Association of inflammatory biomarkers with clinical outcomes in nivolumab-treated patients with advanced hepatocellular carcinoma.

BACKGROUND & AIMS: Nivolumab, a programmed death (PD)-1 (PD-1) inhibitor, led to durable responses, manageable safety, and increased survival in patients with advanced hepatocellular carcinoma (HCC). In our retrospective analysis, we studied the immunobiology and potential associations between biomarkers and outcomes with nivolumab in HCC. METHODS: Fresh and archival tumour samples from dose-escalation and dose-expansion phases of the CheckMate 040 trial were analysed by immunohistochemistry and RNA sequencing to assess several inflammatory gene expression signatures, including CD274 (PD-ligand 1 [PD-L1]), CD8A, LAG3, and STAT1. Biomarkers were assessed for association with clinical outcomes (best overall response by blinded independent central review per RECIST v1.1 and overall survival [OS]). RESULTS: Complete or partial tumour responses were observed in PD-L1-positive and PD-L1-negative patients treated with nivolumab monotherapy. Median OS was 28.1 (95% CI 18.2-n.a.) vs. 16.6 months (95% CI 14.2-20.2) for patients with tumour PD-L1 ≥1% vs. <1% (p = 0.03). Increased CD3 and CD8 showed a non-significant trend towards improved OS (both p = 0.08), and macrophage markers were not associated with OS. Tumour PD-1 and PD-L1 expression were associated with improved OS (p = 0.05 and p = 0.03, respectively). An inflammatory gene signature consisting of 4 genes was associated with improved objective response rate (p = 0.05) and OS (p = 0.01). CONCLUSIONS: PD-1 and PD-L1 expression, biomarkers of inflammation, and inflammatory gene signatures trended with improved survival and response. While further confirmation within a larger phase III trial is needed to evaluate predictive value of these biomarkers, these exploratory analyses suggest that anti-tumour immune response may play a role in the treatment benefit of nivolumab in HCC. LAY SUMMARY: Certain tests may be used to provide a picture of how a tumour is escaping the immune system, allowing it to continue to grow and create more tumours. Therapies such as nivolumab are designed to help the immune system fight the tumour. These tests may be used to determine how effective such therapies will be in the treatment of advanced liver cancer. NCT NUMBER: NCT01658878.

Dichotomal functions of phosphorylated and unphosphorylated STAT1 in hepatocellular carcinoma.

Interferons (IFNs) with antiviral and immune-stimulatory functions have been widely used in prevention and treatment of hepatocellular carcinoma (HCC). Signal transducer and activator of transcription 1 (STAT1) is a key element of the IFN signaling, and the function of STAT1 is critically determined by its phosphorylation state. This study aims to understand the functions of phosphorylated (p-) and unphosphorylated (u-) STAT1 in HCC. We found that u-STAT1 is significantly elevated in patient HCC tumor tissues and predominantly expressed in cytoplasm; while p-STAT1 is absent. Loss of u-STAT1 potently arrested cell cycle and inhibited cell growth in HCC cells. Induction of p-STAT1 by IFN-α treatment effectively triggers the expression of interferon-stimulated genes (ISGs), but has moderate effect on HCC cell growth. Interestingly, both u-STAT1 and p-STAT1 are induced by IFN-α, through with distinct time-dependent process. Furthermore, the ISG induction patterns mediated by p-STAT1 and u-STAT1 are also distinct. Importantly, artificial blocking of the induction of u-STAT1, but not p-STAT1, sensitizes HCC cells to treatment of IFNs. Therefore, p-STAT1 and u-STAT1 exert dichotomal functions and coordinately regulate the responsiveness to IFN treatment in HCC. KEY MESSAGES: STAT1 is upregulated and predominantly presented as u-STAT1 in HCC, while p-STAT1 is absent. U-STAT1 sustains but p-STAT1 inhibits HCC growth. The dynamic change of phosphorylation state of STAT1 control the responsiveness to IFN treatment.

