Mutations In STAT3 Gene Research Articles

Hyper IgE Syndromes.

The Hyper IgE Syndromes are rare primary immunodeficiencies characterized by eczema, recurrent skin and respiratory infections and elevated serum IgE levels. Nowadays a geneticmolecular characterization is possible and allows the distinction in various monogenic pathologies, which share some clinical characteristics but also important differences. In addition to long-known STAT3 and DOCK8 gene mutations, in fact, also ZNF341, CARD11, ERBB2IP, IL6R and IL6ST genes mutations can cause the disease. The main clinical manifestations are represented by newborn rash, eczema similar to atopic dermatitis, bacterial and viral skin infections, cold abscesses, respiratory infections with possible pulmonary complications, allergies, gastrointestinal manifestations, malignancies and connective tissue abnormalities. Diagnosis is still a challenge because, especially in the early stages of life, it is difficult to distinguish from other pathologies characterized by eczema and high IgE, such as atopic dermatitis. Several scores and diagnostic pathways have been developed, but it is essential to seek a genetic diagnosis. Treatment is based on prevention and early treatment of infections, meticulous skincare, intravenous immunoglobulins and HSCT, which, in some HIES subtypes, can modify the prognosis. Prognosis is related to the affected gene, but also to early diagnosis, timely treatment of infections and early HSCT.

Hyper IgE syndrom suspect - Case Report

Introduction: Hyper IgE syndrome (HIES) is a rare condition of the immune system characterized by various types of infections throughout life. This syndrome can be divided into two types, dominant heterozygous related to STAT3 gene mutation and recessive homozygous, which can also be subdivided into two groups related to Tyk2 and DOCK8 gene mutations, each presenting variations in their clinical presentations. Diagnosis is based on genetic sequencing, although there are some symptoms, clinical factors, and hereditary factors that can assist in diagnosis. There is no specific treatment for this pathology, with the main focus being on controlling infections and infusing immunological therapies such as intravenous immunoglobulin G.

Extracellular Vesicles based STAT3 delivery as innovative therapeutic approach to restore STAT3 signaling deficiency

Extracellular Vesicles (EVs) have been proposed as a promising tool for drug delivery because of their natural ability to cross biological barriers, protect their cargo, and target specific cells. Moreover, EVs are not recognized by the immune system as foreign, reducing the risk of an immune response and enhancing biocompatibility. Herein, we proposed an alternative therapeutic strategy to restore STAT3 signaling exploiting STAT3 loaded EVs. This approach could be useful in the treatment of Autosomal Dominant Hyper-IgE Syndrome (AD-HIES), a rare primary immunodeficiency and multisystem disorder due to the presence of mutations in STAT3 gene. These mutations alter the signal transduction of STAT3, thereby impeding Th17 CD4+ cell differentiation that leads to the failure of immune response. We set up a simple and versatile method in which EVs were loaded with fully functional STAT3 protein. Moreover, our method allows to follow the uptake of STAT3 loaded vesicles inside cells due to the presence of EGFP in the EGFP-STAT3 fusion protein construct. Taken together, the data presented in this study could provide the scientific background for the development of new therapeutic strategy aimed to restore STAT3 signaling in STAT3 misfunction associated diseases like AD-HIES. In the future, the administration of fully functional wild type STAT3 to CD4+ T cells of AD-HIES patients might compensate its loss of function and would be beneficial for these patients, lowering the risk of infections, the use of medications, and hospitalizations.

Hyper-IgE syndrome: a case report

Introduction and importance: Hyper-IgE syndrome (HIES), also known as Job syndrome, is a rare primary immunodeficiency disorder characterized by elevated serum IgE levels, recurrent infections, and various clinical features. Early diagnosis, prompt management of infections, and supportive care are essential in improving outcomes for individuals with HIES. Genetic testing, including STAT3 gene sequencing, plays a crucial role in confirming the diagnosis. Further research is needed to enhance our understanding of HIES and develop targeted therapies to improve the quality of life for affected individuals. Case presentation: This case report presents the clinical features and management of a 37-year-old male with HIES, diagnosed at the age of 2 due to recurrent cold abscesses caused by Staphylococcal infections. Clinical discussion: The patient exhibited typical symptoms of HIES, including recurrent eczema, frequent bacterial infections, mucocutaneous candidiasis, and various physical abnormalities. Diagnostic markers such as elevated IgE levels and eosinophilia supported the HIES diagnosis, which was further confirmed by the identification of a STAT3 gene mutation. Treatment primarily involved supportive measures and antibiotics for infections. The patient’s blood test results and imaging findings revealed abnormalities such as low red blood cell count, elevated erythrocyte sedimentation rate, and pulmonary nodules. Conclusion: This case report highlights the importance of early diagnosis, prompt management of infections, and the need for ongoing research to improve our understanding and treatment of HIES.

