietic cells. To address this issue, we have developed a machine learning-based platform for automated identification of optimal regions in whole-slide images of BMA smears. A training dataset was developed by manual annotation of 53 BMA slides across biopsy diagnoses including unremarkable trilineal hematopoiesis, acute leukemia, and plasma cell neoplasms, as well as across differences in total cellularity represented by a spectrum of marrow nucleated cell content and white blood cell counts. 10,537 regions among these 53 slides were manually annotated as either “optimal” (regions near aspirate particles with high proportions of marrow nucleated cells), “particle” (aspirate particles), or “hemodilute” (blood-rich regions with high proportions of red blood cells). Training of a neural network-based classifier on 10x magnification slides with region cropping and image augmentation resulted in a classifier with substantial accuracy on new testing-set BMA slides (one-vs-rest AUROC &amp;gt; 0.999 across 10 training/testing splits for all 3 region classes), with very few particle and hemodilute regions being classified as optimal (particle: 0.83%, hemodilute: 0.39%). Additionally, this classifier accurately classifies BMA regions on slides from hematological disorders not represented in the training data, including Burkitt lymphoma (AUROC &amp;gt; 0.999 across region classes), chronic myeloid leukemia (AUROC &amp;gt; 0.999 across region classes), and diffuse large B-cell lymphoma (AUROC = 1 across region classes), demonstrating the broad applicability of our approach. To assess the performance of our classifier on whole-slide images, tiles from 10x magnification slides were manually annotated by three participants with notable concordance (Krippendorff’s alpha = 0.424); substantial agreement was found between manual annotations and model predictions within whole-slide images (optimal AUROC = 0.958, particle AUROC = 1.0, hemodilute AUROC = 0.947). Based on these promising results, this machine learning-based region classification model is being connected to a previously-developed bone marrow cell classifier to fully automate differential cell counting in whole-slide images. The development of this novel automated pipeline has potential to streamline the diagnostic process for hematological disorders while enhancing accuracy and replicability, as well as decreasing diagnostic turnaround time for improving patient care.

The Role of Amino Acids in the Crosstalk Between Mesenchymal Stromal Cells and Neoplastic Cells in the Hematopoietic Niche.

Within the bone marrow hematopoietic cells are in close connection with mesenchymal stromal cells (MSCs), which influence the behavior and differentiation of normal or malignant lymphoid and myeloid cells. Altered cell metabolism is a hallmark of cancer, and changes in nutrient pools and fluxes are important components of the bidirectional communication between MSCs and hematological cancer cells. Among nutrients, amino acids play a significant role in cancer progression and chemo-resistance. Moreover, selected types of cancer cells are extremely greedy for glutamine, and significantly deplete the extracellular pool of the amino acid. As a consequence, this influences the behavior of MSCs in terms of either cytokine/chemokine secretion or differentiation potential. Additionally, a direct nutritional interaction exists between MSCs and immune cells. In particular, selected subpopulations of lymphocytes are dependent upon selected amino acids, such as arginine and tryptophan, for full differentiation and competence. This review describes and discusses the nutritional interactions existing in the neoplastic bone marrow niche between MSCs and other cell types, with a particular emphasis on cancer cells and immune cells. These relationships are discussed in the perspective of potential novel therapeutic strategies based on the interference on amino acid metabolism or intercellular fluxes.

Long-term immunosuppression and multiple transplants predispose systemic lupus erythematosus patients with cytopenias to hematologic malignancies.