Several genes involved in the JAK-STAT pathway may act as prognostic markers in pancreatic cancer identified by microarray data analysis.

PURPOSE: This study aimed to identify the underlying mechanisms in pancreatic cancer (PC) carcinogenesis and those as potential prognostic biomarkers, which can also be served as new therapeutic targets of PC. METHODS: Differentially expressed genes (DEGs) were identified between PC tumor tissues and adjacent normal tissue samples from a public GSE62452 dataset, followed by functional and pathway enrichment analysis. Then, protein-protein interaction (PPI) network was constructed and prognosis-related genes were screened based on genes in the PPI network, before which prognostic gene-related miRNA regulatory network was constructed. Functions of the prognostic gene in the network were enriched before which Kaplan-Meier plots were calculated for significant genes. Moreover, we predicted related drug molecules based on target genes in the miRNA regulatory network. Furthermore, another independent GSE60979 dataset was downloaded to validate the potentially significant genes. RESULTS: In the GSE62452 dataset, 1017 significant DEGs were identified. Twenty-six important prognostic-related genes were found using multivariate Cox regression analysis. Through pathway enrichment analysis and miRNA regulatory analysis, we found that the 5 genes, such as Interleukin 22 Receptor Subunit Alpha 1 (IL22RA1), BCL2 Like 1 (BCL2L1), STAT1, MYC Proto-Oncogene (MYC), and Signal Transducer And Activator Of Transcription 2 (STAT2), involved in the Jak-STAT signaling pathway were significantly associated with prognosis. Moreover, the expression change of these 5 genes was further validated using another microarray dataset. Additionally, we identified camptothecin as an effective drug for PC. CONCLUSION: IL22RA1, BCL2L1, STAT1, MYC, and STAT2 involved in the Jak-STAT signaling pathway may be significantly associated with prognosis of PC.

Nitric oxide mediated inhibition of antigen presentation from DCs to CD4+ T cells in cancer and measurement of STAT1 nitration.

Myeloid derived suppressor cells (MDSC) produce nitric oxide (NO) and inhibit dendritic cell (DC) immune responses in cancer. DCs present cancer cell antigens to CD4+ T cells through Jak-STAT signal transduction. In this study, NO donors (SNAP and DETA-NONOate) inhibited DC antigen presentation. As expected, MDSC isolated from peripheral blood mononuclear cells (PBMC) from cancer patients produced high NO levels. We hypothesized that NO producing MDSC in tumor-bearing hosts would inhibit DC antigen presentation. Antigen presentation from DCs to CD4+ T cells (T cell receptor transgenic OT-II) was measured via a [3H]-thymidine incorporation proliferation assay. MDSC from melanoma tumor models decreased the levels of proliferation more than pancreatic cancer derived MDSC. T cell proliferation was restored when MDSC were treated with inhibitors of inducible nitric oxide synthase (L-NAME and NCX-4016). A NO donor inhibited OT II T cell receptor recognition of OT II specific tetramers, thus serving as a direct measure of NO inhibition of antigen presentation. Our group has previously demonstrated that STAT1 nitration also mediates MDSC inhibitory effects on immune cells. Therefore, a novel liquid chromatography-tandem mass spectrometry assay demonstrated that nitration of the STAT1-Tyr701 occurs in PBMC derived from both pancreatic cancer and melanoma patients.

Suppressor of cytokine signaling 1 expression during LPS-induced inflammation and bone loss in rats.

