Marrow Serum Research Articles

The Lactate Receptor GPR81 is a Mechanism of Leukemia-Associated Macrophage Polarization in the Bone Marrow Microenvironment.

Interactions between acute myeloid leukemia (AML) and the bone marrow microenvironment (BMME) are critical to leukemia progression and chemoresistance. Altered metabolite levels in the tumor microenvironment contribute to immunosuppression in solid tumors, while this has not been studied yet in the leukemic BMME. Metabolomics of AML patient bone marrow serum detected elevated metabolites, including lactate, compared to age- and sex-matched controls. Excess lactate has been implicated in solid tumors for inducing suppressive tumor-associated macrophages (TAMs) and correlates with poor prognosis. We describe the role of lactate in the polarization of leukemia-associated macrophages (LAMs) using a murine model of blast crisis chronic myelogenous leukemia (bcCML) and mice genetically lacking the lactate receptor GPR81. LAMs were CD206hi and suppressive in transcriptomics and cytokine profiling. Yet, LAMs had a largely unique expression profile from other types of TAMs. We demonstrate GPR81 signaling as a mechanism of both LAM polarization and the direct support of leukemia cell growth and self-repopulation. Furthermore, LAMs and elevated lactate diminished the function of hematopoietic progenitors and stromal support, while knockout of GPR81 had modest protective effects on the hematopoietic system. We report microenvironmental lactate as a critical driver of AML-induced immunosuppression and leukemic progression, thus identifying GPR81 signaling as an exciting and novel therapeutic target for treating this devastating disease.

Comparison of trace elements in peripheral blood and bone marrow of newly diagnosed multiple myeloma patients.

Trace elements are essential micronutrients for the human body. Their roles are indispensable, as they are involved in a wide range of vital biological processes. In this study, we aimed to evaluate alterations in trace elements in the blood and bone marrow serum of patients with newly diagnosed multiple myeloma (NMM). The levels of zinc (Zn), copper (Cu), iron (Fe), manganese (Mn), magnesium (Mg), selenium (Se), arsenic (As), boron (B), nickel (Ni), silicon (Si) and chromium (Cr) were analyzed in the venous blood samples of the patient group comprising 70 patients with NMM (41 males and 29 females) and compared to those in the control group comprising 30 individuals (18 males and 12 females). In addition, trace element levels were analyzed in bone marrow samples from the patient group. Blood and bone marrow serum levels were quantified using inductively coupled plasma optical emission spectrometry. When the blood samples of the patient and control groups were compared: Zn (p = 0.011), Fe (p = 0.008), Mn (p = 0.046), Se (p < 0.001), As (p < 0.001), Ni (p < 0.001) and Cr (p < 0.001) levels were significantly higher in the patient group than in the control group. Higher Zn, Fe, Mn, Se, As, Ni and Cr levels in the NMM patients suggest that alterations of trace elements could be predisposing factor that initiates the malignant process. The relationship between malignancies and trace elements is crucial for the development of adjuvant therapy strategies and preventive medicine and as biomarkers for cancer diagnosis. Therefore, there is a need for studies examining the relationship between hematological malignancies and trace elements.

Differences between Peripheral and Bone Marrow Lipidomics in Patients with Severe Aplastic Anemia and Its Finding in Predicting the Early Immunosuppressive Therapy Response

