Marrow Cells Of Patients Research Articles

Telomere length of various blood and bone marrow cells in patients with aplastic anemia

Background. Aplastic anemia proceeds with bone marrow failure and is associated with immunological suppression of normal blood stem cells’ proliferation, which lead to bone marrow aplasia. autoimmune aggression and internal defects of blood stem cell that cause abnormal hematopoiesis are being actively studied. An important role in the pathogenesis of the aplastic anemia is played by instability of telomere length (TL). determination of the initial TL makes it possible to clearly differentiate between the aplastic anemia and dyskeratosis congenita. also, it helps to identify the group of patients with short telomeres for prediction of therapy response. Aim. To investigation the TL of various blood and bone marrow cells in patients with aplastic anemia before treatment. Materials and methods. The group of patients with aplastic anemia was investigated (n = 45). blood donors (n = 32) and bone marrow donors (n = 10) of different ages were included in the reference group. adult patients with dyskeratosis congenita (n = 5) were included in the comparison group. Relative and absolute tl was identified in peripheral blood and bone marrow mononuclear cells, monocytes, lymphocytes by flow-FISH technique (combination of flow cytometry and fluorescence in situ hybridization). Results. Relative and absolute TL was comparable in different blood and bone marrow cells in patients with aplastic anemia before treatment. TL in peripheral blood and bone marrow mononuclear cells wasn’t significantly differed in groups of patients with aplastic anemia and healthy donors. Telomeres in patients with dyskeratosis congenita were identified as “ultrashort” and were significantly shorter than in patients with aplastic anemia. Conclusion. Determination of TL in patients with aplastic anemia is modern examination method, which is a necessary step of differential diagnosis between aplastic anemia and dyskeratosis congenita, which is the disease from group of constitutional bone marrow aplasia. It is preferred to identify the TL in adult patients with aplastic anemia by the flow-FISH. It is necessary to investigate the TL to predict treatment response and to identify risks of developing adverse experiences, which include relapse and clonal evolution.

Expression and Clinical Significance of miRNA-495 in the Peripheral Blood of Acute Myeloid Leukemia Patients

Objective: To analyze the expression and clinical significance of miRNA-495 in the peripheral blood of patients with acute myeloid leukemia. Methods: Fifty-six patients with acute myeloid leukemia and 56 healthy controls were selected. Fasting venous blood was drawn and centrifuged, and the plasma was collected. The target miRNA was directly amplified and reverse transcribed into cDNA. The expression of plasma miR-495 was detected by qRT-PCR. Results: The expression level of miRNA-495 in newly diagnosed acute myeloid leukemia (AML [ND]) and relapsed/refractory acute myeloid leukemia (AML [RR]) was significantly lower than that in complete remission (AML [CR]) and normal control group (Control) (p < 0.0001). There was no significant difference between AML (ND) group and AML (RR) group (p > 0.05). The area under the ROC curve (AUC) of miRNA-495 was 0.9503, the 95% confidence interval was 0.9113–0.9892 (p < 0.0001), the standard error was 0.020, the sensitivity and specificity were 91.1% and 92.9%, respectively, and the Jordan index was 0.857. There was no significant difference between the expression level of miRNA-495 and gender, age, leukocyte count, hemoglobin, and platelet count (p > 0.05). However, it was found related to the proportion of primitive bone marrow cells in patients (p = 0.017). Conclusion: The decreased expression of plasma miR-495 in AML patients can be used as a new indicator for the diagnosis and prognosis of AML.

Morphological Characteristics of Bone Marrow Cells in Patients with EB Virus Infection

Review and analyze the characteristics of bone marrow cell morphology in patients with Epstein-Barr virus (EBV) infection, and explore the diagnostic value of bone marrow cell morphology for the early identification of EBV infection. A total of 33 patients with EBV-DNA positive detection in the First Affiliated Hospital of Guangxi Medical University from January 2018 to May 2021 were collected as the research objects. Bone marrow cell morphology and peripheral blood cell analysis were performed, and the significance in disease diagnosis was analyzed by statistical methods. The sampling satisfaction of 33 patients with EBV infection was 100%. In the clinical diagnosis of all cases, 7 cases were IM, 17 cases were EBV-HLH, 3 cases were lymphoma, 2 cases were EBV-associated lymphoid hyperplasia, and 4 cases were not diagnosed. Among them, 31 patients had active bone marrow hyperplasia or above, 26 patients had active granulocytic hyperplasia or above, 21 patients had active erythroid hyperplasia or above, and 17 cases of megakaryocyte production platelet function decreased. The abnormal components of bone marrow mainly indude atypical lymphocyte cells (33 cases), hemophagocytic cells (22 cases), abnormal histiocyte (10 cases). According to the proliferation of granulocytes, erythrocytes and megakaryocytes in the bone marrow, and the emergence of abnormal components such as atypical lymphocytes, hemophagocyte, abnormal histiocyte. Bone marrow cell morphological examination can indicate the possibility of EBV infection, which is certain diagnostic value for early identification of EBV infection.