This study aimed to characterize the dynamics of suppressor of cytokine signaling (SOCS1) expression in a rat model of lipopolysaccharide-induced periodontitis. Wistar rats in the experimental groups were injected three times/week with LPS from Escherichia coli on the palatal aspect of the first molars, and control animals were injected with vehicle (phosphate-buffered saline). Animals were sacrificed 7, 15, and 30 days after the first injection to analyze inflammation (stereometric analysis), bone loss (macroscopic analysis), gene expression (qRT-PCR), and protein expression/activation (Western blotting). The severity of inflammation and bone loss associated with LPS-induced periodontitis increased from day 7 to day 15, and it was sustained through day 30. Significant (p < 0.05) increases in SOCS1, RANKL, OPG, and IFN-γ gene expression were observed in the experimental group versus the control group at day 15. SOCS1 protein expression and STAT1 and NF-κB activation were increased throughout the 30-day experimental period. Gingival tissues affected by experimental periodontitis express SOCS1, indicating that this protein may potentially downregulate signaling events involved in inflammatory reactions and bone loss and thus may play a relevant role in the development and progression of periodontal disease.

Recurrent lipoatrophic panniculitis of children.

BACKGROUND: Recurrent panniculitis in children with lipoatrophy has been loosely described and reported under different names, but has never been systematically evaluated by immunohistochemical stains. OBJECTIVE: To depict the profile of children with recurrent idiopathic panniculitis. METHODS: Study of clinical, histopathological and immunohistochemical features in five cases with recurrent idiopathic panniculitis. RESULTS: Five children with repeated attacks of painful subcutaneous nodules in association with fever, malaise and abdominal pain or arthralgia, with subsequent lipoatrophy were reviewed. In two patients, extensive involvement led to loss of the cutaneous fatty tissue. Laboratory abnormalities included increased acute phase reactants, leukocytosis with mild neutrophilia, microcytic anaemia and elevated liver enzymes. Histopathology showed lobar panniculitis without vasculitis and with a mixed infiltrate, composed of neutrophils, mononuclear cells, lymphocytes, macrophages and myeloid cells. Neutrophils and myeloid cells were more prominent in early lesions, whereas macrophages predominated in late stages, leading to lipophagia and lipoatrophy. Immunohistochemistry showed positive staining for myeloperoxidase around the necrotic adipocytes in early stages and CD68/PGM1 macrophages in late stages. Intense STAT1 staining was observed in the inflammatory infiltrate. All patients improved with methotrexate and corticosteroids. CONCLUSION: We present five cases of lobar panniculitis and lipoatrophy in childhood. The clinico-pathologic presentation shares features with other autoinflammatory diseases.

Suppressor of cytokine signaling 1 expression during LPS-induced inflammation and bone loss in rats

Abstract This study aimed to characterize the dynamics of suppressor of cytokine signaling (SOCS1) expression in a rat model of lipopolysaccharide-induced periodontitis. Wistar rats in the experimental groups were injected three times/week with LPS from Escherichia coli on the palatal aspect of the first molars, and control animals were injected with vehicle (phosphate-buffered saline). Animals were sacrificed 7, 15, and 30 days after the first injection to analyze inflammation (stereometric analysis), bone loss (macroscopic analysis), gene expression (qRT-PCR), and protein expression/activation (Western blotting). The severity of inflammation and bone loss associated with LPS-induced periodontitis increased from day 7 to day 15, and it was sustained through day 30. Significant (p < 0.05) increases in SOCS1, RANKL, OPG, and IFN-γ gene expression were observed in the experimental group versus the control group at day 15. SOCS1 protein expression and STAT1 and NF-κB activation were increased throughout the 30-day experimental period. Gingival tissues affected by experimental periodontitis express SOCS1, indicating that this protein may potentially downregulate signaling events involved in inflammatory reactions and bone loss and thus may play a relevant role in the development and progression of periodontal disease.

The combined role of biomarkers and interim PET scan in prediction of treatment outcome in classical Hodgkin's lymphoma: a retrospective, European, multicentre cohort study.