Aplastic anemia (AA) is a common clinical hematological disease, which is characterized by pancytopenia due to bone marrow hematopoietic failure. AA can be divided into severe AA (SAA) and non-severe AA (NSAA) based on disease severity. Anti-thymoglobulin (ATG)-based immunosuppressive treatment (IST) is recommended as the main first-line treatment for AA patients when stem cell transplantation is not available. The mechanism of AA has not been fully clarified. When considering role of the bone marrow microenvironment, current studies showed that the abnormal of quantity, function, and differentiation of mesenchymal stem cells (MSCs) were involved in the occurrence and development of AA, including the boost of adipocyte. Han, et al. [PMID: 35445952] found that the peripheral serum lipid profile may have great meaning in the differentiation diagnosis between transfusion-dependent non-severe AA and hypo- myelodysplastic syndrome (MDS), as well as in predicting their response to cyclosporine (CsA). Our previous study [PMID: 36192750] also revealed that the higher apolipoprotein-A is a potential prognostic biomarker for severe AA patients treated with ATG-based IST. However, the understanding of peripheral serum lipid metabolism may not fully represent the changes in bone marrow. Herein, we try to investigate the difference of lipid metabolomic profile between bone marrow and peripheral serum in SAA patients, and explore its meaning in predicting early IST response. In this study, a total of 11 SAA patients and 15 age and sex matched healthy donors were enrolled. Among them, six SAA received ATG based IST (Table. 1). An orthogonal partial least-squares discrimination analysis (OPLS-DA) model was established to analyze the differences between the subgroups and overfitting was accessed by permutation test (Fig A, B). The following volcano map (Fig. C) and heat map (Fig. D) confirmed that there were differences in subgroups to a certain degree. To explain the difference, we firstly compared lipid metabolism profile between patients (P) and donors (D) in peripheral serum (ps) (P ps Vs D ps) and bone marrow (bm) serum (P bm Vs D bm), and found that the difference in bone marrow was more distinct than that in peripheral serum, which suggested bone marrow may be more reliable as clinical samples in studies (Fig. E). Secondly, bone marrow serum and peripheral serum (P bm Vs P ps) in SAA patients were compared, fatty acids were upregulated (Fig. F), which indicated that there may be hyperproduction or transport disorder of fatty acid in bone marrow than in peripheral serum. Thirdly, comparing patients' baseline bone marrow lipid metabolites with donors' bone marrow lipid metabolites and with patients' bone marrow after treatment respectively, and analyzing the intersections of differences in two comparison groups, there were 11 common different metabolites, including 4 sphingolipids, 2 glycerides, 2 glycerophospholipids, 2 glycolipids, and 1 steroid lipid. Pathway analysis illustrated those metabolites were involved mainly in metabolism of sphingolipid, glycerophospholipid and glycerophospholipid, indicating that IST may restore homeostasis of bone marrow by affecting those pathways. Finally, we compared the bone marrow serum lipidomics between the SAA patients achieved 3-months response (CR/PR) and no response (NR). The difference of lipid metabolism was reduced after 3 months, while the differences in glucuronolipid (GlcADG) and phosphatidylinositol (PI) were significantly increased (Fig. G). In summary, difference of lipid metabolism between SAA and donors was more obvious in bone marrow, and the lipid metabolism process of bone marrow was less affected by external factors. IST may restore the homeostasis of lipid metabolism in the bone marrow by affecting metabolism of sphingolipid and glycerophospholipid, and GlcADG and PI may be used as new predictors for early IST response in SAA patients.

Interleukin-17A Directly Signals to Bone Marrow-Derived Hematopoietic Stem and Progenitor Cells to Promote Their Expansion and Differentiation in Vitro

Rationale: IL-17 is a family of six pro-inflammatory cytokines (named from A to F) with distinct patterns of cellular expression. Among these, IL-17A is secreted by lymphoid subsets such as T-helper 17 cells and innate lymphoid cells type 3. Through its action on many tissues, IL-17A is recognized as a key mediator of response to infection and pathogenic states such as cancer and auto-immunity. Until now, the main effect of IL-17A on hematopoiesis has been attributed to an indirect loop through IL-17RA signaling on bone marrow stromal cells, which stimulates secretion of hematopoietic factors such as G-CSF. However, we hypothesized that IL-17A can directly signal to hematopoietic stem and progenitor cells (HSPCs) to determine their self-renewal and differentiation. Methodology: We analyzed the expression of the IL-17 signaling pathway in publicly available human and mouse hematopoietic single-cell RNAseq datasets. Mouse HSPCs were isolated using FACS to perform clonogenic and differentiation assays in Methocult (StemCell Technologies) and StemPro-34 (Gibco). Spectral flow cytometry (SONY ID7000) was used for analysing IL-17RA expression and differentiation of mouse HSPCs. IL-17A levels were measured in blood and bone marrow serum using ELISA (Invitrogen). Results: IL-17RA is expressed at the surface of mouse HSPCs, with greatest levels in common lymphoid progenitors and granulocyte-monocyte progenitors. mRNA expression of IL-17RA signaling pathway is also detectable in human HSPCs, with greatest levels in young individuals. IL-17A supplementation leads to an increase in clonogenic capacity of mouse HSPCs in methylcellulose colony-formation assays. In liquid culture, supplementation with IL-17A leads to a greater expansion of sorted Lin -/s-kit +/Sca-1 + progenitors, accompanied by an increased proportion of myeloid-committed cells.IL-17A is also detected in the bone marrow serum, with possible production from subsets of cells expressing RORγt, a master regulator in development of T helper 17 (Th17) cells. Conclusions: Our work suggests the ability of IL-17A to directly promote clonogenic potential of HSPCs and drive a myeloid-biased lineage commitment in progenitor subsets. Additional studies are required to characterize the response of HSPCs to direct IL-17A signaling in vivo. Considering the important role of IL-17A as a mediator of immune response, and tissue repair, further studies are warranted to elucidate whether modulation of this pathway may be therapeutic in the context of hematopoietic failure or malignancy.

Postnatal deletion of β-catenin in preosteoblasts regulates global energy metabolism through increasing bone resorption and adipose tissue fibrosis.