Investigation of the mutations in the genes involved in Janus kinase/signal transducer and activator of transcription pathway in canine large cell gastrointestinal lymphoma.

Canine gastrointestinal lymphoma is known to be of T-cell origin in most cases, but the molecular biological aberrations have not been clarified. In human intestinal T-cell lymphoma, the mutations in the genes associated with Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway have been frequently observed. In this study, the gene mutations were investigated in 31 dogs with large cell gastrointestinal lymphoma (LCGIL) by focusing on the genes involved in JAK-STAT pathway. Next-generation sequencing analysis to examine the mutations in STAT3, STAT5B, and JAK1 genes throughout the exon regions revealed the mutations in STAT3 gene in two dogs and JAK1 gene in one dog. In conclusion, this study could not indicate the associations of gene mutations in JAK-STAT pathway with LCGIL in most canine cases.

P20 Job syndrome: hyper IgE syndrome in a young girl with an acneiform rash

Abstract Hyper IgE syndrome (HIES1) is a rare, autosomal dominant, primary immunodeficiency characterized by eczema, recurrent staphylococcal skin infections, pneumonia, increased serum IgE and eosinophilia. It is caused by a heterozygous mutation in the STAT3 gene on chromosome 17q21 with variable expressivity. Nonimmunological findings including characteristic facies, hyperextensibility, long-bone fractures, aneurysms, craniosynostosis and dental abnormalities. We present the case of an 11-year-old girl referred to dermatology with an acneiform facial rash. Examination was consistent with papulo-pustular scarring acne of the face and back. Her history was also significant for recurrent MSSA bacterial lymphadenitis requiring incision and drainage over the proceeding 3 years. Her past medical history was significant for the development of moderate-severity infantile acne at 2 weeks of age, mild childhood eczema, recurrent childhood otitis media Staphylococcus infections resulting in grommet insertion, primary tooth retention requiring extraction and a fractured wrist at age 9 years. Because of the burden of infection the patient was referred to immunology in Crumlin. Investigations revealed mild neutropenia, marginally elevated IgA and IgM, normal eosinophils, marginally elevated IgE, normal lymphocyte subsets, intact complement pathways and normal oxidative burst test. Next-generation sequencing was significant for STAT3 gene mutation consistent with HIES. As there is no family history this likely represents a de novo mutation. The patient is currently on prophylactic azithromycin and low-dose isotretinoin with good effect. This case represents a patient with both immunological and nonimmunological features of HIES1, marginally elevated IgE, absence of eosinophils and negative family history.

Detection of High-Frequency Mutation Genes in HIV-Positive and HIV-Negative Lymphomas

Objective Compare HIV-positive lymphoma with HIV-negative lymphoma mutation genes differences, screen specific mutations genes in HIV-positive lymphoma, analyze high frequency mutation genes in HIV-positive lymphoma patients, and explore the effect of HIV infection on lymphoma gene mutation and disease development. It can provide a theoretical basis for the clincal diagnosis and targeted therapy HIV-positive lymphoma. Methods A total of 7 HIV-positive lymphoma patients and 13 HIV-negative lymphoma patients who were treated in Chongqing University Cancer Hospital from September 2020 to September 2021 were enrolled. Tumor tissue samples were detected by second-generation sequencing technology, and gene mutations in HIV-positive lymphoma patients were analyzed by gene heat map, comparing the gene mutation differences between HIV-positive and HIV-negative lymphoma patients. Results Compared with HIV-negative lymphoma patients, more and more complex gene mutation sites were detected in HIV-positive lymphoma patients. Single nucleotide mutation of MYC gene was more common in HIV-positive lymphoma patients, while deletion of KMT2D gene was more common in HIV-negative lymphoma patients. And TP53 gene is highly mutated in both HIV-positive and HIV-negative lymphoma patients. However, mutations of TET, CCND3, TNFRSF14, ARID1A, TNFAIP3, NFKBIE, CXCR4, STAT3, NOTCH1 and other gene mutation with low mutation abundency occurred only in HIV-positive lymphoma patients. NOTCH2, PRDM1, EZH2, CARD11, EP300, MEF2B, CREBBP and other gene mutations occurred only in HIV-negative lymphoma patients. Conclusion HIV-positive lymphoma patients are more prone to gene mutations, and the gene mutations are more complex and diverse. MYC gene mutations and TP53 gene mutations play an important role in the occurrence and development of HIV-positive lymphoma, while the occurrence and development of HIV-negative lymphoma are more related to KMT2D gene mutations and TP53 gene mutations.

