Cells Of Granulocytic Lineage Research Articles

Inter-Facility Evaluation of Machine Learning Based Deep Hematopoietic Cell Classifiers for Cytological Evaluation of Hematopoiesis

Introduction: Cytology, histology, flow cytometry, cytogenetics and molecular genetics are the cornerstones of diagnosis in hematology. Cytological evaluation of blood and bone marrow is performed by trained experts. However, this task is time consuming and subject to inter- and intra-rater variability. Several studies proposed processing pipelines for peripheral blood and bone marrow smears. Deep learning approaches by neural networks have been evaluated for automated classification of blood and bone marrow cells. Nevertheless, issues concerning the evaluation methodology with respect to real-world applicability remain, e.g., accuracy of designation of cell types, differences in staining and imaging protocols, dataset splitting, number of cell types as well as hierarchy and selection of regions of interest. Methods: We present a slide and facility cross-validated evaluation of machine-learning based automated hematopoietic cell classifiers of bone marrow smears. The analysis is based on the DenseNet121 and ResNet152 architectures of our own database collected at University Hospital RWTH Aachen consisting of 11,899 labeled objects from 24 different classes collected from 6 different patients. In addition, we analyzed 3 publicly available bone marrow and blood cell datasets for staining, color variability and other quality parameters. Coupled with cross-validation we employed model ensembling, augmentation, normalization, and pseudo-labeling to train domain-adapted models for each dataset. Our evaluations are contrasted by an inter-rater analysis performed on a subset of our labeled granulocytic cell line data. Results: A review of performance scores from related works in the field revealed that Convolutional Neural Network (CNN) architectures were used in all analyses, except for Krappe et al. where a tree of polynomial classifiers was employed. Only one publication used slide cross validation (SCV) und the number of cell classes applied was variable as well as the number of cells annotated by experts and used for analysis. Different numbers of cell classes were defined. Accuracy of cell classification was also variable, and F-scores were used to improve interpretation of results also for small cell populations. Fig 1 shows Facility cross-validation results for baseline evaluation (pink) and new model training considering brightness, contrast, saturation, and hue (gold) with DensNet and ResNet CNN for granulopoiesis divided into 5 classes of cells. Dark bars indicate bagging performance. X-axes indicate the evaluation data set. Green shaded regions denote the lower bound for optimal performance, red shaded regions show the upper bound for worst performance. The variations in smear preparation, fixation, and embedding as well as the usage of different staining protocols and scanning devices lead to biases in data collected from different sites. Accuracies of in 13-, 8- and 5-class cross-validation setups were obtained when using different amounts of available data each. Incorporating confusion tolerance fields (one-class difference in granulocytic precursors) yields accuracies of , whilst classifier ensembling increases performance scores similarly well to training on larger combined datasets. The interrater accuracy of experts was 96 % when the one-class difference was applied as confusion tolerance. Conclusion: Deep neural network models as suggested here are most sensitive to color augmentations in the domain of brightness, contrast, hue, and saturation. If configured properly, respective augmentations increase performance scores significantly. However, if not accounted for, the models experience severe performance drops when evaluated on slide data from other sites. Enlarging the corpus of training data via pseudo-labeling improves results only slightly, whilst normalization approaches are outperformed by color augmentation strategies. Even though, our results show inferior accuracy by deep learning approaches compared to medical experts. Deep neural network approaches will become the platform for automated cytological analysis of blood and bone marrow smears in the future. However, harmonization of preanalytical and technical aspects will be necessary to improve accuracy of cell classification and further extension for recognition of specific morphological abnormalities e.g., dysplasia, will be necessary.

Myeloid Sarcoma: Case Series with Unusual Locations

Myeloid sarcoma (MS) or chloroma is a localized mass composed of blastic cells of granulocytic lineage. It is a subtype of acute myeloid leukemia and usually presents as a complication of acute myeloid leukemia, myeloid dysplastic syndrome, or myeloproliferative disorder. MS occurs in 2.5-9.1% of patients with AML, precedes the clinical disease, coincidence with the onset or at relapse and in rare conditions, it can occur with no evidence of hematologic disorders. Here, we presented seven cases of MS in unusual locations or with rare presentations at presentation or relapse. We concluded that MS should be considered in the differential diagnosis of any high-grade tumor, especially in a patient with previous history of any myeloid neoplasm.