Many studies revealed bone can regulate global energy metabolism and our previous study also showed that Wnt/β-catenin pathway is involved in this process. To better understand the participation of canonical Wnt pathway in energy metabolism, we examined the β-catenin knock-out (β-cat KO) mice by crossing the osterix-cre transgenic mice with β-cateninflox/flox mice. First, we identified that postnatal deletion of β-catenin in preosteoblasts led to decreased fat mass and increased energy expenditure in mice. Osteoprotegerin administration largely rescued the decreased fat mass and partly normalized the energy expenditure accompanied by the inhibition of bone resorption. Anti-resorption with alendronate or RANKL-antibody could also partly rescued the decreased bone mass, decreased fat mass and increased energy expenditure in β-cat KO mice. We further found that the adipose cells in the inguinal fat tissue were smaller and the extracellular matrix components around adipocytes accumulated more in β-cat KO mice than their controls by histomorphology. Gene analysis by RT-PCR showed that the expression of collagen VI is 4.8 folds in adipose tissue of the β-cat KO mice compared with the control mice. We further detected the expression of cytokines which were related to fibrosis and the data showed that the level of TGF-beta1 was elevated in both of bone marrow serum and adipose tissue derived from the β-cat KO mice. After administration of TGF-beta1 neutralizing antibody, the impaired energy metabolism was partly rescued in β-cat KO mice. Besides, anti-resorption treatment and TGF-beta1 antibody could partly suppress the increased expression of genes related to fat tissue fibrosis. These results indicate that the abnormal global energy metabolism in β-cat KO mice may be attributed to increasing bone resorption and adipose tissue fibrosis.

Chronic Diarrhoea: A Rare Presentation Of Vitamin B12 Deficiency Anemia In Children

Vitamin B12 deficiency in children often under reported and usually presents with nonspecific manifestations like neuropsychiatric symptoms, anaemia, glossitis and chronic diarrhoea. Vegetarianism, minimal intake of animal products, poverty and malnutrition may lead to vitamin B12 deficiency. Laboratory reports often show pancytopenia, megaloblasts in bone marrow and low serum cynocobalamine. Injectable vitamin B12 is the treatment of choice. We would like to highlight this case report in view of vitamin B12 deficiency presenting as diarrhoea.

Metabolic Profiling during Acute Myeloid Leukemia Progression Using Paired Clinical Bone Marrow Serum Samples.

Cellular metabolic changes reflect the characteristics of patients with acute myeloid leukemia (AML) caused by genetic variations, which are important in establishing AML treatment. However, little is known about the metabolic profile of patients with genetic variation-induced AML. Furthermore, the metabolites differ with disease progression. Here, metabolites in the bone marrow serum of ten patients with AML and healthy individuals were analyzed using gas chromatography–mass spectrometry. Compared with that in healthy individuals, expression of most metabolites decreased in patients with AML; hydroxylamine, 2-hydroxybutyric acid, monomethylphosphate, and ethylphosphate expression was unusually increased in the patients. We further examined serial metabolite changes across the initial diagnosis, postremission, and relapse phases. Patients with relapse showed increased metabolite expression compared with those in the diagnostic phase, confirming that patients with AML had aggressively modified leukemic cells. However, a clear difference in metabolite distribution was not observed between the diagnosis and complete remission phases, suggesting that the metabolic microenvironment did not change significantly despite complete remission. Interestingly, metabolite profiles differed with genetic variations in leukemic cells. Our results, which were obtained using paired samples collected during AML progression, provide valuable insights for identifying vulnerable targets in the AML metabolome and developing new treatment strategies.

The dynamics of human bone marrow adipose tissue in response to feeding and fasting.

BACKGROUNDAdipocytes were long considered inert components of the bone marrow niche, but mouse and human models suggest bone marrow adipose tissue (BMAT) is dynamic and responsive to hormonal and nutrient cues.METHODSIn this study of healthy volunteers, we investigated how BMAT responds to acute nutrient changes, including analyses of endocrine determinants and paracrine factors from marrow aspirates. Study participants underwent a 10-day high-calorie protocol, followed by a 10-day fast.RESULTSWe demonstrate (a) vertebral BMAT increased significantly during high-calorie feeding and fasting, suggesting BMAT may have different functions in states of caloric excess compared with caloric deprivation; (b) ghrelin, which decreased in response to high-calorie feeding and fasting, was inversely associated with changes in BMAT; and (c) in response to high-calorie feeding, resistin levels in the marrow sera, but not the circulation, rose significantly. In addition, TNF-α expression in marrow adipocytes increased with high-calorie feeding and decreased upon fasting.CONCLUSIONHigh-calorie feeding, but not fasting, induces an immune response in bone marrow similar to what has been reported in peripheral adipose tissue. Understanding the immunomodulatory regulators in the marrow may provide further insight into the homeostatic function of this unique adipose tissue depot.FUNDINGNIH grant R24 DK084970, Harvard Catalyst/The Harvard Clinical and Translational Science Center (National Center for Advancing Translational Sciences, NIH, award UL 1TR002541), and NIH grants P30 DK040561 and U19 AG060917S1.