NOVEL HETEROZYGOUS SINGLE NUCLEOTIDE POLYMORPHISM IN STAT-3 GENE IN PATIENT WITH SUSPECTED HYPER IGE SYNDROME

<h3>Introduction</h3> Hyper IgE syndrome (HIES) is a rare genetic immune deficiency disorder affecting multiple organs. Recurrent skin infections, abscesses, atopic dermatitis, and hypereosinophilia are common. Mutations in STAT-3 gene is associated with HIES. We present a novel heterozygous point mutation in STAT-3 gene in a patient with hyper IgE and recurrent abscesses. <h3>Case Description</h3> A 22-year-old male presented with severe atopic dermatitis and recurrent abscesses. Initial labs revealed very high serum IgE 41,559 U/ml and eosinophil count of 1290/cmm. CBC showed mild anemia. Infectious work up was all negative. Patient had normal CMP, troponin, spirometry, tryptase, Vit B12 and ECHO. Skin biopsy was suggestive of eosinophilic fasciitis. Bone marrow biopsy and flow cytometry were normal. CT scans showed inguinal and axillary lymphadenopathy and borderline hepatomegaly. He was hospitalized multiple times for I/D of his recurrent abscesses. He was treated with systemic steroids and started on Bactrim prophylaxis. In addition, Dupilumab was started for his eczema which improved his skin. He longer has abscesses. After Dupilumab his IgE level significantly decreased from >50,000 U/ml to 14,790 U/ml. HIES panel revealed a novel heterozygous point mutation in STAT-3 gene, Exon 19 c1673G>A (p.Gly558Asp). Due to patient's supporting clinical phenotype HIES was suspected. Further genetic and functional studies to confirm this variant are planned. <h3>Discussion</h3> HIES is a complex disease, and it is challenging to confirm the diagnosis. Several mutations in STAT-3 have been described but more studies are still warranted to identify different mutations. Dupilumab may improve patient's atopic dermatitis in suspected cases.

Clinical Case of Job Syndrome in Infant

The article presents clinical case of Job syndrome, variant of primary immune deficiency disease confirmed genetically. This case is interesting for its early diagnosis due to comprehensive assessment of anamnestic, clinical, and laboratory data. Such typical phenotypic features as facial dysmorphisms, recurrent localized purulent infections, and laboratory parameters (absolute neutropenia and eosinophilia) were the major signs of autosomal dominant hyper IgE syndrome in a child with normal IgE levels. The combination of 2 heterozygous mutations in STAT3 gene inherited from his father and his mother has played its role in disease clinical features in the child.

Defective Toll-Like Receptors Driven B Cell Response in Hyper IgE Syndrome Patients With STAT3 Mutations.

Autosomal dominant hyper-IgE syndrome (AD-HIES) is a rare inherited primary immunodeficient disease (PIDs), which is caused by STAT3 gene mutations. Previous studies indicated a defective Toll-like receptor (TLR) 9-induced B cell response in AD-HIES patients, including proliferation, and IgG production. However, the other TLRs-mediated B cell responses in AD-HIES patients were not fully elucidated. In this study, we systematically studied the B cell response to TLRs signaling pathways in AD-HIES patients, including proliferation, activation, apoptosis, cytokine, and immunoglobulin production. Our results showed that the TLRs-induced B cell proliferation and activation was significantly impaired in AD-HIES patients. Besides, AD-HIES patients had defects in TLRs-induced B cell class switch, as well as IgG/IgM secretion and IL-10 production in B cells. Taken together, we first systematically reported the deficiency of TLRs driven B cell response in AD-HIES patients, which help to have a better understanding of the pathology of AD-HIES.