Cytopenias in systemic lupus erythematosus (SLE) require clinical and laboratory workup and bone marrow (BM) examination to determine the cause and for appropriate patient management. Common causes include an increase in SLE activity, immune-mediated hemolysis, iron deficiency, antiphospholipid antibody syndrome, infection, or the effect of medications. We retrospectively evaluated the clinical and laboratory findings of patients with SLE and cytopenias who had undergone BM studies to determine the indicators of malignancy.We retrospectively reviewed medical records of patients with SLE who presented with cytopenias for their disease course, medications, laboratory parameters and documented the spectrum of morphological changes in BM including CD34 expression.Twenty patients with SLE had undergone BM biopsy for evaluation of cytopenias. 14/20 (70%) of the patients had reactive BM, and the rest had hematologic malignancies involving the BM. Of these 14 patients, 8 had hypocellular marrow with loss of precursor cells (low CD34), 4 had left shift in myeloid lineage, 3 had serous atrophy, and 1had multilineage dysplasia. The 6 patients with hematologic malignancies included 2 with diffuse large B cell lymphoma, and one each of natural killer/T cell lymphoma, post-transplant lymphoproliferative disorder, Hodgkin lymphoma, and myelodysplastic syndrome evolving to acute myelogenous leukemia. The presence of autoantibodies, SLE activity, and lupus nephritis were comparable in patients with and without neoplasia. However, the duration of the use of multiple immunosuppressants, years since renal transplant (22 vs 10), multiple transplants, and the presence of other autoimmune diseases were greater in those with neoplasia. Two of the 14 patients with non-neoplastic BM and 1 with the neoplastic BM had nonhematological malignancy.Clinical and laboratory findings, the number of transplants, and the use of immunosuppressive agents can guide physicians to identify patients with a higher risk of developing hematologic malignancy. BM findings of cytopenia in SLE are often due to increased disease activity causing global cell death and dysmaturation. SLE patients presenting with cytopenias, with a history of long-term exposure to immunosuppressive drugs, should be regularly screened for hematologic and nonhematologic malignancies.

Reactive Versus Neoplastic Bone Marrow Lymphoid Nodules—Diagnostic Challenge in a Case with Anemia and IgM Para-proteinemia

Reactive Versus Neoplastic Bone Marrow Lymphoid Nodules—Diagnostic Challenge in a Case with Anemia and IgM Para-proteinemia

Controlling Through-Slice Chemical-Shift Artifacts for Improved Non-Fat-Suppressed Musculoskeletal Turbo-Spin-Echo Magnetic Resonance Imaging at 7 T.

Through-slice chemical shift artifacts in state-of-the-art turbo-spin-echo (TSE) images can be significantly more severe at 7 T than at lower field strengths. In musculoskeletal applications, these artifacts appear similar to bone fractures or neoplastic bone marrow disease. The objective of this work was to explore and reduce through-slice chemical shift artifacts in 2-dimensional (2D) TSE imaging at 7 T. This prospective study was approved by the local ethics board. The bandwidths of the excitation and refocusing radiofrequency (RF) pulses of a prototype 2D TSE sequence were individually modified and their effect on the slice profiles and relative slice locations of water and fat spins was assessed in an oil-water phantom. Based on these results, it was hypothesized that the combination of matched and increased excitation and refocusing RF pulse bandwidths ("MIB") of 1500 Hz would enable 2D TSE imaging with significantly reduced chemical shift artifacts compared with a state-of-the-art sequence with unmatched and moderate RF pulse bandwidths ("UMB") of 1095 and 682 Hz.A series of T1-weighted sagittal knee examinations in 10 healthy human subjects were acquired using the MIB and UMB sequences and independently evaluated by 2 radiologists. They measured the width of chemical shift artifacts at 2 standardized locations and graded the perceived negative effect of chemical shift artifacts on image quality in the bones and in the whole gastrocnemius muscle on a 5-point scale. Similar knee, wrist, and foot images were acquired in a single subject. Signal-to-noise ratios in the femoral bone marrow were computed between the UMB and MIB sequences. Phantom measurements confirmed the expected spatial separation of simultaneously affected water and fat slices between 40% and 200% of the prescribed slice thickness for RF pulse bandwidths between 2500 and 500 Hz. Through-slice chemical shift artifacts at the bone-cartilage interface were significantly smaller with MIB than with UMB (location 1: 0.35 ± 0.20 mm vs 1.27 ± 0.27 mm, P < 0.001; location 2: 0.25 ± 0.13 mm vs 1.48 ± 0.46 mm, P < 0.001; intraclass correlation coefficient = 0.98). The negative effect of chemical shift artifacts on image quality was significantly smaller with MIB than with UMB (bone: 2 ± 0 vs 4 ± 1, P < 0.004 [both readers]; muscle: 3 ± 0 vs 2 ± 0, P < 0.004 [both readers]; κ = 0.69). The signal-to-noise ratio of the UMB and MIB sequences was comparable, with a ratio of 99 ± 7%. Images acquired using the UMB sequence displayed numerous artifactual hyperintensities and diffuse, as well as locally severe, fat signal loss in all examined regions, whereas the MIB sequence consistently yielded high image quality with bright T1-weighted fat signal and excellent depiction of fine tissue structures. On 7 T systems, the selection of high and matched RF bandwidths for excitation and refocusing pulses for 2D TSE imaging without fat suppression showed consistently better image quality than state-of-the-art sequences with unmatched lower RF pulse bandwidths.

