Marrow Cells Research Articles

Differential immune landscapes in appendicular versus axial skeleton.

Roughly 400,000 people in the U.S. are living with bone metastases, the vast majority occurring in the spine. Metastases to the spine result in fractures, pain, paralysis, and significant health care costs. This predilection for cancer to metastasize to the bone is seen across most cancer histologies, with the greatest incidence seen in prostate, breast, and lung cancer. The molecular process involved in this predilection for axial versus appendicular skeleton is not fully understood, although it is likely that a combination of tumor and local micro-environmental factors plays a role. Immune cells are an important constituent of the bone marrow microenvironment and many of these cells have been shown to play a significant role in tumor growth and progression in soft tissue and bone disease. With this in mind, we sought to examine the differences in immune landscape between axial and appendicular bones in the normal noncancerous setting in order to obtain an understanding of these landscapes. To accomplish this, we utilized mass cytometry by time-of-flight (CyTOF) to examine differences in the immune cell landscapes between the long bone and vertebral body bone marrow from patient clinical samples and C57BL/6J mice. We demonstrate significant differences between immune populations in both murine and human marrow with a predominance of myeloid progenitor cells in the spine. Additionally, cytokine analysis revealed differences in concentrations favoring a more myeloid enriched population of cells in the vertebral body bone marrow. These differences could have clinical implications with respect to the distribution and permissive growth of bone metastases.

IL-4 Serum Level Estimation in Myeloproliferative Neoplasm Patients

Back Ground: Myeloproliferative neoplasm (MPN) is a long-term blood disease that has an excess production of mature hematopoietic pluripotent stem cells in the bone marrow. In the early fifties, W. Dameshek structured the Myeloproliferative disorders that are at present the World Health Organization (WHO) changed it to Myeloproliferative Neoplasms (MPNs). According to the Iraqi cancer registry, Chronic Myeloproliferative disorders in the male is 0.62% and the incidence rate is 0.36, in female Chronic Myeloproliferative disorders (45 case) is 0.31% and the incidence rate is 0.24. The JAK2-V617F genetic mutation is approximately seventy percent of the Myeloproliferative Neoplasm cases. Interleukin-4 plasma and serum levels are significantly increased in MPNs different types. 
 Objectives: The goal of this study is to estimate the IL-4 serum levels in the JAK2-V617F negative and positive mutation in the Iraqi MPNs patients.
 Materials and Methods: Total of (60) patients screened by cohort prospective study of having MPN who are patients presented to the National Center of Hematology / Al-Mustansiriyah University. Depending on the JAK2-V617F genetic mutation we classified our MPNs cases into 3 groups: JAK2-V617F negative (N: 20), JAK2-V617F positive (N: 40) and control group (10). Blood sample (5) ml was obtained from each individual in each group, by venipuncture using disposable syringes for IL-4 serum estimation by Enzyme Linked ImmunoSorbent Assay (ELISA) technique. 
 Results: A clear indication of significant differences was observed between IL-4 serum level in JAK2-V617F negative samples and control samples (P < 0.05). 
 Conclusion: The IL-4 serum level is high in MPNs patients, which is one of the immune evading mechanisms of the cancerous acting to imbalance the Th1/Th2 ratio and enhancing the anti-apoptotic activity inside those cells.

Single-cell sequencing reveals MYC targeting gene MAD2L1 is associated with prostate cancer bone metastasis tumor dormancy

BackgroundAmong malignant tumors, bone metastasis is frequently associated with prostate cancer which is seen in about 80% of patients. During cancer treatments, some tumor cells switch to a "dormant mode" to help tumor cells avoid attack from the immune system and anti-tumor therapies. In this dormant mode, tumor cells can be resuscitated, causing cancer to reoccur. The generally accepted explanation for this phenomenon is that the tumor cells have spread to the bone marrow before treatment and are dormant in the bone marrow. However, the key mechanism for inducing and maintaining the dormancy of these prostate cancer disseminated tumor cells in the bone marrow is still unclear. Therefore, studying the dormancy mechanism of tumor cells in bone metastasis is of great significance for the treatment and the prevention of recurrence of prostate cancer.MethodsWe obtained single-cell RNA-seq data of tumors from mouse models of prostate cancer bone metastasis mouse model numbered (GSE147150) from the GEO database, and obtained RNA-seq expression data and clinical information from The Cancer Genome Atlas Program (TCGA) of prostate cancer patients from the USCS Xena database. Screening of differential genes and annotation of GO functions were performed separately. Subsequently, the screened differential genes were compared and analyzed with 50 classic Hallmark signaling pathways, and the prognosis analysis of prostate cancer patients in TCGA data was performed to discover the key genes of the dormant mechanism of tumor cells in bone metastasis, and obtain new biomarkers that can be used to predict the prognosis of patients.ResultsA total of 378 differentially expressed genes were screened, of which 293 were significantly up-regulated and 85 were significantly down-regulated. Among them, the up-regulated genes were mainly related to the immune response, and the down-regulated genes were mainly related to the cell cycle. Through GSVA (Gene set variation analysis), it is found that there are differences in a total of 3 signal pathways: COMPLEMENT, MYC_TARGETS_V1 and MYC_TARGETS_V2. By comparing and analyzing the significantly down-regulated genes in dormant tumor cells with MYC_TARGETS_V1, MYC_TARGETS_V2, three significantly down-regulated genes were obtained: Ccna2, Mad2L1 and Plk1.ConclusionIn summary, our findings indicate that the MYC targeting gene Mad2L1 is potentially related to the dormancy mechanism of prostate cancer. At the same time, Mad2L1, a gene associated with dormant prostate cancer cells, may be used as a biomarker for prognostic survival.