Clinical improvement in Job syndrome following administration of co-trimoxazole, omalizumab and inhaled tobramycin.

Established treatment regimens for the autosomal dominant hyperimmunoglobulin E syndrome, denominated Job syndrome, are lacking. Thus, Job syndrome still exerts a dramatic impact on patients' quality of life. Our aim was to present safety and effectiveness of a regimen including co-trimoxazole, omalizumab and inhaled tobramycin in Job syndrome. A 26-year-old woman diagnosed with Job syndrome since infancy through sequencing revealing G342D mutation in STAT3 gene was initiated in the above mentioned treatment regimen; she was followed for 6 months, and to date, none recurrent pulmonary or skin infection was noticed. Furthermore, a considerable improvement in skin lesions was observed. A combination of anti-IgE and longitudinal use of inhaled antibiotics seems well-founded in Job syndrome.

Penicilliosis marneffei in HIV negative children: three case reports

This study aimed to report the clinical characteristics of penicilliosis marneffei (PSM) in three children negative to HIV. Three children were diagnosed with PSM in the Department of Emergency Medicine, Hunan Children's Hospital between February 2016 to July 2020. The clinical characteristics, laboratory findings, and concomitant diseases were recorded, and the related literatures were reviewed. The clinical characteristics and treatment of PSM were reported according to our experience and literature review. The initial symptom was right lower limb mass in 1 child (first) who developed fever and cough about 1 month later and then was misdiagnosed with tuberculosis. The other child (second) had a fever, reductions in red blood cells, white blood cells and platelets, hepatosplenomegaly and lymphadenectasis. The third child had fever, jaundice, multiple organ dysfunction syndrome (MODS), hepatosplenomegaly and lymphadenectasis. The first child (Case 1) had STAT1 gene mutation on genetic examination, and the second child (Case 2) had history of onychomycosis and oral ulcer, the third child (Case 3) had STAT3 gene mutation on genetic examination, diagnosed with Hyperimmunoglobulin E syndromes (HIES). PSM was confirmed in all cases by the culture bone marrow. All three cases were diagnosed through medulloculture. Case 1 and Case 2 also had lymph node biopsy. Case 3 had sputum culture and bronchoalveolar lavage fluid (BALF). The first child was intravenously administered with voriconazole and amphotericin B liposomes, and orally administered with itraconazole for maintenance therapy, which was discontinued 1 year later. The second child was administered with voriconazole intravenously and thereafter orally for a total of 7 months. Recurrence was not observed. The third child was given amphotericin B for 2 days (discontinued due to liver dysfunction), and intravenous voriconazole for 4 days. The patient gave up therapy finally. In conclusion, HIV negative children can also develop PSM, and may be related to the STAT1/STAT3 gene mutation. For children having no response to antibiotic or antiviral therapy, bacterial/fungal culture or biopsy should be performed as soon as possible to confirm the diagnosis, and physicians should actively identify the underlying diseases of PSM patients, which is beneficial for the early diagnosis, early treatment and improvement of prognosis.

Recurrent STAT3 Mutations in the Lymphocytes of Pure Red Cell Aplasia without T-Cell Large Granular Lymphocytic Leukemia

