Signal Transducer And Activator Of Transcription 3 Mutations Research Articles

A pan-cancer analysis of STAT3 expression and genetic alterations in human tumors.

Combined cancer immunotherapy and targeted therapy have proven to be effective against various cancers and therefore have recently become the focus of cancer research. Signal transducer and activator of transcription 3 (STAT3) is a member of the STAT protein family of transcription factors. Several studies have shown that STAT3 can affect the prognosis of cancer patients by regulating immune microenvironment (IME). Therefore, STAT3 may have high research value for the development of combined immunotherapy/targeted therapy approaches for the treatment of cancer patients. We found differences in STAT3 expression between tumor and normal tissues. Kaplan-Meier survival and Cox regression analyses showed that high expression of STAT3 is associated with poor prognosis in low-grade glioma (LGG) patients. The results of the analysis of the area under the curve of the receiver operating characteristic curve further suggested that the expression of STAT3 is an effective way to evaluate the prognosis of patients with glioma. The results of the IME analysis revealed that the immune and matrix scores of LGGs were positively correlated with the expression of STAT3 (P < 0.05). The results of immune cell infiltration analysis showed that STAT3 was positively correlated with resting dendritic cells, eosinophils, neutrophils, M0 macrophages, M1 macrophages, CD4 memory resting T cells, and CD8 T cells in LGG patients, but negatively correlated with activated mast cells and M2 macrophages (P < 0.05). Our gene set enrichment analysis identified 384 enriched pathways. According to the enrichment scores, the top ten most significantly upregulated pathways were related to immune response. The top ten most significantly downregulated pathways were related to cell signal transduction and the regulation of cell survival, proliferation, and metabolism. Genetic alteration analysis showed that missense mutations in STAT3 account for the majority of mutations, and STAT3 mutations mostly occur in the Src homology domain. In conclusion overexpression of STAT3 can promote the development and growth of tumors by regulating IME, which is significantly related to the poor prognosis of cancer patients. Therefore, targeted inhibition of STAT3 expression may have high research value for the development of combined immunotherapy/targeted therapy approaches for the treatment of cancer patients.

The human Stat1 gain-of-function T385M mutation causes expansion of activated T-follicular helper/T-helper 1-like CD4 T cells and sex-biased autoimmunity in specific pathogen-free mice.

Humans with gain-of-function (GOF) mutations in STAT1 (Signal Transducer and Activator of Transcription 1), a potent immune regulator, experience frequent infections. About one-third, especially those with DNA-binding domain (DBD) mutations such as T385M, also develop autoimmunity, sometimes accompanied by increases in T-helper 1 (Th1) and T-follicular helper (Tfh) CD4 effector T cells, resembling those that differentiate following infection-induced STAT1 signaling. However, environmental and molecular mechanisms contributing to autoimmunity in STAT1 GOF patients are not defined. We generated Stat1T385M/+ mutant mice to model the immune impacts of STAT1 DBD GOF under specific-pathogen free (SPF) conditions. Stat1T385M/+ lymphocytes had more total Stat1 at baseline and also higher amounts of IFNg-induced pStat1. Young mutants exhibited expansion of Tfh-like cells, while older mutants developed autoimmunity accompanied by increased Tfh-like cells, B cell activation and germinal center (GC) formation. Mutant females exhibited these immune changes sooner and more robustly than males, identifying significant sex effects of Stat1T385M-induced immune dysregulation. Single cell RNA-Seq (scRNA-Seq) analysis revealed that Stat1T385M activated transcription of GC-associated programs in both B and T cells. However, it had the strongest transcriptional impact on T cells, promoting aberrant CD4 T cell activation and imparting both Tfh-like and Th1-like effector programs. Collectively, these data demonstrate that in the absence of overt infection, Stat1T385M disrupted naïve CD4 T cell homeostasis and promoted expansion and differentiation of abnormal Tfh/Th1-like helper and GC-like B cells, eventually leading to sex-biased autoimmunity, suggesting a model for STAT1 GOF-induced immune dysregulation and autoimmune sequelae in humans.