RELATIONSHIP BETWEEN HEMOGLOBIN AND PLATELET COUNT AMONG MBBS STUDENTS

Introduction: Haemoglobin is the red colour pigment present inside RBC. All the blood components are derived from the haematopoietic stem cells of the same bone marrow. Factors affecting the bone marrow also affects all the cells including the haemoglobin level and the platelet count. Less is known on the relationship between low or high level of haemoglobin on platelet count. This work was therefore carried out to determine this relationship. Materials And Methods: The study was done in Dept. of Physiology, Jorhat Medical College. Study population included 100 undergraduate medical students of age 18-25 years . Haemoglobin Estimation & Platelet Count will be measured by ACCUREX ACCULAB CBC 360+ HEMATOLOGY Analyser Machine. Ethical Clearance was obtained from Institutional Ethical Committee (H), Jorhat Medical College, Jorhat. Students unpaired t- test was used to compare the mean values of study variables and other parameters in relation to the study. The P value <0.05 was considered as significant. Results: We found that mean Hb count is 10.91±1.05 and 10.22±0.83 respectively when platelet count in between 1.5- 2.5 lakhs/mm3 and >2.5 lakhs/mm3. Mean Platelet count came 2.59±0.58 lakhs/mm3 and 1.89±0.62 respectively when Hb between 9 – 11 gm/dl and >11 gm/dl. Conclusion: The reason for the relationship between low and high platelet count on hemoglobin level may be linked to the normal physiology of hematopoiesis where the origin of all blood components occurs in the bone marrow. This leaves scope for further research to use in therapy like Plateletpheresis where lower hemoglobin concentrations meant higher platelet yield and vice versa.

Abstract P2-02-04: In early breast cancer, the ratios of neutrophils, monocyctes and platelets to lymphocytes significantly correlate with the presence of subsets of circulating tumor cells in blood and disseminated tumor cells in the bone marrow

Abstract Background: Circulating tumor cells (CTCs) are the precursors of metastasis and while travelling through the peripheral blood, they crosstalk with different types of blood cells before they finally reach distant organs to settle down as disseminated tumor cells (DTCs). Proinflammatory markers include the neutrophil lymphocyte ratio (NLR), the platelet lymphocyte ratio (PLR) as well as the monocyte lymphocyte ratio (MLR) and the presence of tumor cells as well as specific proinflammatory markers are two independent predictors of worse outcome in breast cancer (BC). However, little is known about the correlation between NLR, PLR, MLR and subsets of CTCs as well as DTCs in early, non-metastatic BC. Here we evaluated the correlation of NLR, PLR, MLR and the presence of epithelial CTCs (eCTCs), CTCs in epithelial mesenchymal transition (EMT-CTCs) as well as DTCs to better identify patients at risk, to monitor treatment reponse and probably adjust therapeutic options. Methods: The counts of peripheral neutrophils, lymphocytes, monocytes and platelets to determine NLR, PLR and MLR as well as clinicopathological data during diagnosis were retrospectively recorded for 171 patients (pts) diagnosed with BC from July 2006 to December 2012, before the start of therapy. NLR, PLR and MLR were calculated from peripheral blood cell counts and their optimal cutoff levels were determined by the 75% percentile resulting in the following values: NLR: 3.13, MLR: 0.39 and PLR: 222.3, respectively. 170/171 pts were analyzed for DTCs by immunocytochemistry using the pan-cytokeratin antibodyA45-B/B3 (Clodronate intake was recommended in case of DTC-positivity). CTCs were determined in 155/171 pts applying positive immunomagnetic selection using the AdnaTest BreastCancerSelect. The recovered cDNA was tested for the presence of eCTCs and EMT-CTCs using the AdnaTests BreastCancerDetect and EMTDetect. Pts were considered CTC-positive if at least one of the two tests were positive. Statistical analysis included descriptive reporting, U-test for group differences, Rho correlations for dependencies and Fisher's chi-square test for distributional differences. Rho correlations were used to assess the relationship between NLR, PLR, MLR and tumor cells as well as progression-free survival (PFS) and overall survival (OS). Results: DTCs were detected in 34% of the pts and at least one CTC-subtype was found in 28% of the pts. Whereas the presence of DTCs was not associated with PFS or OS, the presence of CTCs significantly correlated with a shorter PFS (p=0.046) and OS (p=0.018). However, neither eCTCs nor EMT-CTSs alone were of prognostic significance. Enhanced lymphocyte (p=0.025) and monocyte counts (p=0.039) as well as a low PLR (p=0.032) significantly correlated with a reduced PFS in pts still alive whereas an enhanced MLR showed a high correlation with a shorter PFS (p=0.007) and OS (p=0.021) in deceased pts. Whereas MLR significantly correlated with the presence of EMT-CTCs (p=0.045), the presence of eCTCs significantly associated with an elevated NLR (p=0.003) and enhanced lymphocyte (p=0.007) as well as monocyte counts (p=0.012). No significant correlations were found for NLR, PLR and MLR with DTCs, however, DTC-positive pts, harboring a lower PLR, had a significant shorter OS (p=0.043). Conclusion: Here we show that proinflammatory markers in blood are closely related to the presence of different CTC subsets in early BC while no direct correlation was found for tumor cell spread to the bone. These findings might improve the prognostication of these pts and probably help to monitor response to therapy and adjust treatment options. Citation Format: Sabine Kasimir-Bauer, Ebru Karaaslan, Olaf Hars, Ann-Kathrin Bittner, Oliver Hoffmann, Rainer Kimmig. In early breast cancer, the ratios of neutrophils, monocyctes and platelets to lymphocytes significantly correlate with the presence of subsets of circulating tumor cells in blood and disseminated tumor cells in the bone marrow [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-02-04.

