Lymphoid Lines Research Articles

Immune Cell Alterations in Psychotic Disorders: A Comprehensive Systematic Review and Meta-Analysis

BackgroundA comprehensive meta-analysis on the composition of circulating immune cells from both the myeloid and the lymphoid lines including specialized subsets in blood and cerebrospinal fluid (CSF) of patients with psychotic disorders compared with healthy control participants has been lacking. MethodsMultiple databases (PubMed, EMBASE, Cochrane Library, Web of Science, ClinicalTrials.gov, and PsycINFO) were searched for eligible studies up until October 18, 2022. All studies investigating circulating immune cells in the blood and CSF from patients with psychotic disorders (ICD-10: F20 and F22-29) compared with healthy control participants were included. ResultsA total of 86 studies were included in the meta-analysis. In the blood, the following categories of immune cells were elevated: leukocyte count (31 studies, standardized mean difference [SMD] = 0.35; 95% CI, 0.24 to 0.46), granulocyte count (4 studies, SMD = 0.57; 95% CI, 0.12 to 1.01), neutrophil granulocyte count (21 studies, SMD = 0.32; 95% CI, 0.11 to 0.54), monocyte count (23 studies, SMD = 0.40; 95% CI, 0.23 to 0.56), and B lymphocyte count (10 studies, SMD = 0.26; 95% CI, 0.04 to 0.48). Additionally, the neutrophil/lymphocyte ratio (23 studies, SMD = 0.40; 95% CI, 0.19 to 0.60), the monocyte/lymphocyte ratio (9 studies, SMD = 0.31; 95% CI, 0.04 to 0.57), and the platelet/lymphocyte ratio (10 studies, SMD = 0.23; 95% CI, 0.03 to 0.43) were elevated. The CSF cell count showed a similar tendency but was not significantly elevated (3 studies, SMD = 0.14; 95% CI, −0.04 to 0.32). ConclusionsThe results indicate a broad activation of the immune system in psychotic disorders, with cells from both the myeloid and the lymphoid line being elevated. However, CSF analyses were lacking in most of the studies, and many studies were hampered by insufficient adjustment for confounding factors such as body mass index and smoking.

Hypereosinophilic Syndrome Complicated by Ischemia of the Lower Limb Clinical Case with Review of the Literature

Hyper eosinophilia is the result of a disorder, which can be of central origin, with an excessive production of polymorphonuclear eosinophils (PNE), or of a peripheral dysfunction with an abundant recruitment of polynuclear neutrophils from the marrow by the tissues. peripheral more frequently the digestive and urogenital respiratory tissue. The diagnostics must be early to avoid complications. Any case of hypereosinophilia requires a very precise etiological investigation, with a balance sheet of visceral repercussions of eosinophilia. Some genetic abnormalities of the eosinophil line or a dysfunction of the lymphoid line, can be discovered, the targeted therapy trials have revolutionized the prognosis, sometimes pejorative of these diseases, we report here the case of hypereosinophilia complicated by ischemia of the lower limbs.

MiR-181a and -b expression in acute lymphoblastic leukemia and its correlation with acute graft-versus-host disease after hematopoietic stem cell transplantation, COVID-19 and torque teno viruses.

Acute lymphoblastic leukemia (ALL), a malignant transformation and proliferation of the lymphoid line of blood cells, is characterized by chromosomal abnormalities and genetic changes. The purpose of this research was the evaluation of expression level of miR-181a and -b in patients with ALL compared to the control group. Furthermore, we examined their expression level in hematopoietic stem-cell transplantation (HSCT) patients who developed acute graft-versus-host disease (aGVHD) in comparison with those without aGVHD and explore the relationship between their expression level and cytogenetic abnormalities. In this cross-sectional study, 76 newly diagnosed adult De novo ALL patients were enrolled who were admitted to our referral hospital. All patients received standard chemotherapy, consisting of daunorubicin. A total of 37 patients underwent HSCT from the related human leukocyte antigen-matched donors. ALL patients have been diagnosed with the coronavirus disease 2019 (COVID-19) and Torque teno viruses (TTVs). We assessed the expression levels of miR-181a and -b in the peripheral blood sample of ALL patients at the time of diagnosis prior to chemotherapy, and healthy matched individuals by RT–PCR. TTVs and COVID-19 load were also determined via RT–PCR. In conclusion, the expression level of miR-181a and -b were significantly higher in ALL patients than healthy controls and also increased in patients who developed aGVHD in comparison with those without aGVHD. MiR-181a and -b can be a useful biomarker in ALL and a useful indicator of aGVHD. The expression level of miR-181a in ALL patients with COVID-19 is significantly up-regulated, while it is reduced in these patients with TTV.

A Phase I/II Study of Arginine Butyrate and Ganciclovir in Patients with Epstein-Barr Virus-Associated Lymphoid Malignancies.