Longitudinal minimal residual disease assessment in multiple myeloma patients in complete remission \u2013 results from the NMSG flow-MRD substudy within the EMN02/HO95 MM trial

BackgroundMultiple myeloma remains an incurable disease with multiple relapses due to residual myeloma cells in the bone marrow of patients after therapy. Presence of small number of cancer cells in the body after cancer treatment, called minimal residual disease, has been shown to be prognostic for progression-free and overall survival. However, for multiple myeloma, it is unclear whether patients attaining minimal residual disease negativity may be candidates for treatment discontinuation. We investigated, if longitudinal flow cytometry-based monitoring of minimal residual disease (flow-MRD) may predict disease progression earlier and with higher sensitivity compared to biochemical assessments.MethodsPatients from the Nordic countries with newly diagnosed multiple myeloma enrolled in the European-Myeloma-Network-02/Hovon-95 (EMN02/HO95) trial and undergoing bone marrow aspiration confirmation of complete response, were eligible for this Nordic Myeloma Study Group (NMSG) substudy. Longitdudinal flow-MRD assessment of bone marrow samples was performed to identify and enumerate residual malignant plasma cells until observed clinical progression.ResultsMinimal residual disease dynamics were compared to biochemically assessed changes in serum free light chain and M-component. Among 20 patients, reaching complete response or stringent complete response during the observation period, and with ≥3 sequential flow-MRD assessments analysed over time, increasing levels of minimal residual disease in the bone marrow were observed in six cases, preceding biochemically assessed disease and clinical progression by 5.5 months and 12.6 months (mean values), respectively. Mean malignant plasma cells doubling time for the six patients was 1.8 months (95% CI, 1.4–2.3 months). Minimal malignant plasma cells detection limit was 4 × 10–5.ConclusionsFlow-MRD is a sensitive method for longitudinal monitoring of minimal residual disease dynamics in multiple myeloma patients in complete response. Increasing minimal residual disease levels precedes biochemically assessed changes and is an early indicator of subsequent clinical progression.Trial registrationNCT01208766

Transcriptional differences between JAK2-V617F and wild-type bone marrow cells in patients with myeloproliferative neoplasms

The JAK2-V617F mutation is the most common cause of myeloproliferative neoplasms. Although experiments have revealed that this gain-of-function mutation is associated with myeloid blood cell expansion and increased production of white cells, red cells, and platelets, the transcriptional consequences of the JAK2-V617F mutation in different cellular compartments of the bone marrow have not yet been fully elucidated. To study the direct effects of JAK2-V617F on bone marrow cells in patients with myeloproliferative neoplasms, we performed joint single-cell RNA sequencing and JAK2 genotyping on CD34+-enriched cells from eight patients with newly diagnosed essential thrombocythemia or polycythemia vera. We found that the JAK2-V617F mutation increases the expression of interferon-response genes (e.g., HLAs) and the leptin receptor in hematopoietic progenitor cells. Furthermore, we sequenced a population of CD34- bone marrow monocytes and found that the JAK2 mutation increased expression of intermediate monocyte genes and the fibrocyte-associated surface protein SLAMF7 in these cells.

Simultaneous Detection of Single Nucleotide Variants and Expression of RNA and Protein in Multiple Myeloma Bone Marrow Aspirates

Introduction: Multiple myeloma (MM) is generally an incurable plasma cell neoplasm that evolves from a premalignant state known as monoclonal gammopathy of undetermined significance (MGUS). Primary genomic events leading to the development of MGUS include hyperdiploidy, translocations involving the immunoglobulin heavy chain genes, and copy number variations. Single-nucleotide variants (SNVs) as well as insertions and deletions are secondary events that trigger progression to active MM. Identifying nucleotide variants that lead to changes in RNA and protein expression is critically important to understanding the biology of the cancer and ultimately identifying therapeutic targets. Analysis of DNA, RNA, and protein has traditionally involved integration of three separate assays and is frequently limited by the availability of sufficient material from MM patient specimens. We used a more efficient approach that simultaneously detects DNA variants, RNA and protein expression in a single sample from as little as 5 ng DNA, 25 ng RNA, and 250 ng protein. We evaluated the NanoString nCounter® Vantage 3D™ DNA:RNA:Protein Heme Assay performed on 1 x 106cells from bone marrow biopsies obtained from patients with MM.Methods: Bone marrow aspirates from 15 patients diagnosed with MM were obtained through an Institutional Review Board approved protocol. Seven patients had newly diagnosed MM and 8 had relapsed disease. DNA, RNA, and protein were extracted from unsorted, cryopreserved, ficoll-hypaque separated bone marrow mononuclear cells and hybridized with optical barcodes designed to detect 124 DNA somatic variants common to hematologic malignancies as well as 180 mRNA transcripts and 38 total and phospho-proteins. We also used the nCounter PanCancer Pathways Panel Assay to measure gene expression of 770 additional genes from 13 cancer-associated canonical pathways. The hybridized probes in each sample were counted using the nCounter Digital Analyzer and statistical analysis was performed using nSolver™ v4.0 alpha software.Results: The percentage of CD138+ bone marrow plasma cells measured by immunohistochemistry at the time of biopsy varied from 44 to 100% in each sample. One or more DNA variants were detected in 3 pre-treatment and 4 relapse samples. Mutations in KRAS, NRAS, and BRAF were most common and 2 patients were found to have variants in both KRAS and NRAS or KRAS and BRAF . Additionally, variants in DNMT3A and IDH2 were detected in 1 patient each. Since KRAS, NRAS, and BRAF are all members of the mitogen-activated protein kinase (MAPK) signaling pathway, a differential gene expression analysis of both RNA and protein was performed comparing samples with and without mutations in these genes. Findings confirmed that a higher level of expression was detectable in MAPK pathway genes, although this result did not reach statistical significance following correction for replicate testing, potentially the consequence of a small sample size.Conclusions: The Vantage 3D DNA:RNA:Protein Heme Assay is a rapid, sensitive and resource sparing platform for the simultaneous detection of DNA variants, RNA transcripts, and protein expression. We show that this method can feasibly be performed on as few as 1 x 106 cryopreserved bone marrow cells in patients with MM and our findings confirm detection of 3 of the most common MM variants. This technology should have broad application in other hematological malignancies and further evaluation of blood and lymph node biopsies from fresh, frozen and formalin-fixed paraffin-embedded (FFPE) samples is warranted. DisclosuresDemirkan:NanoString Technologies: Employment. Meredith:NanoString Technologies: Employment. Meredith:NanoString Technologies: Employment.