▪Background: Activating somatic mutations of STAT3 are one of the most frequently recognized genetic alterations in T-cell lymphoma including T-cell large granular lymphocytic leukemia (T-LGLL). T-LGLL is associated with certain proportions of patients (pts) with acquired aplastic anemia (AA) and/or myelodysplastic syndromes (MDS). Pure red cell aplasia (PRCA), especially acquired PRCA, is a syndrome defined by an anemia with marked reduction or absence of erythroid production associated with immunological mechanisms. PRCA is classified into primary and secondary types including T-LGLL. Previous reports have described that T-LGLL with STAT3 mutations was closely associated with PRCA. However, it is yet to be clear whether STAT3-mutated T cells would be involved in the pathogenesis of other cytopenias, such as AA, MDS, primary PRCA or secondary PRCA without (w/o) T-LGLL. In this study, we analyzed the profiles of STAT3 gene mutations in bone marrow failure syndrome (BMFS)s including AA/MDS and PRCA.Methods: Peripheral blood samples were collected from 94 patients with BMFSs, including AA, MDS, AA/PNH, and PRCA with or w/o T-LGLL. When possible, T-cell receptor (TCR) Vβ repertoire of the patients was determined by flow cytometry. DNA was extracted from mononuclear cells (MNC), and STAT3 Y640F and D661Y, two hot spot mutations, were detected with sensitive allele specific-PCR (AsPCR) methods. Then we performed deep amplicon sequencing analysis of STAT3 in PRCA pts. CD8+ T cells were sorted if possible, and also subjected to the analyses.Results: The recruited pts included 38 AA, 20 MDS, 7 AA/PNH, 11 PRCA with T-LGLL, and 18 PRCA w/o T-LGLL. Among PRCA pts w/o apparent T-LGLL, 10 had primary PRCA and 8 had secondary PRCA including 2 with thymoma. The clinical characteristics of PRCA are shown in Table 1. TCR Vβ repertoire analysis were performed in 8 PRCA w/o T-LGLL and 5 PRCA with T-LGLL. All pts with PRCA associated with T-LGLL showed restricted Vβ repertoire. On the other hand, only 1pt showed monoclonal Vβ pattern in PRCA w/o TLGLL. AsPCR did not detect Y604F or D661Y mutations in any of 65 pts with AA, MDS, or AA/PNH. In contrast, 2 pts with PRCA and 4 pts with PRCA-T-LGLL were positive for either of the mutations, suggesting STAT3 mutations may be unique to PRCA pts. Further analysis with deep amplicon analysis in the 29 PRCA pts revealed STAT3 mutations in 11 pts (61%) of PRCA w/o T-LGLL, which included 6 pts with primary PRCA and 5 pts with secondary PRCA associated with autoimmune diseases (n=2), 2 thymoma (n=2) and thymic cancer (n=1). The median variant allele frequency of STAT3 mutations was 2.3% (0.2-48.9). N538Y and I653V were most frequent mutations in PRCA w/o T-LGLL. Eight pts (73%) with T-LGLL-associated PRCA were also positive for STAT3 mutation. Thirty-nine % and 70% of STAT3 mutation were found in SH2 domain in PRCA w/o T-LGLL and PRCA with T-LGL, respectively. There was no difference in clinical features between pts with and w/o STAT3 mutations.Discussion: The high frequency of STAT3 mutations in PRCA pts regardless of etiology and the absence of the mutation in pts with AA/MDS suggest that STAT3 mutations may serve as a molecular marker of PRCA. The discordant results with regard to AA/MDS pts from a previous report which detected STAT3 mutations in 4.9% of AA/MDS patients by AsPCR methods (Jerez et al. BLOOD 2013) may be due to the difference in ethnic backgrounds of the pts studied. Given very low frequencies of STAT3 mutations in AA and MDS that were revealed by several previous studies, JAK/STAT signaling systems in the axis of STAT3 mutations may have unique roles in the pathogenesis of PRCA.Conclusion: STAT3 mutations are frequently detected in pts with PRCA even in pts w/o T-LGLL and the JAK/STAT pathway may therefore be therapeutic targets of PRCA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical characteristics of T-cell large granular lymphoblastic leukemia with STAT3 gene mutation

目的探讨携带STAT3基因突变的T细胞大颗粒淋巴细胞白血病（T-LGLL）患者的临床特征，为此类患者的临床管理提供参考。方法回顾性分析2009至2019年就诊于江苏省人民医院的T-LGLL患者的临床资料，比较STAT3突变患者与未突变患者的基线临床数据、治疗反应及生存结局。结果共纳入80例患者，STAT3未突变组66例，STAT3突变组14例（17.5％），其中Y640F突变发生频率最高（42.9％）。STAT3突变组与STAT3未突变组相比，HGB减低（67.5 g/L对82.5 g/L，P＝0.018），中性粒细胞计数减少（0.665×109/L对1.465×109/L，P<0.001），乳酸脱氢酶升高（229 U/L对198 U/L，P＝0.041），铁蛋白升高（402.5 g/L对236.0 g/L，P＝0.029），TCR Vβ亚家族表达率升高（89.2％对65.4％，P＝0.014），具备治疗指征患者比例升高（100％对74％，P＝0.033）。STAT3突变组与未突变组一线免疫抑制治疗的完全缓解率分别为38.5％和32.7％，差异无统计学意义（P＝0.748）。STAT3突变组与未突变组一线免疫抑制治疗的总有效率分别为69.2％和69.4％，差异无统计学意义（P＝1.000）。中位随访63（2～121）个月，两组总生存时间（均未达到）的差异无统计学意义（P＝0.170）。结论STAT3基因突变的T-LGLL患者可能有更高的肿瘤负荷和治疗需求，一线应用免疫抑制剂疗效良好。STAT3基因突变对T-LGLL患者预后的意义尚需进一步验证。

