QuantiGene Plex Research Articles

Analysis of the sensitivity to endocrine therapy (SET) assay in the PALLAS adjuvant trial of palbociclib in HR+/HER2- breast cancer (ABCSG-42/AFT-05/BIG-14-13).

538 Background: The phase 3 PALLAS trial compared 2 years of the CDK4/6 inhibitor palbociclib with endocrine therapy of provider choice, versus endocrine therapy alone, as adjuvant treatment for patients (pts) with Stage II-III hormone receptor-positive HER2-negative (HR+/HER2-) breast cancer. The SET2,3 index adjusts the measurement of SETER/PR index of endocrine receptor-related transcriptional activity with a baseline prognostic index (BPI) derived from tumor size (T), number of involved lymph nodes (N) and gene expression levels of ESR1, PGR, ERBB2 and AURKA (RNA4 subtype score). High SET2,3 (index ≥ 2.10) represents high endocrine sensitivity with favorable prognosis. Clinical outcome results from PALLAS were previously published, and the trial now has 5-year median follow-up. Methods: Participants in PALLAS provided a tumor tissue block prior to randomization. RNA was extracted from samples with sufficient tumor tissue and cellularity (>25mm2 with ≥20% cancer nuclei). SET2,3 index was measured from 300ng total RNA using the QuantiGene Plex platform (ThermoFisher, Waltham, MA) following the manufacturer’s protocol and using pre-defined quality control (QC) parameters and cut point. Results from primary tumor samples prior to systemic therapy were analyzed. Invasive disease-free survival (iDFS) was summarized with Kaplan-Meier plots using log-rank test. Cox models were adjusted for treatment arm assignment, with a treatment arm by SET index value interaction term to test for prediction of palbociclib benefit. The pre-defined level of significance was a two-sided 0.05. Results: There were 4075 pts from the total PALLAS population (N=5796) who provided a primary tumor sample that met pre-analytical requirements to perform the SET assay, from which 3388 (83%) results passed quality control, including 3093 from a sample obtained prior to receiving systemic therapy (1559 in palbociclib arm and 1534 in the control arm). SET2,3 index could not be calculated for 12 pts due to missing tumor or nodal information. There was no evidence of an interaction between treatment arm and SET indices (continuous) for iDFS: SET2,3 index interaction p=0.12 and SETER/PR index interaction p=0.92. Estimated 5-year iDFS was superior for those with high SET2,3: iDFS was 91.4% (95%CI 89.9–92.8) in 1719 (55.8%) pts with high SET2,3, versus 78.5% (95%CI 76.0–80.8) in 1362 (44.2%) pts with low SET2,3 (HR 0.38, 95%CI 0.31–0.47, p<0.0001). The SETER/PR index was also significantly associated with iDFS (HR 0.70 per 0.5 units, 95%CI 0.65–0.77, p<0.0001). Conclusions: There was no evidence that the SET2,3 or SETER/PR index of endocrine-related transcriptional activity predicts benefit from palbociclib treatment. SET2,3 index was strongly prognostic for pts with Stage II-III breast cancer, with excellent 5-year IDFS for pts classified as SET2,3 high. Clinical trial information: NCT02513394 .

Combination of predicted sensitivity to endocrine therapy (SET2,3 index) and the Recurrence Score in node-positive breast cancer: Independent validation in the PACS-01 trial.