P302 Precision medicine for a case with monogenic inflammatory bowel disease

Abstract Background Here we report the case of a 33-year-old patient suffering from recurring infections since childhood, and Crohn’s disease (CD)-like inflammation in the ileum and colon for 5 years. The patient was transferred to our hospital due to severe pneumonia with Pseudomonas aeruginosa and Mycobacterium chimaera infection, recurrent fever, bronchiectasis, cachexia (BMI 12.9) and severe gastrointestinal symptoms (diarrhoea with blood 15 times per day). Colonoscopy revealed a peculiar inflammatory pattern in the ileum and the colon without healthy sections and deep punched lesions which did not fit to a diagnosis of CD. Methods Exome sequencing was performed and pathogenic variants identified by in silico prediction. Peripheral blood mononuclear cells (PBMCs) were isolated and Signal Transducer And Activator Of Transcription 1 (STAT1) activation was assessed by immunoblot after treatment with JAK-inhibitors. STAT1 activation was quantified in leukocytes of whole blood samples by flow cytometry. Mucosal sections were used to quantify STAT1 activation within mucosal cells by multicolour immunofluorescence. Two healthy individuals served as controls. Results Exome sequencing revealed a pathogenic mutation (c.1256C&amp;gt;G) in the STAT1 gene, which was predicted to cause a gain-of-function. STAT1 is part of the JAK/STAT1 pathway which is induced by different stimuli and orchestrates proliferation and responses of immune cells as a transcription factor. Isolated PBMCs from the patient exhibited increased STAT1 activation, and in whole blood samples increased levels were particularly observed in Monocytes and T-helper cells. In mucosal sections activated STAT1 was especially expressed in immune cells but also epithelial cells. Stimulation of PBMCs with Upadacitinib or Tofacitinib and partially with Filgotinib decreased STAT1 levels. We initiated treatment with Tofacitinib due to the in vitro results and its approval to treat ulcerative colitis. After two weeks, clinical symptoms improved drastically and endoscopy performed after 3 month indicated full remission, which was paralleled by a normalisation of STAT1 in PBMCs. Conclusion We present a case with a rare form of Inflammatory bowel disease (IBD) caused by a gain-of-function mutation affecting the STAT1 gene. IBD-like symptoms are unusual for STAT1 mutations, which usually present as immunodeficiency, and this specific mutation was only identified twice before and listed in data bases without describing the clinical phenotype. Our results and approach demonstrate how delineating the specific pathomechanism of a case with IBD enables targeted and precision treatment, a currently intensively investigated concept which might be implemented into clinical routine to treat IBD in the future.

Prevalence of STAT3 mutations in patients with rheumatoid arthritis-associated T-cell large granular lymphocytic leukaemia and Felty syndrome.

Neutropenia is a key presentation of Felty syndrome (FS) and rheumatoid arthritis (RA)-associated T-cell large granular lymphocytic (T-LGL) leukaemia. Clonal rearrangement of T-cell receptor (TCR) gene supports the diagnosis of T-LGL leukaemia but not FS. Mutations in the signal transducer and activator of transcription 3 (STAT3) gene are highly specific for T-LGL leukaemia, but their prevalence in FS remains poorly clarified. The study included 100 patients with RA and unexplained neutropenia. TCR rearrangements were examined in blood (100 cases), bone marrow (47 cases), and spleen (12 cases) using the BIOMED-2 protocol. Patients were stratified into RA-associated T-LGL leukaemia cohort if a clonal TCR rearrangement was identified in any of the tested patient samples, and into FS cohort in other cases. Mutations in the STAT3 were examined using next-generation sequencing (NGS) technology in blood (100 cases), bone marrow (37 cases), and spleen (7 cases). STAT3 mutations were identified in 71% (49/69) patients with RA-associated T-LGL leukaemia and in 10% (3/31) patients with FS (p=4.7×10-8). Three samples from the RA-associated T-LGL leukaemia cohort and 5 samples from the FS cohort had STAT3 mutations in the absence of clonal TCR rearrangement. The results suggest that STAT3 mutations are significantly less common in FS than in RA-associated T-LGL leukaemia. Moreover, NGS can detect clones undetectable by fragment analysis. We speculate that in patients with RA and neutropenia, the detection of STAT3 mutations can point to T-LGL leukaemia even in the absence of clonal TCR rearrangement.

Diagnostic challenge in a series of eleven patients with hyper IgE syndromes.

