Cell Disease Research Articles

Male sickle cell patients, compensated transpubertal hypogonadism and normal final growth.

Investigate the gonadal hormonal function in sickle cell individuals. Sickle cell disease (SCD) is associated with delayed physical and sexual development, and it has been related to both primary testicular failure and hypothalamo-pituitary-gonadal axis abnormalities. The study of the pituitary gonadotrophin reserve was done evaluating the hormonal levels before and after stimulation by gonadoliberin. Male patients with homozygous SCD (18-39years, median=29.5years). Gonadal function was evaluated through clinical parameters and the hormonal quantification. Although low body weight and other clinical signs of undernutrition such as clinical hypoandrogenism and the extreme retardation of puberty were seen in these patients, final stature and hormonal testicular reserve to hCG stimulation were proved to be normal according to our previous data. In the present investigation, the basal luteotropic gonadotropin (LH), follicle-stimulating hormone (FSH) and testosterone (T) levels were similar between the patients and controls. Prostate-specific antigen (PSA) levels-used as a biochemical marker of androgenicity, mainly in puberty-were lower in the patients than in the controls and were only correlated with T. A subtle abnormality in the pituitary responsivity to gonadotropin-releasing hormone (GnRH) was disclosed, with a higher response to LH 60minutes after stimulation in patients than in controls. These data, in addition to both the clinical and biochemical signs of hypoandrogenism associated with normal to elevated T levels strongly suggest a peripheral origin of hypogonadism, which is probably due to androgen resistance in the patients with SCD.

Expression of carcinoma, apoptosis, and cell-death-related genes are determinants for sensitivity of pediatric cancer cell lines to lysis by natural killer cells.

Natural killer (NK) cells have potential utility in pediatric cancer immunotherapy for their ability to lyse diverse tumor targets, lack of dependence on mutation-associated tumor antigens, and for their relative safety demonstrated so far in clinical trials. Here, we evaluate the cytotoxic potential of expanded NK cells against a well-characterized panel of pediatric cancer cell lines representing Ewing sarcoma, rhabdomyosarcoma, neuroblastoma, lymphoma, leukemia, and brain tumors. We correlate their sensitivity NK cell lysis with tumor phenotypic, transcriptomic, and genetic determinants, and correlate known immunogenetic determinants with donor NK cell potency. Although ligand expression on cell lines stratified according to hematologic versus nonhematologic cancer types, the sensitivity to NK cell lysis varied widely and did not correlate with cancer type, expression of individual activating or inhibitory ligands, gene-expression clusters of NK cell ligands, disease status (newly diagnosed or relapsed), or MYCN amplification. Rather, sensitivity to NK cell-mediated lysis was associated with a novel 96-gene cluster of predominantly carcinoma-, apoptosis-, and cell death-related pathways, and with functional p53 status. NK cell potency was strongly associated with activating KIR gene content, but not with KIR/KIR-ligand mismatch. This study suggests that adoptive immunotherapy with expanded NK cells has the potential for a wide range of pediatric cancers, identifies potential biomarkers of efficacy and response, and establishes a foundation for using this cell line panel for the preclinical evaluation of immunotherapies.

SEMEN CHARACTERISTICS IN HIV-INFECTED MEN

The goal of the study was to evaluate the impact of antiretroviral therapy (ART) on the semen characteristics in HIV-infected men.Materials and methods. A prospective case-control study enrolled 66 HIV-infected male patients who presented for fertility assessment. Group 1 included 51 male patients treated with ART; Group 2 included 15 males who were not receiving ART. 97 and 25 semen samples were analyzed, respectively. HIV status was assessed based on the data regarding the stage and phase of the disease, viral load, CD3+, CD4+, CD8+ counts and the ART duration.Study results. At enrollment in the study, the duration of HIV infection in Group 1 was longer than that in Group 2 (the difference being statistically significant): 5 years (2–9 years) versus 2,5 years (0,4–4,5), respectively (р=0,0004). Median duration of treatment in Group 1 was 1,5 years. The most common type of abnormal semen morphology in ART-treated patients was teratozoospermia (40%), and the percentage of abnormally shaped spermatozoa in this group was higher compared to Group 2 (the difference being statistically significant): 97 (96–98) versus 96 (94,5–96,5); р=0,006). Group 1 demonstrated a negative relationship between the count of abnormally shaped spermatozoa and CD4+ cell count (r=–0,362; р=0,026), disease duration (r=0,173; р=0,173) and sperm count (r=–0,242; р=0,020), progressively motile (category B) sperm count (r=–0,241; р=0,024). The ART duration showed an inverse correlation with the progressively motile sperm count (category B) (r=–0,224; р=0,036). Group 2 demonstrated a statistically significant positive relationship between the CD4+ cell count, the semen volume (r=0,778; p=0,014), and the count of progressively motile (category B) spermatozoa (r=0,667; р=0,05). We also revealed a statistically significant inverse relationship between the viral load and the semen volume (r=–0,669; р=0,035). Sperm DNA fragmentation was found to be higher in HIV-infected patients treated with ART compared to HIV-infected men not receiving ART (15,8% (12,4–23,0) and 14% (10,9–20,5); р=0,533), respectively. Thus, HIV-infected patients treated with ART demonstrated abnormal sperm morphology (increased abnormal sperm count), decreased sperm motility and increased sperm DNA fragmentation level. The percentage of abnormally shaped sperm was higher in patients with lower CD4+ cell counts. In patients with a long history of HIV infection, the sperm count decreases and the count of immotile spermatozoa increases. HIV-infected individuals who are not receiving ART and have high viral loads show decreased semen volume. This value increases upon an increase in the CD4+ cell count.