565 Background: SET2,3 index measures hormone receptor-related transcriptional activity (SETER/PR) adjusted for a baseline prognosis index (BPI) derived from tumor size, nodes involved and a 4-gene molecular subtype (RNA4). SET2,3 added prognostic information to the 21-gene Breast Recurrence Score (RS) in the SWOG 8814 trial (1). To confirm this, we independently evaluated both tests performed on node-positive, hormone receptor-positive (HR+) cancers from the PACS-01 trial (Roche et al, JCO 2006, PMID: 17116941) that evaluated the addition of adjuvant docetaxel to anthracycline-based chemotherapy. Adjuvant endocrine therapy became standard for premenopausal patients during PACS-01, justifying a sensitivity analysis in the population who received adjuvant endocrine therapy. Methods: SET2,3 was measured with the QuantiGene Plex bead-based hybridization assay (ThermoFisher) using an aliquot of residual RNA from prior RS testing in 791 HR+ tumor samples. Pre-defined cut points defined higher risk if RS >25 or SET2,3 <2.10. SETER/PR index and BPI, individual components of SET2,3 index, were evaluated as continuous variables. The primary endpoint was distant recurrence-free interval (DRFI). Multivariable Cox proportional hazards models were used to calculate hazard ratios with 95% confidence interval (HR, 95%CI) adjusted for treatment arm, and the likelihood ratio (LR) of benefit from addition of SET2,3 to RS with LR test for significance at p <0.05. Results: Of 791 tumor samples, 760 (96.1%) passed quality control (QC) for the SET assay; 724 had pathologic information to calculate SET2,3 index; and 659 had results for RS and SET2,3. At a median follow-up of 8 years, distant recurrence occurred in 144 of 659 (21.9%) patients overall, and 94 of 490 (19.2%) patients in the sensitivity population. Overall, high RS status (HR 2.33, 1.60-3.39) and high SET2,3 (HR 0.43, 0.30-0.62) were independently prognostic. SET2,3 was high in 303/377 (80.4%) patients with RS ≤25, with 5-year DRFI of 93.7% [95%CI 90.2;95.9]; and SET2,3 was low in 171/282 (60.6%) patients with RS >25, with 5-year DRFI 64.6% [95%CI 56.9;71.3]. SETER/PR index of endocrine transcriptional activity and the BPI, each contributed additional prognostic information to RS status in the sensitivity population (Table). Conclusions: SET2,3 index added prognostic information to the Recurrence Score in this independent blinded validation study of the PACS-01 trial for node-positive breast cancer. 1. Speers et al, JCO 2023, PMID: 36649570. [Table: see text]

Pilot Study of the DRD3, GHRL, FTO, LEPR, INSIG2, GSTP1, and ABCB1 Gene Expression in Peripheral Blood Leukocytes in Schizophrenic Patients with Metabolic Syndrome

—Many individuals with schizophrenia also suffer from metabolic syndrome (MetS), which is a major risk factor for the development of cardiovascular disroders associated with a heavy burden of disease, as well as with premature death of patients. This study investigated the expression of 7 genes potentially important for the development of metabolic syndrome. QuantiGene Plex 2.0 technology was used to measure how 7 studied genes (DRD3, GHRL, FTO, LEPR, INSIG2, GSTP1, and ABCB1 (MDR1)) were expressed in leukocytes in 60 recently admitted patients with schizophrenia who had been on treatment with antipsychotic drugs. The preliminary results of our study show a change in the expression of the FTO gene in schizophrenic males with metabolic disorders, however, further studies are needed to determine the role of disturbances in the expression of this gene in the development of the metabolic syndrome in patients with schizophrenia.

MZB1-expressing cells are essential for local immunoglobulin production in chronic rhinosinusitis with nasal polyps

BackgroundThe expression of MZB1 genes is significantly elevated in patients who have chronic rhinosinusitis with nasal polyp (CRSwNP) disease compared with healthy controls. ObjectiveTo characterize MZB1-positive B cells in CRSwNP and to estimate the contribution of distinct subsets of B cells to the local overproduction of immunoglobulins. MethodsSingle-cell RNA-sequencing with Cellular Indexing of Transcriptomes and Epitopes by Sequencing technology, Switching Mechanism At the 5′ end of RNA Template sequencing, flow cytometry, immunohistochemistry and immunofluorescence staining, Western blot, QuantiGene Plex assay, B-cell ImmunoSpot assay, Luminex assay, and enzyme-linked immunosorbent assay were performed. ResultsSignificantly higher mRNA expression of MZB1 and HSP90B1 was found in type 2 CRSwNP compared with controls. In CRSwNP, MZB1 expression correlated with the local production of IgE. MZB1 could be colocalized with plasma and mature B cells, especially marginal zone (MZ) B cells. Single-cell transcriptome and epitope studies revealed prominent populations of B cells in type 2 CRSwNP with unexpectedly high MZB1 gene expression. The MZ B-cell population was significantly increased in CRSwNP compared with healthy controls in both peripheral blood mononuclear cells and nasal tissue single-cell suspensions. When those single cells were cultured overnight, the MZ B-cell numbers were positively correlated with local IgE production but negatively correlated with local IgM production. In vitro, MZB1 stimulation up-regulated the mRNA expression of IgE. ConclusionMZB1 was primarily expressed by plasma and mature B cells in nasal mucosa. MZB1 expression level was increased in CRSwNP compared with controls. MZB1 contributed to the local IgE production in type 2 CRSwNP.