CXCR2, a novel target to overcome tyrosine kinase inhibitor resistance in chronic myelogenous leukemia cells

Chronic myeloid leukemia (CML) is a reciprocal translocation disorder driven by a breakpoint cluster region (BCR)-Abelson leukemia virus (ABL) fusion gene that stimulates abnormal tyrosine kinase activity. Tyrosine kinase inhibitors (TKIs) are effective in treating Philadelphia chromosome (Ph) + CML patients. However, the appearance of TKI-resistant CML cells is a hurdle in CML treatment. Therefore, it is necessary to identify novel alternative treatments targeting tyrosine kinases. This study was designed to determine whether C-X-C chemokine receptor 2 (CXCR2) could be a novel target for TKI-resistant CML treatment. Interleukin 8 (IL-8), a CXCR2 ligand, was significantly increased in the bone marrow serum of initially diagnosed CML patients and TKI-resistant CML cell conditioned media. CXCR2 antagonists suppressed the proliferation of CML cells via cell cycle arrest in the G2/M phase. CXCR2 inhibition also attenuated mTOR, c-Myc, and BCR-ABL expression, leading to CML cell apoptosis, irrespective of TKI responsiveness. Moreover, SB225002, a CXCR2 antagonist, caused higher cell death in TKI-resistant CML cells than TKIs. Using a mouse xenograft model, we confirmed that SB225002 suppresses tumor growth, with a prominent effect on TKI-resistant CML cells. Our findings demonstrate that IL-8 is a prognostic factor for the progression of CML. Inhibiting the CXCR2-mTOR-c-Myc cascade is a promising therapeutic strategy to overcome TKI-sensitive and TKI-insensitive CML. Thus, CXCR2 blockade is a novel therapeutic strategy to treat CML, and SB225002, a commercially available CXCR2 antagonist, might be a candidate drug that could be used to treat TKI-resistant CML.

MO560IS SERUM ERYTHROPOIETIN CORRELATED WITH IRON METABOLISM AND INFLAMMATION IN NON-DIALYSIS CHRONIC KIDNEY DISEASE PATIENTS WITH ANEMIA?

Abstract Background and Aims Both the relative erythropoietin (Epo) deficiency and its relationship with serum hemoglobin (Hb) are widely postulated in chronic kidney disease (CKD), but the influence of chronic inflammation and iron status on serum Epo levels is still a matter of debate, with yet divergent reported results. Therefore, we aimed to assess the determinants of serum Epo in non-dialysis CKD patients. Method Fifty-two adults with CKD and anemia (defined as Hb &amp;lt;12g/dL), in stable clinical condition, never treated with erythropoiesis-stimulating agents (ESA) entered this cross-sectional, single-center study. Diabetes mellitus, active infectious and inflammatory diseases, malignancy, anemia of other causes than CKD, current immunosuppressive therapy, iron supplementation and blood transfusions in the previous six months were exclusion criteria. The subjects were mostly men (56%), elderly (two thirds over 60 years), with advanced CKD [71% in CKD stages G4-G5, median estimated glomerular filtration rate – eGFR 14.5 (95%CI 16 to 25) mL/min], moderate anemia [Hb 9.8 (95%CI 9.2 to 9.9) g/dL], and mild to moderate inflammation [C-reactive protein 6 (95%CI 9.2 to 18.4) mg/L]. Serum Epo was assessed by ELISA (Abcam® 119522). Complete blood count, reticulocyte index, peripheral blood smear, bone marrow aspiration (Perls’ stain), serum ferritin, and transferrin saturation, were used to investigate anemia and iron metabolism. Parameters of kidney disease (CKD etiology, eGFR and proteinuria), demographic data (age, gender), C-reactive protein, serum albumin, and serum hepcidin-25 (Hep-25, Bachem® commercial ELISA kit) were also analyzed. Results The median serum Epo of the whole cohort was 4.8 (95%CI 5.1 to 9.9) mU/mL. According to median Epo, subjects were clustered in Group 1 (below median, G1) and Group 2 (above median, G2). Estimated GFR and serum Hep-25 were lower in G1 than in G2 [10.6 (95%CI 9.7 to 20.8) vs. 26 (95%CI 19.1 to 32.8) mL/min, p=0.004, and 62.6 (95%CI 51.0 to 85.1) vs. 95.4 (95%CI 77.0 to 108.5) ng/mL, p=0.03, respectively]. All the other investigated parameters were similar in the two groups. In bivariate analysis (Spearman rank correlation), serum Epo was positively associated only with eGFR (rs=0.40, p=0.003). Marginal associations with the percentage of bone marrow sideroblasts, as marker of the iron available for erythropoiesis (rs=0.25, p=0.08), erythrocyte mean corpuscular hemoglobin concentration (rs=−0.26, p=0.07), and reticulocyte index (rs=0.24, p=0.09) were observed. Conversely, serum Epo was not related to hemoglobin, indices of iron stores (e.g. serum ferritin and iron content in bone marrow macrophages), inflammation and nutritional status (e.g. C-reactive protein and serum albumin). In a model of multiple linear regression which explained 14% of serum Epo variation, eGFR was the only determinant: Beta 0.14 (95%CI 0.05 to 0.23), p=0.004. Also, a binary logistic multiple regression model predicting serum Epo lower or higher than the median retained the eGFR as an independent predictor, while serum hepcidin showed only borderline significance: Conclusion Kidney function is the main determinant of endogenous erythropoietin level in moderately anemic patients with advanced CKD, ESA or iron naive, while serum hepcidin-25 seems to exert a limited influence.