Abstract 1056: Xenograft-associated B cell lymphoproliferative disease (XABLD) as a surrogate model to study Epstein-Barr virus (EBV) driven B cell Diseases

Abstract Background: Patient-derived tumor xenografts (PDX) are powerful tools to study cancer biology, cancer genomics and developmental therapeutics. A common problem in the development of PDX models is proliferation of atypical lymphocytes at the implantation site, which often overtake or limit the growth of the original tumor. This atypical lymphocyte proliferation has been described as XABLD in our PDX models. In this study, we characterized XABLD cases by morphology, immunophenotyping and genomic profiling. We hypothesize that XABLD tumors are morphologically and phenotypically similar to EBV-driven post-transplant lymphoproliferative disease (PTLD) and diffuse large B cell lymphoma (DLBCL). XABLD is a surrogate model to study EBV-driven PTLD and DLBCL. Materials and Methods: Models were generated from patient tissue collected under NCI Tissue Procurement Protocol (clinicaltrials.gov: NCT00900198) and CIRB Tissue Procurement Protocol 9846 for development of models for NCI’s Patient-Derived Models Repository (https://pdmr.cancer.gov). Specimens were implanted subcutaneously in NOD/SCID/IL2Rg null (NSG) mice and animal health was monitored throughout the study. Tumors in mice with suspected XABLD were harvested and reviewed by histology and immunohistochemical analysis for CD45, B and T cell markers, EBV status, B-cell clonality assay. All samples were also classified by the Lymph2Cx NanoString cell of origin assay and transcriptome profiling. Results: XABLD cases were found to originate from both solid tumor and circulating tumor cell implants. XABLD is a rapidly growing tumor positive for CD45, CD20, and LMP1 stains, 36 of 42 cases are strongly positive for PD-L1 stain. 39 of 42 cases exhibited an activated B cell (ABC) phenotype with evidence of elevated NF-kB signaling. Most cases were monoclonal for IGK/IGH and contained high numbers of tumor infiltrating CD8-positive T-cells with associated high mRNA expression of activated T cell markers. Conclusion: The clinical presentation, morphology and molecular characteristics of XABLD cases were similar to EBV-driven DLBCL. As the XABLD models exhibited frequent PD-L1 expression and marked infiltration of CD8-positive T cells, they may be useful for in vitro evaluation of checkpoint inhibitor response and T cell antitumor activity. Grant Support: This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Citation Format: Tomas Vilimas, Gloryvee Rivera, Brandie Fullmer, Wiem Lassoued, Lindsay Dutko, Amanda Peach, Corinne Camalier, Li Chen, Rajesh Patidar, Suzanne Borgel, John Carter, Howard Stotler, Raymond Divelbiss, Jesse Stottlemyer, Michelle M. Gottholm-Ahalt, Michelle Crespo-Eugeni, Sean McDermott, William Jacob, Liqiang Xi, Pallavi Galera, Yvonne A. Evrard, Melinda G. Hollingshead, Elaine S. Jaffe, Mark Raffeld, Biswajit Das, Chris Karlovich, Vivekananda Datta, James H. Doroshow, P. Mickey Williams. Xenograft-associated B cell lymphoproliferative disease (XABLD) as a surrogate model to study Epstein-Barr virus (EBV) driven B cell Diseases [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 1056.

Apport des examens de biologie dans la prise en charge des hémopathies lymphoïdes B matures. L’intérêt de l’intégration des données clinicobiologiques

Diagnosis of mature B cell malignancies is highly multidisciplinary. Biological tools provide diagnostic, prognostic and theranostic information. Biological hematology allows considering mature B cell diseases from two perspectives: cellular and molecular approaches. Cytomorphology and flow cytometry are tools from cell hematology. Conventional cytogenetics, FISH and molecular biology are tools from molecular hematology. NGS is a new technique that could dramatically change diagnostic and therapeutic management of B cell malignancies in the near future. Integration of clinical, pathological and biological data allows for personalized management of these diseases.

Abstract 458: Precision systems medicine in acute myeloid leukemia: real-time translation of tailored therapeutic opportunities arising from ex-vivo drug sensitivity testing and molecular profiling