The Reduction of the Cell Nuclear Size in the Cell Body Space During the Differentiation Might Be Cell Lineage Specific (A Retrospective Morphological Note).

The cell body space occupied by the nucleus decreased during the cell differentiation of the granulocytic cell lineage in CML (Chronic Myeloid Leukemia) patients. In contrary, in patients suffering from CLL (Chronic Lymphocytic Leukemia), the cell body space occupied by the nucleus during the cell differentiation of the lymphocytic lineage did not decrease despite the reduction of the cell size. Thus, the cell body space occupied by the cell nucleus during the differentiation was characteristic for each of these cell lineages.

How we monitor the therapy effectiveness in patients with chronic myeloid leukemia - A Single center experience

Chronic myeloid leukemia (CML) is a hematological disorder characterized by increased proliferation of the granulocytic cell lineage. We diagnose CML by presence of the breakpoint cluster region-abelson (BCR-ABL1) oncogene using the quantitative real-time polymerase chain reaction (QRT-PCR). This method provides an accurate and unambiguous follow-up of the treatment response to tyrosine kinase inhibitors (TKIs). This study aimed to determine the molecular response to the first and second generation of TKIs in first and second line of treatment using the QRT-PCR method. We conducted a retrospective study on 48 CML patients treated with the first and second generation of TKIs in first and second-line treatment. Treatment responses have been followed-up every 3 months using the QRT-PCR method. Patients were divided into three groups according to molecular responses to the first line of TKIs. Results obtained in this study showed that the first group of patients did not achieve major molecular response (MMR) in the first 18 months of TKI treatment. The second and third group of patients achieved MMR and deep molecular response (DMR) in the first 18 months of TKI treatment. These results indicate that patients with MMR and DMR in the first 18 months of TKIs treatment had a favourable clinical course of the disease. Inadequate molecular responses to the first line of TKIs can be improved with in increase of the dose of TKIs or by switching to other TKIs. Continuous and timely molecular monitoring of TKI’s response in CML patients provides a careful observation of the disease's course and a proper treatment approach.

Rab44 Deficiency Induces Impaired Immune Responses to Nickel Allergy.

Rab44 was recently identified as an atypical Rab GTPase that possesses EF-hand and coiled-coil domains at the N-terminus, and a Rab-GTPase domain at the C-terminus. Rab44 is highly expressed in immune-related cells such as mast cells, macrophages, osteoclasts, and granulocyte-lineage cells in the bone marrow. Therefore, it is speculated that Rab44 is involved in the inflammation and differentiation of immune cells. However, little is known about the role of Rab44 in inflammation. In this study, we showed that Rab44 was upregulated during the early phase of differentiation of M1- and M2-type macrophages. Rab44-deficient mice exhibited impaired tumor necrosis factor alpha and interleukin-10 production after lipopolysaccharide (LPS) stimulation. The number of granulocytes in Rab44-deficient mice was lower, but the lymphocyte count in Rab44-deficient mice was significantly higher than that in wild-type mice after LPS stimulation. Moreover, Rab44-deficient macrophages showed impaired nickel-induced toxicity, and Rab44-deficient mice showed impaired nickel-induced hypersensitivity. Upon nickel hypersensitivity induction, Rab44-deficient mice showed different frequencies of immune cells in the blood and ears. Thus, it is likely that Rab44 is implicated in immune cell differentiation and inflammation, and Rab44 deficiency induces impaired immune responses to nickel allergies.

Biology of amphibian granulocytes - From evolutionary pressures to functional consequences

Granulocyte-lineage cells are important innate immune effectors across all vertebrates. Named for conspicuous secretory granules, granulocytes have historically been studied for their antimicrobial roles. Although versions of these cells are found in all vertebrate species examined to date, disparate environmental and physiological pressures acting on distinct vertebrate classes have shaped many of the facets dictating granulocyte biology. Immune pressures further determine granulopoietic constraints, ultimately governing granulocyte functions. For amphibians that inhabit pathogen-rich aquatic environments for some or all their lives, their unique granulocyte biologies satisfy many of their antimicrobial needs. Amphibians also occupy an intermediate position in the evolution of vertebrate immune systems, using combinations of primitive (e.g., subcapsular liver) and more recently evolved (e.g., bone marrow) tissue sites for hematopoiesis and specifically, granulopoiesis. The last decade of research has revealed vertebrate granulocytes in general, and amphibian granulocytes in particular, are more complex than originally assumed. With dynamic leukocyte phenotypes, granulocyte-lineage cells are being acknowledged for their multifaceted roles beyond immunity in other physiological processes. Here we provide an overview of granulopoiesis in amphibians, highlight key differences in these processes compared to higher vertebrates, and identify open questions.