Alpha-Smooth Muscle Actin (a-SMA) Gene, a Possible Derive of Myofibroblasts in Bone Marrow Fibrosis

Background: Alpha-smooth muscle actin (α-SMA) is used as a marker for a subset of activated fibrogenic cells, myofibroblasts, which are regarded as important effector cells of tissue fibrogenesis. Purpose: We addressed whether ASMA gene (actin alpha 2 gene or ACTA2) expression is up regulated in cases with BM fibrosis compared to those with no bone marrow (BM) fibrosis in order to evaluate its role in the pathogenesis of BM fibrosis. Subjects and methods: ASMA expression was detected by quantitative RT-PCR in formalin fixed paraffin embedded (FFPE) bone marrow trephine biopsy samples of cases with neoplastic bone marrow diseases as well as reactive bone marrow disorders cases. Both groups included cases with and without bone marrow fibrosis. Results: Results indicated that there was no statistically significant difference in ASMA (ACTA2) gene expression between neoplastic fibrotic and non-fibrotic cases as well as between reactive fibrotic and non-fibrotic cases. Also the level of α-SMA expression does not correlate positively with the grade of bone marrow fibrosis. Conclusion: We conclude that α-SMA is not a functional marker of fibrogenic cells in bone marrow fibrosis. Exploration of other related genetic pathway is recommended.

Integrative Study of EZH2 Mutational Status, Copy Number, Protein Expression and H3K27 Trimethylation in AML/MDS Patients

Introduction Enhancer of Zeste Homolog 2 (EZH2), the catalytic domain of Polycomb Repressive Complex 2 (PRC2), mediates the repressive mark of trimethylation of histone H3 lysine 27 (H3K27me3). The EZH2 gene is located on chromosome (chr.) 7q36.1 (frequently deleted in AML/MDS). Its role in tumorigenesis appears to be context-dependent since both EZH2 overexpression and loss of function are associated with different types of cancer. In patients (pts) with MDS or MDS/MPN, loss-of-function EZH2 mutations (mut) are recurrently found, associated with a poor prognosis, and EZH2 is considered a tumor suppressor gene (TSG). In AML, the incidence of EZH2-mut is lower and less well-studied. We wished to determine the effects of EZH2-mut and copy number (CN) on EZH2 expression, and the consequences for H3K27me3 levels in vivo. Methods Fifty-eight pts (51 AML, 3 MDS, 4 MDS/MPN), median age 63.5 years (yr, range 20-86, almost all diagnosed between 2015 and 2017), with available sequencing data, EZH2 CN status and EZH2 expression data were studied. Mutation status was determined by next-generation sequencing (NGS) including a panel of 54 genes (Illumina Myeloid NGS panel). Metaphase cytogenetics and/or fluorescence in situ hybridization and single nucleotide polymorphism arrays (selected pts) were conducted to determine EZH2 CN status. Protein expression of EZH2 and H3K27me3 was assessed semi-quantitatively by immunohistochemistry (IHC) on formalin-fixed EDTA-decalcified paraffin-embedded bone marrow (BM) core biopsies. The Kruskal-Wallis test and Dunn's multiple comparison test were used to address whether EZH2 protein expression was associated with EZH2-mut and CN. Results The EZH2 gene showed mutations (mostly missense or nonsense, median variant allele frequency (VAF) 45%, range 7-63%) in 13/58 pts and was unmutated in 45/58 pts. EZH2-mut pts had a median of 4 mutations. Additional mutations were most frequently found in ASXL1 (10/13; median VAF 35%, range 19-48%), less frequently in TET2, RUNX1, STAG2 (3/13 each), NRAS (2/13), and DNMT3A (1/13). In contrast, EZH2-wt pts had a median of 2 mutations, most frequently in DNMT3A (12/45), followed by NRAS (10/45), IDH1 (9/45), FLT3 (8/45), and NPM1 (7/45). Regarding chr. 7, 43 pts had no detectable deletion, 15 had 7q-/-7. Notably, the incidence of EZH2-mut was similar in pts with 7q-/-7 lesions (3/15, i.e. 20%) and pts with normal chr. 7 (10/43, 23%). EZH2 expression in neoplastic BM cells ranked from no (score 0) to strong expression (score 3). While the hematopoietic BM cells of healthy donors usually showed a moderate EZH2 expression (score 2), in our cohort the score was 0 in 3, 1 in 13, 2 in 23, and 3 in 19 pts, respectively. We next asked whether EZH2 protein expression differed between pts depending on EZH2-mut and chr. 7 status. In Figure 1, 4 subgroups are depicted, showing highest expression in pts with EZH2-wt and either no chr. 7 abnormalities (group A, n=33) or 7q-/-7 (group C, n=12). In comparison, expression was significantly reduced in pts with EZH2-mut and no chr. 7 abnormalities (group B, n=10), and lowest in pts with EZH2-mut and chr. 7 abnormalities (group D, n=3), p&amp;lt;0.05 (indicated by asterisks). Since functional EZH2 protein is necessary for the trimethylation of H3K27, the presence of this histone mark was also determined semi-quantitatively by IHC in 40 pts. H3K27me3 levels were variable, and a test for a possible association between EZH2 and H3K27me3 levels by linear regression analysis did not show an association (R²=0.13). Sixty-two % of EZH2-mut (median age 71 yr) and 51% of EZH2-wt pts (median age 61 yr) received allografting. Median overall survival in EZH2-mut pts was 16.1 months compared to 23.5 in EZH2-wt pts (p=0.16). Conclusions Inactivating EZH2 mutations are infrequent events in AML. In our study, they were strongly associated with mutations of ASXL1 (also involved in PRC2 function). We did not observe overrepresentation of EZH2-mut in 7q-/-7 pts, in line with Bejar et al. (N Engl J Med 2011); this is in contrast to the frequent cooperative events between TSG mutations and deletions, e.g. of TP53. Functionally, EZH2 mutations appeared to have a stronger effect on decreased EZH2 expression than hemizygosity. Global H3K27me3 levels were not altered by EZH2 reduction, which could be due to compensatory upregulation of EZH1, supporting the clinical development of EZH1/2 inhibition. Disclosures No relevant conflicts of interest to declare.