Automated Classification of Breast Cancer Across the Spectrum of ERBB2 Expression Focusing on Heterogeneous Tumors With Low Human Epidermal Growth Factor Receptor 2 Expression

PURPOSE Although pharmaceutical companies conduct clinical trials of novel human epidermal growth factor receptor 2 (HER2)-low–directed drugs, diagnosing HER2-low cancer by immunohistochemistry (IHC) and in situ hybridization (ISH) remains challenging. This study investigates the performance of first-in-kind computerized intelligence to classify samples across gene expression levels and differentiate HER2-low tumors. MATERIALS AND METHODS We classified 251 samples: 142 primary invasive breast cancers (IBCs), 75 ductal carcinomas in situ (DCIS), and 34 mammaplasties (reference) using mRNA expression data from the QuantiGene Plex 2.0 assay. We used g3mclass probabilistic software to assess the number of classes in the assay data, the mean and the variance in each class, diagnostic cutoffs, and the prevalence of each class in the study population. RESULTS HER2-low (IHC score of 1+ or 2+/ISH–) accounted for 31% of IBC. First, we discovered that HER2-low tumors were represented by cases with normal ERBB2 transcript levels that were expected to produce physiologic levels of HER2 (70%) and cases with abnormally upregulated unamplified ERBB2 (30%). We termed the latter cancers ERBB2-up as they do not meet the standard definitions for ERBB2 overexpression and amplification. Second, HER2-low IBC classified as ERBB2-up had not only abnormally increased luminal growth and adhesion markers ( ERBB2, ESR1, PGR, IGF1R, VAV2, VAV3, KRT8, CDH1) but also downregulated myoepithelial marker ( KRT5). The vascularization ( RAP1 and C3G), immune cell infiltration ( VAV1), and mesenchymal transition ( CDH2) markers were dysregulated. Finally, in the independent cohort of DCIS, 40% of HER2-low DCIS shared similar traits with HER2-low IBC except for rare downregulation of KRT5 and no change in C3G, VAV1, and CDH2. CONCLUSION We demonstrated how innovative bioinformatic tools could help diagnose cancer across the spectrum of ERBB2 expression to aid decision making for HER2-low.

PCSK9 Inhibitors Reduce PCSK9 and Early Atherogenic Biomarkers in Stimulated Human Coronary Artery Endothelial Cells

Despite reports on the efficacy of proprotein convertase subtilisin-Kexin type 9 (PCSK9) inhibitors as a potent lipid-lowering agent in various large-scale clinical trials, the anti-atherogenic properties of PCSK9 inhibitors in reducing PCSK9 and atherogenesis biomarkers via the NF-ĸB and eNOS pathway has yet to be established. This study aimed to investigate the effects of PCSK9 inhibitors on PCSK9, targeted early atherogenesis biomarkers, and monocyte binding in stimulated human coronary artery endothelial cells (HCAEC). HCAEC were stimulated with lipopolysaccharides (LPS) and incubated with evolocumab and alirocumab. The protein and gene expression of PCSK9, interleukin-6 (IL-6), E-selectin, intercellular adhesion molecule 1 (ICAM-1), nuclear factor kappa B (NF-ĸB) p65, and endothelial nitric oxide synthase (eNOS) were measured using ELISA and QuantiGene plex, respectively. The binding of U937 monocytes to endothelial cell capacity was measured by the Rose Bengal method. The anti-atherogenic effects of evolocumab and alirocumab were contributed to by the downregulation of PCSK9, early atherogenesis biomarkers, and the significant inhibition of monocyte adhesion to the endothelial cells via the NF-ĸB and eNOS pathways. These suggest the beyond cholesterol-lowering beneficial effects of PCSK9 inhibitors in impeding atherogenesis during the initial phase of atherosclerotic plaque development, hence their potential role in preventing atherosclerosis-related complications.

Measurement of endocrine activity (SET2,3) related to prognosis and prediction of benefit from dose-dense (DD) chemotherapy in estrogen receptor-positive (ER+) cancer: CALGB 9741 (Alliance).