A Phase I/II, Prospective Multi-Center Study of the Safety and Preliminary Efficacy of Intravenous Allogeneic Mesenchymal Bone Marrow Cells in Chronic Ischemic Stroke

Abstract INTRODUCTION Stroke is a leading cause of disability, affecting patients for years. We examined safety and preliminary efficacy for a cellular therapy targeting patients with chronic disabling stroke, a population for whom limited treatment options currently exist. METHODS Entry criteria included ischemic stroke > 6 mo prior, substantial disability (subject confined to wheelchair, had home-nursing care, or needed assistance with activities of daily living), and NIH Stroke Scale (NIHSS) score = 6 to 20. Enrollees received a single intravenous dose of allogeneic mesenchymal bone marrow cells. Phase 1 used a dose escalation design (3 tiers, n = 5 each). Phase 2 (n = 21) was an expanded safety cohort. The primary endpoint was safety over 1-yr. Secondary endpoints examined behavior, with a pre-specified focus at 6-mo. RESULTS In phase 1, each dose (0.5, 1.0, and 1.5 million cells/kg body weight) was found safe, so phase 2 subjects received 1.5 million cells/kg. At baseline, subjects (n = 36) averaged 4.2 ± 4.6 yr poststroke, age 61.1 ± 10.8 yr, NIHSS score 8.7 ± 3.0, and Barthel Index 65 ± 29. Two were lost to follow-up, one was withdrawn, and two died (unrelated to study treatment). There were 15 serious adverse events, none possibly or probably related to study treatment. Two adverse events were possibly related to study treatment, a UTI and IV site irritation. Treatment was safe based on serial exams, EKGs, laboratory tests, and pan-computed tomography scans. Change from baseline to 6-mo post-transfusion was significant for all 4 secondary endpoints: Barthel Index (6.8 ± 11.4 points, P = .002), NIHSS (−1.25 ± 1.7 points, P < .001), MiniMental Status Exam (1.8 ± 2.8 points, P < .001), and Geriatric Depression Scale (−1.6 ± 3.8 points, P = .015). At baseline 11.4% (4/35 subjects) had Barthel Index = 95 to 100 (favorable outcome); at 6-mo, 27.3% (9/33); by 12-mo, 35.5% (11/31). CONCLUSION Transfusion of mesenchymal bone marrow cells in patients with disabling chronic stroke was safe and significantly improved several behavioral measures. The initial results were potentially equivalent to or exceeded those of prior studies utilizing stereotactic implantation. These data support proceeding to a randomized, placebo-controlled study.