The cell body space occupied by the nucleus during the cell differentiation in human lymphocytic, granulocytic and erythroid cell lineages.

The present nuclear and cell body diameter measurements demonstrated size differences of the approximate cell space estimate occupied by the cell nucleus during the cell differentiation in lymphocytic, granulocytic and erythroid cell lineages. These lineages were used as convenient models because all differentiation steps were easily identified and accessible in diagnostic peripheral blood or bone marrow smears of blood donors (BDs), patients suffering from chronic lymphocytic leukemia (CLL), patients with chronic myeloid leukemia (CML) and refractory anemia (RA) of the myelodysplastic syndrome (MDS). The cell space occupied by the nucleus was constant and did not change during the cell differentiation in the lymphocytic cell lineages of BDs and CLL patients despite the decreased cell size. In contrary, the cell space occupied by the nucleus markedly decreased in differentiating cells of granulocytic and erythroid lineages of patients suffering from CML. In the erythroid cell lineage in patients with RA of MDS the small reduction of the cell space occupied by the nucleus during the differentiation was not significant. The measurements also indicated that in progenitor cells of all studied cell lineages nuclei occupied more than 70 % of the cell space. Thus, the nucleus-cytoplasmic morphological and functional equilibrium appeared to be characteristic for each differentiation step and each specific cell lineage.

Leukemic retinopathy, a rare entity: A case report

Chronic myelogenous leukemia (CML), also known as chronic myeloid leukemia, is a myeloproliferative disorder characterized by increased proliferation of the granulocytic cell line without the loss of their capacity to differentiate. Consequently, the peripheral blood cell profile shows an increased number of granulocytes and their immature precursors, including occasional blast cells. CML accounts for 20% of all leukemias affecting adults. Retinal lesions are the most common ocular manifestation of leukemia. They are found most often in adults and in patients with myeloid leukemia. Despite the significant efforts made by different groups to optimize treatment and outcome, there are still unmet needs and unanswered questions. Ophthalmologic manifestations are among the therapeutic challenge. Here we present a case of CML (chronic phase) with ophthalmologic manifestations as initial presentation, trying to shed light on this important type of presentation.

Dermatofibrosarcoma Protuberans and Chronic Myeloid Leukemia: A Rare Co-incidence

Dermatofibrosarcoma protuberans (DFSP) is an uncommon, low grade, malignant fibroblastic tumor. Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by dysregulated production of granulocytic cell line without the loss of their capacity to differentiate. To our knowledge, there is no described association of DFSP with CML in literature. The authors have encountered a first case of DFSP coexisting with chronic myeloid leukemia.

Taro Lectin Can Act as a Cytokine-Mimetic Compound, Stimulating Myeloid and T Lymphocyte Lineages and Protecting Progenitors in Murine Bone Marrow.

Taro (Colocasia esculenta) corm is traditionally consumed as a medicinal plant to stimulate immune responses and restore a health status. Tarin, a taro lectin, is considered responsible for the immunomodulatory effects of taro. In the present study, in order to investigate the effects of tarin on bone marrow hematopoietic population, murine cells were stimulated with tarin combined with a highly enriched conditioned medium containing either IL-3 or GM-CSF. Cells challenged with tarin proliferated in a dose-dependent manner, evidenced by the increase in cell density and number of clusters and colonies. Tarin exhibited a cytokine-mimetic effect similar to IL-3 and GM-CSF, increasing granulocytic cell lineage percentages, demonstrated by an increase in the relative percentage of Gr-1+ cells. Tarin does not increase lymphocytic lineages, but phenotyping revealed that the relative percentage of CD3+ cells was increased with a concomitant decrease in CD19+ and IL-7Rα+ cells. Most bone marrow cells were stained with tarin-FITC, indicating non-selective tarin binding, a phenomenon that must still be elucidated. In conclusion, taro corms contain an immunomodulatory lectin able to boost the immune system by promoting myeloid and lymphoid hematopoietic progenitor cell proliferation and differentiation.