Hyper IgE syndromes (HIES) is a heterogeneous group of Inborn Errors of Immunity characterized by eczema, recurrent skin and lung infections associated with eosinophilia and elevated IgE levels. Autosomal dominant HIES caused by loss of function mutations in Signal transducer and activator of transcription 3 (STAT3) gene is the prototype of these disorders. Over the past two decades, advent in genetic testing allowed the identification of ten other etiologies of HIES. Although Dedicator of Cytokinesis 8 (DOCK8) deficiency is no more classified among HIES etiologies but as a combined immunodeficiency, this disease, characterized by severe viral infections, food allergies, autoimmunity, and increased risk of malignancies, shares some clinical features with STAT3 deficiency. The present study highlights the diagnostic challenge in eleven patients with the clinical phenotype of HIES in a resource-limited region. Candidate gene strategy supported by clinical features, laboratory findings and functional investigations allowed the identification of two heterozygous STAT3 mutations in five patients, and a bi-allelic DOCK8 mutation in one patient. Whole Exome Sequencing allowed to unmask atypical presentations of DOCK8 deficiency in two patients presenting with clinical features reminiscent of STAT3 deficiency. Our study underlies the importance of the differential diagnosis between STAT3 and DOCK8 deficiencies in order to improve diagnostic criteria and to propose appropriate therapeutic approaches. In addition, our findings emphasize the role of NGS in detecting mutations that induce overlapping phenotypes.

Clinical Study of the Relationship between Sjögren Syndrome and T-Cell Large Granular Lymphocytic Leukemia: Single-Center Experience.

The relationship between Sjögren syndrome (SS) and T-cell large granular lymphocytic (T-LGL) leukemia remains unclear. In this paper, we report for the first time a large case series of 21 patients with primary and secondary SS associated with T-LGL leukemia. Our results suggest the importance of considering T-LGL leukemia in the diagnostic evaluation of SS patients, particularly when neutropenia occurs. We also postulate that elevated antinuclear antibody titers in patients with T-LGL leukemia indicate the need for the clinical assessment of SS. To assess whether SS affects the frequency of the signal transducer and activator of transcription 3 (STAT3) gene mutations in T-LGL leukemia, we examined STAT3 mutations by next-generation sequencing in two cohorts of patients: with SS-associated T-LGL leukemia and T-LGL leukemia in the setting of rheumatic diseases but without SS. While our results suggest that SS, per se, is not associated with an increased frequency of STAT3 mutations in T-LGL leukemia, further studies are needed to better assess the role of the STAT pathway in the development of concomitant SS and T-LGL leukemia.

RECURRENT FUNGAL INFECTIONS FROM BIRTH TO ADULTHOOD: UNKNOWN DIAGNOSIS UNTIL GENETIC TESTING FOR STAT-1 VARIANTS

<h3>Introduction</h3> Signal transducer and activator of transcription 1 (STAT-1) is a transcription factor that plays key roles in cell viability and pathogen responses. Mutations in STAT-1 result in differing degrees of susceptibility to viral, fungal and bacterial infections, vascular alterations, failure to thrive, and autoimmunity. We report a case of STAT-1 variant presenting with a life-long history of recurrent fungal infections where the diagnosis required genetic testing. <h3>Case Description</h3> A 33-year-old man presented for an evaluation of recurrent fungal infections. He experienced oral thrush, tinea pedis, tinea corporis, and onychomycosis since infancy. He was hospitalized twice for Candida esophagitis requiring IV antifungals. He required treatments with oral antifungals every few months, as he was refractory to topical treatments. He had no bacterial or viral infections, and otherwise unremarkable past medical history. He had thorough workups through multiple specialists with no diagnosis made as his immunologic laboratory studies were normal. However, his 4-year-old son was diagnosed with chronic mucocutaneous candidiasis (CMCC) and had genetic testing showing a STAT-1 variant (c.1159A>G (p.Thr387Ala)). The patient then underwent genetic testing which revealed the same pathogenic STAT-1 variant. Further evaluation showed normal T-cell mitogen studies, total IgG/A/M/E, IgG subclass levels, and vaccine responses. Flow cytometry showed mild B-cell lymphopenia and normal T/NK cells. <h3>Discussion</h3> Patients with pathogenic STAT-1 variants can present with variable infection histories and normal immunologic testing. This case highlights the importance of genetic testing for patients presenting with CMCC regardless of normal immunologic testing to identify a specific diagnosis and defects associated with CMCC.