PS1327 PRO‐NECROPTOTIC RIPK3 AS A STAGE‐DEPENDENT MARKER IN THE BONE MARROW OF PATIENTS WITH MDS AND CMML

Background:Chronic inflammation plays a central role in the development of hematological neoplasms. In Myelodysplastic syndromes (MDS) pro‐inflammatory microenvironment is suspected to drive disease development. Several studies have shown increased rates of programmed cell death in marrow cells of patients with early stage MDS. In these patients improvement of cytopenia is a major therapeutic approach. Overcoming resistance to programmed cell death is a central therapeutic aim in patients with high‐risk MDS/sAML. RIPK3 is a crucial player of regulated necrosis (necroptosis). Necroptosis has a strong inflammatory capacity and pro‐inflammatory processes trigger necroptosis.Aims:The aim of this project is to identify pro‐necroptotic proteins in the bone marrow of patients with MDS, CMML and sAML and to correlate the necroptotic capacity with the stage of the disease. By pharmacological modulation of necroptosis we would like to evaluate the effects of necroptotic signaling on inflammation, cell death and cell differentiation.Methods:We therefore analyzed the impact of pro‐necroptotic signaling on bone marrow bulk and the subset of CD34+stem/progenitor cells of patients with MDS and chronic myelomonocytic leukemia (CMML) in vitro. Bone marrow mononuclear cells (BMMNCs) were isolated from BM aspirates of 5 patients with early MDS (MDS‐MLD, MDS‐RS‐SLD and MDS‐RS‐MLD), 5 patients with late MDS (MDS‐EB1 and MDS‐EB2), 5 patients with CMML‐1, 7 patients with CMML‐2 and 4 patients with sAML after a history of MDS and 4 patients with sAML after a history of CMML. Bulk of BMMNCs and purified CD34+stem/progenitor cells were stained intracellular for RIPK3 and analyzed by flow cytometry. The patient samples were compared to 12 age‐matched BM samples obtained from hip replacement surgery from otherwise healthy individuals. Furthermore, immunohistochemistry was performed in an enlarged cohort of patients. Therefore paraffin embedded bone marrow biopsies of individuals with MDS or CMML‐1/2 were analyzed over time and disease progression. Additionally BMMNCs of patients with MDS were treated with specific RIPK1 (Nec1 s) and RIPK3 (GSK 843A) inhibitors for 72 h. Short term viability analysis by flow cytometry were performed and long‐term analysis using growth factor enriched methylcellulose.Results:Expression of RIPK3 was significantly increased in sAML after a history of MDS compared to late MDS and compared to healthy controls. Furthermore patients with sAML after a history of CMML showed a significantly higher expression of RIPK3 than patients with CMML‐1, CMML‐2 and healthy controls. These findings were confirmed by immunohistochemistry in an enlarged cohort of patients. Here paraffin embedded bone marrow biopsies of individuals with MDS or CMML‐1/2 were analyzed over time and disease progression.Additionally BMMNCs of patients with MDS or CMML were treated with specific RIPK1 (Nec1 s) and RIPK3 (GSK 843A) inhibitorsfor 72 h. Short term viability analysis by flow cytometry showed no impact of inhibitor treatment. However after culture in growth factor enriched methylcellulose for 10–14 days colony forming capacity was significantly increased in low‐risk MDS patients after inhibition of pro‐necroptotic signaling. Here increase in colony numbers was not restricted to a specific lineage.Summary/Conclusion:We conclude that RIPK3 plays a critical stage‐dependent role in patients with MDS and CMML. Disease progression to sAML is associated with an increased RIPK3‐Expression. We further hypothesize that pro‐inflammatory processes like necroptosis have an impact on aberrant myeloid homeostasis.

SS20. Medium-term Effect of Intramuscular Injection of Autologous Bone Marrow Cells in Patients With Critical Limb Ischemia: Two-Year Follow-up of the MOBILE Trial

SS20. Medium-term Effect of Intramuscular Injection of Autologous Bone Marrow Cells in Patients With Critical Limb Ischemia: Two-Year Follow-up of the MOBILE Trial

Choice of proper approach for the assessment of plasma cells in the bone marrow of patients with monoclonal gammapathies

Choice of proper approach for the assessment of plasma cells in the bone marrow of patients with monoclonal gammapathies

Intramyocardial bone marrow cell injection does not lead to functional improvement in patients with chronic ischaemic heart failure without considerable ischaemia.

BackgroundIt has been suggested that bone marrow cell injection may have beneficial effects in patients with chronic ischaemic heart disease. However, previous trials have led to discrepant results of cell-based therapy in patients with chronic heart failure. The aim of this study was to evaluate the efficacy of intramyocardial injection of mononuclear bone marrow cells in patients with chronic ischaemic heart failure with limited stress-inducible myocardial ischaemia.Methods and resultsThis multicentre, randomised, placebo-controlled trial included 39 patients with no-option chronic ischaemic heart failure with a follow-up of 12 months. A total of 19 patients were randomised to autologous intramyocardial bone marrow cell injection (cell group) and 20 patients received a placebo injection (placebo group). The primary endpoint was the group difference in change of left ventricular ejection fraction, as determined by single-photon emission tomography. On follow-up at 3 and 12 months, change of left ventricular ejection fraction in the cell group was comparable with change in the placebo group (P = 0.47 and P = 0.08, respectively). Also secondary endpoints, including left ventricle volumes, myocardial perfusion, functional and clinical parameters did not significantly change in the cell group as compared to placebo. Neither improvement was demonstrated in a subgroup of patients with stress-inducible ischaemia (P = 0.54 at 3‑month and P = 0.15 at 12-month follow-up).ConclusionIntramyocardial bone marrow cell injection does not improve cardiac function, nor functional and clinical parameters in patients with severe chronic ischaemic heart failure with limited stress-inducible ischaemia.Clinical Trial Registration: NTR2516Electronic supplementary materialThe online version of this article (10.1007/s12471-018-1213-2) contains supplementary material, which is available to authorized users.