Malignancies associated with latent Epstein-Barr virus are resistant to nucleoside-type antiviral agents because the viral enzyme target of these drugs, thymidine kinase (TK), is not expressed. Short-chain fatty acids, such as butyrate, induce EBV-TK expression in latently-infected B cells. In preclinical studies, we have shown that butyrate sensitizes EBV(+) lymphoid cells and tumor lines to apoptosis induced by ganciclovir. We conducted a Phase I/II trial of Arginine Butyrate in combination with ganciclovir in patients with refractory EBV(+) lymphoid malignancies to evaluate toxicity, pharmacokinetic parameters, and clinical responses. Fifteen patients with refractory EBV(+) lymphoid malignancies, including monoclonal lymphoproliferative disease (PTLD), B cell non-Hodgkin's lymphomas (NHL), T cell NHL (including one panniculytic subcutaneous lymphoma), T/NK cell lymphomas, and Hodgkin's disease were studied. The patients had been relatively heavily pretreated with chemotherapy and/or radiation before entry into this study. In this study, ganciclovir was administered twice daily at approved doses and Arginine Butyrate (AB) was administered in an intra-patient dose-escalation. Arginine Butyrate was instituted at 500 mg/kg/day by continuous infusion, and escalated to 2000 mg/kg/day, as tolerated. Pharmacokinetic studies were performed. The MTD for Arginine Butyrate when administered in combination with ganciclovir was established as 1000 mg/kg/day by continuous infusion. Overall, the combination was well-tolerated, with the most common toxicities being nausea, headache. Complications from rapid tumor lysis occurred in three patients, including acute hepatic necrosis in one patient. Reversible somnolence or stupor occurred in three patients at Arginine Butyrate doses of greater than 1000 mg/kg/day. Ten of fifteen patients showed significant anti-tumor responses within 10 days of initiation of treatment, with 5 CR and 5 PR. In some of the patients who demonstrated a CR, subsequent pathological analysis showed no residual tumor cells. The combination of Arginine Butyrate and ganciclovir was reasonably well-tolerated and appears to have significant biological activity in vivo against EBV(+) lymphoid malignancies. Further trials, especially in patients with earlier stage disease, appear warranted.

Improvement in Survival Outcomes of Acute Lymphoblastic Leukemia Patients Treated With Berlin-Frankfurt-Munster 76/79 Protocol

Introduction: Acute lymphoblastic leukemia (ALL) is a malignancy of the white blood cells and lymphoid lines of hematopoietic cells in the blood and bone marrow while lymphomas mostly involve the lymph nodes. This study showed the response rate of ALL treated with BFM (Berlin-Frankfurt-Munster) 76/79 protocol.
 Aims & Objectives: To study the effects of WBC (White Blood Cells), age, and treatment protocols on the survival of ALL patients to BFM 76/79 protocol and other chemotherapy protocol.
 Place and duration of study: 6 months retrospective study on 2015-2017 indoor patient data from INMOL Hospital Lahore, Pakistan.
 Material & Methods: Data of 129 patients was assessed for eligibility, and 84 patients of ALL selected. Clinical information of patients regarding Gender, Age, WBC, Fever, Hepatosplenomegaly, Lymphadenopathy, Bleeding, Bone Pain, Immunophenotype, Relapse, Death, and survival rates were recorded. Patients were grouped according to Age, WBC, Gender, and Immunophenotype. Patients were treated with BFM 76/79 Protocols and the remaining patients were treated with other protocols (CALGB, FLAG-TDA, Hyper-CVAD, EURO, COG, T-Cell ALL protocol, and ALL protocol 9111).
 Results: Out of 84 patients, 55 (65.5%) patients were treated with Berlin-Frankfurt-Munster 76/79 protocol and the remaining 20 (23.81%) patients with other protocols. After the end of induction 64 (73.8%) patients achieved complete remission and 5 (5.95%) patients did not achieve Remission.
 Conclusion: Response rate of ALL (Acute Lymphoblastic Leukemia) patients treated with BFM 76/79 protocol was better than other protocols and possibly when risk adapted for male gender, WBC > 50 x 109/L and age> 30 years.