Abstract Acute myeloid leukemia (AML) is an aggressive disease of clonal hematopoietic progenitor cells. Here, we applied ex-vivo drug sensitivity and resistance testing on AML patient cells with 362 emerging and 153 approved cancer drugs together with genomic and transcriptomic profiling to identify and tailor therapies for patients with advanced disease. Ex-vivo testing with freshly isolated patient cells revealed cancer-specific efficacies of approved drugs in 97% of the 164 patient cases, including 47% of the cases with no actionable driver mutations. We identified 142 statistically significant associations between drug responses and somatic mutations, including increased sensitivity to JAK inhibitors in patients with NPM1 mutations. Transcriptomic profiles predicted drug responses better than genomics and helped to identify additional response markers, especially beyond mutations. For example, overexpression of HOX family genes was associated with sensitivity to JAK inhibitors in patients with NPM1 mutation. In a prospective study, we translated the functional drug response and molecular profile data to the clinic and suggested tailored therapy with targeted drugs for 26 relapsed or refractory AML patients. In an observational intervention study, acting on these recommendations resulted in a temporary complete clinical remission or leukemia-free state in 39% of the cases. In summary, we conclude that ex-vivo testing of drugs on patient AML cells i) revealed clinically actionable drug efficacies in almost all AML patients, including patients with no actionable mutations, ii) predicted cases with actionable driver mutations with no pharmacological dependency on the target, and iii) enabled real-time tailoring of therapy with 39% clinical response rate in chemorefractory advanced AML. Taken together, we believe this real-time systems medicine approach could become a powerful strategy for tailoring therapies for individual patients in the future. Citation Format: Disha Malani, Ashwni Kumar, Bhagwan Yadav, Mika Kontro, Swapnil Potdar, Oscar Bruck, Säri Kytölä, Jani Saarela, Samuli Eldfors, Riikka Karjalainen, Muntasir M. Majumder, Imre Västrik, Pekka Ellonen, Matti Kankainen, Minna Suvela, Siv Knappila, Alun Parson, Aino Palva, Pirkko Mattila, Evgeny Kulesskiy, Laura Turunen, Karoliina Laamanen, Elina Lehtinen, Maria Nurmi, Katja Suomi, Astrid Muruimägi, Bjorn T. Gjertsen, Satu Mustjoki, Simon Anders, Maija Wolf, Tero Aittokallio, Krister Wennerberg, Caroline Heckman, Kimmo Porkka, Olli Kallioniemi. Precision systems medicine in acute myeloid leukemia: real-time translation of tailored therapeutic opportunities arising from ex-vivo drug sensitivity testing and molecular profiling [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 458.

Abstract 5206: Role of super-enhancers in HPV+ head and neck squamous cell carcinoma

Abstract Head and neck squamous cell carcinomas (HNSCC) are the sixth leading cause of cancer worldwide with different incidences, mortalities, and prognosis for different subsites. Infection by Human Papilloma Viral (HPV) can cause the development of HPV+ HNSCC, the most rapidly growing population of cancer patients. The molecular biology of HPV+ HNSCC is related to abnormal transcriptional regulation. Super-enhancers (SEs) were recently identified as critical regulators of gene expression during cell differentiation and disease development. SEs are long clusters of enhancers that recruit master transcription factors (TFs) and coactivators to regulate the expression of target genes. We have recently developed a new whole-genome analytical pipeline to optimize ChIP-Seq procedures on primary surgical samples, and this protocol helped us to define the whole-genome distribution of H3K27ac, the hallmark of SEs, in HPV+ HNSCC samples. To detect SEs, we used a novel LILY algorithm that corrects the ChIP-Seq signal for copy number variation and CpG density. The analysis revealed that HPV+ HNSCC-specific SEs regulate genes critical for head and neck cancer development, such as EGFR, TP63, JAG1, IRF6, SMAD3, MAP4K2, SNAI1, TNFAIP3, ANO2, and TNFRSF1A. These genes are located in the vicinity of HNSCC-specific SEs, and the expression of those genes was altered by JQ1, the inhibitor of BRD4, the main component of SE machinery, supporting the role of SE in their regulation. The following Cistrome analysis allowed us to elucidate the TF composition of the SE machinery. Thus, we have found out that P63, P53, SOX2, FOSL1, SMAD3, and SNAI2 are TFs that are the most overrepresented in the HPV+ HNSCC-specific SEs. These comprehensive analyses have revealed novel insight into the HPV+ HNSCC biology and paved the basis for the implications for epigenetic therapeutics for this tumor types, and potentially other virus-related malignancies. Citation Format: Fernando T. Zamuner, Ilya Vorontsov, Emily Flam, Vera Mukhina, Ludmila Danilova, Tingting Ou, Elena Stavrovskaya, Theresa Guo, Eric Windsor, Dylan Z. Kelley, Michael Parfenov, Michael Considine, Elana J. Fertig, Alexander Favorov, Daria A. Gaykalova. Role of super-enhancers in HPV+ head and neck squamous cell carcinoma [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 5206.

Abstract SY21-01: Analyzing tumor metabolism in patients

Abstract Metabolism is dynamic and responds to a wide range of cell-intrinsic and cell-extrinsic factors. Many human diseases involve perturbations of metabolism at the cellular level, and in many cases normalizing the metabolic state has proven to be therapeutically valuable. Cancer is one such disease, in which factors both intrinsic and extrinsic to the malignant cell impact tumor biology and disease progression. In cancer, cell-intrinsic influences on metabolism include somatically-acquired mutations in oncogenes and tumor suppressor genes, many of which regulate metabolic activity. Cell-extrinsic factors include nutrient access, which may become limiting due to inadequate vasculature and intense fuel utilization, and metabolic interactions with stromal and immune cells. A major challenge in cancer metabolism research is to understand how these various factors culminate in the metabolic phenotype of an intact tumor, and ultimately to identify which altered pathways represent potential therapeutic targets. Among the many metabolic pathways reprogrammed in cancer, nucleotide metabolism and responses to DNA damage are highly integrated with aberrant signaling and gene regulatory networks in cancer. We have taken two parallel approaches to understand metabolic complexity and identify potential therapeutic liabilities in human cancer. The first uses a combination of multi-parametric imaging and intraoperative stable isotope infusions to assess metabolic fluxes in patients with solid tumors, and to compare fluxes between tumors and adjacent tissue. Genomic, histological and metabolic analysis of tumor samples allows us to correlate various intrinsic and extrinsic factors to specific aspects of the metabolic phenotype. The second approach uses standardized culture conditions to assess cell-intrinsic heterogeneity of metabolic preferences and dependencies in large panels of human cancer cell lines. This approach has uncovered liabilities associated with specific molecular subtypes of non-small cell and small cell lung cancerm ncluding several related to nucleotide and nucleic acid metabolism. I will discuss the application of these approaches to metabolic heterogeneity in human cancer, with an emphasis on features relevant to intact tumors and therapeutic liabilities and the role of metabolic imaging in studying cancer metabolism. Citation Format: Ralph J. Deberardinis. Analyzing tumor metabolism in patients [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 SY21-01.