Study on the Relativity between Cytogenetics and Cytomorphology and its Prognosis Significance in Children with Acute Myelogenous Leukemia

Objective: The main objective of this study was to retrospectively evaluate that the cytogenetic abnormalities is an important prognostic factor for the cure of acute myeloid leukemia (AML).Methods: This retrospective study enrolled newly diagnosed 70 cases (37 males and 33 females, aged 10.1 months to 14.5 years) of pediatric patients with AML during 2010 January - 2016 February from the Second Affiliated Hospital of Anhui Medical University. Excluding criteria were cases secondary to treatment-related MDS and AML. Samples were obtained from bone marrow cells in patients after treatment on the anterior superior iliac spine, blood diseases laboratory by direct culture or 24/48 hour short-term culture, G -banding technique for testing. Follow-up of 1 - 60 months, the analysis of treatment response rates of different karyotypes, distribution ratios in various subtypes, normal karyotype and abnormal karyotype. ISPSS17.0 software statistics was used for statistical analysis. Groups were compared using chi-square test; Survival rate was calculated by method of Kaplan Meier and survival difference between groups were compared with breslow test.Results: Among 70 cases, 42 cases were detected for chromosomal abnormalities (i.e. 60% of the total number of cases), M3 abnormal karyotype distortion rate of 78.5%, M2 abnormal karyotype aberrations 63.3%, M4 60.0%, M1 50%, M5 lowest 38.9 %, M7 nuclear aberrations highest rate was 100%. Total chromosomal aberration rate was 60%. Acute myeloid leukemia cases, t (8; 21) at most, there are 15 cases, and the presence of abnormal karyotype 86.7% in the original part of differentiated myeloid leukemia (M2); t (15; 17) has 11 cases, exists only in acute promyelocytic cell leukemia (M3). After treatment, the remission rate of t (8; 21) was 80%; the remission rate of t (15; 17) was 90%; the remission rate of other abnormal karyotype abnormalities was 50%; the remission rate of total abnormal karyotype was 71.4%. The event free survival rate was significantly different between normal karyotype, t (8; 21), t (15; 17) and other abnormal karyotype groups (P<0.05).Conclusions: Acute myeloid leukemia karyotype abnormalities among FAB subtypes are different; M3 is the highest rate of abnormal karyotype aberrations, M2, M4 medium, M5 minimum. t (15; 17) seen in acute promyelocytic leukemia (APL), prognosis is good; t (8; 21) is more common in M2, prognosis is good, also found in M4 and M5, worse prognosis; +8 Abnormalities found in AML M2, M3, M4, M5 and M6 subtypes, prognosis medium; inv (16) high white blood cells, low platelet poor prognosis, AML patients with normal karyotype prognosis medium. J-GMC-N | Volume 11 | Issue 01 | January-June 2018, Page: 35-41

Abstract 3916: Disease-specific markers may be used for the identification of disseminated tumor cells in the bone marrow of patients with prostate cancer

Abstract Introduction: The detection of microscopic disseminated disease in prostate cancer has largely been attempted with the study of circulating tumor cells (CTCs). Little is known about the character and clinical significance of disseminated tumor cells (DTCs) that have reached the bone marrow (BM) niche, and prior studies has relied on epithelial markers. Yet, it is known that such markers are also expressed on normal erythroid precursor cells in the BM, thus we developed an assay for prostate-specific DTC identification. Materials and Methods: BM aspirates from 18 men with localized and 2 men with metastatic prostate cancer were collected at time of RP or clinic visit respectively, and processed with the AccuCyte system (RareCyte, Inc., Seattle, WA). Slides were immunostained with DAPI (nuclear), anti-pan-cytokeratin (epithelial), anti-CD45/CD66b/CD11b/CD14/CD34 (white blood cell), and the prostate-specific markers HOXB13 and NKX3.1 (prostate). DTCs were detected using the CyteFinder system. DTCs were required to have positive prostate channel staining and no white blood cell signal. Results: DTCs were present in 89% (16/18) of patients at time of RP, with range 0 to 3592 cells/4 mL sample (average 270, median of 39.5). Two patients with metastatic prostate cancer had 52 and 105 DTCs/4mL BM (Table 1). Notably, only 5% of all DTCs were epithelial marker positive (CK+), and only 44% (8/18) of patients had any CK+ DTCs. Conclusions: The RareCyte system is a promising selection-free system for DTCs detection that does not rely on epithelial markers. Here we report on a novel assay to distinguish DTCs from other cells in the bone marrow using the presumed prostate-specific markers HOXB13 and NKX3.1. Furthermore, we show that the majority of prostate-specific marker positive DTCs did not express the epithelial marker CK. Analyses of the association of DTC count with clinicopathologic variables are ongoing. Table 1.Clinicopathologic Data and DTC Counts of the CohortBxRP GleasonMetastatic/LocalizedPSA# of DTCsCK+ DTCsCK- DTCs% of CK- DTCsGleason3+3=63+3=6Localized4.3000N/A3+3=63+3=6Localized6.3909100%3+3=64+3=7Localized3.948123675%3+3=63+4=7Localized3.4615015100%3+4=73+4=7Localized525025100%3+4=73+4=7Localized4.527918100%3+4=73+4=7Localized8.112012100%3+4=73+4=7Localized7.534034100%3+4=73+4=7Localized6.781216074%4+3=74+3=7Localized16.3114249079%4+3=74+3=7Localized5.91352710880%4+3=73+4=7Localized8.345045100%4+3=73+4=7Localized3.12254817779%4+3=74+3=7Localized942911731273%4+3=73+4=7Localized4.3359203592100%4+5=94+5=9Localized10.1000N/A4+5=94+4=8Localized9.1606100%4+5=94+5=9Localized9.46095185%4+5=9N/AMetastatic5.79105159086%4+5=9N/AMetastatic655284485% Citation Format: Emma E. van der Toom, Stephanie A. Glavaris, Michael A. Gorin, James A. Verdone, Kenneth J. Pienta, Heather J. Chalfin. Disease-specific markers may be used for the identification of disseminated tumor cells in the bone marrow of patients with prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3916. doi:10.1158/1538-7445.AM2017-3916