Effect of Selected Diets/Xanthone Derivatives on Morphotic Image of Bone Marrow and Other Tissues

Introduction: The diet as well as some xanthone derivatives can influence the processes occurring in the bone marrow, which is a hematopoietic organ and plays an important role in the immune system. Changes in the proportions of individual cell lines may lead to illnesses such as anemia or blood coagulation disorders. Aim: The aim of the study is to identify the effect of selected xanthone derivatives and high-fat diet on the morphological construction of individual cell lines in the bone marrow (erythrocyte line, myeloblastic line, monocytic line, megakaryocytic line, lymphoid line). Material and Methods: The studies were carried out on 4 groups of CD1 mice (n = 10): control (c), diabetes induced by a high-fat diet (f) and two derivatives of xanthone (gr. 1 and 2). Five-week-old animals received a standard or an enriched in fat diet for another 5 weeks. After decapitation of animals the bone marrow smear was prepared. The stained preparations were observed under a microscope and hematopoietic cells were counted. Results: Based on the results and preliminary statistical treatment, it was found that there is a relationship between the diet used and changes in the proportion of cells of particular hematopoietic lines. The strongest changes were observed in the number of lymphocytes. The control group has statistically significantly more, compared to the group receiving the fat diet. It was also found that there is a relationship between the dietary xanthone derivatives used and the changes in the proportion of cells of particular hematopoietic lines. Conclusion: The use of a high-fat diet and xanthone derivatives can have a significant impact on the change in the number of lymphocytes in the bone marrow.

The Association of Apolipoprotein A1 Gene Polymorphisms with Acute Lymphocytic Leukemia in Tehran Province

Background: Acute lymphocytic leukemia (ALL) is a cancer of the lymphoid line of blood cells. It is characterized by the development of large numbers of immature lymphocytes. It is one of the most common cancers among children. Molecular markers can play an important role in determining the nature of leukemia-related tumors and can be used as a diagnostic adjuvant along with precise pathological methods. Some research has shown that apolipoprotein A1 gene polymorphisms were associated with the formation of various types of diseases. Objectives: This study aimed to evaluate the association of ApoA1 gene insertion/deletion polymorphisms with ALL disease. Methods: The salting-out method was used for DNA extraction from the blood samples. Questionnaires were prepared with the approval of a demographic data expert. The genotype distribution and allele frequencies for the ApoA1 gene insertion/deletion polymorphisms were determined by Gap-polymerase chain reaction (Gap-PCR) to compare patient’s cases with ALL disease and healthy subjects. This case-control study was performed by odds ratio (OR, with a confidence interval of 0.95) to reveal the association of these polymorphisms with ALL disease. Results: In this study, two ApoA1 gene polymorphisms, including II and DD genotypes, were observed, which II genotype had the highest frequency in both groups of healthy subjects and patients. However, no significant differences were found between the two groups (P > 0.05). ALL and both genotypes had a higher frequency in males than females. Despite the high frequency of both genotypes in males, there were no significant differences between healthy subjects and patients in terms of age and sex (P > 0.05). Conclusions: Based on the results of this study, it can be concluded that ApoA1 gene polymorphisms were not associated with ALL disease. It was not effective in the formation or exacerbation of it in this population. Also, it could not be identified as a prognostic marker. It should be noted that these findings are the first report of the degree of association of ApoA1 polymorphisms with ALL.

Glucocorticoids can induce BIM to trigger apoptosis in the absence of BAX and BAK1

Cells from two murine lymphoid lines died 24–48 h after treatment with the glucocorticoid dexamethasone. Deletion of Bax and Bak1 prevented rapid apoptosis, but treatment with dexamethasone for greater 6 days still led to cell death that was characterized by release of cytochrome c into the cytosol, activation of caspases, and loss of cell membrane integrity. In WEHI7 thymoma cells, this did not occur when Bcl2l11 (Bim) was deleted in addition to Bax and Bak1. When these triple mutant lines were exposed to dexamethasone for 10 days, they arrested, but after dexamethasone was removed, they had 10-fold higher clone forming efficiency than Bax/Bak1 double knock-out cells. Although induced over-expression of BIMs alone was not sufficient to induce the death of Bax−/−Bak1−/−Bim−/− cells, they did die when BIMs was induced in the presence of dexamethasone. These results suggest that dexamethasone induces production of BIM together with other, as yet unidentified proteins, that cause release of cytochrome c and apoptosis in the absence of BAX and BAK1.

Enhancing the sensitivity of the thymidine kinase assay by using DNA repair-deficient human TK6 cells.

The OECD guidelines define the bioassays of identifying mutagenic chemicals, including the thymidine kinase (TK) assay, which specifically detects the mutations that inactivate the TK gene in the human TK6 lymphoid line. However, the sensitivity of this assay is limited because it detects mutations occurring only in the TK gene but not any other genes. Moreover, the limited sensitivity of the conventional TK assay is caused by the usage of DNA repair‐proficient wild‐type cells, which are capable of accurately repairing DNA damage induced by chemicals. Mutagenic chemicals produce a variety of DNA lesions, including base lesions, sugar damage, crosslinks, and strand breaks. Base damage causes point mutations and is repaired by the base excision repair (BER) and nucleotide excision repair (NER) pathways. To increase the sensitivity of TK assay, we simultaneously disrupted two genes encoding XRCC1, an important BER factor, and XPA, which is essential for NER, generating XRCC1 −/− /XPA −/− cells from TK6 cells. We measured the mutation frequency induced by four typical mutagenic agents, methyl methane sulfonate (MMS), cis‐diamminedichloro‐platinum(II) (cisplatin, CDDP), mitomycin‐C (MMC), and cyclophosphamide (CP) by the conventional TK assay using wild‐type TK6 cells and also by the TK assay using XRCC1 −/− /XPA −/− cells. The usage of XRCC1 −/− /XPA −/− cells increased the sensitivity of detecting the mutagenicity by 8.6 times for MMC, 8.5 times for CDDP, and 2.6 times for MMS in comparison with the conventional TK assay. In conclusion, the usage of XRCC1 −/− /XPA −/− cells will significantly improve TK assay.