BACKGROUND: Early-interim fluorodeoxyglucose (FDG)-PET scan after two ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) chemotherapy courses (PET-2) represents the most effective predictor of treatment outcome in classical Hodgkin's lymphoma. We aimed to assess the predictive value of PET-2 combined with tissue biomarkers in neoplastic and microenvironmental cells for this disease. METHODS: We enrolled 208 patients with classical Hodgkin's lymphoma and treated with ABVD (training set), from Jan 1, 2002, to Dec 31, 2009, and validated the results in a fully matched independent cohort of 102 patients with classical Hodgkin's lymphoma (validation set), enrolled from Jan 1, 2008, to Dec 31, 2012. The inclusion criteria for both the training and validation sets were: the availability of a representative formalin-fixed, paraffin-embedded tissue sample collected at diagnosis; treatment with ABVD with or without radiotherapy; baseline staging and interim restaging after two ABVD courses with FDG-PET; no treatment change based solely on interim PET result; and HIV-negative status. We used Cox multivariate analysis classification and regression tree (CART) to compare the predictive values of these markers with that of PET-2 and to assess the biomarkers' ability to correctly classify patients whose outcome was incorrectly predicted by PET-2. FINDINGS: In multivariate analysis, PET-2 was the only factor able to predict both progression-free survival (hazard ratio [HR] 33·3 [95% CI 13·6-83·3]; p<0·0001) and overall survival (HR 31·3 [95% CI 3·7-58·9]; p=0·002). In the training set, no factor had a stronger adverse predictive value than a positive PET-2 scan and none was able to correctly reclassify PET-2 positive patients. In PET-2 negative patients, expression of CD68 (≥25%) and PD1 (diffuse or rosetting pattern) in microenvironmental cells, and STAT1 negativity in Hodgkin Reed Sternberg cells identified a subset of PET-2 negative patients with a 3 year progression-free survival significantly lower than that of the remaining PET-2 negative population (21 [64%] of 33 [95% CI 45·2-79·0] vs 130 [95%] of 137 [95% CI 89·4-97·7]; p<0·0001). These findings were reproduced in the validation set. INTERPRETATION: The CART algorithm correctly predicted the response to treatment in more than a half of patients who had a relapse or disease progression despite a negative PET-2 scan, thus increasing the negative predictive value of PET-2. In keeping with preliminary results from interim PET response adapted clinical trials of patients with advanced Hodgkin's lymphoma, there might be a non-negligible proportion of treatment failures in the interim PET negative group treated with standard ABVD. FUNDING: Italian Association for Cancer Research, Bologna Association against leukaemia, lymphoma and myeloma, and Bologna University.

Respiratory Syncytial Virus Persistence in Murine Macrophages Impairs IFN-ß Response but Not Synthesis.

Type-I interferon (IFN-I) production is an early response to viral infection and pathogenic viruses have evolved multiple strategies to evade this cellular defense. Some viruses can establish and maintain persistent infections by altering the IFN-I signaling pathway. Here, we studied IFN-I synthesis and response in an in vitro model of persistent infection by respiratory syncytial virus (RSV) in a murine macrophage-like cell line. In this model, interferon regulatory factor 3 was constitutively active and located at nuclei of persistently infected cells, inducing expression of IFN-beta mRNA and protein. However, persistently infected macrophages did not respond in an autocrine manner to the secreted-IFN-beta or to recombinant-IFN-beta, since phosphorylated-STAT1 was not detected by western blot and transcription of the interferon-stimulated genes (ISGs) Mx1 and ISG56 was not induced. Treatment of non-infected macrophages with supernatants from persistently infected cells induced STAT1 phosphorylation and ISGs expression, mediated by the IFN-I present in the supernatants, because blocking the IFN-I receptor inhibited STAT1 phosphorylation. Results suggest that the lack of autocrine response to IFN-I by the host cell may be one mechanism for maintenance of RSV persistence. Furthermore, STAT1 phosphorylation and ISGs expression induced in non-infected cells by supernatants from persistently infected macrophages suggest that RSV persistence may trigger a proinflammatory phenotype in non-infected cells as part of the pathogenesis of RSV infection.

IL-6, through p-STAT3 rather than p-STAT1, activates hepatocarcinogenesis and affects survival of hepatocellular carcinoma patients: a cohort study.