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

Endothelial cell-specific expression of serine/threonine kinase 11 modulates dendritic cell differentiation

In the bone marrow, classical and plasmacytoid dendritic cells (DC) develop from the macrophage-DC precursor (MDP) through a common DC precursor (CDP) step. This developmental process receives essential input from the niche in which it takes place, containing endothelial cells (EC) among other cell types. Here we show that targeted deletion of serine/threonine kinase 11 (Stk11) encoding tumor suppressor liver kinase b1 (Lkb1) in mouse ECs but not DCs, results in disrupted differentiation of MDPs to CDPs, severe reduction in mature DC numbers and spontaneous tumorigenesis. In wild type ECs, Lkb1 phosphorylates polypyrimidine tract binding protein 1 (Ptbp1) at threonine 138, which regulates stem cell factor (Scf) pre-mRNA splicing. In the absence of Lkb1, exon 6 of Scf is spliced out, leading to the loss of Scf secretion. Adeno-associated-virus-mediated delivery of genes encoding either soluble Scf or the phosphomimetic mutant Ptbp1T138E proteins rescued the defects of MDP to CDP differentiation and DC shortage in the endothelium specific Stk11 knockout mice. In summary, endothelial Stk11 expression regulates DC differentiation via modulation of Scf splicing, marking the Stk11-soluble-Scf axis as a potential cause of DC deficiency syndromes.

Gene Mutation and Overexpression of Newly Diagnosed Multiple Myeloma Patients

To analyze the characteristics of gene mutation and overexpression in newly diagnosed multiple myeloma (NDMM) patients. Bone marrow cells from 208 NDMM patients were collected and analyzed. The gene mutation of 28 genes and overexpression of 6 genes was detected by DNA sequencing. Chromosome structure abnormalities were detected by fluorescence in situ hybridization (FISH). Gene mutations were detected in 61 (29.33%) NDMM patients. Some mutations occurred in 5 or more cases, such as NRAS, PRDM1, FAM46C, MYC, CCND1, LTB, DIS3, KRAS, and CRBN. Overexpression of six genes (CCND1, CCND3, BCL-2, CCND2, FGFR3, and MYC) were detected in 83 (39.9%) patients, and cell cycle regulation gene was the most common. Single nucleotide polymorphisms (SNP) changes were detected in 169 (81.25%) patients, the TP53 P72R gene SNP (70.17%) was the most common. Abnormality in chromosome structure was correlated to gene overexpression. Compared to the patients with normal chromosome structure, patients with 14q32 deletion showed higher proportion of CCND1 overexpression. Similarly, patients with 13q14 deletion showed higher proportion of FGFR3 overexpression, whereas patients with 1q21 amplification showed higher proportion of CCND2, BCL-2 and FGFR3 overexpression. There are multiple gene mutations and overexpression in NDMM. However, there is no dominated single mutation or overexpression of genes. The most common gene mutations are those in the RAS/MAPK pathway and the genes of cyclin family CCND are overexpression.