505 Background: We investigated the clinical utility of SET2,3, a novel biomarker designed to measure to endocrine sensitivity. SET2,3 measures nonproliferative hormone receptor-related transcription (SETER/PR) adjusted for a baseline prognosis index derived from tumor size, nodes involved and a 4-gene molecular subtype (RNA4). CALGB 9471 is a seminal phase III study that showed improved DFS and OS from 2-weekly dose-dense (DD) vs 3-weekly chemotherapy in ER-negative cancers. Risk of recurrence (ROR-PT) score (intrinsic subtype, proliferation score and tumor size) measured by Nanostring assay was reported to be prognostic in CALGB 9741, but did not predict benefit from DD chemotherapy. Methods: SET2,3 was performed using an aliquot of 200-300 ng RNA (residual from prior ROR-PT testing) from 682 ER+ tumor samples and tested using the QuantiGene Plex bead-based hybridization assay (ThermoFisher, Luminex). We report results for the primary and two secondary objectives of the NCI/CTEP-approved correlative science proposal CSC0154) to evaluate SET2,3 in CALGB 9741 for prognosis (primary endpoint: 95%CI for 5-year (yr) DFS > 75% for High SET2,3 using the predefined prognostic cutpoint 2.10), SET2,3 prognostic independence from ROR-PT, and prediction of outcome according to chemotherapy regimen. We used Cox models to estimate hazard ratios (HR) for prognosis and comparison with ROR-PT results (using c-indices) and for prediction according to chemotherapy schedule using an interaction term (prespecified significance level for interaction: p < 0.10). Results: The study met its primary endpoint with a 5-yr DFS of 85.6% (95%CI 81.3-90.2) in the High-SET subset (244/613, 40%). High-SET vs Low-SET was significantly associated with favorable outcomes at 5 yr (DFS 85.6% vs 69%, p <.0001; OS 95.3% vs 84.6%, p <.0001) and 10 yr (DFS 77.7% vs 58.2%, p <.0001; OS 86.9% vs 65.9%, p <.0001). PAM50 ROR-PT and SET classification were available for 596 tumors. In multivariate models for DFS and OS, SET2,3 remained an independent prognostic variable for DFS (SET high vs low HR = 0.46, 95% CI, 0.34 – 0.63, p < 0.0001; PAM50 ROR-PT high vs low HR = 1.22, 95% CI, 0.91 – 1.64, p = 0.18) and for OS (SET high vs low HR = 0.36, 95% CI, 0.25 – 0.53, p < 0.0001; PAM50 ROR-PT high vs low HR = 1.26, 95% CI, 0.91 – 1.75, p = 0.16). Similar observations were seen in models including SET and PAM50 ROR-PT as continuous variables. Lower SET2,3 values predicted improved outcomes from DD vs 3-weekly chemotherapy (interaction p=0.0998 for DFS, 0.042 for RFS and 0.027 for OS). This was unrelated to menopausal status and lower SET2,3 values favored DD concurrent treatments. Conclusions: SET2,3 index was strongly prognostic, independent of ROR-PT, and predicted survival benefit from DD chemotherapy in pre- and postmenopausal women with ER+ cancer. Clinical trial information: NCT00003088.

P234 The effect of calcitriol on the expression of interferon signature genes in dendritic cells and macrophages in systemic lupus erythematosus

Abstract Background/Aims A number of studies have demonstrated the inverse association between vitamin D and (i) disease activity and (ii) interferon (IFN) signature gene expression in systemic lupus erythematosus (SLE). The aim of the study was to establish the effect of calcitriol supplementation to cultured macrophages and dendritic cells (DC) from a SLE patient and control, on the expression of 12 IFN signature genes, interferon regulatory factor (IRF)7 and IRF8. Methods Blood samples were collected from a 41-year-old female SLE patient who fulfilled the SLICC classification criteria for SLE and from a 36-year-old healthy female control after obtaining informed consent. Both patients had a serum 25-hydroxyvitamin D level of 25ng/ml. Peripheral blood mononuclear cells (PBMC) were isolated by using histopaque®-1077. CD83 and CD14 expression was measured by flow cytometry and cell count and viability were assessed. The PBMC suspension was cultured in 6-well plates using: (i) ImmunoCultTM DC culture kit (following kit protocol) for DC differentiation, (ii) culture medium for macrophage differentiation containing RPMI 1640 Medium, DMEM, fetal bovine serum and LPS. Following 7 days of incubation, differentiation of cells was confirmed using microscopy and flow cytometry to measure CD83 and CD14 expression. Calcitriol at a concentration of 20nM was added to half of the wells of each sample category. Following 24 and 48 hours, RNA extraction was carried out on treated and untreated samples. QuantiGene Plex technology was used to measure the expression of 12 IFN signature genes, IRF7, IRF8 and four housekeeping genes in the extracted RNA. The normalised median florescence intensity for the 12 IFN signature genes was used to calculate the IFN score. Results A significantly lower IFN score was noted at 24 hours in SLE patient dendritic cell and macrophage cell cultures and in control macrophage cell cultures treated with calcitriol when compared with respective untreated samples (p = 0.014, p = 0.034, p = 0.031 respectively). On comparing IRF8 expression in treated and untreated cell cultures, a significantly lower IRF8 expression was noted in treated macrophage cell cultures obtained from both SLE patient and control (p = 0.034, p = 0.041). No difference in IRF7 expression was noted in treated and untreated cultures for all samples. Conclusion The suppression of the interferon signature genes in dendritic cell and macrophage cell cultures with vitamin D was confirmed in the in vitro experiment carried out. In this experiment a decrease in the expression of IRF8 (known to have a VDR binding site) was also observed in macrophage cultures from both SLE patient and control. Disclosure R. Magro: None. E. Seria: None. G. Grech: None. A.A. Borg: None. C. Scerri: None.