Watermelon Reduces the Toxicity of Cisplatin Treatment in C57BL/6 Mice with Induced Melanoma

An alternative to reduce the undesirable effects of antineoplastic agents has been the combination of classical treatments with nutritional strategies aimed at reducing systemic toxicity without decreasing the antitumor activity of already used drugs. Within this context, this study evaluated the possible reduction of toxicity when cisplatin treatment is combined with watermelon pulp juice supplementation in C57BL/6 mice with melanoma. Watermelon is a fruit rich in vitamins, minerals, proteins, lycopene, carotene, and xanthophylls, which has shown effectiveness in the treatment of cardiovascular diseases, weight loss, urinary infections, gout, hypertension, and mutagenicity. The following parameters were analyzed: animal survival, bone marrow genotoxicity, serum creatinine and urea, histopathological features of the tumor tissue, tumor weight and volume, and weight of non-tumor tissues (kidney, liver, spleen, heart, and lung). The results showed that watermelon had no antitumor effect but reduced the toxicity of cisplatin, as demonstrated by an increase in the number of bone marrow cells and a decrease in serum creatinine and urea levels. The data suggest that watermelon pulp juice can be an alternative for reducing the side effects of antineoplastic agents.

P0861THE RELATIONSHIP BETWEEN HEPCIDIN-25 AND BONE MARROW IRON IN ANEMIC, NON-DIALYSIS, CHRONIC KIDNEY DISEASE PATIENTS

Abstract Background and Aims Hepcidin-25 (Hep25) is a key known regulator of iron metabolism and its interactions with inflammation, iron stores and erythropoietic activity were involved in the pathogenesis of chronic kidney disease (CKD)-associated anemia. Therefore, our aim was to assess the determinants of serum Hep25 level in non-dialysis CKD patients. Method In this cross-sectional, single-center study, 52 subjects (56% men, 65±13 years) with CKD [estimated glomerular filtration rate, eGFR 14.5 (95%CI 16 to 25) mL/min] and anemia [hemoglobin, Hb 9.8 (95%CI 9.2 to 9.9) g/dL], not treated with erythropoiesis-stimulating agents (ESA) or iron in the previous 6 months, were enrolled. Patients with anemia of other causes than CKD, active infectious and inflammatory diseases, malignancy, severe hyperparathyroidism, transfusions during the last 3 months, and immunosuppressive therapy were excluded. The iron status was evaluated using both peripheral and central parameters. The iron stores were assessed by serum ferritin (Fer) and iron content in bone marrow macrophages (iMf, measured quantitively on a scale from 0 to 6). The iron available for erythropoiesis was assessed by transferrin saturation (TSAT) and the percentage of sideroblasts (%Sb). Anemia was further evaluated by a peripheral blood smear, erythrocytes indices and reticulocyte index. Serum Hep25 and erythropoietin (Epo) were assessed by ELISA (Bachem®, and Abcam® 119522, respectively). C-reactive protein (CRP), albumin, and parameters of kidney disease (eGFR, proteinuria) were also measured. Mann-Whitney, Kruskal-Wallis, Chi2 tests, Spearman bivariate correlation and multiple linear regression were used for statistical analysis. Results The median serum Hep25 of the whole cohort was 82.1 (95%CI 68.7 to 92.1) ng/mL. According to median Hep25, subjects were clustered in Group 1 (below median - G1) and Group 2 (above median - G2). %Sb and reticulocyte index had higher levels in G2 than in G1 [9 (95%CI 5 to 14) vs. 5 (95%CI 4 to 7) %, p=0.003 and 0.55 (95%CI 0.39 to 0.77) vs. 0.41 (95%CI 0.32 to 0.58), p=0.05, respectively], while the proportions of subjects with iMf suggestive for iron deficiency or iron overload were similar in G2 and G1 (38% vs. 50%, p=0.40, and 26% vs. 23%, p= 0.75, respectively). Peripheral blood smear, peripheral iron indices and all the other studied parameters were also alike. In bivariate analysis, Hep25 was positively associated both with indices of iron stores, i.e. Fer (rs = 0.30, p=0.03) and iMf (rs = 0.34, p=0.01) and indices of iron available for erythropoiesis, i.e. %Sb (rs = 0.55, p&amp;lt;0.001) and (marginally) with TSAT (rs = 0.26, p=0.06). Meanwhile, Hep25 was not related to serum Epo, CKD parameters or inflammation markers. In a multivariate linear regression model that explained 28% of Hep25 variation, the percentage of bone marrow sideroblasts, i.e. the tissue iron available for erythropoiesis, was the only independent determinant of Hep25: Variables entered in the first step: reticulocyte index, percentage of medullary sideroblasts (%Sb), iron content in the bone marrow macrophages (iMf), serum ferritin, and transferrin saturation Conclusion In stable patients with advanced CKD, not treated with ESA or iron, with low to moderate inflammation, serum hepcidin was related only to bone marrow iron available for erythropoiesis, suggesting that in this clinical setting the need of iron for erythropoiesis prevails over inflammation in regulation of hepcidin synthesis.