Hyperoxia Alters Ultrastructure and Induces Apoptosis in Leukemia Cell Lines

Oxygenation conditions are crucial for growth and tumor progression. Recent data suggests a decrease in cancer cell proliferation occurring after exposure to normobaric hyperoxia. Those changes are associated with fractal dimension. The purpose of this research was to study the impact of hyperoxia on apoptosis and morphology of leukemia cell lines. Two hematopoietic lymphoid cancer cell lines (a T-lymphoblastoid line, JURKAT and a B lymphoid line, CCRF-SB) were tested under conditions of normobaric hyperoxia (FiO2 > 60%, ± 18h) and compared to a standard group (FiO2 = 21%). We tested for apoptosis using a caspase-3 assay. Cell morphology was evaluated by cytospin, microphotography after coloration, and analysis by a fractal dimension calculation software. Our results showed that exposure of cell cultures to transient normobaric hyperoxia induced apoptosis (elevated caspase-3) as well as significant and precocious modifications in cell complexity, as highlighted by increased fractal dimensions in both cell lines. These features are associated with changes in structure (pycnotic nucleus and apoptosis) recorded by microscopic analysis. Such morphological alterations could be due to several molecular mechanisms and rearrangements in the cancer cell, leading to cell cycle inhibition and apoptosis as shown by caspase-3 activity. T cells seem less resistant to hyperoxia than B cells.

Caso Clínico: Citopénia Autoinmune como complicación inmunológica de Trasplante Alogénico de Donante no emparentado, en paciente pediátrico con Anemia Drepanocítica

Introducción: La enfermedad de células falciformes es una condición heredada en la que se produce una hemoglobina anómala que desfavorece a la oxigenación tisular, crisis vaso-oclusivas y reacciones hemolíticas. Los pacientes con esta enfermedad presentan una activación anómala de la vía del complemento llevándolos al aumento en frecuencia de infecciones y enfermedades autoinmunes. Presentamos un caso de asociación de una enfermedad autoinmune en un paciente con enfermedad de células falciforme.
 Caso clínico: Niño de 10 años con Anemia drepanocítica (2009) con esplenectomía y crisis veno-oclusivas recurrentes, fue sometido a trasplante Alogénico en abril del 2019 fuera de la institución con donante isogrupo O+ no emparentado (10/10). Tratado con: Fludarabina – Busulfan, Timoglobulina+ y Metotexate. Desarrolló Bicitopenia autoinmune y síndrome febril al día +165 post TPH. Glóbulos blancos: 360 uL, neutrófilos: 14 %, hemoglobina: 7.90 g/dL, plaquetas: 25000 uL, ferritina: 4695 ng/ml, IgG total: 9.88 gr/l, LDH: 190 UI/l. Proteína C reactiva: 2.79 mg/dL, procalcitonina 0.13 ng/mL.
 Evolución: posterior a descartar infección viral, se completó un tratamiento antibiótico de amplio espectro y se realizó la suspensión del tratamiento inmunosupresor por sospecha de toxicidad, sin respuesta. Se realizó un estudio medular por citometría de flujo determinando una disminución de la línea linfoide B, y se concluye Citopénia autoinmune como complicación inmunológica del trasplante.
 Desenlace: recibió terapia transfusional (plaquetoferesis + glóbulos rojos concentrados). Se utilizó metilprednisolona IV por 3 días y prednisona 30 mg por 14 días con reducción posterior gradual para inicio de Rituximab y ciclosporina. Se completó el tratamiento con Imnunoglobulina 6g IV por 5 días. Al alta glóbulos blancos: 5080 uL, neutrófilos: 67%, hemoglobina: 9.20 g/dL, plaquetas: 20000 uL, después de 18 días de ingreso hospitalario.
 Conclusión: Los resultados con el tratamiento en este caso sugieren que puede ser razonable considerar las citopenias autoinmunes como una manifestación hematológica diagnóstica de la EICH crónica. Alternativamente, es posible que el tratamiento de citopenia inmune con esteroides, rituximab y otros inmunosupresores.