Ferret Lung Transplantation Models Differential Lymphoid Aggregate Morphology Between Restrictive and Obstructive Forms of Chronic Lung Allograft Dysfunction.

Long-term survival after lung transplantation remains limited by chronic lung allograft dysfunction (CLAD). CLAD has 2 histologic phenotypes, namely obliterative bronchiolitis (OB) and restrictive alveolar fibroelastosis (AFE), which have distinct clinical presentations, pathologies, and outcomes. Understanding of OB versus AFE pathogenesis would improve with better animal models. We utilized a ferret orthotopic single-lung transplantation model to characterize allograft fibrosis as a histologic measure of CLAD. Native lobes and "No CLAD" allografts lacking aberrant histology were used as controls. We used morphometric analysis to evaluate the size and abundance of B-cell aggregates and tertiary lymphoid organs (TLOs) and their cell composition. Quantitative RNA expression of 47 target genes was performed simultaneously using a custom QuantiGene Plex Assay. Ferret lung allografts develop the full spectrum of human CLAD histology including OB and AFE subtypes. While both OB and AFE allografts developed TLOs, TLO size and number were greater with AFE histology. More activated germinal center cells marked by B-cell lymphoma 6 Transcription Repressor, (B-cell lymphoma 6) expression and fewer cells expressing forkhead box P3 correlated with AFE, congruent with greater diffuse immunoglobulin, plasma cell abundance, and complement 4d staining. Furthermore, forkhead box P3 RNA induction was significant in OB allografts specifically. RNA expression changes were seen in native lobes of animals with AFE but not OB when compared with No CLAD native lobes. The orthotopic ferret single-lung transplant model provides unique opportunities to better understand factors that dispose allografts to OB versus AFE. This will help develop potential immunomodulatory therapies and antifibrotic approaches for lung transplant patients.

Replacement per- and polyfluoroalkyl substances (PFAS) are potent modulators of lipogenic and drug metabolizing gene expression signatures in primary human hepatocytes

Per- and polyfluoroalkyl substances (PFAS) are a class of environmental toxicants, and some, such as perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), have been associated with hepatic steatosis in rodents and monkeys. It was hypothesized that perfluorosulfonic acids (C4, 6, 8), perfluorocarboxylic acids (C4–14), perfluoro(2-methyl-3-oxahexanoic) acid (HFPO-DA), 1H, 1H, 2H, 2H-perfluorooctanesulfonic acid (6:2 FTS) along with 3 PFOS precursors could induce expression of lipid metabolism genes and lipid deposition in human hepatocytes. Five-donor pooled cryopreserved human hepatocytes were cultured and treated with 0.1% DMSO vehicle or various PFAS (0.25 to 25 μM) in media. After a 48-h treatment, mRNA transcripts related to lipid transport, metabolism, and synthesis were measured using a Quantigene Plex assay. After 72-h treatments, hepatocytes were stained with Nile Red dye to quantify intracellular lipids. Overall, PFAS were transcriptionally active at 25 μM. In this model, lipid accumulation was not observed with C8-C12 treatments. Shorter chain PFAS (C4-C5), 6:2 FTS, and PFOS precursor, metFOSA, induced significant liver lipid accumulation, and gene activation at lower concentrations than legacy PFAS. In summary short chain PFAS and other alternative PFAS were more potent gene inducers, and potential health effects of replacement PFAS should be critically evaluated in humans.

Vitamin D supplementation in systemic lupus erythematosus: relationship to disease activity, fatigue and the interferon signature gene expression