THBS1 Is a Novel Serum Prognostic Factors of Acute Myeloid Leukemia.

Dysregulation of cytokines and growth factors is a general feature of tumor microenvironment, and unraveling the expression spectrum of cytokine and growth factor in niche is of utmost importance. Here, we evaluated cytokine profiling of bone marrow serum samples in AML patients and healthy controls. Protein expression profiling of serum from nine AML patients and five healthy controls was obtained using a biotinylated antibody chip. A total of 507 cytokines and growth factors were analyzed. Compared with healthy people, AML patients expressed 31 signature proteins, among which, 27 were significantly higher expressed and 4 proteins were lower. When patients were divided into favorable and poor prognosis, 12 signature proteins were significantly differentially expressed between these two groups. Furthermore, in order to identify the accuracy of cytokine expression profiles, we verified and analyzed the expression of THBS1 (Thrombospondin 1) in 116 patients and 9 healthy people. We found that THBS1 was lowly expressed in AML patients, which might be induced by promoter methylation, and patients with low THBS1 possessed shorter survivor time. Our data indicated that we successfully unveil differentially expressed proteins in AML patients using a biotinylated antibody chip; among them, THBS1 may be a potential therapeutic target for AML patients' treatment.

Analysis of clinical characteristics and prognosis factors in patients with angioimmunoblastic T-cell lymphoma

Objective To investigate the clinical characteristics and prognostic factors of patients with angioimmunoblastic T-cell lymphoma (AITL). Methods From January to December, 2012, a total of 44 AITL patients admitted to Department of Hematology, First Affiliated Hospital of Air Force Medical University were included as study subjects. There were 36 males and 8 females, with age of 18-78 years and median age of 57.5 years. All 44 patients were treated with CHOP (cyclophosphamide + pirarubicin + vincristine + prednisone) regimen, 4 of them were combined with chidamide or bortezomib, 6 of them were combined with autologous hematopoietic stem cell transplantation (auto-HSCT). Clinical characteristics of AITL patients were retrospectively analyzed. Kaplan-Meier method was used to map overall survival (OS) curves of patients treated with and without auto-HSCT. And univariate analysis of OS rate was performed in patients treated without auto-HSCT using Log-rank test. Influencing factors included gender, age, International Prognostic Index (IPI) score, Prognostic Index for peripheral T-cell lymphoma (PIT) score, B symptoms, skin rashes, chest/abdominal cavity effusion, bone marrow involvement, white blood cell count (WBC), hemoglobin (Hb) value, platelet count, serum lactate dehydrogenase (LDH) level, ferritin level, β2-microglobulin (MG) level. Factors with statistical significance in univariate analysis and clinical guiding significance were included in COX proportional hazards regression model for multivariate analysis. The procedure of this study was accordance with the requirement of the revised World Medical Association Declaration of Helsinki in 2008 and 2013. Results ① Among 44 AITL patients, 34 cases (77.3%) had B symptoms. Five cases (11.4%) were Ann Arbor Ⅰ-Ⅱ stage, 39 cases (88.6%) were Ⅲ-Ⅳ stage. Twenty cases (45.5%) had Eastern Cooperative Oncology Group, performance status (ECOG-PS) score 60 years, and the difference was statistically significant (χ2=0.139, P=0.023). The 5-year OS rate in patients with serum β2-MG level 60 years (HR=2.716, P=0.031), bone marrow involvement (HR=2.696, P=0.042), serum β2-MG level≥4 mg/L (HR=4.927, P=0.004) were independent risk factors for AITL prognosis. Conclusions Majority of AITL patients are middle-aged and elderly males, often accompanied by skin rash and serous effusion. The disease was first diagnosed late-stage in Ann Arbor. Age>60 years, bone marrow involvement and serum β2-MG level≥4 mg/L, can be used as independent indicators of poor prognosis of patients treated without auto-HSCT. New drugs such as chidamide and bortezomib could improve curative effect of AITL patients. auto-HSCT coukl effectively improve the OS of patients. Key words: Lymphoma; Immunoblastic lymphadenopathy; Drug therapy; Hematopoietic stem cell transplantation; Prognosis; Angioimmunoblastic T-cell lymphoma; Restrospective studies

Megaloblastic anemia in a 9-weeks old infant

Megaloblastic anaemia due to vitamin B12 and folic acid deficiency is uncommon in infancy and rarely reported in infants below 3 months of age. We hereby report a case of megaloblastic anaemia in a 9-weeks old infant having fever from 7th week of life. Blood picture showed pancytopenia and diagnosis was confirmed on bone marrow biopsy and serum level of vitamins. Patient positively responded to vitamin B12 and folic acid supplementation. Infants with pancytopenia even younger than 2 months, should also be investigated for vitamin B12 and folate deficiency. Mother of the baby was not antenatally investigated for anaemia. Prompt antenatal diagnosis and treatment of mothers can reduce the incidence in the infants.