Fluorescence spectral detection of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML): A novel photodiagnosis strategy.

Acute lymphoblastic leukemia (ALL) is a cancer of the lymphoid line of blood cell, showing a rapid growth of lymphoblastic immature cells. Acute myeloid leukemia (AML) is a cancer of the myeloid line of blood cells. Early diagnosis is crucial for the effective treatment of these patients. The current standard procedure of diagnosis is extensive blood count analysis, microscopic morphological investigations, bone marrow biopsy and flow-cytometer which are all time- consuming and expensive. Here we demonstrate the advantage a new technique for the diagnosis of ALL and AML based on the fluorescence spectra of blood plasma and RBCs samples from the above patients. Based on the 85 patients analyzed the results reveal that our approach could discriminate the two malignancies unambiguously from the normal with 88 % sensitivity and 80 % specificity. The abnormal decrease in the level of amino acid, tryptophan and coenzyme NADH and abnormal increase in the other amino acid, tyrosine and another coenzyme FAD, (both in comparison to the normal control) act as malignancy indicative biomarkers. Since the contrast parameter between the normal and malignant samples is four- fold, the potential for early detection of leukemia is high. The instrumentation and technique are new and simple; hence may have significant supplementary or complementary value with the existing diagnostic methods.

Bacterial pore-forming toxin pneumolysin: Cell membrane structure and microvesicle shedding capacity determines differential survival of cell types.

Bacterial infectious diseases can lead to death or to serious illnesses. These outcomes are partly the consequence of pore-forming toxins, which are secreted by the pathogenic bacteria (eg, pneumolysin of Streptococcus pneumoniae). Pneumolysin binds to cholesterol within the plasma membrane of host cells and assembles to form trans-membrane pores, which can lead to Ca2+ influx and cell death. Membrane repair mechanisms exist that limit the extent of damage. Immune cells which are essential to fight bacterial infections critically rely on survival mechanisms after detrimental pneumolysin attacks. This study investigated the susceptibility of different immune cell types to pneumolysin. As a model system, we used the lymphoid T-cell line Jurkat, and myeloid cell lines U937 and THP-1. We show that Jurkat T cells are highly susceptible to pneumolysin attack. In contrast, myeloid THP-1 and U937 cells are less susceptible to pneumolysin. In line with these findings, human primary T cells are shown to be more susceptible to pneumolysin attack than monocytes. Differences in susceptibility to pneumolysin are due to (I) preferential binding of pneumolysin to Jurkat T cells and (II) cell type specific plasma membrane repair capacity. Myeloid cell survival is mostly dependent on Ca2+ induced expelling of damaged plasma membrane areas as microvesicles. Thus, in myeloid cells, first-line defense cells in bacterial infections, a potent cellular repair machinery ensures cell survival after pneumolysin attack. In lymphoid cells, which are important at later stages of infections, less efficient repair mechanisms and enhanced toxin binding renders the cells more sensitive to pneumolysin.

NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase

BackgroundHIV-1 integration results in genomic DNA gaps that are repaired by cellular DNA repair pathways. This step of the lentiviral life cycle remains poorly understood despite its crucial importance for successful replication. We and others reported that Ku70 protein of the non-homologous end joining pathway (NHEJ) directly binds HIV-1 integrase (IN). Here, we studied the importance of this interaction for post-integrational gap repair and the recruitment of NHEJ factors in this process.ResultsWe engineered HIV-based pseudovirus with mutant IN defective in Ku70 binding and generated heterozygous Ku70, Ku80 and DNA-PKcs human knockout (KO) cells using CRISPR/Cas9. KO of either of these proteins or inhibition of DNA-PKcs catalytic activity substantially decreased the infectivity of HIV-1 with native IN but not with the mutant one. We used a recently developed qPCR assay for the measurement of gap repair efficiency to show that HIV-1 with mutant IN was defective in DNA post-integrational repair, whereas the wild type virus displayed such a defect only when NHEJ system was disrupted in any way. This effect was present in CRISPR/Cas9 modified 293T cells, in Jurkat and CEM lymphoid lines and in primary human PBMCs.ConclusionsOur data provide evidence that IN recruits DNA-PK to the site of HIV-1 post-integrational repair due to Ku70 binding—a novel finding that explains the involvement of DNA-PK despite the absence of free double stranded DNA breaks. In addition, our data clearly indicate the importance of interactions between HIV-1 IN and Ku70 in HIV-1 replication at the post-integrational repair step.