BackgroundIn addition to the well-known role of vitamin D in calcium homeostasis and bone metabolism, vitamin D is important in the modulation of the immune system and inflammatory processes. Vitamin D deficiency is common in patients with systemic lupus erythematosus (SLE), possibly as a result of sun avoidance. The aim of this prospective open-label study was to assess the effect of the treatment of vitamin D deficiency and insufficiency in SLE patients, particularly with regards to disease activity, fatigue and interferon signature gene expression.Methods31 SLE patients, 13 with vitamin D deficiency and 18 with vitamin D insufficiency were treated with vitamin D3. They were supplemented with vitamin D3 8000 IU daily for 8 weeks if they were vitamin D deficient, or 8000 IU daily for 4 weeks if they were insufficient. This was followed by 2000 IU daily maintenance. They were assessed at baseline, after 6 and 12 months by means of an interview, filling in questionnaires and blood tests. The expression of 12 interferon signature genes in RNA extracted from whole blood was measured by using QuantiGene Plex technology.ResultsAn improvement in disease activity measured by systemic lupus erythematosus disease activity index-2K (SLEDAI-2K; p = 0.028) and fatigue measured by fatigue severity scale (FSS; p = 0.071) at 12 months were noted. A significant decrease in anti-double stranded deoxyribonucleic acid (dsDNA) titre (p = 0.045) was also noted. The mean interferon signature gene expression score decreased from baseline to 6 months, however statistical significance was not achieved (p = 0.165).ConclusionsImproved disease activity and fatigue have been noted when Vitamin D has been supplemented in vitamin D deficient/insufficient SLE patients. One possible mechanism could be the suppression of the interferon signature gene expression.Trial registration: The study was registered with the ISRCTN registry on 12/04/2021 (Trial ID: ISRCTN59058825).

Intra- and Interlaboratory Reproducibility of the Sensitivity to Endocrine Therapy Assay for Stage II/III Breast Cancer.

The sensitivity to endocrine therapy assay (SET2,3) predicts treatment outcomes in Stage II-III breast cancer. SET2,3 measures transcription related to estrogen and progesterone receptors (SETER/PR index) and the molecular subtype (RNA4: ESR1, PGR, ERBB2, AURKA) from formalin-fixed paraffin-embedded (FFPE) tissue sections. We designed a nested study across 3 pathology laboratories, each testing 60 breast cancers twice in controlled batches. Laboratories macrodissected and directly homogenized the unstained FFPE tumor sections, then performed the QuantiGene Plex bead-based hybridization assay. SET2,3 was calculated centrally using predefined statistical R-scripts and applying pre-defined cutpoints. Concordance correlation coefficient (CCC) was calculated from continuous measurements and Kappa statistic from categorical results. A mixed-effects model estimated contributions to bias (fixed effects) and variance (random effects) from the replicated design. Intralaboratory (CCC 0.96-0.99) and interlaboratory (CCC 0.98-0.99) SET2,3 results were concordant, with rates of agreement for high/low categorization within (Kappa 0.83-0.93) and between laboratories (Kappa 0.87-0.88). The relative contributions to overall variance of SET2,3 measurements were 96.90% from biological differences between cancers, 0.67% from interlaboratory variability, and 2.44% from residual causes including intralaboratory replicates. Similar results were obtained with SETER/PR, the baseline prognostic index calculated using pathological or clinical tumor and nodal staging information, and the 4 individual genes (ESR1, PGR, ERBB2, and AURKA). Intra- and interpathology laboratory measurements of SET2,3 and its components were highly reproducible when tested from FFPE tumor sections.

Interferon-Induced Transmembrane Protein 3 Expression Upregulation Is Involved in Progression of Hepatocellular Carcinoma.

Purpose Interferon-induced transmembrane protein 3 (IFITM3) is a key signaling molecule regulating cell growth in some tumors, but its function and mechanism in hepatocellular carcinoma (HCC) remain unknown. Our study investigated the relationship between the expression of IFITM3 and HCC development. Material and Methods. IFITM3 expression was identified via multiple gene expression databases and investigated in HCC tissue samples. Then, PLC/PRF/5 cells were transfected with lentivirus to knock down and overexpress the expression of IFITM3. IFITM3 expression, cell proliferation, and migration were detected by qRT-PCR, western blotting, QuantiGene Plex 2.0 assay, immunohistochemistry, CCK-8, and wound healing tests. RNA-seq technology identified the PI3K/AKT/mTOR pathway as an IFITM3-related signaling pathway for investigation. Results IFITM3 expression was higher in HCC tissues than in adjacent normal tissues, and the level of IFITM3 was higher in HCC tissues with low differentiation and metastatic potential than in those with high/medium differentiation and without metastatic potential. A higher RNA level of IFITM3 was found in samples with IFITM3 rs12252-CC genotype rather than the TT genotype. Knockdown of IFITM3 in PLC/PRF/5 cells inhibited cell proliferation and migration, blocked the expression of the PI3K/AKT/mTOR signaling pathway, and decreased the expression of vimentin. The results were opposite with the overexpression of IFITM3. Conclusion Upregulation of IFITM3 plays a role in the development of HCC. Possibly through regulating HCC cell proliferation and migration, these effects are associated with the PI3K/AKT/mTOR signaling pathway. Upregulation of IFITM3 is also associated with the IFITM3 rs12252-CC genotype.