Disruption of hematopoiesis attenuates the osteogenic differentiation capacity of bone marrow stromal cells

BackgroundThe homeostasis of mesenchymal stem cells (MSCs) is modulated by both their own intracellular molecules and extracellular milieu signals. Hematopoiesis in the bone marrow is maintained by niche cells, including MSCs, and it is indispensable for life. The role of MSCs in maintaining hematopoietic homeostasis has been fully elucidated. However, little is known about the mechanism by which hematopoietic cells reciprocally regulate niche cells. The present study aimed to explore the close relationship between MSCs and hematopoietic cells, which may be exploited for the development of new therapeutic strategies for related diseases.MethodsIn this study, we isolated cells from the offspring of Tie2Cre + and Ptenflox/flox mice. After cell isolation and culture, we investigated the effect of hematopoietic cells on MSCs using various methods, including flow cytometry, adipogenic and osteogenic differentiation analyses, quantitative PCR, western bloting, and microCT analysis.ResultsOur results showed that when the phosphatase and tensin homolog deleted on chromosome 10 (Pten) gene was half-deleted in hematopoietic cells, hematopoiesis and osteogenesis were normal in young mice; the frequency of erythroid progenitor cells in the bone marrow gradually decreased and osteogenesis in the femoral epiphysis weakened as the mice grew. The heterozygous loss of Pten in hematopoietic cells leads to the attenuation of osteogenic differentiation and enhanced adipogenic differentiation of MSCs in vitro. Co-culture with normal hematopoietic cells rescued the abnormal differentiation of MSCs, and in contrast, MSCs co-cultured with heterozygous null Pten hematopoietic cells showed abnormal differentiation activity. Co-culture with erythroid progenitor cells also revealed them to play an important role in MSC differentiation.ConclusionOur data suggest that hematopoietic cells function as niche cells of MSCs to balance the differentiation activity of MSCs and may ultimately affect bone development.

Impacts of Angelica Polysaccharide on Proliferation and Differentiation of Mesenchymal Stem Cells of Rat Bone Marrow.

In literature, antiosteoporotic effects of Angelica sinensis root have been confirmed, but the impact of Angelica sinensis polysaccharide (ASP) on osteoblastic or adipogenic distinction of BMSCs is limited. This paper aimed to explore the role of ASP on proliferation and differentiation of rat BMSCs. Rat BMSCs were subjected to isolation and identification through flow cytometry. The proliferation of rat BMSCs under ASP was performed by CCK-8 kit. Measures of osteogenesis under different concentrations of ASP were detected by using alizarin red staining for mesenchymal cells differentiation and ALP activity assay to identify ALP activity. Quantitative RT-PCR was selected to identify osteoblastic or adipogenic biomarkers from a genetic perspective. Likewise, we have evaluated measures of indicators of Wnt/β-catenin signal. ASP significantly promoted the proliferation, increased osteogenesis, and decreased adipogenesis of rat BMSCs within the limit of 20–60 mg/L in a dose-dependent manner but was suppressed at 80 mg/L. The expression of cyclin D1 and ß-catenin showed a considerable rise over the course of ASP induced osteogenesis. Dickkopf 1 (DKK1) suppressed the regulation of rat BMSCs differentiation through the mediation of ASP. We have observed that ASP upregulated the osteogenic but downregulated adipogenic differentiation of BMSCs, and our findings help to contribute to effective solutions for treating bone disorders.

Patient's knowledge and Believes Regarding the Risk Factors of Leukemia in Marjan Hospital

Background: Leukemia is a cancerous changes begin in a marrow cell and blood. It is divided into a cute type, acute leukemia which affects the cells that are not completely developed, and Chronic which is slowly progressed, There is an increasing rate in the risk factors regarding leukemia cases particularly in ages 65 to 69 years old and this represented (1 case per 100,000) while patients aging 70 and over constituting about (10 cases per 100,000). Knowledge of patients and believes regarding the risk factors may include chemical substances, radiation, exposure to benzene comes from cigarette smoke. 
 Aims of the study: The aim of the present study was to assess patient's knowledge and believes about the risk factors of leukemia, and to find out the relationships between some of socio demographic variables and patient's knowledge and believes regarding the risk factors. 
 Methodology: A descriptive design study was carried out to assess the knowledge and believes of patients with leukemia regarding the risk factors of this disease, the period of this study was Jan \ 2018 to June \ 2018, Convenient non probability sampling method was used, the sample consisted of (50) patients who admitted to Marjan Hospital. Questionnaire developed after comprehensive review of literatures related to this topic, it was completed during patient's interview. It consisted of 3 parts: the socio demographic data, Knowledge of patients and believes regarding the risks factors of Leukemia. 
 Results: Most of the patients age (40%) between (31-36) years old Male patients were more than female (60%). The majority of them (80%) were married. Half of them lived in urban area, in addition their level of education (52%) of them had primary school level, while (76%) of them had sufficient economic status. the result declared that most of patient believed that smoker is risk factor of leukemia. The majority of them did not believed that fear and anxiety, life events (fatigue and frustration), the poverty, contaminated food, stress, and the disease can't be cured. There were significant relationships between all the variables and patients believes and knowledge scores at P > 0.001. 
 Conclusion: Although it is impossible to prevent leukemia there are various risk factors which may cause this disease, and patients may have different knowledge and believes toward those factors. The current research findings declared that there were significant relationship between patients knowledge and believes with the their sociodemographic data. 
 Recommendations: Increase Patients awareness about risk factors of leukemia and how to minimize the exposure to them through the ministry of health effort