Abstract 2467: Heme-CMap: Generation and characterization of ~20K L1000 profiles across 11 hematologic malignant lines

Abstract The Next Generation Connectivity Map (CMap) provides a resource to explore the relationships between diseases, genetic perturbations and compound treatments at the transcriptome level. Current CMap focuses on 9 tumor cell lines of various cancer types, but none of hematological malignancies. Considering the distinctive features of hematologic malignancies at both transcriptome and genomics alteration level, we recognized the pressing need for a similar resource of hematologic malignancies to better understand their unique characteristics of compound treatment response. Here we generated the “Heme-CMap” which included 7 lymphoid and 4 myeloid lines. In order to better understand the compound effects on hematological malignant lines, especially lineage specificity of lymphoid versus myeloid lines, we selected 100 compounds targeting B-cell lymphoma and 38 compounds targeting cancer pathways and hallmarks in our reference compound list. Each compound was tested at 4 time points and 6 doses. To this end, the Heme-CMap encompasses 20,472 compound perturbations. The size of this dataset and compound collection allowed us not only to globally compare the classic CMap to the newly generated Heme-CMap, but also to explore cell lineage effects of lymphoid versus myeloid cell lines within Heme-CMap. We found that several perturbation classes showed differential activity between classic CMap and Heme-CMap while some pathways show coherent activity. For example, EGFR inhibitors showed coherent activity in both classic CMap and Heme-CMap. On the other hand, BTK inhibitors showed coherent effects only in a subset of the lymphoid lines, but not the myeloid lines, consistent with the function in altering B cell signaling. We further investigated the compounds in Heme-CMap and observed samples clustered into two major groups by mechanism of action (MoA) of the compounds. One group contained samples treated with compounds targeting growth and survival, and B-cell signaling pathways, while the other group contained samples treated with compounds causing cell killing. Interestingly, we found both groups in the lymphoid lines but only the cell killing group in the myeloid lines. This may reflect our compound selection targeting signaling pathways implicated B cell lymphoma. These results suggest cell lineage plays an important role in compound response, and the experimental design of Heme-CMap allowed us to observe the differential compound effect in lymphoid and myeloid lines. We also found different types of compounds showed maximum effects at different timepoints and doses, suggesting various timepoints and doses can provide insights of compound MoA. In summary, our results demonstrate that Heme-CMap with lineage specific cell lines can elucidate compound MoAs complementary to classic CMap. Additionally, this effort also serves as a roadmap for generating data collaboratively with CMap on a large scale. Citation Format: Chia-Ling Huang, Andrew Yang, Ted Natoli, Lev Litichevskiy, Frederic Vaillancourt, Alan Rolfe, Yonghong Xiao, Aravind Subramanian, Lihua Yu. Heme-CMap: Generation and characterization of ~20K L1000 profiles across 11 hematologic malignant lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2467.

PB1712 PROGNOSTIC IMPACT OF ABERRANT EXPRESSION IN ACUTE MYELOID LEUKEMIA

Background:Immunophenotyping allows identification of aberrant antigen expression in acute myeloid leukemia (AML). These antigens are related with particular and genetic abnormalities and recognized as factors of poor prognosis in AML.Aims:To assess prognostic impact of aberrant expression in treated AML.Methods:Our study is retrospective (January 2017‐June 2018) concerning 97 cases of AML (AML 3 and mixed phenotype acute leukemia were excluded) of the adult from 18 to 60 years old diagnosed and treated at one institution. The immunophenotyping was performed by flow cytometry (BD Facs Canto II®) after incubation with monoclonal antibodies labeled with different flurochromes. Aberrant expression correspond to the expression of antigens of the lymphoid line B or T in AML. We studied the impact of aberrant expression on complete remission (CR) and relapse in a delay of 1 year. We excluded the cases with missing data.Results:We had collected 33 cases of treated AML. Aberrant lymphoid antigen were seen in 18 cases (54.54%). It is about CD 56 in 6 cases (33%), CD7 in 6 cases (33%), CD4 in one case (5.5%) and CD19 in 2 cases (11%). Association between lymphoid B and T antigens or T and NK antigens was seen in 3 cases (CD19 + CD7, n= 1; CD7 + CD56, n=2). We didn’t find any correlation between expression of lymphoid antigen and lower CR rates (93% vs 54%; r= 0.17, p = 0.42). Analysed individually, no lymphoid antigen was associated with more induction failure. Relapse seems to be associated with aberrancy expression without statistically significant association (p = 0.29); CD7 was the most aberrant antigen expressed in cases of relapses (75%).Summary/Conclusion:The expression of aberrant antigens in AML is correlated with poor prognosis in literature. This result was not statistically prouved in our study and that's can be explained by the reduced size of our sample. Otherwise, our study shows the pejorative impact of the expression of CD7 on cases of relapses. Expression of CD56 (33% of our cases), similar to literature (10‐30% of cases), was not associated with higer relapse's rate in the limit of our sample (12.5% of AML CD56 + ).Despite the limited number of cases, this study allow us to better understand the impact of aberrant antigens expression in prognosis of AML. A study on a larger series is underway.