PS1361 BIDIRECTIONAL MITOCHONDRIAL TRANSFER BETWEEN MYELOMA CELLS AND BONE MARROW STROMAL CELLS CONTRIBUTES TO THE CHEMORESISTANCE OF MYELOMA CELLS

Background:Neoplastic cells and bone marrow (BM) stromal cells interact intimately in the marrow and communicate with each other in numerous ways such as paracrine signaling via cytokines and chemokines, cell‐to‐cell interactions through tunneling nanotubes (TNTs) or gap junctions, and the secretion and incorporation of microvesicles (MVs). As the acquisition of cancer drug resistance was associated with mitochondrial transfer in several malignancies, we investigated the possibility of a similar phenomenon in multiple myeloma (MM) using an in vitro stromal cell–malignant plasma cell co‐culture system.Aims:Our primary goal was to establish whether mitochondrial transfer occurs between BM stromal cells and primary myeloma cells, and if so, exactly by what mechanism. We also wanted to clarify if this phenomenon could contribute to the drug resistance of myeloma cells.Methods:Mitochondrial exchange between BM stromal cells and myeloma cells was quantified by flow cytometry and visualized by confocal laser scanning microscopy. Cellular toxicity of different drugs was assessed by high throughput screening.Results:We found a relatively fast, bidirectional mitochondrial transfer between BM stromal cells and malignant plasma cells. As mitochondria can be transferred through TNTs, gap junctions, or via MVs, we selectively inhibited these pathways to dissect the exact mechanisms involved. According to our results, only cytochalasin D, a potential blocker of TNT formation, was able to reduce significantly the amount of transferred mitochondria in a dose‐dependent manner; however, the transmission of mitochondria has also been slightly reduced by inhibiting the MV uptake or gap junctions, suggesting a dominant but non‐exclusive role of TNTs in this process. In further experiments we demonstrated that stromal cells accumulate substantial amounts of mitochondria from myeloma cells delivered by MVs, whereas myeloma cells incorporate much less MVs and thus less mitochondria from stromal cells. Confocal microscopic analysis also confirmed bidirectional mitochondrial transport between stromal and myeloma cells through TNTs. Finally, we evaluated the possible contribution of stromal cell‐derived and myeloma cell‐derived mitochondria to the drug resistance of malignant plasma cells. Several drugs including apoptosis inducers, proteasome inhibitors, and epigenetic modifiers were tested, and it was clearly demonstrated that drug treatment always enhances the transfer of mitochondria from stromal cells to myeloma cells in a dose‐dependent manner (see figure for data on carfilzomib).Summary/Conclusion:Our cytotoxic assays demonstrated that myeloma cells are more resistant to drug treatment in co‐cultures than alone, and it is most likely caused by bi‐directional mitochondrial transfer. Our results provide additional clues for understanding the biological processes that underlie cell adhesion‐mediated drug resistance upon myeloma therapy.imageThis work was supported by grant NVKP_16–1–2016–0005 from the NKFIA, Hungary