BACKGROUND: Biologic activities of functional mediators activate downstream transducers regulating inflammation and carcinogenesis. Correlation among mediators (IL-6, IL-27, TNF-α, and VEGF) with STAT proteins at diverse clinical-pathologic stages of hepatocellular carcinoma (HCC) remains limited. METHODS: Serum mediators assayed from 147 untreated HCC cases (HCC-total group) included 70 HBV-infected (HCC-HBV group), 64 HCV-infected (HCC-HCV group), and 13 without HBV-/HCV-infection (HCC-NBNC group). Another 156 non-HCC individuals comprised 54 healthy individuals (HG) and 102 chronic hepatitis patients (CH-total group) as control group. To correlate with serum mediators, 86-paired liver tissues (CH: 52 and HCC: 34 cases) served for p-STATs proteins immunostain. RESULTS: Although four mediators (IL-6, IL-27, TNF-α, and VEGF) significantly over-expressed, IL-6 presented the strongest correlation in HCC-total versus CH-total or HG groups (HCC-total versus CH-total: P < 0.001; HCC-total versus HG: P < 0.001). Over-expressed IL-6 concentration linked with poor liver function (Albumin: r = -0.383, P < 0.001; Bilirubin: r = 0.280, P = 0.001; INR: r = 0.299, P < 0.001; AST: 0.212, P = 0.016), tumor progression (TNM system: r = 0.370; P < 0.001), clinical condition severity (BCLC system: r = 0.471; P < 0.001; terminal- versus early-stage HCC, P = 0.001; advanced- versus early-stage HCC, P = 0.007; terminal- versus intermediate- stage HCC P = 0.003; advanced- versus intermediate-stage HCC P = 0.019), and 6-month mortality (P = 0.024). Likewise, serum IL-6 (r = 0.501, P = 0.003) as compared to IL-27 (r = 0.052, P = 0.770), TNF-α (r = 0.019, P = 0.917), and VEGF (r = 0.096, P = 0.595) expression reflected positive correlation with activation of tissues p-STAT3 rather than p-STAT1. CONCLUSIONS: Serum IL-6, through p-STAT3 rather than p-STAT1 signal pathway, affected hepatic function, tumor progression, and determine HCC patient survival.

Different associations of CD45 isoforms with STAT3, PKC and ERK regulate IL-6-induced proliferation in myeloma.

In response to interleukin 6 (IL-6) stimulation, both CD45RO and CD45RB, but not CD45RA, translocate to lipid rafts. However, the significance of this distinct translocation and the downstream signals in CD45 isoforms-participated IL-6 signal are not well understood. Using sucrose fractionation, we found that phosphorylated signal transducer and activator of transcription (STAT)3 and STAT1 were mainly localized in lipid rafts in response to IL-6 stimulation, despite both STAT3 and STAT1 localizing in raft and non-raft fractions in the presence or absence of IL-6. On the other hand, extracellular signal-regulated kinase (ERK), and phosphorylated ERK were localized in non-raft fractions regardless of the existence of IL-6. The rafts inhibitor significantly impeded the phosphorylation of STAT3 and STAT1 and nuclear translocation, but had little effect on (and only postponing) the phosphorylation of ERK. This data suggests that lipid raft-dependent STAT3 and STAT1 pathways are dominant pathways of IL-6 signal in myeloma cells. Interestingly, the phosphorylation level of STAT3 but not STAT1 in CD45+ cells was significantly higher compared to that of CD45- cells, while the phosphorylation level of ERK in CD45+ myeloma cells was relatively low. Furthermore, exogenously expressed CD45RO/RB significantly enhanced STAT3, protein kinase C (PKC) and downstream NF-κB activation; however, CD45RA/RB inhibited IL-6-induced ERK phosphorylation. CD45 also enhanced the nuclear localization of STAT3 but not that of STAT1. In response to IL-6 stimulation, CD45RO moved into raft compartments and formed a complex with STAT3 and PKC in raft fraction, while CD45RA remained outside of lipid rafts and formed a complex with ERK in non-raft fraction. This data suggests a different role of CD45 isoforms in IL-6-induced signaling, indicating that while CD45RA/RB seems inhibit the rafts-unrelated ERK pathway, CD45RO/RB may actually work to enhance the rafts-related STAT3 and PKC/NF-κB pathways.