MAKING A STAT DIAGNOSIS IN A PATIENT PRESENTING WITH VILLOUS ATROPHY

<h3>Introduction</h3> Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) gene mutations are highly penetrant, resulting in enhanced STAT1-dependent responses to interferon-alpha/beta and interferon-gamma signaling, and impaired Th17 development. This manifests as recurrent infections, cytopenias, and autoimmune disease. Herein, we present a case of a child diagnosed with a STAT1 mutation. <h3>Case Description</h3> A 3-year-old female presented for secondary evaluation of persistent gastrointestinal and respiratory symptoms, recurrent sinopulmonary and skin infections, failure to thrive, and developmental delay. An esophagogastroduodenoscopy performed elsewhere showed increased duodenal intraepithelial lymphocytes with crypt hyperplasia and villous atrophy. Testing for Celiac disease and autoimmune enteritis were negative. Chest computed tomography showed bilateral bronchiectasis with mucous plugging. Bronchoalveolar lavage culture was positive for Streptococcus pneumoniae. Laboratory workup was notable for anemia and T-cell lymphopenia. Quantitative lymphocytes showed a low CD3+ count of 446 cells/mcL, low CD4+ count of 185 cells/mcL, and low CD8+ count of 194 cells/mcL. Lymphocyte proliferation to antigens showed essentially absent total CD45+ lymphocytes and CD3+ T cell responses to Candida and Tetanus toxoid antigens. Genetic testing later revealed a heterozygous, pathogenic c.1154C>T (p.Thr385Met) variant in the STAT1 gene. Additional testing for Th17 enumeration showed a decreased IL17 and Th17 cell percentage. She was treated with supportive care, antibiotics, and referred to Medical Genetics for additional education and counseling. <h3>Discussion</h3> STAT1-GOF gene mutations are rare and associated with variable phenotypes and disease severity. Genetic testing can help determine the cause of complex, multisystem diseases. Stem cell transplant, immunoglobulin replacement therapy, and JAK inhibitors are potential treatment considerations.

STAT3 mutations in "gray-zone" cases of T-cell large granular lymphocytic leukemia associated with autoimmune rheumatic diseases.

A persistently increased T-cell large granular lymphocyte (T-LGL) count in the blood of more than 2 × 109/L for at least 6 months is necessary for a reliable diagnosis of T-LGL leukemia. In cases with LGL counts of approximately 0.5–2 × 109/L, a diagnosis of T-LGL leukemia can be made if clonal rearrangement of T-cell receptor (TCR) genes is present and if the patient shows typical manifestations of T-LGL leukemia, such as cytopenia, splenomegaly, or concomitant autoimmune disease. However, in cases with LGL counts of less than 0.5 × 109/L, the diagnosis of T-LGL leukemia is questionable (termed as “gray-zone” cases). Although mutations in signal transducer and activator of transcription 3 (STAT3) gene are the molecular hallmark of T-LGL leukemia, their diagnostic value in the “gray-zone” cases of T-LGL leukemia has not been evaluated – our study has been aimed to examine the prevalence of STAT3 mutations in these cases. Herein, we describe 25 patients with autoimmune rheumatic diseases, neutropenia, clonal rearrangement of TCR genes, and circulating LGL count of less than 0.5 × 109/L. Splenomegaly was observed in 19 (76%) patients. Mutations in the STAT3 were detected in 56% of patients using next-generation sequencing. Importantly, in 3 patients, no involvement of the blood and bone marrow by malignant LGLs was noted, but examination of splenic tissue revealed infiltration by clonal cytotoxic T-lymphocytes within the red pulp, with greater prominence in the cords. We suggest using the term “splenic variant of T-LGL leukemia” for such cases.

Genetic and Functional Identifying of Novel STAT1 Loss-of-Function Mutations in Patients with Diverse Clinical Phenotypes.