Abstract 1739: Diminished SKP1 or CUL1 expression induces chromosome instability in high-grade serous ovarian cancer precursor cells

Abstract High-grade serous ovarian cancer (HGSOC) is the most common ovarian cancer subtype as well as the most lethal. High mortality rates associated with HGSOC are in part due to a lack of reliable early detection methods and effective therapies. Importantly, chromosome instability (CIN; an increased rate of chromosome gains or losses), is causally implicated in cancer development, progression, and resistance to treatment, and is just beginning to be evaluated in an HGSOC context. Overexpression of Cyclin E1 protein induces CIN and genomic amplification contributes to HGSOC pathogenesis in 20% of HGSOC patients. Cyclin E1 protein levels are normally regulated in a cell cycle-dependent manner by the SCF (SKP1-CUL1-FBOX) complex, an E3 ubiquitin ligase that targets substrates for proteolytic degradation. In cases where genomic amplification is absent, we hypothesize that loss of SCF complex function stemming from diminished expression of individual SCF complex components underlies increases in Cyclin E1 levels and induces CIN. The current study focuses on the SCF complex components SKP1 and CUL1 and characterizes their role in early HGSOC pathogenesis within a fallopian tube secretory epithelial cell model (FT; a cell of origin for HGSOC). Using siRNA-based approaches, we show that diminished SKP1 and CUL1 expression results in increased Cyclin E1 protein levels in FT cells. Using quantitative imaging microscopy techniques, we further identify statistically significant changes in CIN-associated phenotypes within the silenced populations, including changes in nuclear areas, increases in micronucleus formation (i.e. small DNA-containing bodies outside of the primary nucleus), and increases in numerical chromosome abnormalities identified in mitotic chromosome spreads. Thus, the current study explores the early origins of HGSOC and identifies SKP1 and CUL1 as two promising new molecular players that may contribute to CIN to drive FT cell transformation, drug resistance and disease recurrence. Citation Format: Chloe C. Lepage, Mark W. Nachtigal, Kirk J. McManus. Diminished SKP1 or CUL1 expression induces chromosome instability in high-grade serous ovarian cancer precursor cells [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 1739.

Abstract 1823: SNORD78 promotes prostate cancer progression and regulates AR expression and signaling

Abstract While small nucleolar RNAs (snoRNAs) have long been thought of as housekeeping genes, emerging evidence now suggests that snoRNAs are involved in more than previously thought and also in the development of disease. In cancer, it is understood that snoRNA expression and function can be altered, but little is known regarding the role of snoRNAs in these diseases. SNORD78 is a C/D box snoRNA which has been shown to be overexpressed in prostate cancer and associated with disease progression. However, there are no studies functionally characterizing SNORD78 in prostate cancer. In this study we sought to understand the functional contribution of SNORD78 in prostate cancer progression. Oncomine was used to assess SNORD78 expression in clinical prostate cancer samples. LNCaP and C4-2B prostate cancer cell lines were used. We used siRNAs to inhibit SNORD78 expression and cell viability assays to assess cell growth. ELISAs were used to assess PSA secretion and cell death. Western blots and quantitative PCR were used to assess cell death (PARP-cleavage) and AR expression and signaling. We found that SNORD78 expression was up-regulated in prostate tumors and associated with metastatic progression. Cell viability assays demonstrated that inhibition of SNORD78 expression resulted in a drastic reduction in prostate cancer cell growth and lower secretion of PSA into the media. Cell death ELISAs and western blots for cleaved-PARP showed that SNORD78 inhibition induced robust apoptosis in both LNCaP and C4-2B cells. As the androgen receptor (AR) is the primary target for prostate cancer therapy, we tested whether SNORD78 regulated AR expression and signaling. Inhibition of SNORD78 in both LNCaP and C4-2B cells resulted in reduced AR mRNA and protein levels. We also found that SNORD78 knockdown led to decreased levels of PSA and NKX3.1, suggesting that AR downstream signaling was inhibited. Our findings suggest that SNORD78 promotes prostate cancer cell viability and disease progression. We also demonstrate that SNORD78 regulates the AR and may be involved in promoting AR signaling needed for prostate cancer cell survival. Collectively, these data suggest that SNORD78 plays a critical role in prostate cancer progression and may represent a novel therapeutic target. Citation Format: Alan P. Lombard, Chengfei Liu, Cameron M. Armstrong, Leandro S. D'Abronzo, Wei Lou, Christopher P. Evans, Allen C. Gao. SNORD78 promotes prostate cancer progression and regulates AR expression and signaling [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 1823.