THBS1 Is a Novel Serum Prognostic Factors of Acute Myeloid Leukemia

Background: Dysregulation of cytokines and growth factor is a general hallmark of AML patients, unraveling the mysteries of cytokine and growth factor interaction in the context of AML is of utmost importance. Here, we evaluated bone cytokines profiling in bone marrow serum sample in AML patients and healthy controls. Methods: Serum expression profiles of 507 proteins in the serum samples of 14 patients (9 AML patients and 5 healthy controls) using RayBiotech biotinylated antibody chip. THBS1 expression in 116 patients and nine healthy control was verified via ELISA. Findings: Compared with healthy people, 31 signature proteins were found to be significantly expressed in AML patients, among these proteins, 27 proteins were highly expressed. When divided into well prognosis and poor prognosis, 12 signature proteins were significantly and differently expressed. Furthermore, in order to identify the results of expression profiles, we verified and analysis the expression of THBS1 (Thrombospondin 1) in the 116 patients and nine healthy control. We found that THBS1 was lowly expressed in AML patients, patients with low expressed THBS1 possessed shorter survivor time, and promoter methylation was the cause of the low expression of THBS1. Interpretation: Our data indicated that RayBiotech biotinylated antibody chip analysis can unveil differentially expressed proteins in AML patients, and THBS1 may be a novel therapeutic approach for AML patients. Funding: Supported by grants from the National Natural Science Fund for Youth (No. 81600166) Declaration of Interest: All authors declare no competing interests. Ethical Approval: This study was approved by the Xinqiao hospital Ethics committees and all patients provided a signed informed consent. All human specimens used in the experiments were approved by Ethics committee of Army Medical University.

Expression of IL-17 in Bone Marrow of Patients with Multiple Myeloma and Its Effect in Pathogenesis

To investigate the expression and pathogenesis of IL-17 in bone marrow blood of multiple myeloma (MM) patients. Expression levels of IL-6, TNF-α and IL-17 in bone marrow serum of 20 MM patients and 20 control subjects were detected by ELISA, and correlation analysis was performed to analyze the correlation IL-17 with IL-6 and TNF-α. The effect of IL-17 on the proliferation of MM cells treated with different concentration of IL-17 was detected by cell prollferation and toxicity tesis. The morphological changes of RAW264.7 cells treated with IL-17 were observed by tartrate resistant acid phosphatase (TRAP) staining. The levels of IL-17, IL-6 and TNF- in the bone marrow of MM patients were all higher than those of the normal control group (P＜0.05). The IL-17 level positively correlated with IL-6 and TNF-α levels (r=0.6045, P＜0.01 and r=0.627, P＜0.01). Cell proliferation and toxicity tests confirmed that IL-17 can promote the proliferation of multiple myeloma cells. TRAP staining revealed that IL-17 could induce differentiate of RAW264.7 cells into multinuclear giant cells. IL-17 may be involved in the pathogenesis of MM and promotes the proliferation of tumor cells, and induces the activation of osteoclasts leading to increased bone destruction.

RETROSPECTIVE ANALYSIS OF MULTIPLE MYELOMA PATIENTS IN A TERTIARY CARE CENTRE OF UTTARAKHAND

Introduction: Multiple myeloma (MM) is a hemato-lymphoid malignancy of B-cell type. It occurs due to the accumulation of malignant monoclonal plasma cells. The exact incidence of MM in India is not well-known. The current study presented the clinical characteristics, radiological findings and laboratory findings of MM patients who were initially treated at the tertiary care centre, Dehradun, Uttarakhand (India).&#x0D; Material and methods: Retrospective analysis of the medical records of 123 consecutive patients with MM who were initially presented to Hemato-Oncology department during the period from January 2014 to December 2018. Peripheral blood finding, bone marrow diagnosis, flow-cytometry analysis, serum protein and immunofixation electrophoresis finding, biochemical parameter, histo-pathological and cytological diagnosis, if any, urine examination finding and radiological examination of cases shall be compiled and tabulated. Diagnosis of symptomatic multiple myeloma was done based on The International Myeloma Working Group criteria for the diagnosis of MM.&#x0D; Result: The study included 123 cases of multiple myeloma with male: female ratio of 2:1, mean age of 59.88 ±11.08 years and range of 32-87 years. The back pain (n=106, 86.2%) was the common presenting complaint followed by inability to walk (n=90,73.2%). CT scan and/or MRI scan finding of MM patients, the lytic lesion was found in 107 patients (87%) and was found significant with its correlation with ISS/DS plus staging system (pvalue 0.013). The most location was dorso-lumbar spine (n=72, 67.28%) followed by skull (n=37, 34.58%) and ribs (n=19, 17.75%). Hemoglobin (Hb) analysis showed 92.7 % (n=114) cases were anemic with mostly had normocytic (n=95,77.2%). 62.6%(n=77) of cases showed rouleaux formation while 23.57% (n=29) cases were circulating plasmacytoid lymphocytes or plasma cells. The raised ESR values was found in 89.33% (n=67) of cases. Diagnosis of cases shows 99.2% (n=122) of cases diagnosed as secretory MM while 1 (0.8%) case diagnosed as non-secretory MM.&#x0D; Conclusion: MM is a disease with a inconsistent clinical presentation with multiple system involvement. Younger age of disease onset is some noteworthy features of myeloma in Uttarakhand state of India. Bony pain associated with generalized weakness is the commonest presentation, normocytic normochromic anemia with rouleaux formation, raised ESR, raised total protein with hypoalbuminemia, hypercalcemia, presence of M band and presence of &gt;10% plasma cell in bone marrow is clue to diagnosis in MM cases .&#x0D; Keywords: Multiple Myeloma, Uttrakhand, Hemato-lymphoid malignancy