Waldenstrom Macroglobulinaemia with Signet Ring Cells: A Case Report

Waldenstrom Macroglobulinemia (WM) is an indolent lymphoproliferative disorder characterised by Immunoglobulin M (IgM) production and neoplastic lymphoid infiltrate. The morphology of the neoplastic cells show spectrum of maturation characterised by small lymphocytes, lymphoplasmacytoid cells and plasma cells. The infiltrate is predominantly lymphoid with relatively small population of plasma cells. The plasma cell morphology is commonly of mature type with basophilic cytoplasm, eccentric nucleus with clumped chromatin and perinuclear halo (hof). But incidence of signet ring cells in WM is very rare and only one case has been reported till date. Here, authors report an interesting case of a 61-year-old man who presented with on and off chest pain and later diagnosed as WM with signet ring cells. On examination, the patient was found to have anaemia and lymphadenopathy. Bone marrow aspirate cytology showed hypercellular particles and scattered signet ring like plasma cells with large globular cytoplasmic inclusions. Flow Cytometry (FCM) established the diagnosis by demonstrating the clonal nature of plasma cells and B-lymphoid cells. Immunofixation confirmed the presence of monoclonal protein of IgM lambda type. The cytoplasmic content in these signet ring plasma cells is probably the accumulated IgM due to the defect in releasing. Clustering of these signet ring cells in marrow may raise a suspicion of metastatic adenocarcinoma and should not be misinterpreted.

Basic concepts in clinical immunology: A review

Immunity is the state of protection against foreign pathogens or substances(antigens). Host defence mechanisms consist of innate immunity (natural immunity), which mediates the initial protection against infections, and adaptive immunity (specific/acquired immunity), which develops more slowly and provides more specialized and more effective defence against infections. The immune system evolved to protect multicellular organisms against pathogens. The body is protected against pathogens by a variety of effector cells and molecules that together make up the immune system. All the cellular elements of blood, including the red blood cells, platelets and white blood cells of the immune system, ultimately derive from the hematopoietic stem cells of the bone marrow. The cells of the adaptive immune system consist of lymphocytes, antigen-presenting cells and effector cells that eliminate microbes. Strategies of avoidance, and tolerance represent different ways of dealing with pathogens. Anatomic barriers and chemical barriers e.g complement and antimicrobial proteins may be considered as primary forms of avoidance.Macrophages,neutrophils and dendritic cells are important cells that that detect infection. Pattern recognition receptors (PRRs) recognize simple molecules and regular patterns of molecular structures called pathogen associated molecular patterns. Some PRR are transmembrane proteins e.g Toll like receptors (TLRs). Vaccination is a simple, safe, and effective way of protecting people against harmful diseases, before they come into contact with them. Immunization is the process whereby a person is made immune or resistant to an infectious disease, typically by the administration of a vaccine. Inappropriate immune responses can result into hypersensitivity, autoimmune disease or immune deficiency.

АНАЛИЗ ПОЯВЛЕНИЯ МИКРОЯДЕР В ЭРИТРОЦИТАХ И АКТИВНОСТИ ПРОЛИФЕРАЦИИ КЛЕТОК КОСТНОГО МОЗГА ПОСЛЕ ПРОЛОНГИРОВАННОГО ОБЛУЧЕНИЯ МЫШЕЙ БЫСТРЫМИ НЕЙТРОНАМИ В НИЗКИХ ДОЗАХ