CFTR regulates B cell activation and lymphoid follicle development

BackgroundCystic fibrosis (CF) is an inherited disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that promotes persistent lung infection and inflammation and progressive loss of lung function. Patients with CF have increased lung lymphoid follicles (LFs) and B cell-activating factor of tumor necrosis factor family (BAFF) that regulates B cell survival and maturation. A direct role for CFTR in B cell activation and disease pathogenesis in CF remains unclear.MethodsThe number of LFs, BAFF+, TLR4+ and proliferation marker Ki67+ B cells in lung explants or resections from subjects with CF and normal controls was quantified by immunostaining. The role of CFTR in B cell activation and LF development was then examined in two independent cohorts of uninfected CFTR-deficient mice (Cftr−/−) and wild type controls. The number of lung LFs, B cells and BAFF+, CXCR4+, immunoglobulin G+ B cells was examined by immunostaining. Lung and splenocyte B cell activation marker and major histocompatibility complex class II (MHC class II) expression was quantified by flow cytometry. Inflammatory cytokine levels were measured in supernatants from isolated B cells from Cftr−/− and wild type mice stimulated in vitro with Pseudomonas aeruginosa lipopolysaccharide (LPS).ResultsThere was a significant increase in well-formed LFs in subjects with CF compared to normal controls. Increased B cell activation and proliferation was observed in lung LFs from CF subjects as was quantified by a significant increase in B cell BAFF, TLR4 and Ki67 expression. Uninfected Cftr−/− mice had increased lung LFs and BAFF+ and CXCR4+ B cells compared to wild type controls. Lung B cells isolated from uninfected Cftr−/− mice demonstrated increased MHC class II expression. In vitro, isolated B cells from Cftr−/− mice produced increased IL-6 when stimulated with LPS compared to wild type controls.ConclusionsThese data support a direct role for CFTR in B cell activation, proliferation and inflammatory cytokine production that promotes lung LF follicle development in cystic fibrosis.

PB2314 CHRONIC NEUTROPHILIC LEUKEMIA IN A SICKLE CELL PATIENT WHO SURVIVED CBF‐ACUTE MYELOID LEUKEMIA

Background:Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm, for which current WHO diagnostic criteria include leukocytosis of ≥25 × 109 /L of which ≥80% are neutrophils, with <10% circulating neutrophil precursors with blasts rarely observed. In addition, there is no dysplasia, nor clinical or molecular criteria for other myeloproliferative neoplasms. The true incidence of CNL is unknown, but only 200 cases have been reported.Sickle cell disease (SCD) is a hereditary hemoglobinopathy characterized by abnormal hemoglobin production(HbS) which leads to hemolytic anemia and intermittent occlusion of small blood vessels.Aims:Case report of a 55 years old patient with double heterozygous sickle cell/β + thal disease, first diagnosed at age of 45, who now suffers from CNL, ten years post treatment for acute myeloid leukemia (AML).Methods:A 55 years old male patient was referred to our clinic in 2012 with the diagnosis of double heterozygous sickle cell/β + thal disease. He was initated on red cell exchange transfusions due to sickle cell hepatopathy. Ten years earlier he had received intensive chemotherapy for AML with myelomonocytic differentiation, CBFB/MYH11(+). He attained a complete morphological and cytogenetic remission after 1st induction. He remained in CR until 2015 when he presented with worsening anemia that could not be attributed to his hemoglobinopathy(Hb = 8 g/dl). A complete work up including bone marrow aspiration, biopsy, molecular testing for CBFB/MYH11 and cytogenetics were normal. In 2018, he presented with a severe sickle cell crisis with multi‐organ failure during an infection and he started hydroxycarvamide at a dose of 500 mg. During the next months a progressive increase of WBC counts with elevated neutrophils was noticed and was attributed to dental abscess. Leukocytosis insisted despite appropriate treatment so he was admitted to the clinic. A complete work up, including imaging, virology, blood and urine cultures revealed no sign of infection or malignancy. Despite hydroxycarbamide treatment leukocytosis persisted (WBC: 90 × 109/L, ≥95% neutrophils, Hb: 8 g/dl, PLT:180000), with no circulating neutrophil precursors or myeloblasts and no evidence of dysplasia.Bone marrow biopsy was repeated. This time an hypercellular marrow with increased neutrophils, normal maturation and blasts <5% raised the question of a myeloproliferative neoplasm. The patient did not fulfill WHO criteria for Ph + CML, PMF, PV or ET. Analysis of a myeloid panel by Next generation sequencing(NGS) revealed the presence of the following mutations: CSF3R T618I, VAF:38% and SETBP1 G870Ser, VAF:54%.Results:A diagnosis of Chronic Neutrophilic Leukenmia was made according to the WHO classification. Hydroxycarbamide at a dose of 2gr was started,since patient was not eligible for bone barrow transplantation. Five months later,patient remains in good condition and has no evidence of disease progression.Further investigation will involve the detection of driver mutations in the bone marrow sample from the primary AML diagnosis using NGS.Summary/Conclusion:Although the clinical course of CNL is variable, the overall prognosis is generally reserved with a substantial portion of patients eventually progressing to blast crisis,The median overall survival is 24 months. Effective management has been hampered by the rarity of the disease, the lack of prospective trials, and the limited therapeutic options.The most common therapeutic approaches are Hydroxyurea, Interferon and Ruxolitinib. Our patient shows an initial response in Hydroxyurea therapy.