Peripheral immune profile of children with Talaromyces marneffei infections: a retrospective analysis of 21 cases

BackgroundTalaromyces marneffei (T. marneffei) is an opportunistic pathogen that infects immunodeficient children. The aim of the study is to determine the clinical features and peripheral immune state of Talaromyces marneffei (T. marneffei) infections in children for early detection and diagnosis.MethodsWe retrospectively reviewed 21 pediatric patients who were diagnosed with T. marneffei infections and were followed up in the Guangzhou Women and Children’s Medical Center from January 2010 to January 2020. For each patient, we collected and analyzed clinical characteristics, peripheral immunological results, genetic tests, complications and prognosis.ResultsCommon clinical features of the patients included fever (20/21, 95.24%), cough (17/21, 80.95%) and hepatomegaly (17/21, 80.95%). Severe complications included septic shock (12/21, 57.14%), hemophagocytic lymphohistiocytosis (HLH) (11/21, 52.38%), acute respiratory distress syndrome (ARDS) (10/21, 47.62%), multiple organ dysfunction syndrome (MODS) (9/21, 42.86%), and disseminated intravascular coagulation (DIC) (7/21, 33.33%). Eleven children (11/21, 52.38%) eventually died of T. marneffei infections. All patients were HIV negative. Seven cases revealed reduced antibody levels, especially IgG. Higher levels of IgE were detected in 9 cases with an obvious increase in two patients. Ten patients showed decreased complement C3 levels, some of whom had low C4 levels. Three patients displayed decreased absolute T lymphocyte counts, including the CD 4+ and CD 8+ subsets. A reduction in NK cells was present in most patients. No patient had positive nitro blue tetrazolium (NBT) test results. Nine patients were screened for common genetic mutations. Of the cases, one case had no disease-specific gene mutation. Four children had confirmed hyperimmunoglobulin M syndrome (HIGM) with CD40LG variation, one case had severe combined immunodeficiency (SCID), and one case had hyper-IgE syndrome (HIES). One patient was identified as having a heterozygous mutation in STAT3 gene; however, he showed no typical clinical manifestations of HIES at his age. One patient had a mutated COPA gene with uncertain pathogenic potential. Another patient was diagnosed with HIES that depended on her clinical features and the National Institutes of Health (NIH) scoring system.ConclusionsT. marneffei infections in HIV-negative children induced severe systemic complications and poor prognosis. Children with T. marneffei infections commonly exhibited abnormal immunoglobulin levels in peripheral blood, particularly decreased IgG or increased IgE levels, further suggesting possible underlying PIDs in these patients.

Autosomal dominant hyper IgE syndrome from a single centre in Chongqing, China (2009-2018).

Autosomal dominant hyper IgE syndrome (AD-HIES) caused by STAT3 gene mutation is a rare primary immunodeficiency disease. To better understand the disease, we described the clinical characteristics of 20 AD-HIES patients in Chongqing, China and explored the effect of mutations in different domains of STAT3 gene on the function of STAT3 protein by Western blot and confocal microscopy. The mean age at onset was 0.12years. The mean age at diagnosis was 5.31years. The most common presentation was eczema, pneumonia, skin abscesses and chronic mucocutaneous candidiasis. Seven patients suffered from BCG complications. R382W/Q were identified in 12 patients, V637M mutation in three patients. Three patients have died. The phosphorylated STAT3 was expressed more in wild-type(WT) and R382W mutant STAT3 in the cytoplasm of COS7 cells with epidermal growth factor(EGF) stimulation, less in the V637M mutation and T620S mutation. Dynamic observation showed that STAT3 cytoplasmic accumulation and nuclear translocation occurred rapidly after EGF stimulation in WT-STAT3-GFP, the time of accumulation and nuclear translocation was later and the expression was less in R382W-STAT3-GFP compared with WT-STAT3-GFP, followed by V637M and T620S mutation. These results suggested that our patients had earlier onset, diagnostic age and higher rate of BCG complications. However, our patients had higher incidence of mortality though the earlier diagnostic age. We did not find a significant genotype/phenotype correlation, but Src homology 2 domain mutations (V637M and T620S) had a greater effect on STAT3 phosphorylation and nuclear translocation than DNA-binding domain mutation (R382W) in vitro.