Hematopoietic Bone Marrow Cells of Rat after Intravenous Administration of Chitosan-Modified Magnetite Nanoparticles

Chitosan-modified magnetic nanoparticles are a promising basis for the creation of new diagnostic agents and therapeutic drugs. The cells of erythroid, granulocytic, monocytic, lymphocytic, and megakaryocytic lineages of bone marrow of mature rats were investigated for 120 days after single injection of 0.14 g per 1 kg body weight chitosan-modified magnetic nanoparticles (Fe3O4). Light microscopy was applied to describe the structure of hematopoietic cells and morphometric study was performed on Romanowsky—Giemsa stained smears to measure cells size (µm) and relative number (%). Comparison of the biological effects of nonmodified and modified magnetite nanoparticles on hematopoietic cells of bone marrow revealed the advantages of modification. Chitosan-modified magnetite nanoparticles did not affect the structure of hematopoietic cells in the rat bone marrow, and their injection produced a reversible increase in the relative number of monocytes, stab neutrophils and segmented neutrophils, as well as polychromatic and oxyphilic normoblasts.

The Morphology of Cell Differentiation, Terminal Differentiation and Ageing Seems To Reflect the Same Process: a Short Note

Based on simple microscopic cell morphology in blood and bone marrow smear preparations, it seems to be likely that the cell differentiation and terminal differentiation in human blood cells, and particularly in erythroid or granulocytic lineages, simultaneously reflect ageing of the lineage progenitors and terminal differentiation steps. The terminal differentiation stages of both these lineages actually appear as senescent cells. Abnormal ageing of progenitor cells may represent one of the “dysplastic” phenomena of the premature terminal differentiation state. Such state is characterized by heterochromatin condensation and nucleolar morphology similar to that in fully differentiated terminal cells of granulocytic or erythroid lineages. It should also be mentioned that in some known erythropoietic disorders, less differentiated erythroblasts may lose nuclei similarly as “normal” fully terminally differentiated cells of the erythroid cell lineage. It seems to be clear that cells in both abnormal less differentiated and terminally differentiated stages of erythroid or granulocytic lineages lose the ability to multiply similarly as senescent cells. On the other hand, the background of cell ageing and differentiation is very complicated and requires a different approach than the simple microscopic morphology at the single cell level. However, the morphology and clinical cytology at the single cell level might still contribute with complementary data to more sophisticated complex studies of that topic. In addition, the morphological approach facilitates the study of the main components of single cells in various states, including the differentiation steps or ageing.

RNA Cytosine Methyltransferases NSUN1 and NSUN2 Mediate the Lineage-Associated Resistance to Venetoclax in Leukemia