Ultrastructure of bone marrow cells in patients with autoimmune pathologies

Objective: To determine the morpho-functional state of bone marrow autologous cells at the ultrastructural level. Material and Methods: For the electron microscopic examination, bone marrow cells taken from patients with autoimmune diseases were fixed in a 2.5% solution of glutaraldehyde with post fixation in a 1% solution of osmium tetroxide, was performed according to the conventional method and enclosed in epon. Ultrathin sections were prepared on a Leica ultramicrotome, contrasted with lead citrate by Reynolds, and examined in an electron microscope called Libra 120 (C. Zeiss). Results: Morpho-functional state of autologous bone marrow cells in severe chronic pathology was characterized by mitochondrial dysfunction and signs of insufficiency of microvesicular secretory function. Conclusion: Precultivation of bone marrow cells of patients with severe autoimmune diseases restored the energetic function of mitochondria, increased the secretory activity of endosomes.

Abstract 3233: Macrophages orchestrate early dissemination of HER2+ cancer cells

Abstract Metastatic dissemination was proposed to occur only in invasive cancer. However, detection of cancer cells in the bone marrow of patients with pre-invasive breast ductal carcinoma in situ (DCIS) showed that dissemination could, through unknown mechanisms, take place earlier. Late evolved invasive breast cancer cells disseminated by recruiting circulating monocyte-derived tumor associated macrophages (TAMs). We therefore investigated whether macrophages might actively participate in early dissemination. We show that pre-malignant cancer cells in MMTV-HER2 mice produced CCL2 in an NFkB dependent manner and thereby recruited resident mammary tissue macrophages into the duct, months before monocyte-derived TAMs arrived. Intra-epithelial mammary tissue macrophages induced an epithelial-to-mesenchymal transition in early HER2+ cells in a Wnt-dependent manner and thereby drove early dissemination. Consequently, depletion of macrophages from pre-malignant MMTV-HER2 mice significantly reduced early dissemination of pre-malignant cancer cells as measured by reduction in levels of circulating cancer cells and disseminated cancer cells in lungs. Interestingly, macrophage depletion during pre-malignant stages alone was sufficient to reduce the onset of late metastasis in MMTV-HER2 mice, indicating that early disseminating cancer cells contributed to late metastasis formation. In humans DCIS samples we frequently found intra-epithelial macrophages inside of ducts and their presence correlated with reduced E-Cadherin levels. Finally, a first pilot study indicated that those patients whose DCIS lesions contained macrophage +/E-Cadherin low microenvironments frequently had DCCs in the bone marrow. Taken together, we reveal that resident mammary tissue macrophages can promote early dissemination, and unravel a mechanism how early cancer spread might proceed in breast cancer patients and in other cancers where metastasis are diagnosed and the primary tumor is never detected (cancer of unknown primary). We also propose that early DCCs play a long-term causal role in metastasis development when patients are diagnosed in late stages, implying that the heterogeneity of metastases is higher than we might anticipate. Citation Format: Nina Linde, Arthur Mortha, Nicole Saenger, Maria Soledad Sosa, Kathryn Harper, Ethan Tardio, Tanja Fehm, Thomas Karn, Miriam Merad, Julio A. Aguirre-Ghiso. Macrophages orchestrate early dissemination of HER2+ cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3233.

Abstract P4-04-07: Association between tumor infiltrating immune cells and disseminated tumor cells in the bone marrow of patients with primary breast cancer: A ten year follow-up