Mutations in signal transducer and activator of transcription 1 (STAT1) cause a broad spectrum of disease phenotypes. Heterozygous STAT1 loss-of-function (LOF) mutations cause Mendelian susceptibility to mycobacterial diseases (MSMD) infection, which is attributable to impaired IFN-γ signaling. The identification of novel mutations may extend the phenotypes associated with autosomal dominant (AD) STAT1 deficiency. Five patients with heterozygous STAT1 variations were recruited and their clinical and immunologic phenotypes were analyzed, with particular reference to JAK-STAT1 signaling pathways. Four, heterozygous STAT1 deficiency mutations were identified, three of which were novel mutations. Two of the mutations were previously unreported mRNA splicing mutations in AD STAT1-deficient patients. Patients with heterozygous STAT1 deficiency suffered not only mycobacterial infection, but also intracellular non-mycobacterial bacterial infection and congenital multiple malformations. AD-LOF mutation impaired IFN-γ-mediated STAT1 phosphorylation, gamma-activated sequence (GAS), and IFN-stimulated response element (ISRE) transcription activity and IFN-induced gene expression to different extents, which might account for the diverse clinical manifestations observed in these patients. The infectious disease susceptibility and phenotypic spectrum of patients with AD STAT1-LOF are broader than simply MSMD. The susceptibility to infections and immunological deficiency phenotypes, observed in AD-LOF patients, confirms the importance of STAT1 in host-pathogen interaction and immunity. However, variability in the nature and extent of these phenotypes suggests that functional analysis is required to identify accurately novel, heterozygous STAT1 mutations, associated with pathogenicity. Aberrant splice of STAT1 RNA could result in AD-LOF for STAT1 signaling which need more cases for confirmation.

STAT3 gain-of-function is not responsible for low total IgE levels in patients with autoimmune chronic spontaneous urticaria.

BackgroundThe pathogenesis of chronic spontaneous urticaria (CSU) has not been clarified entirely. Type IIb autoimmune chronic spontaneous urticaria (CSUaiTIIb) is a distinct subtype of CSU that is often difficult to treat and is connected to low levels of total IgE. Previous findings indicate that an enhanced signal transducer and activator of transcription 3 (STAT3) may be responsible for reduced IgE serum levels.ObjectiveOur aim was to investigate a possible underlying gain-of-function mutation or activating polymorphism in STAT3 that could be responsible for the low levels of IgE in patients with CSUaiTIIb.MethodsWe included 10 patients with CSUaiTIIb and low levels of IgE and sequenced selected single nucleotide polymorphisms (SNP) in STAT3 associated with common autoimmune diseases. Exon sequencing was performed for the most relevant exons of STAT3. To test for a gain-of-function of STAT3, we performed a phospho-specific flow cytometry analysis of STAT3 in peripheral blood mononuclear cells before and after stimulation with interleukin-6.ResultsNo differences were found in the prevalence of the tested SNPs between our patients and a control population. Moreover, we could not find any mutations or variants on the tested exons of STAT3. The function of STAT3 was also not altered in our patients.ConclusionIn total, we could not find any evidence for our hypothesis that low IgE in patients with CSUaiTIIb is linked to mutations in STAT3 or altered activity of STAT3. Thus, it remains to be discovered what causes the low serum levels of IgE in patients with CSUaiTIIb.

Longitudinal study of 2 patients with cyclic thrombocytopenia, STAT3 and MPL mutations

AbstractCyclic thrombocytopenia (CTP) is a rare disease of periodic platelet count oscillations. The pathogenesis of CTP remains elusive. To study the underlying pathophysiology and genetic and cellular associations with CTP, we applied systems biology approaches to 2 patients with stable platelet cycling and reciprocal thrombopoietin (TPO) cycling at multiple time points through 2 cycles. Blood transcriptome analysis revealed cycling of platelet-specific genes, which are in parallel with and precede platelet count oscillation, indicating that cyclical platelet production leads platelet count cycling in both patients. Additionally, neutrophil and erythrocyte-specific genes also showed fluctuations correlating with platelet count changes, consistent with TPO effects on hematopoietic progenitors. Moreover, we found novel genetic associations with CTP. One patient had a novel germline heterozygous loss-of-function (LOF) thrombopoietin receptor (MPL) c.1210G>A mutation, and both had pathogenic somatic gain-of-function (GOF) variants in signal transducer and activator of transcription 3 (STAT3). In addition, both patients had clonal T-cell populations that remained stable throughout platelet count cycles. These mutations and clonal T cells may potentially involve in the pathogenic baseline in these patients, rendering exaggerated persistent thrombopoiesis oscillations of their intrinsic rhythm upon homeostatic perturbations. This work provides new insights into the pathophysiology of CTP and possible therapies.