PB2052 PHENOTYPIC IDENTIFICATION OF EBV‐INFECTED LYMPHOCYTES FOR THE DIFFERENTIAL DIAGNOSIS OF HLH, CAEBV AND PTLD; A CLINICALLY APPLICABLE, FLOW‐CYTOMETRY‐ BASED PERIPHERAL BLOOD ASSAY

Background:Epstein–Barr virus (EBV) is a ubiquitous lymphotropic virus which preferentially infects B‐cells1,2, but can also infect T‐cells and NK‐cells. EBV can cause B‐cell or T/NK lymphoproliferative (LPD) disorders and EBV‐related haemophagocytic lymphohistiocytosis (HLH)2. These rare but distinct diseases share clinical features of lymphadenopathy, fever, malaise, organomegaly and night sweats1. High levels of EBV DNA, as assessed by qPCR on whole blood or plasma are typical but, per se, have limited clinical value. EBV copy number in individual lymphocyte subsets offers much greater clinical value but is technically challenging and hence rarely investigated.Aims:We have applied a dual in‐situ hybridisation‐flow cytometry technique (EBER‐FlowRNA) allowing the detection and phenotypic characterisation of specific lymphocyte subsets infected by EBV, impacting the clinical management of patients with EBV‐associated lymphoproliferative disease.Methods:Following informed consent, blood samples from patients with suspected EBV‐related LPD were obtained at diagnosis. All patients were part of the observational ‘Studies of EBV‐associated NK/T‐cell diseases and formation of a registry’ (07/H1208/62). All analyses were performed at the Institute for Immunology and Immunotherapy (Birmingham University, UK). Clinical information for the cases was retrospectively obtained from clinical records.Peripheral blood mononuclear cells (PBMC) were isolated from fresh patient blood and subjected to multicolour cell surface staining using up to 10 individual fluorophores. The cells were then fixed/permeabilised and subjected to EBER in‐situ hybridisation (PrimeFlowRNA) using pairs of 20‐mer specific probes, previously amplified and loaded with up to 400 fluorescent molecules; Beta‐2‐microglobulin probe pairs were used as positive control. Samples were processed by multicolour flow cytometry (BD Fortessa) and analysed with FlowJo software.Results:We present 4 representative clinical cases. Clinical characteristics described in Table 1. Flow cytometry of the samples was performed within 48 h of sample collection. Case 1 reflected a CD3+/CD4+/EBER+ population compatible with relapsed CAEBV, excluding the alternative diagnosis of EBV‐related B‐cell PTLD. Case 2 was an unusual CAEBV with gastric involvement and a CD3lo/CD4+/EBER+ population in peripheral blood, consistent with EBER positive malignant clone in the gastric biopsy. Case 3 presented with florid HLH and a CD19+/EBER+ clone consistent with primary EBV infection in the context of immunosuppression, treated successfully with Rituximab. Case 4 presented with a systemic illness and hepatic impairment, with identification of a CD56+/CD8+/EBER+ clone representing aggressive NK cell leukaemia. In cases 1, 2 and 4, EBER‐flowRNA prevented inappropriate use of empirical rituximab for raising EBV DNA‐aemia, whereas in case 3, EBER‐flowRNA excluded a T/NK LPD and supported rituximab use.Summary/Conclusion:In line with previously reported methodology4,5, our customised flow‐cytometry approach successfully identified the EBV‐infected lymphocyte subsets with a short turnaround time (48 hours sample‐to‐result). Additionally, successful EBER fluorescent labelling allowed for cell sorting and subsequent genomic and transcriptomic studies of EBV infected cells. Importantly, flow cytometry findings directed appropriate therapeutic approaches. Application of this technique in a larger cohort is warranted. We propose EBER‐FlowRNA as a relevant, rapid and feasible differential diagnostic tool for EBV‐related LPD.image

1736-P: Significance of Anti-insulin Immune Responses in Autoimmune Diabetes with HLA DR9

Previously we reported that CXCL10 in type 1 diabetes (T1D) is higher than controls, and the level reflects islet associated antigen specific T cell responses and disease activity in T1D (Diabetes Care, 2001). However, little is known regarding the relationship between CXCL10 level and autoantibody levels in T1D. Therefore, we investigated relationship between the marker of cellular immunity including CXCL10 levels and islet associated autoantibody levels in T1D. We obtained informed consent and blood samples from 101 T1D patients, and examined HLA (DRB1*04:05, *09:01; susceptible HLA types in Japanese), serum CXCL10, insulin peptide (B9-23, 10-24, 11-25, 12-26) and PPD reactive T cell responses by using ELISPOT (IFNg), GAD, IA-2, ZnT8 Abs and insulin antibody. Because it has been reported that BCG administration might affect the autoimmune responses in T1D, we assessed the PPD responses in these patients as well. Among them, we focused to GAD Ab positive T1D including acute onset (n=28, 13 male and 15 female, mean age 43.1 years, disease duration 5.9 years), and slowly progressive type (n=17, 9 male and 8 female, mean age 56.9 years, disease duration 9.1 years); i.e., autoimmune diabetes subjects. We found significant positive correlation between serum CXCL10 and insulin antibody levels in the autoimmune diabetes with DRB1*09:01 (p<0.05), but it was not the case in those with DRB1*04:05. Interestingly, there was significant positive correlation between insulin peptide reactive T cells and PPD responses in the autoimmune diabetes with DRB1 09:01 (p<0.05), but it was not the case in those with DRB1 04:05. The observation was still true when we focused to acute onset subjects. These results suggest that insulin antibody levels seem to reflect higher disease activity in the autoimmune diabetes with DR9, and modulation of PPD responses may affect insulin peptide T cell responses in those patients. Disclosure A. Shimada: Advisory Panel; Self; Terumo Medical Corporation. Speaker's Bureau; Self; Abbott, Eli Lilly and Company, Novo Nordisk A/S, Sanofi-Aventis. A. Satomura: None. A. Haisa: None. Y. Oikawa: None.