Background: Combinations of venetoclax/ABT-199, a small molecule that selectively inhibits anti-apoptotic protein BCL2, with hypomethylating agents (HMAs), such as 5-azacytidine (5-AZA, azacidtine) and decitabine have demonstrated remarkable synergistic effects and resulted in high response rates and significant overall survivals in patients with refractory MDS/AML (Ram, et al. Annual Hematology 2019; DiNardo et al. Blood 2019). However, resistance to venetoclax-based therapies has emerged as a major therapeutic barrier and been linked to monocytic clones in leukemia (Kuusanmaki et al. Haematologica 2020; Pei et al. Cancer Discovery 2020). Our recent study demonstrated that specific RNA cytosine methyltransferases (RCMTs), namely NSUN1 and NSUN2, mediate the lineage-associated resistance to 5-AZA through formation of a drug-resistant elongating RNA-Polymerase-II (eRNAPII) complex at nascent RNA (Cheng et al. Nature Communications 2018). This study aims to address the role of NSUN1 and NSUN2 in mediating venetoclax resistance in leukemia. Experimental Design and Methods: Experiments, including drug-induced cell growth inhibition, western blot, and co-immunoprecipitation, were performed on leukemia cell lines with various lineages to assess lineage-associated venetoclax resistance and identify the key factors/proteins involved in such resistance. Venetoclax-resistant cell lines were established from drug sensitive lines in order to elucidate mechanisms underlying resistance and cell lineage plasticity. Knockdown of NSUN1 and NSUN2 expression was performed to determine their roles in venetoclax-resistant cell lines. Results: Our experimental results have demonstrated monocyte differentiation-associated resistance to venetoclax in leukemia cell lines of different lineages (Figure 1A), which is consistent with previous published studies (Kuusanmaki et al. Haematologica 2020; Pei et al. Cancer Discovery 2020). The degree of lineage-associated venetoclax resistance is closely correlated with the expression of eRNAPII, NSUN2 and NSUN1. Importantly, venetoclax strongly induces expression of eRNAPII and NSUN1 (Figure 1B). We established venetoclax-resistant leukemia cell line (K1VR) from original venetoclax-sensitive granulocytic leukemia cell line Kasumi-1 and confirmed the importance of NSUN1 and NSUN2 in mediating venetoclax resistance in those leukemia cells. siRNA knockdown of NSUN1 or NSUN2 expression inhibits growth of leukemia cells and re-sensitizes the venetoclax-resistant K1VR leukemia cells to a low dose of venetoclax (Figure 1C). Conclusion: Our study has demonstrated that RNA cytosine methyltransferases NSUN1 and NSUN2 mediate monocyte-associated resistance to venetoclax in leukemia cells. We are currently extending our study to clinical specimens. Our study may lead to development of novel RNA epigenetics-driven strategies to predict and overcome the resistance to venetoclax-based therapies. Disclosures No relevant conflicts of interest to declare.

CME: Polycythemia vera

CME: Polycythemia vera Abstract. Polycythemia vera is a myeloprolifere disease which is characterized by proliferation of all three (erythroid, megakaryocytic and granulocytic) cell lines. The causative mutation is in the JAK2-tyrosine kinase gene. The symptoms are related to the increased red blood cells. Common signs are itching (pruritus) and pain in the hands or feet. The most common complications are thrombotic events. Risk factors are age over 60 years and a thrombotic event in the patient's history. The treatment consists of phlebotomy combined with acetylsalicylic acid 100 mg a day. The goal of the therapy is the prevention of the common thrombotic events. During the course of the disease, cytoreductive treatment is indicated in most of the patients.

To the approximate size of the nuclear region occupied by nucleolar bodies during cell differentiation and maturation using the human leukemic granulocytic lineage as a convenient model.

The present study was undertaken to estimate the approximate size of nuclear regions occupied by nucleolar bodies during the cell differentiation and maturation. The differentiation and maturation of human leukemic granulocytic cells in patients suffering from the chronic phase of the chronic granulocytic leukemia (CML) represented a convenient model for such study because of the large number of cells for the diameter measurements at the single cell level. Early and advanced differentiation or maturation stages of these cells are well defined and nucleolar bodies and nuclear outlines are easily seen by simple cytochemical methods for the visualization of RNA and silver stained proteins in smear preparations. During the cell differentiation and maturation, the estimated size of the nuclear region occupied by nucleolar bodies decreased in both untreated and treated patients with the anti-leukemic therapy. However, the size reduction of nucleolar bodies in differentiated and mature cells was larger than that of the nucleus. In addition, the results also indicated that the nuclear region occupied by nucleolar bodies was characteristic for each differentiation and maturation stage of the granulocytic cell lineage and was not substantially influenced by the anti-leukemic therapy of CML patients.