Subleukaemic acute myeloid leukaemia with myelodysplasia in a horse

SummaryAcute myeloid leukaemia (AML) is rarely reported in horses and myelodysplastic syndrome (MDS) has been described only in one case. Acute myeloid leukaemia is defined as the presence of at least 20% blasts in the marrow or blood. On the other hand, MDS is characterised by morphologic abnormalities in one or more cell lineages with hypercellular marrow and peripheral cytopenias due to ineffective haematopoiesis. We report a case of acute myelomonocytic leukaemia with myelodysplasia‐related features in a horse. A supposed diagnosis was based on abnormal morphology of circulating neoplastic cells and bone marrow cytology. A final diagnosis was made by using flow cytometry (FC) in conjunction with cytochemistry (CC) rarely reported in the haematopoietic neoplasms of the horse.

CD7 is expressed on a subset of normal CD34-positive myeloid precursors.

To improve monitoring of myeloid neoplasms by flow cytometry-based minimal residual disease (MRD) analysis, we analyzed the significance of leukemia-associated immunophenotype (LAIP) markers in 44 patients. In a pilot study cohort, peripheral blood or bone marrow samples from 13 patients with myeloid neoplasms and one case of B lymphoblastic leukemia in complete hematologic remission after allogeneic bone marrow or stem cell transplantation were subjected to selection for leukemia-specific phenotypes by fluorescence-activated cell sorting using individual marker combinations, followed by PCR-based chimerism analysis. The feasibility of this method could be demonstrated, with selection being successful in 12 cases, including two cases where mixed chimerism was found exclusively in sorted cells. Interestingly, four specimens displayed full donor chimerism in cells expressing the presumably aberrant combination CD34+ /CD7+ . Further analyses, including assessment of an independent cohort of 25 patients not affected by neoplastic bone marrow infiltration, revealed that normal myeloid precursors usually include a population coexpressing CD34, CD13, CD33, and CD7. We conclude that the combination CD34+ /CD7+ might not be suitable as an LAIP for MRD diagnostics and that a subset of normal myeloid precursors in the bone marrow expresses CD7.

PAN3\u2013PSMA2 fusion resulting from a novel t(7;13)(p14;q12) chromosome translocation in a myelodysplastic syndrome that evolved into acute myeloid leukemia

BackgroundAcquired primary chromosomal changes in cancer are sometimes found as sole karyotypic abnormalities. They are specifically associated with particular types of neoplasia, essential in establishing the neoplasm, and they often lead to the generation of chimeric genes of pathogenetic, diagnostic, and prognostic importance. Thus, the report of new primary cancer-specific chromosomal aberrations is not only of scientific but also potentially of clinical interest, as is the detection of their gene-level consequences.Case presentationRNA-sequencing was performed on a bone marrow sample from a patient with myelodysplastic syndrome (MDS). The karyotype was 46,XX,t(7;13)(p14;q12)[2]/46,XX[23]. The MDS later evolved into acute myeloid leukemia (AML) at which point the bone marrow cells also contained additional, secondary aberrations. The 7;13-translocation resulted in fusion of the gene PAN3 from 13q12 with PSMA2 from 7p14 to generate an out-of-frame PAN3–PSMA2 fusion transcript whose presence was verified by RT-PCR together with Sanger sequencing. Interphase fluorescence in situ hybridization analysis confirmed the existence of the chimeric gene.ConclusionsThe novel t(7;13)(p14;q12)/PAN3–PSMA2 in the neoplastic bone marrow cells could affect two key protein complex: (a) the PAN2/PAN3 complex (PAN3 rearrangement) which is responsible for deadenylation, the process of removing the poly(A) tail from RNA, and (b) the proteasome (PSMA2 rearrangement) which is responsible for degradation of intracellular proteins. The patient showed a favorable response to decitabine after treatment with 5-azacitidine and conventional intensive chemotherapy had failed. Whether this might represent a consistent feature of MDS/AML with this particular gene fusion, remains unknown.