Asthma Promotes Choroidal Neovascularization via the Transforming Growth Factor Beta1/Smad Signalling Pathway in a Mouse Model

Introduction: The association between age-related macular degeneration and asthma is controversial. Transforming growth factor beta (TGF-β), which plays a critical role in asthma, has been extensively studied with regard to its function in choroidal neovascularization (CNV). In the present study, we aimed to investigate the role of TGF-β and the possible mechanism of CNV formation complicated with asthma and to explore the effect of a TGF-β inhibitor on CNV development in asthma mouse models. Methods: Laser-induced CNV and ovalbumin-induced asthma mouse models were divided into 5 groups: control group, acute asthma group, chronic asthma group, inhibitor-treated acute asthma group, and inhibitor-treated chronic asthma group. The gene expression patterns of angiogenic cytokines, vascular endothelial growth factor (VEGF) receptors and inflammasomes in the control group, acute asthma group, and chronic asthma group were detected using a QuantiGene Plex 6.0 Reagent System. Fundus fluorescein angiography and histology of CNV lesions stained with haematoxylin-eosin were performed to evaluate CNV formation. Quantitative real-time PCR and western blotting were used to assess TGF-β1, TGF-β2, and VEGF expression and Smad2/3, AKT, p38 MAPK, and ERK1/2 signal transduction and phosphorylation in retinal and choroidal tissues from each group. Results: In this study, we verified that laser treatment led to more CNV and vascular leakage in asthmatic mice than that in control mice. The changes were particularly notable in the chronic asthma group. The respective TGF-β1, VEGF, and phosphorylated Smad2/3 (p-Smad2/3) mRNA and protein levels in retinal and choroidal tissues were significantly upregulated in both the acute and chronic asthma groups. After injection of a TGF-β inhibitor, a distinct decline in VEGF, TGF-β1, and p-Smad2/3 protein and mRNA levels was observed, and the mean CNV area also decreased. Conclusion: We provide new evidence that asthma could be a risk factor for CNV development via the TGF-β1/Smad signalling pathway. A TGF-β inhibitor can be applied as a useful, adjunctive therapeutic strategy for preventing CNV formation in asthmatic patients.

CDH2/N-cadherin and early diagnosis of invasion in patients with ductal carcinoma in situ.

This proof-of-concept study investigates gene expression in core needle biopsies (CNB) to predict whether individuals diagnosed with ductal carcinoma in situ (DCIS) on CNB were affected by invasion at the time of diagnosis. Using a QuantiGene Plex 2.0 assay, 14 gene expression profiling was performed in 303 breast tissue samples. Preoperative diagnostic performance of a gene was measured by area under receiver-operating characteristic curve (AUC) with 95% confidence interval (CI). The gene mRNA positivity cutoff was computed using Gaussian mixture model (GMM); protein expression was measured by immunohistochemistry; DNA methylation was evaluated by targeted bisulfite sequencing. mRNA from 69% (34/49) mammoplasties, 72% (75/104) CNB DCIS, and 89% (133/150) invasive breast cancers (IBC) were analyzed. Based on pre-and post-surgery DCIS chart reviews, 21 cases were categorized as DCIS synchronous with invasion and 54 DCIS were pure DCIS without pathologic evidence of invasive disease. The ectopic expression of neuronal cadherin CDH2 was probable in 0% mammoplasties, 6% pure DCIS, 29% synchronous DCIS, and 26% IBC. The CDH2 mRNA positivity in preoperative biopsies showing pure DCIS was predictive of a final diagnosis of invasion (AUC = 0.67; 95% CI 0.53-0.80; P = 0.029). Site-specific methylation of the CDH2 promoter (AUC = 0.76; 95% CI 0.54-0.97; P = 0.04) and measurements of N-cadherin, a pro-invasive cell-cell adhesion receptor encoded by CDH2 (AUC = 0.8; 95% CI 0.66-0.99; P < 0.005) had a discriminating power allowing for discernment of CDH2-positive biopsy. Evidence of CDH2/N-cadherin expression, predictive of invasion synchronous with DCIS, may help to clarify a diagnosis and direct the course of therapy earlier in a patient's care.

Transcriptomic analysis of hidradenitis suppurativa skin suggests roles for multiple inflammatory pathways in disease pathogenesis.