Purpose: To analyze the level of cytogenetic damage and the activity of bone marrow cells proliferation in C57BL/6 mice after prolonged fast neutrons low dose irradiation at 10–500 mGy. 
 Material and methods: Male C57BL/6 mice at the age of 7–8 and 16 weeks were used in the experiments. Irradiation was carried out on an OR-M installation in the field of fast neutrons and gamma quanta using five Pu(α,n)Be radionuclide sources with a high fast neutron yield at a dose rate of 2.13 mGy/h. The frequency of polychromatophilic (PCE) and normochromic (NCE) erythrocytes with micronuclei (MN) and the ratio of PCE and NCE were analyzed using light microscopy after cytochemical staining of the bone marrow cells of control and irradiated mice. The proliferation activity of bone marrow cells was determined by the number of Ki-67+-cells. The parameters of the cell cycle and the level of apoptosis were studied after DNA staining with DAPI using flow cytometry. Statistical processing of the results was carried out according to the Student’s method using the computer program Origin.
 Results: It was found that prolonged irradiation of mice with fast neutrons at a low dose rate (2.13 mGy/h) at doses from 10 to 500 mGy after 24 h led to statistically significant increase in the frequency of PCE with MN at all studied doses. No dose dependence of this parameter was observed in the studied range. The increase in the frequency of PCE with MN at a dose of 500 mGy was prolonged and persisted for at least 72 h. A significant increase in the frequency of NCE with MN 24 h after irradiation was found only at a dose of 500 mGy, which persisted up to 48 h. At this dose, there was also a decrease in the number of nucleated cells in the bone marrow 24 – 72 h after exposure, a decrease in the number of Ki-67+-cells 24 h after irradiation of mice, a block of the cell cycle in the G2/M phase, and a decrease of cells in the G0/G1 phase, but after 48 h, there were no disturbances in the cell cycle. 
 Conclusion: It has been shown that after a single total prolonged irradiation of mice at low doses (10–500 mGy), when analyzing the frequency of PCE with MN, cytogenetic damage is recorded in the bone marrow, which indicates the genetic danger of exposure to even such low levels of fast neutron irradiation. A decrease in Ki67+ cells and cell cycle arrest at the G2/M phase were found only after irradiation of mice at a dose of 500 mGy and only 24 h after exposure, while the number of nucleated cells in the bone marrow at this dose was reduced, at least to 72 h.

Inhibition of Cytosolic Phospholipase A2α Induces Apoptosis in Multiple Myeloma Cells.

Cytosolic phospholipase A2α (cPLA2α) is the rate-limiting enzyme in releasing arachidonic acid and biosynthesis of its derivative eicosanoids. Thus, the catalytic activity of cPLA2α plays an important role in cellular metabolism in healthy as well as cancer cells. There is mounting evidence suggesting that cPLA2α is an interesting target for cancer treatment; however, it is unclear which cancers are most relevant for further investigation. Here we report the relative expression of cPLA2α in a variety of cancers and cancer cell lines using publicly available datasets. The profiling of a panel of cancer cell lines representing different tissue origins suggests that hematological malignancies are particularly sensitive to the growth inhibitory effect of cPLA2α inhibition. Several hematological cancers and cancer cell lines overexpressed cPLA2α, including multiple myeloma. Multiple myeloma is an incurable hematological cancer of plasma cells in the bone marrow with an emerging requirement of therapeutic approaches. We show here that two cPLA2α inhibitors AVX420 and AVX002, significantly and dose-dependently reduced the viability of multiple myeloma cells and induced apoptosis in vitro. Our findings implicate cPLA2α activity in the survival of multiple myeloma cells and support further studies into cPLA2α as a potential target for treating hematological cancers, including multiple myeloma.

Study of the effects of ultra-low intensity electromagnetic fields on biological objects

The object of research is the efficiency of exposure to electromagnetic field (EMF) of ultra-low intensity on biological objects, which is formed by a generator of broadband radiation. The principle of action of the generator is based on formation of electromagnetic radiation induced by periodic pulsed gas discharge in coaxial system of electrodes, which is loaded on a dielectric rod antenna. The method of selection of signals and corresponding equipment, which energy characteristics of radiation correspond to the criterion of non-thermal influence on bioobjects, is developed for obtaining a comparative assessment of influence bioefficiency. The proposed new method for processing experimental data using statistical calculations that meet the requirements for the processing and interpretation of the results. The seeds of wheat and interaction of millimeter range electromagnetic oscillations with bone marrow cells of rats were used as biological objects for investigating the effect of millimeter range electromagnetic oscillations. A biosensory effect was obtained when exposed to broadband radiation of ultra-low intensity, compared to the control sample. A change in the properties of the seeds, in particular, heat resistance, is observed. According to the experimental data, seeds turn out to be less susceptible to heat as a result of their pretreatment with EMF. The biological response is observed to depend on the frequency and time of irradiation. Also, the dependence of the decrease in the number of dead cells on the time of EMF irradiation was experimentally proved. The equation of dependence of selective average proportion of dead cells in rat bone marrow on irradiation time was calculated. Biosensory effect of exposure to broadband ultra-low intensity EMF of the developed emitter was revealed. It was established and statistically proved that the minimum time with the maximum positive effect of exposure to electromagnetic radiation of millimeter range on bone marrow cells of rats is 30 minutes, compared with an unirradiated sample. The results make it possible to evaluate the positive effect of electromagnetic oscillations on biological objects and propose the results of studies for practical use in the development of medical systems.