Screening and insilico analysis of deleterious nsSNPs (missense) in human CSF3 for their effects on protein structure, stability and function

Human granulocyte colony stimulating factor (hG-CSF), known as CSF3, plays an important role in the growth, differentiation, proliferation, survival, and activation of the granulocyte cell lineage such as neutrophils and their precursors. Functional reduction in native CSF3 protein results in reduced proliferation and activation of neutrophils and leads to neutropenia. Single nucleotide polymorphisms (SNPs) in the CSF3 gene may have deleterious effects on the CSF3 protein function. This study was undertaken to find the functional SNPs in the human CSF3 gene. Results suggest that 18.9% of all the SNPs in the dbSNP database for CSF3 gene were present in the coding region. Out of 59 non-synonymous SNPs (nsSNPs), 26 nsSNPs were predicted to be non-tolerable by SIFT whereas 18 and 7 nsSNPs were predicted as probably damaging and possibly damaging, respectively by PolyPhen. Among this 31 nsSNPs, 16 nsSNPs were identified to be potentially deleterious by PANTHER server, and 4 nsSNPs were found to be neutral by PROVEAN. SNPAnalyzer predicted 7 nsSNPs to be neutral phenotype and the remaining 24 nsSNPs to be associated with diseases. Among the predicted nsSNPs, rs144408123, rs144408123, rs145136406, rs145311241, rs373191696, rs762945096, rs763688260, rs767572172, rs775326370, rs777777864, rs777983866, rs781596455, rs139072004, rs757612684, rs772911210, rs139072004, rs746634544, rs749993200, rs763426127, rs772466210 were identified as deleterious and potentially damaging. I-Mutant analysis revealed that th 20 nsSNPs were important for protein stability of CSF3. Therefore, th 20 nsSNPs may be used for the wider population-based genetic studies and also should be taken into account while engineering the recombinant CSF3 protein for clinical use.

Label-free optical hemogram of granulocytes enhanced by artificial neural networks

An outstanding challenge for immunology is the classification of immune cells in a label-free fashion with high speed. For this purpose, optical techniques such as Raman spectroscopy or digital holographic microscopy have been used successfully to identify immune cell subsets. To achieve high accuracy, these techniques require a post-processing step using linear methods of multivariate processing, such as principal component analysis. Here we demonstrate for the first time a comparison between artificial neural networks and principal component analysis (PCA) to classify the key granulocyte cell lineages of neutrophils and eosinophils using both digital holographic microscopy and Raman spectroscopy. Artificial neural networks can offer advantages in terms of classification accuracy and speed over a PCA approach. We conclude that digital holographic microscopy with convolutional neural networks based analysis provides a route to a robust, stand-alone and high-throughput hemogram with a classification accuracy of 91.3 % at a throughput rate of greater than 100 cells per second.

PTBP2 exon 10 inclusion is associated with the progression of CML and it is BCR-ABL1 dependent.

Altered or aberrant expression of several splicing factors leads to the progression of different cancers. Though there are several ongoing studies underscoring the role of the splicing regulator polypyrimidine tract binding protein 2 (PTBP2) in neuronal cells, we unveil the role of PTBP2 in chronic myeloid leukemia (CML). Different RNA binding proteins (RBP's) earlier reported in chronic myeloid leukemia blast crisis (CML-BC) cases (n = 28) from Radich Oncomine leukemia dataset, were compared. We observed increased expression of MSI2 followed by PTBP2 in BC cases and increased PTBP2 expression in relapsed cases (n = 10) from the same dataset compared to other RBPs. We also observed increased PTBP2 exon 10 inclusion in KCL22, a granulocytic lineage CML cell line when compared to other CML cell lines of different lineages. As PTBP2 protein expression is associated with PTBP2 exon 10 inclusion, we observed in cell lines and in a set of progressed cases (n = 4) that increased BCR-ABL1 expression potentiates PTBP2 exon 10 inclusion and thus confers the existence of a functional protein. Inhibition of BCR-ABL1 with imatinib not only blocks the inclusion of exon 10 but also deregulates PTBP2 expression in CML cells. Knockdown of PTBP2 in KCL22 cells leads to reduced cell proliferation, increased G2/M cell cycle arrest and increased apoptosis. Taken together our study portrays PTBP2 as a new possible target for CML and progressive inclusion/exclusion of PTBP2 exon 10 might play an important role in CML progression.