Abstract Background: Disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer patients (pts) were shown to be an independent prognostic factor with regard to reduced PFS and OS. Although this negative prognostic impact has been related to chemotherapy resistant and stem cell-like cells, the microenvironment of the primary tumor might also influence tumor cell spread and thus, outcome of the disease. After a ten year follow-up, we here analyzed the prognostic significance of infiltrating leukocytes in the tumor (TILs) and stroma (SILs) with regard to PFS and OS as well as their association with DTCs in 163 pts with primary breast cancer, diagnosed between 2001 and 2004. Patients and Methods: Tissue microarrays (TMA) were stamped from paraffin embedded specimens and 5µm thick sections were subjected to immunohistochemistry using antibodies against CD3 (DCS, Hamburg, Germany), CD68, CD20, CD8 (all Dakocytomation, Glostrup, Denmark), CD4 (Zytomed Systems, Berlin, Germany) and CD45R0 (Labvision, Fremont, USA), respectively. Each TMA has been evaluated for TILs and SILs assessing intraepithelial and stromal tumor areas occupied by respective immune cells separately: score 0: no staining; score 1: ≤ 10%; score 2: 11-60%; score 3: ≥61%, respectively, using light microscopy at magnifications of x100-200. Statistical analysis was conducted with the R programming language (version 3.20) and Cox proportional hazard regression and the Kruskal-Wallis test. DTCs were studied at the time of primary diagnosis using immunocytochemistry, applying the pan cytokeratin (CK) antibody A45-B/B3. Results: Univariate analyses showed significant associations between prolonged OS and high rates of CD8TIL (p=0.01) and CD4TIL (p=0.028). In the subgroup of triple-negative carcinomas, CD3 TIL and CD8SIL were positively correlated with OS (p=0.038 and p=0.046, respectively), whereas CD8TIL was predictive in HR+/HER2- tumors (p=0.029). In a multivariate model, only CD4TIL was significantly associated with longer OS (p=0.049) in the total group of 163 pts, whereas in the triple-negative subgroup, CD3TIL was significantly correlated with favorable outcome (p=0.044). In contrast, no significant associations were found between TILs/SILs and PFS. The results for DTCs were available for 92/163 pts resulting in a positivity rate of 53%. Intriguingly, despite failing overall associations between DTCs and TILs/SILs, an unfavorable CD4SIL/CD8SIL ratio in the tumors of DTC-positive pts predicted significantly shorter PFS and OS (p=0.009 and p=0.018, respectively). In the DTC-negative subgroup, high CD4SILs were a prognostic marker for shorter OS. Notably, no significant associations were found between CD20, CD45R0 and CD68 and any parameter tested. Conclusion: Analyzing a variety of leukocyte subtypes, we here demonstrate that especially the distribution of CD4 and CD8 in the primary tumor and their ratio seems to be involved in the prognosis of DTC-positive as well as DTC-negative primary breast carcinomas. Since the results for DTCs were only available in a subgroup of pts, further studies will have to prove these findings. Citation Format: Kasimir-Bauer S, Spiekermann A, Friedrich CM, Hoffmann O, Schmid KW, Kimmig R, Bankfalvi A. Association between tumor infiltrating immune cells and disseminated tumor cells in the bone marrow of patients with primary breast cancer: A ten year follow-up. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-07.

A Comparison of Myelopoiesis from Induced Pluripotent Stem Cells with a Mutation in ELANE between Cyclic Neutropenia and Severe Congenital Neutropenia

The ELANE is known as the responsible gene for both cyclic neutropenia (CyN) and severe congenital neutropenia (SCN). However, relations between mutations in the ELANE gene and abnormal myelopoiesis in the different phenotype of these diseases still remain unclear. It has been reported that induced pluripotent stem cell (iPSC) from an individual patient with SCN (SCN-iPSC) demonstrated maturation arrest of myeloid progenitor cells and poor response to granulocyte-colony stimulating factor as similarly observed in patient's bone marrow cells. Thus, the study on myelopoiesis using disease specific iPSC seems to provide disease pathogenesis as a novel in vitro experimental model.In this study, we established iPSC line from an individual patient with CyN (CyN-iPSC) with heterozygous mutation in ELANE gene (Exon5, R191Q point mutation). Then we compared myelopoiesis among healthy Control-iPS (253G1), SCN-iPS (Exon5, C194X point mutation) , and CyN-iPSC. Undifferentiated colonies derived from CyN-iPSC were staind with pluripotency markers (OCT3/4 and NANOG). CyN-iPSC retained a normal karyotype and ELANE locus mutation of the original samples.In vitro myelopoiesis was examined by using a serum- and feeder-free monolayer hematopoietic culture system. iPSC colonies were cultured on growth factor-reduced Matrigel-coated cell culture dishes in modified Tenneille Serum Replacer 1 (mTeSR™1) medium (StemCell Technologies, Inc.), containing BSA, rh bFGF, rh TGFβ, Lithium Chloride, Pipecolic acid, GABA. Medium was replaced every four days. Then medium was changed to StemPro®-34 SMF Complete Medium plus nutrient supplement (Life technologies Corp.). The iPSC were cultured with BMP4 (80 ng/mL) for four days, and then replaced with VEGF165 (80 ng/mL), bFGF (25.7 ng/mL), and SCF (100 ng/mL) on Day 4. On Day 6, cytokines were replaced with a combination of SCF (50 ng/mL), IL-3 (50 ng/mL), and G-CSF (50 ng/mL). Medium was replaced every 3 - 4 days.No significant difference in the ratio of proliferating CD33+ cells were noted between CyN-iPSCs and Control-iPSCs. CyN-iPSCs showed less capability in the proliferation and maturation for CD15+ cells on days 20 to 40 than Control-iPSCs. The decreased number of CD15+ cells derived from CyN-iPSc implies the defect in mature neutrophil survival. In contrast, CD15+ / CD33+ cells derived from SCN-iPSCs were hardly observed in this culture condition, suggesting the defects of proliferation and maturation in SCN-iPSCs.We next examined the colony formation of CD34+ cells derived from CyN-iPSCs, Control-iPSCs, and SCN-iPSCs. CD34+ cells were obtainded at the day 12 of primary culture of iPSCs and purified by cell sorting using FACS-Aria®. No significant differences in the number of G-colony and GM-colony between CD34+ cells from CyN-iPSCs and Control-iPSCs. In contrast, CD34+ cells from SCN-iPSCs gave rise to the significantly decreased number of G-colony and GM-colony.The observations of myeloid proliferation/maturation and colony formation of CD34+ cells were almost compatible with those obtained from bone marrow cells in patients with SCN and CyN. Furthermore, neutrophils differentiated from CyN-iPSCs showed the excessive cell death, whereas SCN-iPSCs presented the defective myelopoiesis. These results suggest that the analyses using CyN-iPSCs and SCN-iPSCs may be useful tool for investigating the relation of gene mutation and pathophysiology in both diseases. DisclosuresNo relevant conflicts of interest to declare.