Abstract LBA002: Targeting GSPT1 by a novel cereblon E3 ligase modulator for the treatment of Acute Lymphoblastic Leukemia

Abstract Acute Lymphoblastic Leukemia (ALL), the most common childhood cancer and the second most common acute leukemia in adults, arises from clonal expansion of undifferentiated lymphoid precursor cells in bone marrow. Although the majority of childhood ALL cases harbor clonal genetic alterations of transcription factor (TF) gene mutations or rearrangements, TF alterations remain difficult therapeutic targets. Small molecule induced protein degradation is a novel strategy that can be applied to currently undruggable targets such as TF and fusion oncoproteins. In this paradigm, small molecule degraders (either PROTAC or Molecular Glue (MG)) redirect the cell’s endogenous ubiquitin proteasome system and induce ubiquitination of the target protein or non-native substrate of an E3 ligase (neosubstrate) and its subsequent proteasomal degradation. Recently, the CRBN E3 ligase modulator, CC90009 was reported to show potent antitumor activity in acute myeloid leukemia, leading to the identification of GSPT1 (G1 to S phase transition factor) as a CRBN neosubstrate. These findings suggest the potential of MGs to target unanticipated vulnerabilities in different malignancies. Here, using a structurally diverse and unique set of MGs (Molecular Glue Library (MGL)) with confirmed CRBN binding affinity, we sought to identify novel CRBN modulators through phenotypic and proteomic methods. Our screening of the MGL in a panel of representative acute leukemia cell lines, including the CRLF2-rearranged ALL cell line MHH-CALL-4 identified several active MGs with EC50 <5 μM. A lenalidomide competition assay and MHH-CALL-4 CRBN knock down cells confirmed the CRBN dependency of these MGs. Among these compounds, SJ6986, a thalidomide-driven sulfonamide showed potent cytotoxicity in over 10 ALL cell lines tested in vitro. TMT-MS proteomic analyses identified GSPT1/2 as the primary targets for this compound with high selectivity. We next tested SJ6986 activity in a panel of patient derived ALL xenografts (PDX) harboring rearrangement of IGH-CRLF2, EPOR, ATF7IP-JAK2 ex vivo. All the tested tumors were highly sensitive to SJ6986 with IC50s at nanomolar range. PK analyses in NSG mice indicated rapid absorption and over 80% oral bioavailability for SJ6986. PD studies in an IGH-CRLF2 PDX showed dose-dependent degradation of GSPT1 within 48 hours of treatment. Finally, we examined the antitumor activity of SJ6986 in 6 different PDX representative of high-risk subtypes of ALL including near haploid, low-hypodiploid, CRLF2-rearranged and EPOR-rearranged, in vivo for 28 days. SJ6986 was able to dramatically decrease the tumor burden at 1 mg/kg dose in most of the tumor models. Collectively, these results affirm that SJ6986 is a novel CRBN modulator and a potential therapeutic agent by targeting GSPT1 protein with high selectivity and potency for the treatment of ALL. Citation Format: Fatemeh Keramatnia, Yunchao Chang, Gisele Nishiguchi, Jaeki Min, Charles Mullighan, Marcus Fischer, Zoran Rankovic, Fatemeh Keramatnia. Targeting GSPT1 by a novel cereblon E3 ligase modulator for the treatment of Acute Lymphoblastic Leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA002.

Abstract LBA013: Phosphoproteomics reveals active drug targets on pathways of resistance and predicts response to midostaurin plus chemotherapy in FLT3 mutant-positive acute myeloid leukemia

Abstract Background: Midostaurin (mido) is approved for treatment of FLT3 mutant-positive (FLT3+) acute myeloid leukemia (AML). However, FLT3 mutation is not the only determinant of mido sensitivity. Here we report phosphoprotein signatures which predict response to chemotherapy (chemo) plus mido, and identify active drug targets on potential resistance pathways. Methods: Samples collected at diagnosis, post-treatment and relapse from FLT3+ patients treated with chemo+mido were obtained from the Leukemia Tissue Bank at the Princess Margaret Cancer Centre. Peptides and enriched phosphopeptides from bone marrow (BM) and peripheral blood (PB) mononuclear cells were quantified using liquid chromatography-tandem mass spectrometry. Signatures for BM/PB diagnosis samples were analyzed independently and used to train a classification machine learning algorithm to group patients (n=54) based on response to treatment. Additional features (e.g. genetic mutations) were also analyzed. Kaplan-Meier and Log-Rank test methods were used to assess differential survival between patient groups. To investigate pathways potentially driving resistance to chemo+mido, differential protein phosphorylation indexes were identified through comparison of post-treatment or relapse samples to paired diagnosis samples. To account for population heterogeneity, a filter was applied based on frequency of observation. Activated pathways potentially driving resistance were identified with functional enrichment tools and kinase-substrate enrichment analysis. Statistical significance of enrichment were determined using parametric methods and p-values adjusted for multiple testing using the Benjamini-Hochberg method. Results: Patients positive for a signature consisting of 26 phosphorylation sites showed a markedly longer survival time than negative patients (PB: 269 vs 76 weeks, Log-Rank p=1.30e-05; BM: 241 vs 56, Log-Rank p=2.13e-09). This signature partially overlapped with an ex-vivo signature of response to mido, described previously by Casado et al (Leukemia, 2018). A proteomic signature was also identified, with positive patients showing a longer survival time than negative patients (PB: 330 vs 173 weeks, Log-Rank p=5.0e-04; BM: 460 vs 156, Log-Rank p=5.2e-06). Key, diverging phosphorylation site signatures were identified between patients with refractory disease/early relapse and patients with complete response and no relapse or death within 2 years post-treatment. Pathways with increased activity in post-treatment or relapse specimens were associated with molecular functions such as regulation of cell proliferation, migration, differentiation and anti-apoptosis. Conclusions: We identified phosphoproteomic and proteomic signatures that differentiate survival mediated by response to chemo+mido. While the former was more predictive, both may enable further stratification of FLT3+ AML receiving mido treatment. Drug targets on pathways demonstrating increased activity in relapse/refractory cases may play a role in resistance; this will be determined in follow-up inhibitor studies. Citation Format: Luis Veiga Nobre, Celia Colomina Basanta, Salvatore Federico Pedicona, Arran David Dokal, Andrea Arruda, Ryan Smith, Calum Greenhalgh, Francesca Patella, Pedro Maria Casado-Izquierdo, Bela Wrench, Jane Theaker, Andrew Thompson, Mark D. Minden, John G. Gribben, David James Britton, Pedro Rodriguez Cutillas. Phosphoproteomics reveals active drug targets on pathways of resistance and predicts response to midostaurin plus chemotherapy in FLT3 mutant-positive acute myeloid leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA013.