High NCALD expression predicts poor prognosis of cytogenetic normal acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML) is a heterogeneous disease in terms of genetic basis, clinical, biological and prognostic, and is a malignant clonal disease of leukemia stem cells (LSCs). Nearly half of adult AML patients exhibit a cytogenetic normal acute myeloid leukemia (CN-AML). The expression level of NCALD gene was associated with the prognosis of ovarian cancer and non-small cell lung cancer (NSCLC). The expression level of NCALD gene is still unclear in the prognosis of patients with AML.MethodWe integrated 5 independent datasets totally 665 AML patients (497 CN-AML patients) to analyzed relation between NCALD gene expression and the clinical FAB classification, gene mutation, therapy, prognosis of CN-AML. We analyzed the NCALD gene expression with the prognosis and LSC of 165 AML patients from The Cancer Genome Atlas (TCGA) dataset and 78 AML patients from GEO dataset.ResultsHigh NCALD-expressing CN-AML patients were associated with poor event-free survival (EFS) and overall survival (OS) compared to low NCALD expression (EFS, P < 0.0001, OS, P < 0.0001). In AML patients of allogeneic hematopoietic stem cell transplantation (allo-HSCT), high NCALD expression was associated with poor survival prognosis in EFS and OS (EFS, P < 0.0051, OS, P = 0.028). Post-chemotherapy in AML patients, high NCALD expression led a worse prognosis in EFS and OS (EFS, P = 0.011; OS, P = 0.0056). In multivariate analysis, high NCALD expression was an independent prognostic factor that predicts shorter EFS and OS (EFS, P = 3.84E−05, OS, P = 8.53E−05) of CN-AML.ConclusionOur results indicate that high expression of NCALD gene is a poor prognostic factor for CN-AML. NCALD can be considered as independent predictors of CN-AML patients and can be used as a biomarker for the prognosis of CN-AML.

CD38hi B-regulatory (B-reg) cells maintain pathological immune tolerance in chronic lymphocytic leukemia (CLL)/B cell diseases: Potential therapeutic considerations

Abstract Chronic Lymphocytic Leukemia (CLL) is considered a disease of antigen-naive CD5+CD23+CD19+κ/λ+ B-lymphocytes, in which the tumor microenvironment is highly immunosuppressive. Among several human B cell subsets, B10 [CD24hiCD27+CD38hi]/B regulatory cells (Bregs) are functionally discernable in patients with aggressive CLL as they support and regulate immunological tolerance via production of IL-10 and transforming growth factor β (TGF-β). In an ex vivo setting, we noted CD38hi CLL Bregs to promote transformation and expansion of CD4+CD25+FoxP3+Tregs; an effect that was abrogated in the presence of IL10/TGF-β neutralizing antibodies. In patients with CLL, Bregs also prohibit the expansion of cytotoxic T cells (CD8+T cells; Tc), natural killer (NK) cells and other pro-inflammatory lymphocytes (CD4+ helper T cells; Th1 and Th17). We noted that patients with CLL had a significantly higher % of Tregs (11.8±2.0) compared to healthy donors (1.9±0.3) and similar to Bregs, a substantial proportion of CLL Tregs had high CD38 expression (MFI=616.8±36.2). We demonstrated that an anti-CD38 therapeutic antibody was able to eliminate CD38+ Bregs and Tregs from CLL patients by immune effector mechanisms (ADCC, CDC, ADCP) as well as direct mitochondrial/FcγR-mediated apoptosis. Furthermore, in a PDX model of CLL, mice treated with anti-CD38 therapy had decreased % Bregs and Tregs but increased Th17 and CD8+ T-cells (vs. vehicle; p&amp;lt;0.05). These observations carry implications beyond CLL for any Breg/Treg-dependent cancer or disease (i.e. Systemic Lupus Erythematosus, Rheumatoid arthritis, Sjögren’s syndrome), wherein CD38-targeted therapy could potentially rescue/improve an immune tolerant microenvironment by eradication of Bregs.