Correlation of STAT1 with apoptosis and cell-cycle markers in esophageal squamous cell carcinoma.

We recently found evidence that STAT1 in esophageal squamous carcinoma (ESCC) cells exerts tumor suppressor function, and it regulates five key regulators of apoptosis or cell-cycle progression, including Bcl-2, Bcl-xL, survivin, cyclin D1 and p21. In this study, we confirmed these findings in four ESCC cell lines. Using immunohistochemistry, we also assessed the expression of these proteins in 62 primary tumors. The expression of these markers was heterogeneous, ranging 39 to 69% of the cohort. Significant correlation was found between STAT1 and three proteins (p21, Bcl-xL and survivin), whereas only a trend was identified for cyclin D1 and Bcl-2. We then correlated the expression of these proteins with several clinicopathologic parameters including lymph node metastasis, depth of invasion, clinical stage and overall survival. Significant correlations were found between Bcl-2 and deep invasion (p = 0.033), survivin and lymph node metastasis (p = 0.006), as well as cyclin D1 and clinical stage (p = 0.014). Patients with p21-positive tumors had a significantly longer survival compared to those with p21-negative tumors (p = 0.031). To conclude, our findings support the concept that STAT1 exerts its tumor suppressor effects in ESCC via modulating the expression of key regulators of apoptosis and cell-cycle progression.

STAT1 drives tumor progression in serous papillary endometrial cancer.

Recent studies of the interferon-induced transcription factor STAT1 have associated its dysregulation with poor prognosis in some cancers, but its mechanistic contributions are not well defined. In this study, we report that the STAT1 pathway is constitutively upregulated in type II endometrial cancers. STAT1 pathway alteration was especially prominent in serous papillary endometrial cancers (SPEC) that are refractive to therapy. Our results defined a "SPEC signature" as a molecular definition of its malignant features and poor prognosis. Specifically, we found that STAT1 regulated MYC as well as ICAM1, PD-L1, and SMAD7, as well as the capacity for proliferation, adhesion, migration, invasion, and in vivo tumorigenecity in cells with a high SPEC signature. Together, our results define STAT1 as a driver oncogene in SPEC that modulates disease progression. We propose that STAT1 functions as a prosurvival gene in SPEC, in a manner important to tumor progression, and that STAT1 may be a novel target for molecular therapy in this disease.

Differential expression of STAT1 and p21 proteins predicts pancreatic cancer progression and prognosis.

OBJECTIVE: The transcription factor Stat1 is a member of the family of signal transducers and transcription activators and responds to interferon-γ stimulation. p21 is a p53-responsive gene for cell cycle regulation and mediates Stat1 antitumor activity. The aim of this study was to analyze their expression for prediction of pancreatic cancer progression and prognosis. METHODS: A total of 100 pancreatic adenocarcinoma tissue specimens were used for construction of a pancreatic cancer tissue microarray for immunohistochemical staining of Stat1 and p21 expression. RESULTS: Stat1 and p21 proteins were expressed in 88% (88/100) and 82% (82/100) of pancreatic adenocarcinoma tissue specimens, and the expression was inversely associated with tumor differentiation, clinical stages, and lymph node metastasis of pancreatic cancer. There was no association with age, tumor size, or invasion. Moreover, the Kaplan-Meier curve analysis showed that patients with higher Stat1 and p21 expression had better overall survival rates than those with low expression of Stat1 and p21 proteins. CONCLUSIONS: The loss of expression of Stat1 and p21 proteins corresponded to lymph node metastasis, advanced stage, tumor dedifferentiation, and poor prognosis in patients with pancreatic cancer.

Innate immunity but not NLRP3 inflammasome activation correlates with severity of stable COPD.