Evaluation of the Hamburg-Glasgow Classification in Pancreatic Cancer: Preoperative Staging by Combining Disseminated Tumor Load and Systemic Inflammation.

Simple SummaryDespite the great achievements in pancreatic ductal adenocarcinoma (PDAC), identification of patients who will suffer rapid disease relapse and progression is not perfect, when definitive histology for tumor staging is not available. The Hamburg Glasgow Classification combines tumor cell dissemination in the bone marrow and systemic inflammatory response into a preoperative staging system. In this work, we assessed the Hamburg Glasgow Classification in potentially resectable PDAC as a prognostic classification for overall and progression-free survival and compared it to the UICC-TNM classification with promising results.This study aims to compare the Hamburg Glasgow Classification (HGC) to Union for International Cancer Control (UICC) classification in patients with pancreatic ductal adenocarcinoma (PDAC). As adequate tumor classification is only possible after tumor resection and histological evaluation, only 20% of patients with PDAC receive accurate tumor staging. Thus, an accurate preoperative staging system is still missing but urgently needed. Systemic inflammation and tumor dissemination are important factors regarding the oncological outcome. HGC integrates both into a preoperative staging system, by combining C-reactive protein (CRP), albumin, and disseminated tumor cells (DTC) in the bone marrow. In this prospective study, 109 patients underwent surgical exploration for suspected PDAC. All patients underwent a preoperative bone marrow aspiration for DTC detection. HGC showed significant preoperative risk stratification for overall survival (OS) (p-value < 0.001) and progression-free survival (PFS) (p-value < 0.001). These results were comparable to the UICC survival stratification for OS and PFS (p-value = 0.001 and 0.006). Additionally, in non-metastatic PDAC, HGC III-IV was associated with shorter OS and PFS (p-value < 0.001, respectively) when compared to HGC I-II. Therefore, the HGC is a promising preoperative prognostic staging classification for accurate and simple outcome stratification in patients with PDAC.

Granulocytes Negatively Regulate Secretion of Transforming Growth Factor β1 by Bone Marrow Mononuclear Cells via Secretion of Erythropoietin Receptors in the Milieu.

In my previous study, I demonstrated that bone marrow-derived mononuclear cells (BM MNCs) secrete copious amounts of Transforming Growth Factor β1 (TGFβ1) in response to erythropoietin (EPO). In this study, I investigated the principal cell type involved in the process. I found that a large percentage of various marrow cells, but not their mature counterparts present in the peripheral blood, express EPO-receptors (EPO-R). Cell depletion experiments showed that depletion of Glycophorin positive erythroblasts and CD41+ megakaryocytes - the prime suspects - did not affect the EPO-mediated TGFβ1 secretion by the BM MNCs. However, individual depletion of CD2+ T lymphocytes, CD14+ monocyte/macrophages, and CD19+ B cells affected the TGFβ1 secretion by EPO-primed MNCs: depletion of CD2+ cells had the most striking effect. Unexpectedly, and most interestingly, depletion of CD15+ granulocytes led to a significant increase in the TGFβ1 secretion by both naïve and EPO-primed BM MNCs, suggesting that these cells negatively regulate the process. Mechanistically, I show that the CD15+ cells exert this regulatory effect via secretion of both full-length and soluble EPO-R in the milieu. Overall my results, for the first time, unravel an in-built regulatory mechanism prevailing in the BM microenvironment that regulates the secretion of TGFβ1 by controlling EPO-EPO-R interaction.My data could be relevant in understanding the pathophysiology of several conditions associated with deregulated production of TGFβ1 in the marrow compartment.