Non-linear Dose Response of Lymphocyte Cell Lines to Microtubule Inhibitors.

Microtubule (MT) inhibitors show anti-cancer activity in a wide range of tumors in vitro and demonstrate high clinical efficacy. To date they are routinely included into many chemotherapeutic regimens. While the mechanisms of MT inhibitors’ interactions with tubulin have been well-established, the relationship between their concentration and effect on neoplastic cells is not completely understood. The common notion is that tumor cells are most vulnerable during division and all MT inhibitors block them in mitosis and induce mitotic checkpoint-associated cell death. At the same time multiple evidence of more subtle effects of lower doses of MT inhibitors on cell physiology exist. The extent of efficacy of the low-dose MT inhibitor treatment and the mechanisms of resulting cell death currently present a critical issue in oncology. The prospect of MT inhibitor dose reduction is promising as protocols at higher concentration have multiple side effects. We assessed cell cycle changes and cell death induced by MT inhibitors (paclitaxel, nocodazole, and vinorelbine) on human lymphoid B-cell lines in a broad concentration range. All inhibitors had similar accumulation effects and demonstrated “trigger” concentrations that induce cell accumulation in G2/M phase. Concentrations slightly below the “trigger” promoted cell accumulation in sub-G1 phase. Multi-label analysis of live cells showed that the sub-G1 population is heterogeneous and may include cells that are still viable after 24 h of treatment. Effects observed were similar for cells expressing Tat-protein. Thus cell cycle progression and cell death are differentially affected by high and low MT inhibitor concentrations.

Abstract 17: Increasing Ischemia Time Diminishes Chimerism After Vascularized Bone Marrow Allotransplantation

PURPOSE: Vascularized composite allotransplantation (VCA) can include vascularized bone marrow (VBM) to potentially generate chimerism and subsequent tolerance in the recipient. While increasing ischemia time is known to be detrimental to free flaps and solid organ transplants, it is unknown whether increasing VCA ischemia time may diminish VBM’s capacity to achieve chimerism and tolerance. This study determines whether increasing cold ischemia time results in decreased peripheral chimerism levels, increasing loss of the donor bone marrow compartment, and increased skin paddle rejection in a murine VCA model. METHODS: Twenty-three adult Lewis rats received osteomyocutaneous hind limb allografts from 3 groups of Brown-Norway rats. The groups underwent 0, 6, and 12 hours of cold ischemia at 4°C (n=8, 6, and 9 respectively). Immunosuppressive conditioning consisted of anti-lymphocyte serum 1cc 3 days pre-operatively and on post-op day (POD) 1, tacrolimus 2mg/kg/day for PODs 1–7, and then concluded with rapamycin 3mg/kg/day for PODs 8–28. Peripheral chimerism levels were drawn after POD 30. Skin paddle was observed for 30 days after immunosuppression cessation for rejection or tolerance. Allograft was collected at POD 60 for flow cytometry and histologic analysis. RESULTS: Peripheral chimerism levels in both myeloid and lymphoid lines were decreased in the 12 & 6 hours groups versus 0 hours (p<0.01). At POD 60, the amount of donor cells in the allograft bone marrow was significantly decreased at 12 & 6 hours versus the 0 hours group (p<0.05). At 12 hours, hematoxylin & eosin histology demonstrated significantly increased reactive lymphocytes, increased stroma, significantly decreased hematopoetic cells, and significant myelofibrosis. At 6 hours, moderate stromal increases, moderately decreased hematopoetic cells, and moderate myelofibrosis was observed. Trichrome staining revealed viable bone at all time points though with there were increasing amounts of collagen deposition with increasing ischemia and osteoslerosis was noted at 12 hours. Microvasculature was preserved at all time points. By POD 60, 0% of skin paddles survived in the 12 hours group, 17% at 6 hours, and 73% at 0 hours. CONCLUSION: With increasing ischemia time prior to VCA re-vascularization, there is decreased peripheral chimerism levels, increased loss of donor hematopoetic cells in the donor bone marrow, and increased skin paddle rejection. These findings indicate that ischemia time may significantly limit the capacity for VBM to facilitate chimerism and subsequent VCA tolerance and thus strategies using VBM to induce tolerance may need to consider the additional obstacle of time. Prolonged ischemia time may be a contributing factor to the absence of stable chimerism and tolerance in human VBM-bearing VCA recipients.

Collagen-Embedded Tumor Transplantations in Xenopus laevis Tadpoles.

The Xenopus laevis tadpole provides a valuable model for studying tumorigenesis and tumor immunity by intravital real-time microscopy. Using well-characterized thymic lymphoid tumor lines (15/0 and ff-2) that are transplantable into their compatible hosts (LG-15 isogenic clones and the F inbred strain, respectively), a system of semisolid tumor engraftment has been designed. Because these lymphoid tumor cell lines are not adherent and grow in suspension, they are first immobilized in a matrix of type I rat tail collagen before transplantation as a semisolid tumor graft under the transparent dorsal skin in the head region of a tadpole. This semisolid tumor engraftment is amenable to manipulation and permits real-time visualization of tumor growth, neovascularization, collagen rearrangements, immune cell infiltration, and formation of the tumor microenvironment.