Chronic mucocutaneous Candidiasis caused by a novel STAT1 mutation: a report of 4 patients

Background: Chronic mucocutaneous Candidiasis (CMCC) is characterized by recurrent or persistent fungal infections of the skin, nails, and oral and genital mucosae. There are several underlying genetic causes for CMCC, with mutations in Signal Transducer and Activator of Transcription-1 (STAT1) accounting for the majority of cases. Aim: To broaden the genotypic spectrum of CMCC caused by STAT1 mutations. Methods: We evaluated a young patient and her family with CMCC. Immune workup and targeted gene sequencing were performed. Results: The proband presented at 7 years of age with persistent oral thrush. Immune evaluation revealed her cellular and humoral immunity to be within normal range. Given that her family history was significant for oral lesions in father, siblings, and paternal family members, STAT1 gene sequencing was performed. A novel heterozygous missense c.G799A, predicting a p. Ala267Thr amino acid change within the coiled-coil domain, was identified in our patient and 3 of her family members. Conclusion: Gain-of-function mutations in STAT1 have been associated with a variety of phenotypes, ranging from isolated CMCC to severe fatal combined immunodeficiency, mycobacterial infections, autoimmune disorders, as well as malignancy and aneurysms. Here, we describe a novel STAT1 mutation, c.G799A, resulting in a very mild phenotype of isolated CMCC in 4 members of one kindred. Statement of novelty: We describe 4 patients with a mild phenotype of CMCC caused by a novel STAT1 heterozygous mutation.

STAT1 gain-of-function heterozygous cell models reveal diverse interferon-signature gene transcriptional responses

Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) is an autosomal dominant immune disorder marked by wide infectious predisposition, autoimmunity, vascular disease, and malignancy. Its molecular hallmark, elevated phospho-STAT1 (pSTAT1) following interferon (IFN) stimulation, is seen consistently in all patients and may not fully account for the broad phenotypic spectrum associated with this disorder. While over 100 mutations have been implicated in STAT1 GOF, genotype–phenotype correlation remains limited, and current overexpression models may be of limited use in gene expression studies. We generated heterozygous mutants in diploid HAP1 cells using CRISPR/Cas9 base-editing, targeting the endogenous STAT1 gene. Our models recapitulated the molecular phenotype of elevated pSTAT1, and were used to characterize the expression of five IFN-stimulated genes under a number of conditions. At baseline, transcriptional polarization was evident among mutants compared with wild type, and this was maintained following prolonged serum starvation. This suggests a possible role for unphosphorylated STAT1 in the pathogenesis of STAT1 GOF. Following stimulation with IFNα or IFNγ, differential patterns of gene expression emerged among mutants, including both gain and loss of transcriptional function. This work highlights the importance of modeling heterozygous conditions, and in particular transcription factor-related disorders, in a manner which accurately reflects patient genotype and molecular signature. Furthermore, we propose a complex and multifactorial transcriptional profile associated with various STAT1 mutations, adding to global efforts in establishing STAT1 GOF genotype–phenotype correlation and enhancing our understanding of disease pathogenesis.

The Positivity of Phosphorylated STAT3 Is a Novel Marker for Favorable Prognosis in Germinal Center B-Cell Type of Diffuse Large B-Cell Lymphoma.

On the basis of immunohistochemistry, diffuse large B-cell lymphoma (DLBCL) is categorized as a germinal center B-cell (GCB) or non-GCB subtype. Recent integrated genomic analyses have highlighted the importance of the JAK-STAT3 pathway in the molecular pathogenesis of DLBCL. However, its relevance to clinical outcomes remains controversial. Therefore, we evaluated the extent of the nuclear expression of phosphorylated STAT3 (pSTAT3), a surrogate marker of signal transducer and activator of transcription 3 (STAT3) activation, by immunohistochemistry. We also analyzed the potential relationship between pSTAT3 positivity (defined as ≥40% positive neoplastic cells) and clinicopathologic characteristics in 294 patients with DLBCL. pSTAT3 was detected in 122 patients (42%), with a higher rate in the non-GCB subtype than in the GCB subtype (57% vs. 28%, P<0.001). Factors potentially activating STAT3, MYD88L265P, and Epstein-Barr virus-encoded small RNA were identified in the pSTAT3-positive non-GCB subtype, whereas the pSTAT3-positive GCB subtype often showed STAT3 mutations and lacked EZH2 mutations and the rearrangements of BCL2 and MYC. Multivariate analyses revealed that the pSTAT3-positive GCB subtype showed a favorable prognosis (HR: 0.17; 95% confidence interval, 0.04-0.7; P=0.014). These findings suggest that pSTAT3 positivity may have a unique impact on the clinicopathologic characteristics of DLBCL, making it a promising novel marker for the favorable prognosis of patients with the GCB subtype.