Recent progress in the diagnosis and management of acquired pure red cell aplasia

Acquired pure red cell aplasia (PRCA) is characterized by normocytic anemia, reticulocytopenia, and a marked decrease in erythroid cell count in the bone marrow. PRCA develops in the context of various backgrounds, including recently recognized immune checkpoint inhibitor-associated PRCA, that need careful differential diagnoses. Besides humoral abnormalities such as major ABO-incompatible allogeneic hematopoietic stem cell transplantation-related PRCA, dysregulations of T cells have been shown. STAT3 gene mutations of cytotoxic T cells were identified in 40% of PRCA patients, which might suggest their use as novel molecular markers for PRCA. As initial management options for PRCA, red blood cell transfusion and immunosuppressive therapy (IST) drugs, such as cyclosporin, are usually selected. Roughly 80% of patients respond to IST; however, some relapse afterward or are refractory to IST. When patients with PRCA become refractory to two or three lines of IST, allogeneic hematopoietic stem cell transplantation (HCT) would become an appropriate choice, although the optimal procedures for allogeneic HCT have not been determined. A prospective study of PRCA in Japan has been ongoing since 2016 to solve the myriad clinical issues of PRCA.

PF367 MOLECULAR EVIDENCE THAT ATOVAQUONE IS A NEW STAT3‐INHIBITOR COUNTERBALANCING LEUKEMIC CELL PRO‐SURVIVAL IN LARGE GRANULAR LYMPHOCYTE LEUKEMIA

Background:Large Granular Lymphocyte Leukemia (LGLL) is a rare heterogeneous lymphoproliferative disorder characterized by the chronic proliferation of clonal Large Granular Lymphocytes (LGLs) with cytotoxic activity. The hallmark of leukemic LGLs is a constitutive activation of STAT3 leading to enhanced triggering of many pro‐survival genes. Moreover, 30–40% of patients carry mutations in STAT3 gene bringing to the constitutive activation of the malignant clone.No specific treatment is now available for the patients affected by LGLL and the in‐use therapy is based on low doses of immunosuppressive drugs, i.e. Methotrexate (MTX), Cyclophosphamide (CTX) and Cyclosporin A (CyA). Anyway, these therapies show limited efficacy, induce many side effects with the Overall Response Rate (ORR) being around 50% and a high rate of relapse in the responding patients.The JAK/STAT signaling pathway, which is central to LGL leukemia pathogenesis, includes several players that provide the basis for new targeted treatments. This offers the rationale for using Atovaquone (ATQ, Mepron™), a recently FDA‐approved anti‐microbial agent characterized by a low adverse effect profile that has been recently demonstrated (Xiang et al., 2016) to work as a powerful STAT3 inhibitor with anti‐cancer efficacy in both animal models and human cell lines.Aims:Our study focuses on in vitro tests to evaluate the effects of ATQ as inhibitor of STAT3 phosphorylation and of LGLs survival, with the goal to develop a targeted approach to LGLL, likely devoid of adverse effects.Methods:Patients’ Peripheral Blood Mononuclear Cells (PBMC) were purified to perform cultures to test serial drug concentrations. Using in vitro cells culture, the inhibition of STAT3 protein phosphorylation and the expression of its target proteins, MCL1 and BCL2, were evaluated by Western Blot assay (WB). In addition, the cytotoxicity and the apoptotic selectivity of ATQ was investigated by Annexin V test; a staining with anti‐CD57, CD56 or CD16 was used to identify leukemic LGLs from PBMCs. Finally, by RT‐PCR, STAT3 activity was analyzed along its transcriptional levels and target genes.Results:Through in vitro experiments we initially tested the best concentration inhibiting cell viability and STAT3 activation on patients’ PBMCs. We observed that ATQ was efficiently able to inhibit STAT3 phosphorylation at a concentration of 25 μM. The results of the apoptosis analysis showed that ATQ has a dose and time dependent cytotoxic effect. The IC50 was reached at a dose of 25 μM after 24 h of culture. ATQ demonstrated a selective cytotoxicity against leukemic LGLs (reaching 3.9 ± 0.7 fold increase of apoptosis vs untreated condition after 48 h of culture) sparing non‐leukemic cells (ATQ treated apoptosis vs untreated in non‐LGL PBMC = 1.18 ± 0.3 fold change). By comparing the in vitro cell effect of ATQ with that of the commonly used therapies, ATQ showed a cytotoxic effect consistent with CTX and CyA, but higher than MTX (p &lt; 0.01). Concerning anti‐apoptotic factors under the STAT3 control, after 6 h ATQ treatment the expression of MCL1 and BCL2 showed only minimal changes, thereby suggesting that different mechanisms of action take place.Summary/Conclusion:These in vitro data indicate that ATQ has a promising action on the inhibition of the JAK/STAT pro‐survival pathway in leukemic LGLs, resulting in a selective cytotoxic effect against LGLs through MCL1‐ and BCL2‐independent mechanisms.