Investigation of the Significance of Son Isoform mRNA Expression in Patients with Blood Cytopenias with/without Evidence of Myeloid Neoplasia

Myelodysplastic Syndrome (MDS) refers to hematopoietic neoplasms characterized by defective development of myeloid progenitors (erythrocytic, granulocytic, and megakaryocytic cell lines) and bone marrow. Clinical signs are characterized by blood cytopenias that can affect one or all three myeloid cell lines and bone marrow irregularities. Because of this heterogeneity, patients can present with anemia, thrombocytopenia, and/or neutropenia and it is important to determine prognosis, severity, and progression. Currently, the standard method for determining a diagnosis of MDS is based on metaphase cytogenetics which is limited in its scope. Other newer karyotyping procedures such a fluorescent in situ hybridization and single nucleotide polymorphism assay have improved the ability to evaluate patients. However, these tests still do not establish conclusive key fundamental biomolecular-genetic origins. It is hoped that in the future diagnostics will include information that further supports development of clinical prognostic and therapeutic options.Many mutational abnormalities have been discovered in MDS. The mutation of interest in this investigation is a shortened isoform of the SON protein which has been implicated to contribute to the pathogenesis of MDS, acute myelogenous leukemia (AML), and other malignancies. SON is a splicing cofactor that regulates the cell cycle and stability by interacting with genes and proteins known to contribute to tumorigenesis. Samples were obtained from patients being evaluated for cytopenia, MDS, AML, and other myeloid neoplasms and compared SON mRNA expression levels to control samples. Prior studies found that alternative splicing of the SON gene produces SON B and SON E both of which are truncated and aberrantly upregulated in patients with hematopoietic malignancies. The short SON isoforms antagonize the normal functions of full-length SON (SON F) leading to repression and splicing dysregulation of key cell cycle genes such as mixed lineage leukemia (MLL) family methyltransferases. SON regulates MLL complex-mediated histone methylation; this process requires the binding of menin, a tumor suppressor. The truncated SON isoform prevents this binding of meninto the MLL complex while opposing the transcription repressing function of full-length SON. A collaborative study between the laboratory of Dr. E.Y. Ahn and the hematology clinic of the Mitchell Cancer Institute further investigates the relationship between SON expression and hematopoietic disorders. SON E was found to be upregulated patients with anemias, MDS, and other myeloid neoplasms, a finding consistent with preceding research. Iron replete subjects and subjects taking erythropoietin did not follow his pattern. This new insight has yet to be explored due to limited data.In this study, samples were obtained from consented participants with cytopenias, MDS, or myeloproliferative disorders and analyzed for SON mRNA expression. Investigation revealed SON expression is abnormal in the majority of patients tested with cytopenias from either iron deficiency or myeloproliferative neoplasms or myelodysplastic syndrome. The differential expression of SON E appears to increase in patients with myeloproliferative neoplasms, myelodysplastic syndrome but differ in expression among patients. Deviations from the expected SON E short isoform expression were discovered for iron replete patients and a patient taking erythropoietin, leading to the conclusion that further clinical correlation of the SON expression in patients with iron deficiency and no MPD or MDS may further elucidate SON expression relevance in this disease and the relation to iron metabolism. In the future, longitudinal inspection of laboratory values and SON expression in patients with and without treatment is expected with specific emphasis on total body iron status as well as comparison to other mutational data (RUNX1 and GATA1). Overall, the results displayed potentiate SON isoform expression may be important in understanding the pathogenesis of malignant and non-malignant diseases of the blood. DisclosuresNo relevant conflicts of interest to declare.

The Effect Of Temperature On Recombinant Human Granulocyte Colony Stimulating Factor Production By Pichia pastoris Expression System

Granulocyte colony stimulating factor (G-CSF) is a haematopoetic growth factor that functions as specific stimulator of the survival, proliferation, and differentiation of precursor cells of the neutrophilic granulocyte cell lineage as well as an activator of mature neutrophil function. The main objective of this work is to compare the effect of different temperature on the production of extracellular recombinant G-CSF in Pichia pastoris. Cells were cultured for 72h in baffled shake-flasks at 20°C, 25°C, and 30°C in two different medium; buffered glycerol/methanol-complex medium (BMGY/BMMY) and buffered minimal glycerol/methanol (BMGH/BMMH) after methanol induction every 12h. Expressed recombinant hG-CSF in the methylotrophic yeast P. pastoris was analyzed with SDS-PAGE. The 23 kDa protein was secreted into the culture supernatant when induced with methanol. Production of recombinant G-CSF protein in P. pastoris at 30°C at 48h incubation after methanol induction every 12h is the highest in both complex and minimum medium.