Clinical Relevance of Gain- and Loss-of-Function Germline Mutations in STAT1: A Systematic Review.

Background: Germline mutations in signal transducer and activator of transcription 1 (STAT1), which lead to primary immunodeficiency, are classified as defects in intrinsic and innate immunity. To date, no comprehensive overview comparing GOF with LOF in early-onset immunodeficiency has been compiled.Objective: To collect and systematically review all studies reporting STAT1 GOF and LOF cases, and to describe the clinical, diagnostic, molecular, and therapeutic characteristics of all the conditions.Methods: A systematic review of the PubMed, EMBASE, Web of Science, Scopus, and Cochrane to identify articles published before May 23, 2020. Data pertaining to patients with a genetic diagnosis of STAT1 GOF or LOF germline mutations, along with detailed clinical data, were reviewed.Results: The search identified 108 publications describing 442 unique patients with STAT1 GOF mutations. The patients documented with chronic mucocutaneous candidiasis (CMC; 410/442), lower respiratory tract infections (210/442), and autoimmune thyroid disease (102/442). Th17 cytopenia was identified in 87.8% of those with GOF mutations. Twenty-five patients with GOF mutations received hematopoietic stem cell transplantation (HSCT), and 10 died several months later. Twelve of 20 patients who received JAK inhibitor therapy showed improved symptoms. Twenty-one publications described 39 unique patients with STAT1 LOF mutations. The most common manifestations were Mendelian susceptibility to mycobacterial diseases (MSMD) (29/39), followed by osteomyelitis (16/39), and lymphadenopathy (9/39). Missense, indel, and frameshift mutations were identified as LOF mutations. There were no obvious defects in lymphocyte subsets or immunoglobulin levels. Eighteen patients required antimycobacterial treatment. Three patients received HSCT, and one of the three died from fulminant EBV infection.Conclusions: STAT1 GOF syndrome is a clinical entity to consider when confronted with a patient with early-onset CMC, bacterial respiratory tract infections, or autoimmune thyroid disease as well as Th17 cytopenia and humoral immunodeficiency. HSCT is still not a reasonable therapeutic choice. Immunoglobulin replacement therapy and JAK inhibitors are an attractive alternative. STAT1 LOF deficiency is a more complicated underlying cause of early-onset MSMD, osteomyelitis, respiratory tract infections, and Herpesviridae infection. Anti-mycobacterial treatment is the main therapeutic choice. More trials are needed to assess the utility of HSCT.

Analysis of a single-institution cohort of patients with Felty's syndrome and T-cell large granular lymphocytic leukemia in the setting of rheumatoid arthritis

T-cell large granular lymphocytic leukemia (T-LGLL) is a lymphoproliferative disorder characterized by a persistent increase in the number of large granular lymphocytes (LGLs), neutropenia, and splenomegaly. Clinical manifestations of T-LGLL in the setting of rheumatoid arthritis (RA) are often identical to those in which one would suspect Felty's syndrome (FS). These disorders are distinguished by the presence of T-cell clonality, which is present in T-LGLL but not in FS. Mutations in the signal transducer and activator of transcription 3 (STAT3) and 5b (STAT5b) genes can be used as molecular markers of T-LGLL, but their prevalence in FS is unknown.Eighty-one patients with RA and unexplained neutropenia or/and an increase in the number of LGLs above 2 × 109/L were stratified into RA-associated T-LGLL (N = 56) or FS (N = 25) groups based on the presence or absence of T-cell clonality. STAT3 and STAT5b gene mutations were assessed in each group by means of allele-specific polymerase chain reaction assays. Clinical, immunological, laboratory data and the results of immunophenotyping of blood and bone marrow lymphocytes were also evaluated.Mutations of the STAT3 gene and an increase in the number of LGLs above 2 × 109/L were detected in RA-associated T-LGLL, but not in FS (39% vs 0% and 21% vs 0%, respectively). Mutations in the STAT5b gene were not observed in either group. Expression of CD57, CD16, and CD5−/dim on CD3+CD8+ T-lymphocytes was observed in both RA-associated T-LGLL and FS.STAT3 gene mutations or LGL counts over 2 × 109/L in RA patients are indicative of T-LGLL.