3200 Effect of chemotherapy drugs in vitro on the death of bone marrow cells in patients with multiple myeloma

3200 Effect of chemotherapy drugs in vitro on the death of bone marrow cells in patients with multiple myeloma

Bone Marrow Transplantation Followed by Growth Factors Injection in a Patient with Femoral Neck Nonunion: A Case Report

Background: Most fractures of femoral neck typically heal, but a significant proportion of fractures fail to heal, resulting in aseptic bone necrosis, delayed union or persistent nonunion. Specially, nonunion of fractured neck of femur in young adults is a serious problem. Some preclinical evidences show the therapeutic potential of autologous bone marrow for bone fracture healing; however, clinical outcome following of transplantation of bone marrow cells in patients with femoral neck nonunion has never been reported. Case description: We describe a 57-year-old male who had a femoral neck nonunion and received autologous bone marrow transplantation, followed by growth factors injection. The primary endpoint of this study is to achieve radiological fracture healing (union) by evaluating anteroposterior images taken after and on 6th month, following bone marrow therapy and no serious adverse event occurred. Literature review: To our knowledge, this is the first case in the literature of nonunion of femoral neck fracture treated with percutaneous autologous bone marrow transplantation. Clinical relevance: This case suggests that autologous bone marrow transplantation, followed by plasma rich in growth factors (PRGF) could be considered as an exciting option to treat nonunion in femoral neck fracture.

Characterization of the Hematopoietic Stem and Progenitor Cell Hierarchy in Myelodysplastic Syndromes Patients with Monosomy 7 As the Sole Cytogenetic Abnormality

Deletions involving chromosome 7 constitute the second most common cytogenetic abnormality in myelodysplastic syndromes (MDS), associated with an overall poor outcome and high risk of transformation to acute myeloid leukemia (AML). Previous studies focusing on low to intermediate risk MDS, including cases with del(5q) as the only cytogenetic abnormality, have revealed a remarkably preserved stem and myeloid progenitor cell hierarchy, more characteristic of normal hematopoiesis than that of AML. Herein we explored this in a group of seven higher-risk MDS cases, all with an isolated deletion of the entire chromosome 7 (-7; monosomy 7), as the only cytogenetic abnormality.In all investigated isolated monosomy 7 cases, the expression of cell surface markers used to define the normal hematopoietic stem and myeloid progenitor cell hierarchy in bone marrow resembled more the expression pattern observed in AML than in low-risk MDS, irrespectively of the -7 MDS patients blast counts. Specifically, when compared to healthy age-matched controls, we observed a 15-fold reduction of LIN-CD34+CD38-CD45RA-CD90+ cells (hematopoietic stem cells; HSCs, p=0.010), a 15-fold increase in LIN-CD34+CD38+CD123+CD45RA+ cells (Granulocyte-macrophage progenitors; GMPs, p=0.038) and a 300-fold increase of LIN-CD34+CD38-CD90-CD45RA+ cells (lymphoid-primed multipotent progenitors; LMPPs, p=0.050) within total bone marrow mononuclear cells of MDS patients with isolated monosomy 7. Fluorescence in situ hybridization (FISH) analysis revealed high -7 clonal involvement (>80%) for all investigated progenitor populations, whereas it was much lower (<20%) in the HSC compartment. Functional assays (colony forming unit assays, long term culture initiating cell assays and mouse xenotransplantation in-vivo) revealed dramatic reductions in stem and progenitor activities in the investigated -7 patients, more resembling that previously reported in AML than in low risk MDS cases. Various differentiation assays supported that the -7 clone was much less affected in its myeloid than erythroid differentiation pathway. Similar to -7, also identifiedconcomitant recurrent driver mutations showed high clonal involvement. Notably, while a 3- to 4-fold reduction of -7 clonal involvement was observed in total bone marrow cells in patients achieving good partial remissions in response to azacytidine, the treatment failed to significantly impact on the high -7 clonal involvement in expanded GMPs and LMPPs.In conclusion, investigations of the stem and progenitor cell compartments of MDS patients with loss of chromosome 7 as the sole cytogenetic abnormality, is associated with a loss of HSC phenotype and function, and an expansion of cells with a GMP and LMPP phenotype, remarkably similar to what is frequently observed in AML, and in contrast to reported low-risk MDS cases. While azacytidine treatment significantly reduces the overall clonal involvement in bone marrow, it fails to significantly affect the -7 clonal involvement in notably expanded progenitor cell populations, providing an explanation for the typically short duration of remissions in these patients, and identifying distinct and potentially important target cells for future treatments. DisclosuresNo relevant conflicts of interest to declare.