The first cohort of Iranian patients with hyper immunoglobulin E syndrome: A long-term follow-up and genetic analysis.

Hyper-IgE syndromes (HIES) are distinct diseases characterized by recurrent cutaneous and lung infections, eczema, and elevated serum IgE level. In this study, clinical manifestations, immunologic findings, and genetic studies of all patients with HIES in the Iranian national registry database were evaluated. A total of 129 HIES patients with a median age of 14.0 (9.0-24.0) years were followed up for a total of 307.8 patient-years. Genetic studies showed heterozygous STAT3 mutations in 19 patients and homozygous DOCK8 mutation in 16 patients. The mean of National Institutes of Health score in STAT3-deficient patients was higher than in patients with DOCK8 mutation (P=0.001). It was shown that the presence of pneumatocele and hematologic complication were significantly frequent in STAT3-deficient cases compared to patients with DOCK8 deficiency (P=0.001 and P=0.002, respectively). Moreover, the median IgE serum levels were higher in patients with STAT3 gene mutation than in patients with DOCK8 gene mutation (P=0.02). The eosinophils' count was enhanced in patients with DOCK8 deficiency than in patients with STAT3 gene defects (P=0.02). Specific molecular study of STAT3 and DOCK8 mutations in patients with HIES clinical phenotype could help the physician to definitively characterize the disease. Since HIES showed the highest rate of unsolved combined immunodeficiency, investigation of other genetic and environmental factors could also help in understanding the mechanism of remaining patients as well as providing strategy into therapeutic modalities.

Variation of STAT3 Gene in Myleproliferative Neoplasms and Its Significance

To detect the mutation and single nucleotide polymorphisms of STAT3 gene in the patients with myeloproliferative neoplasms (MPN), and to analyze the correlation between STAT3 gene and the subtypes of MPN. A total of 147 patients with MPN were selected, including 28 patients with polycythaemia vera (PV), 46 patients with essential thrombocythemia (ET), 10 patients with primary myelofibrosis (PMF), and 63 patients with chronic myeloid leukemia (CML); and 88 healthy persons were used as normal control. DNA of all cases was extracted from bone marrow or peripheral blood, and JAK2V617F gene mutation was detected by allele-specific PCR, then 23 exons of STAT3 gene were amplified by PCR. Mutation and single nucleotide polymorphism of Rs2293152 of STAT3 gene were identified by DNA sequencing. STAT3 gene mutation was found in 8 patients with CML. The mutation rate was 12.7%. the missense mutation（S629T）as found in 3 cases, the synonymous mutation was found in 5 cases (Q469Q 3 cases, G618G 2 cases). One case had mutations at the both sites of S629T and G618G. No mutation of STAT3 gene was found in the normal control group. Rs2293152: detection showed that the G allele of CML group was significantly higher than that of normal control, PV, ET and PMF group (P<0.01), suggesting that the patients with Rs2293152 G allele were more likely to develop CML. The C allele of PV, ET and PMF group was significantly higher than that of CML group (P<0.05), suggesting that the patients with Rs2293152 C allele were more likely to develop PV, ET and PMF. The G allele fiequency of JAK2V617F-negative group was significantly lower than that of the normal control and JAK2V617F positive group (P<0.01), suggesting that the Rs2293152 G allele may be a factor protecting against JAK2V617F mutation. In MPN patients, STAT3 gene is unstable and prone to mutation. The different alleles of the Rs2293152 locus of the STAT3 gene are relates with different subtypes and JAK2V617F-negative MPN.