CODA-ML: context-specific biological knowledge representation for systemic physiology analysis

BackgroundComputational analysis of complex diseases involving multiple organs requires the integration of multiple different models into a unified model. Different models are often constructed in heterogeneous formats. Thus, the integration of the models requires a standard language format that can effectively represent essential biological information. However, the previously introduced formats have limitations that prevent from adequately representing essential biological information, particularly specifications of bio-molecules and biological contexts.ResultsWe defined an XML-based markup language called context-oriented directed association markup language (CODA-ML), which better represents essential biological information. The CODA-ML has two major strengths in designating molecular specifications and biological contexts. It can cover heterogeneous entity types involved in biological events (e.g. gene/protein, compound, cellular function, disease). Molecular types of entities can have molecular specifications which include detailed information of a molecule from isoforms to modifications, enabling high-resolution representation of molecules. In addition, it can distinguish biological events that vary depending on different biological contexts such as cell types or disease conditions. Especially representation of inter-cellular events as well as intra-cellular events is available. These two major strengths can resolve contradictory associations when different models are integrated into one unified model, which improves the accuracy of the model.ConclusionsWith the CODA-ML, diverse models such as signaling pathways, metabolic pathways, and gene regulatory pathways can be represented in a unified language format. Heterogeneous entity types can be covered by the CODA-ML, thus it enables detailed description for the mechanisms of diseases or drugs from multiple perspectives (e.g., molecule, function or disease). The CODA-ML is expected to help integrate different models into one systemic model in an efficient and effective. The unified model can be used to perform computational analysis not only for cancer but also for other complex diseases involving multiple organs beyond a single cell.

Successful mast-cell-targeted treatment of chronic dyspareunia, vaginitis, and dysfunctional uterine bleeding

Dyspareunia, vaginitis and dysfunctional uterine bleeding (DUB) are common problems which, despite their polygenicity, commonly appear idiopathic and treatment-refractory. Mast cell (MC) activation syndrome (MCAS) is a newly-recognised, prevalent, chronic multisystem polymorbidity of general themes of inflammation ± allergic-type phenomena ± aberrant growth/development in assorted tissues. MCs produce significant quantities of heparin, too. As such, MCAS may underlie some cases of chronic dyspareunia, vaginitis or DUB. We report five such patients; all who responded well to MC-targeted treatment. We review aspects of MC biology and pathobiology of potential relevance to otherwise idiopathic persistent inflammatory or coagulopathic genital tract problems. Diagnostic testing for MCAS may be warranted in some patients with chronic dyspareunia, vaginitis or DUB (especially patients whose histories well fit the general profile of MCAS), and prospective therapeutic trials of MC-directed topical and/or systemic therapies may be warranted in such populations. Impact statement What is already known on this subject? Chronic, idiopathic, treatment-refractory female genital tract inflammation or bleeding are common problems for which mast cell (MC) disease, previously generally thought to consist of just rare cases of mastocytosis, and is seldom considered in the differential diagnosis. What do the results of this study add? The substantial prevalence of the newly recognised ‘mast cell activation syndrome’ (MCAS), featuring chronic inappropriate MC activation with little-to-no MC neoplasia, and its clinical presentation with chronic multisystem inflammation ± allergic-type phenomena ± aberrant growth/development in assorted tissues, raises the possibility that MCAS might underlie the aforementioned genital tract problems, especially in patients whose larger clinical presentations fit the MCAS profile. We report five example patients (among many more we have similarly treated) who enjoyed excellent responses to safe, inexpensive MC-targeted treatments, often given just intravaginally. What are the implications of these findings for clinical practice and/or further research? Our report identifies a potentially significant new MC-focused direction, of relevance to millions of affected women worldwide, for clinical treatment as well as for basic and clinical research, which historically has yielded major advancements disappointingly disproportionate to the scope of the affected population.

Chronic lymphocytic leukaemia: the role of T cells in a B cell disease.

Chronic lymphocytic leukaemia (CLL) has long been thought to be an immunosuppressive disease and abnormalities in T-cell subset distribution and function have been observed in many studies. However, the role of T cells (if any) in disease progression remains unclear and has not been directly studied. This has changed with the advent of new therapies, such as chimeric antigen receptor-T cells, which actively use retargeted patient-derived T cells as "living drugs" for CLL. However complete responses are relatively low (~26%) and recent studies have suggested the differentiation status of patient T cells before therapy may influence efficacy. Non-chemotherapeutic drugs, such as idelalisib and ibrutinib, also have an impact on T cell populations in CLL patients. This review will highlight what is known about T cells in CLL during disease progression and after treatment, and discuss the prospects of using T cells as predictive biomarkers for immune status and response to therapy.

MiR-1180 from bone marrow MSCs promotes cell proliferation and glycolysis in ovarian cancer cells via SFRP1/Wnt pathway

BackgroundThe ovarian cancer microenvironment is responsible for cancer cell growth and disease relapse. Bone marrow mesenchymal stem cells (BM-MSCs) play important roles in ovarian cancer, however, the mechanism of BM-MSCs inducing cell proliferation and glycolysis needs further research.MethodsmiRNA array was used to analyze the significant miRNAs. RT-qPCR was used to examine the level of miR-1180 and SFRP1. The western blotting was used to detect the protein level of SFRP1 and Wnt signal pathway. We utilized luciferase reporter assay to confirm the direct interaction of SFRP1 with miR-1180. MTT assay were employed to investigate the proliferation of ovarian cancer cells. ECAR, ATP assay were used to measure the glycolysis state of ovarian cancer cells.ResultsIt was demonstrated that BM-MSCs promoted ovarian cancer cell proliferation and glycolysis. The miRNA profile from the BM-MSCs indicated that miR-1180 was up-regulated in the conditioned medium of BM-MSCs. MiR-1180 could accelerate ovarian cancer cell proliferation and glycolysis. We also found that up-regulation of miR-1180 activated Wnt signaling by targeting SFRP1 in ovarian cancer cells.ConclusionThe study demonstrated that miR-1180 was a critical miRNA mediating BM-MSCs induced cell proliferation and glycolysis and could be a new target in ovarian cancer therapy.