Staphylococcus aureus and Hyper-IgE Syndrome.

Hyper-immunoglobulin E syndrome (HIES) is a primary immunodeficiency disease characterized by recurrent Staphylococcus aureus (S. aureus) infections, eczema, skeletal abnormalities and high titers of serum immunoglobulin E. Although the genetic basis of HIES was not known for almost a half century, HIES most frequently exhibits autosomal dominant trait that is transmitted with variable expressivity. Careful genetic studies in recent years identified dominant-negative mutations in human signal transducer and activator of transcription 3 (STAT3) gene as the cause of sporadic and dominant forms of HIES. The STAT3 mutations were localized to DNA-binding, SRC homology 2 (SH2) and transactivating domains and disrupted T helper 17 (TH17) cell differentiation and downstream expression of TH17 cytokines IL-17 and IL-22. Deficiency of IL-17 and IL-22 in turn is responsible for suboptimal expression of anti-staphylococcal host factors, such as neutrophil-recruiting chemokines and antimicrobial peptides, by human keratinocytes and bronchial epithelial cells. TH17 cytokines deficiency thereby explains the recurrent staphylococcal lung and skin infections of HIES patients.

The pathogenic T387A missense mutation in the gene encoding signal transducer and activator of transcription 1 exhibits a differential gene expression profile

Heterozygous gain-of-function (GOF) mutations in the interferon-driven transcription factor STAT1 (signal transducer and activator of transcription 1) cause chronic mucocutaneous candidiasis (CMC). In this study, we characterized the molecular basis of a CMC-associated missense mutation by introducing a threonine-to-alanine exchange in the STAT1 DNA-binding domain at position 387. This substitution had previously been described in a CMC patient with suppurative eyelid infection and cutaneous abscesses, which are both unusual symptoms in this immunodeficiency. The STAT1-T387A mutant generated was compared to the wild-type protein and, in addition, to the missense mutant in the neighbouring position 386. Our results showed that the T387A mutant displayed distinct properties different from the wild-type molecule, namely elevated levels of tyrosine phosphorylation in conjunction with increased DNA-binding activity, hyperactive transcriptional regulation, and prolonged nuclear accumulation. The elevated tyrosine phosphorylation of the T387A mutant did not result in an increased mRNA production above the level of the wild-type molecule for all transcripts tested, indicating that the transcriptional activity of this mutant is largely gene-dependent. Nonetheless, these data demonstrate that the pathogenic T387A mutation associated with an atypical CMC symptomatology is biochemically similar to other well-characterized GOF mutants, while the H386A mutant was indistinguishable from the wild-type molecule. Our findings are in line with the assumption that the phenotype of this dominant STAT1 GOF mutation probably results from a disturbed shift in the equilibrium between the parallel and antiparallel dimer conformation, which is required for physiological gene activation.

Helicobacter pylori infection induces STAT3 phosphorylation on Ser727 and autophagy in human gastric epithelial cells and mouse stomach

Helicobacter pylori (H. pylori) infection is considered as one of the principal risk factors of gastric cancer. Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) plays an important role in inflammation-associated gastric carcinogenesis. In the canonical STAT3 pathway, phosphorylation of STAT3 on Tyr705 is a major event of STAT3 activation. However, recent studies have demonstrated that STAT3 phosphorylated on Ser727 has an independent function in mitochondria. In the present study, we found that human gastric epithelial AGS cells infected with H. pylori resulted in localization of STAT3 phosphorylated on Ser727 (P-STAT3Ser727), predominantly in the mitochondria. Notably, H. pylori-infected AGS cells exhibited the loss of mitochondrial integrity and increased expression of the microtubule-associated protein light chain 3 (LC3), the autophagosomal membrane-associated protein. Treatment of AGS cells with a mitophagy inducer, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), resulted in accumulation of P-STAT3Ser727 in mitochondria. In addition, the elevated expression and mitochondrial localization of LC3 induced by H. pylori infection were attenuated in AGS cells harboring STAT3 mutation defective in Ser727 phosphorylation (S727A). We also observed that both P-STAT3Ser727 expression and LC3 accumulation were increased in the mitochondria of H. pylori-inoculated mouse stomach.