Immunomodulatory Drugs in the Context of Autologous Hematopoietic Stem Cell Transplantation Associate With Reduced Pro-tumor T Cell Subsets in Multiple Myeloma.

Immunomodulatory drugs (IMiDs) are effective therapeutics for multiple myeloma (MM), where in different clinical settings they exert their function both directly on MM cells and indirectly by modulating immune cell subsets, although with not completely defined mechanisms. Here we studied the role of IMiDs in the context of autologous hematopoietic stem cell transplantation on the T cell subset distribution in the bone marrow of newly diagnosed MM patients. We found that after transplantation pro-tumor Th17-Th1 and Th22 cells and their related cytokines were lower in patients treated with IMiDs during induction chemotherapy compared to untreated patients. Of note, lower levels of IL-17, IL-22, and related IL-6, TNF-α, IL-1β, and IL-23 in the bone marrow sera correlated with treatment with IMiDs and favorable clinical outcome. Collectively, our results suggest a novel anti-inflammatory role for IMiDs in MM.

Potential Metabolite Markers of Multiple Myeloma

E-mail: wenzhou@csu.edu.cnBackground: Metabolism reprogramming is one of ten features in cancer. It is well known that metabolites in tumor microenvironment contribute to the survival and proliferation of cancer cells. Currently, a lack of detailed information about the metabolites profiling in bone marrow microenvironment limits us to understand the roles of metabolites associated with multiple myeloma(MM) and its diagnosis and treatment. Here we report a serum untargeted metabolomics study of MM patients, together with healthy donors(HD), with the aim of discovering metabolite markers associated with MM.Materials and Methods: Gas chromatography-time-of-flight mass spectrometry (GC-TOFMS)-based metabolomics was used to analyze 140 serum subjects, including 81 bone marrow subjects(22 HD, 59 MM patients) and 59 peripheral blood subjects(27 HD, 32 MM patients). The bone marrow subjects were divided into training set(11 HD, 32 MM patients) and testing set(11 HD, 27 MM patients). SIMCA-14.1 software package was used to visualize the metabolite alterations between MM patient and HD through Principal component analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). Both the T-test and the receiver operating characteristic curve(ROC) analysis were performed by SPSS software. Metabolites in serum with higher fold change(FC) and variable importance in the projection(VIP) value(VIP > 1.5, P < 0.05 and FC > 1.5, P < 0.05, FDR < 0.05) were considered as biomarker candidates.Results: A total of 117 and 123 metabolites were annotated from the detected spectral features in bone marrow serum subjects derived from training set and testing set, respectively. Based on multivariate statistical analysis(PCA and OPLS-DA) and univariate statistical analysis(T-test), a panel of 6 and 10 metabolites were identified as differential metabolites(VIP > 1.5, P < 0.05 and FC > 1.5, P < 0.05, FDR < 0.05) between MM patients and HD in training set and testing set, respectively, among of which 5 metabolites were found significantly altered in both sets. Creatinine and glycine were significantly elevated in MM patients compared with HD, while fatty acid consists of palmitic acid, petroselinic acid and stearic acida were found decreased in MM patients compared with HD. ROC analysis of these 5 metabolites resulted in an area under the receiver operating characteristic curve (AUC) of 0.922(95% confidence interval=0.748-1) in the training set and 0.923(95% confidence interval=0.853-1) in the testing set. Furthermore, the diagnostic potential of the metabolite signatures was assessed in peripheral blood subjects. Consistent with bone marrow subjects, metabolite signatures were significantly changed(VIP > 1.5, P < 0.05 and FC > 1.5, P < 0.05, FDR < 0.05) in peripheral blood subjects derived from MM patients compared with HD. The AUC of this metabolites signatures was 0.901(95% confidence interval=0.748-1) in peripheral blood subjects, implying that this panel of metabolites could be of potential clinical significance for the diagnosis of MM.Conclusion: We conclude that a panel of 5 metabolites, including creatinine, glycine, palmitic acid, petroselinic acid and stearic acid, in serum has great potential in discriminating MM patient from HD. This metabolite signatures provides a novel and promising molecular diagnostic approach for the detection of MM. DisclosuresNo relevant conflicts of interest to declare.