ETV6 Regulates Pax5 Expression in Early B Cell Development

Recent studies from our group and others have revealed a role for ETV6 germline mutations in the predisposition to ALL. Although ETV6 is among the most commonly mutated genes in ALL, its mechanistic role in leukemogenesis remains unclear. ETV6 is an ETS family transcription factor. ETV6 regulates gene transcription through homo- and hetero- oligomerization with other ETS family members and transcriptional repressors. The germline mutation (P214L amino acid change) identified by our group and others impairs the transcriptional activity and nuclear localization of ETV6 in a dominant negative fashion. The goal of this project is to determine the role of ETV6 in early B cell development and define how germline ETV6 mutations result in predisposition to leukemia.To identify functions of ETV6 in B cell development, we queried the gene expression commons database for evidence of Etv6 expression during B cell development. Etv6 is highly expressed in hematopoietic stem and lymphoid progenitor cells through the pre-pro-B stage (FrA), but its expression is significantly reduced in fraction B and thereafter (P<0.0001). To confirm relative patterns of Etv6 and Pax5 expression in developing B cells, we isolated bone marrow (BM) from wild type (WT) mice and fractionated cells committed to the B cell lineage via B220+ and CD43+ staining by flow cytometry and then separated into the following fractions: Fraction A (CD24low, CD19-), Fraction B (CD19+, CD24+, BP1-) and Fraction C (CD19+ CD24+ BP1+). Etv6 expression decreases as B cells develop and is negatively correlated with Pax5 expression (r2=.9993; P= 0.0167). We next confirmed the expression patterns of ETV6 and PAX5 during B cell development in human samples. We found that ETV6 expression was higher in the early B cell fraction (CD10+, CD34+, CD19-, and CD20-) compared to the preB cell fraction (CD10+, CD34-, CD19+, CD20-). Conversely, we observed that PAX5 expression was higher in the preB cell fraction compared to the early B cell fraction.To determine if a function relationship exists between ETV6 and Pax5 we overexpressed an empty vector (MiG), wild type (WT) ETV6 and ETV6 P214L in a murine lymphoid progenitor line (Ba/F3). ETV6, but not ETV6 P214L overexpression significantly decreased Pax5 expression (P≤0.05). To further interrogate the role of ETV6 in regulating Pax5 transcription we measured the association of ETV6 with putative ETS factor binding sites (GGAA sequence) within the Pax5 transcription start site (TSS) using ChIP-PCR. ETV6 is associated with the proximal GGAA site 72 base pairs upstream of the Pax5 TSS, but not GGAA sites further from the TSS. In addition, the transcriptional repressors SIN3A and HDAC3 were detected on the same regions of the Pax5 locus. We next determined the consequences of ETV6 mutation on the recruitment of ETV6, SIN3A, and HDAC3 to the Pax5 locus by performing ChIP-PCR in Ba/F3 cells that express a FLAG-tagged WT ETV6 or ETV6 P214L. We detected association of ETV6, SIN3A and HDAC3 with the proximal GGAA site upon expression of WT ETV6, but not ETV6 P214L. We conclude that ETV6, SIN3A and HDAC3 are responsible for the repression of Pax5 transcription. Moreover, mutant ETV6 inhibits the ability of normal ETV6 to bind and recruit SIN3A and HDAC3 to the Pax5 locus. Finally, we determined if the recruitment of SIN3A and HDACs to the Pax5 locus was essential to repression of Pax5 by WT ETV6 by knocking out SIN3A and inhibiting HDACs using pan HDAC inhibitor, SAHA and measuring Pax5 expression by RT-PCR. We found that upon SIN3A knockout or HDAC inhibition Pax5 expression was no longer repressed upon WT ETV6 overexpression.To determine the consequences of ETV6 P214L expression on B cell development, we generated a transgenic mouse expressing the P214L mutation in the endogenous ETV6 gene. Preliminary data suggests that these mice have thrombocytopenia, similar to patients with germline ETV6 mutation. In addition, mice with the ETV6 P214L mutation displayed reduced level of cKIT expression on the FrA B cell population. Further studies will be necessary to understand the consequences of reduced cKIT expression to overall B cell development and if this cKIT reduction is linked to aberrant Pax5 expression.In conclusion, ETV6 regulates Pax5 expression through the recruitment of SIN3A and HDAC3 to the Pax5 locus. These findings are significant because Pax5 misregulation results in a B cell development halt, lineage infidelity and leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.