BACKGROUND: In models of COPD, environmental stressors induce innate immune responses, inflammasome activation and inflammation. However, the interaction between these responses and their role in driving pulmonary inflammation in stable COPD is unknown. OBJECTIVES: To investigate the activation of innate immunity and inflammasome pathways in the bronchial mucosa and bronchoalveolar lavage (BAL) of patients with stable COPD of different severity and control healthy smokers and non-smokers. METHODS: Innate immune mediators (interleukin (IL)-6, IL-7, IL-10, IL-27, IL-37, thymic stromal lymphopoietin (TSLP), interferon γ and their receptors, STAT1 and pSTAT1) and inflammasome components (NLRP3, NALP7, caspase 1, IL-1ß and its receptors, IL-18, IL-33, ST2) were measured in the bronchial mucosa using immunohistochemistry. IL-6, soluble IL-6R, sgp130, IL-7, IL-27, HMGB1, IL-33, IL-37 and soluble ST2 were measured in BAL using ELISA. RESULTS: In bronchial biopsies IL-27+ and pSTAT1+ cells are increased in patients with severe COPD compared with control healthy smokers. IL-7+ cells are increased in patients with COPD and control smokers compared with control non-smokers. In severe stable COPD IL-7R+, IL-27R+ and TSLPR+ cells are increased in comparison with both control groups. The NALP3 inflammasome is not activated in patients with stable COPD compared with control subjects. The inflammasome inhibitory molecules NALP7 and IL-37 are increased in patients with COPD compared with control smokers. IL-6 levels are increased in BAL from patients with stable COPD compared with control smokers with normal lung function whereas IL-1ß and IL-18 were similar across all groups. CONCLUSIONS: Increased expression of IL-27, IL-37 and NALP7 in the bronchial mucosa may be involved in progression of stable COPD.

Fatal combined immunodeficiency associated with heterozygous mutation in STAT1.

BACKGROUND: Mutations in the gene for the signal transducer and activator of transcription 1, STAT1, have been shown to be associated with death at an early age due to overwhelming viral infection (complete STAT1 deficiency) or, more commonly, selective deficiencies to mycobacterial or fungal infection (typically heterozygous STAT1 mutations). OBJECTIVES: To define the molecular basis of progressive combined immunodeficiency in a group of patients with fatal infections. METHODS: We studied a group of unrelated patients who displayed an unusual progressive form of combined immunodeficiency. Whole exome sequencing assisted in confirming a common genetic defect in this group, which consisted of a heterozygous mutation of the STAT1 gene. STAT1 protein level as well as function was assessed, and a detailed evaluation of the immune system, including analysis of thymus tissue, was performed. RESULTS: Patients were found to carry de novo heterozygous mutations in STAT1 encoding T385A, I294T, or C284R amino acid substitutions. STAT1 expression appeared significantly decreased as a result of these changes but not completely absent, with diminished signaling responses. This group display progressive loss in lymphocyte number and function accompanied by increasing autoimmune features as well as severe, fatal infections. CONCLUSIONS: These findings show that some heterozygous aberrations of STAT1 can be associated with progressive combined immunodeficiency, quite distinct from the limited susceptibilities to infection previously reported for heterozygous STAT1 mutations. These mutations were not inherited, rather, arose de novo in each case. Accompanied by significant patient mortality, this finding suggests that this class of STAT1 mutation is ultimately fatal due to overwhelming infection.

Phosphorylated signal transducer and activator of transcription-1 immunohistochemical expression is associated with improved survival in patients with oral squamous cell carcinoma.

PURPOSE: To estimate whether the immunohistochemical (IHC) expression patterns of the tumor suppressor gene signal transducer and activator of transcription-1 (STAT1) and its active phosphorylated form (PSTAT1) serve as potential prognostic and predictive markers in patients with oral squamous cell carcinoma (OSCC). MATERIALS AND METHODS: STAT1 and PSTAT1 protein expressions were examined immunohistochemically in OSCC tumor tissues and adjacent normal mucosa from 49 patients who underwent primary surgery. The IHC scores were correlated with all available clinicopathologic parameters that were obtained from a maximum of 7 years of follow-up, including survival and response to adjuvant therapy treatment. RESULTS: There was a shift toward lower percentages of cells with STAT1 (P < .014) and PSTAT1 (P < .001) detected in OSCC tumors compared with adjacent normal tissue sites. No association with patients' clinicopathologic characteristics was shown. However, for the group of patients who received adjuvant chemotherapy, increased PSTAT1 intensity of staining in OSCC tumors was strongly associated with better overall survival (P = .008). CONCLUSIONS: This is the first study to concurrently evaluate STAT1 and PSTAT1 IHC expression patterns and their prognostic significance in patients with OSCC, highlighting the potential role of PSTAT1 as a biomarker in therapeutic decision making. Large prospective studies are needed to verify these findings.

Differential expression of STAT1 and IFN-γ in primary and invasive or metastatic wilms tumors.

BACKGROUND AND OBJECTIVES: IFN/STAT1 signaling has been found to be not only associated with an aggressive tumor phenotype but also activated and functional during metanephric development. This study was undertaken to evaluate STAT1 and IFN-γ expression and its relation to histopathological features of primary and invasive/metastatic Wilms tumors. METHODS: Immunohistochemistry was used to determine the expression and cellular distribution of STAT1 and IFN-γ in 18 pairs of primary and corresponding invasive/metastatic Wilms tumors and 40 primary tumors without invasion or metastasis. RESULTS: Positive rate of STAT1/IFN-γ expression was 66.7%/61.1% and 72.2%/77.8% in 18 pairs of primary and associated invasive/metastatic Wilms tumor tissues, while 35.0%/27.5% in 40 primary tumors without invasion or metastasis. The expression of STAT1 and IFN-γ was significantly associated with invasion/metastasis (P = 0.025; P = 0.015). There was a positive correlation between STAT1 and IFN-γ expression in all Wilms tumor tissues (χ(2) = 23.408, P = 0.05, r = 0.555). The expression of STAT1 and IFN-γ between primary and matched invasive/metastatic tissues was concordance, respectively (P = 0.710 and P = 0.375). CONCLUSIONS: These results suggest that IFN-γ/STAT1 signaling might have clinical potential as a promising predictor to identify individuals with poor prognostic potential and as a possible novel target molecule of therapy for Wilms tumor.

Construction of a novel oligonucleotide array-based transcription factor interaction assay platform and its uses for profiling STAT1 cofactors in mouse fibroblast cells.

Here, we describe a novel oligonucleotide array-based transcription factor (TF) interaction assay platform that can directly identify cointeracting TF complexes following binding to their regulatory DNA elements. This platform that combines immuno-coprecipitation technology with our previously reported oligonucleotide array-based TF assay (OATFA), is named targeted immuno-coprecipitation OATFA (TIC-OATFA). We illustrate use of the system to identify interaction partners of STAT1 (signal transducer and activator of transcription proteins 1) in mouse fibroblasts. Several previously known partners of STAT1, as well as new partners, were identified by TIC-OATFA, including the upstream stimulatory factors 1 and 2 (USF1, USF2), nuclear factor of activated T cells, TATA box-binding protein, nuclear factor erythroid-derived 2, nuclear factor-kappa B, and nuclear factor 1. Both USF1 and nuclear factor-kappa B are well known to interact with STAT1, but the other five TFs are previously unreported STAT1 interaction partners. We examined interactions between one new TF, USF2, and STAT1 in detail. USF2 belongs to the group of bHLH-zip transcription factors, which in a number of diseases including cancers, has enhanced activity. In summary, a novel oligonucleotide array-based assay platform was developed and used to study interactions between STAT1 and functional TF binding partners, revealing that USF2 and potentially four other new TFs are partners of STAT1 in an IFN-γ stimulated mouse fibroblast cell line.