Hidradenitis suppurativa (HS) is a chronic inflammatory disease with limited treatment options; therefore, the current study investigated the downstream signaling pathways that are differentially expressed in HS subjects and may drive disease pathogenesis. The expression of 144 genes was evaluated in the skin of 16 healthy subjects and 34 subjects with mild to severe HS using QuantiGene Plex assay. One hundred and twenty-nine genes were significantly elevated in lesional HS skin as compared to the skin of healthy controls including pro-inflammatory cytokines (IL-1α, IL-6, TNF-α), IL-17-associated cytokines (IL-17A, IL-17F, IL-23A), the IL-10 family of cytokines (IL-10, IL-19, IL-20, IL-22, IL-24), and IFN family members (IFNA1, IFNB1, IFNG, IL-12B). This corresponded with increased expression of tyrosine kinases (JAK1, JAK3, BTK, SYK) and their downstream signaling partners (STAT1, STAT2, STAT3, STAT5A, STAT5B, STAT6). These data illustrate the diverse immune activation in lesional HS skin and suggest that deeper interrogation of the disease heterogeneity may reveal unique opportunities for targeted therapies in designated subpopulations.

Technical Validity of a Customized Assay of Sensitivity to Endocrine Therapy Using Sections from Fixed Breast Cancer Tissue.

We translated a multigene expression index to predict sensitivity to endocrine therapy for Stage II-III breast cancer (SET2,3) to hybridization-based expression assays of formalin-fixed paraffin-embedded (FFPE) tissue sections. Here we report the technical validity with FFPE samples, including preanalytical and analytical performance. We calibrated SET2,3 from microarrays (Affymetrix U133A) of frozen samples to hybridization-based assays of FFPE tissue, using bead-based QuantiGene Plex (QGP) and slide-based NanoString (NS). The following preanalytical and analytical conditions were tested in controlled studies: replicates within and between frozen and fixed samples, age of paraffin blocks, homogenization of fixed sections versus extracted RNA, core biopsy versus surgically resected tumor, technical replicates, precision over 20 weeks, limiting dilution, linear range, and analytical sensitivity. Lin's concordance correlation coefficient (CCC) was used to measure concordance between measurements. SET2,3 index was calibrated to use with QGP (CCC 0.94) and NS (CCC 0.93) technical platforms, and was validated in two cohorts of older fixed samples using QGP (CCC 0.72, 0.85) and NS (CCC 0.78, 0.78). QGP assay was concordant using direct homogenization of fixed sections versus purified RNA (CCC 0.97) and between core and surgical sample types (CCC 0.90), with 100% accuracy in technical replicates, 1-9% coefficient of variation over 20 weekly tests, linear range 3.0-11.5 (log2 counts), and analytical sensitivity ≥2.0 (log2 counts). Measurement of the novel SET2,3 assay was technically valid from fixed tumor sections of biopsy or resection samples using simple, inexpensive, hybridization methods, without the need for RNA purification.

Effects of prexasertib, a CHK1 inhibitor, in the immune microenvironment of head and neck squamous cell carcinoma (HNSCC).

e18541 Background: Despite considerable advancements in the standard therapies including surgery, chemotherapy and radiotherapy, the clinical outcome for head and neck squamous cell carcinoma (HNSCC) remains poor due to tumor recurrence and metastasis. Molecular-targeted therapies have evolved as novel and promising treatment for HNSCC patients. Herein, we identified a CHK1 inhibitor, prexasertib, as a therapeutic target that enhanced the infiltration of innate and adaptive immune markers in the mice tumor immune microenvironment (TIME) and subsequently sensitizes the tumors. Methods: For this study we used syngeneic mouse model of HNSCC developed using mouse tonsil epithelial cells transformed with HRAS expression and PTPN13 knockdown to represent tobacco-induced HNSCC (MTE-Ras). In vitro drug activity of prexasertib was determined using immunoblotting, flow cytometry, cell proliferation and colony formation assays. The effect of prexasertib on TIME was quantified by Quantigene Plex panel and multiplex immunohistochemistry (mIHC). Results: We found that in vitro treatment with prexasertib increased the amount of DNA damage in the cancer cells and eventually lead to their death. Similarly, our in vivo data showed that treatment with prexasertib resulted in significant tumor regression and increased mice survival. At the molecular level, prexasertib treatment resulted in an upregulation of transcripts associated with T-cell activation, cytokines, chemokines and macrophages indicating an upregulation of inflammatory gene signature. This was further supported by our findings from mIHC staining of mice tumors showing increased infiltration of natural killer cells, natural killer T cells and dendritic cells, following prexasertib treatment. Interestingly, we also saw an increase in the Tregs after prexasertib treatment, which is indicative of an immunosuppressive environment, and we speculate this could be as a result of negative feedback following immune activation by prexasertib treatment. Conclusions: Our results uncover a previously unidentified role of prexasertib in regulating the innate and adaptive immune response in HNSCC, and therefore further corroborate CHK1 as a promising therapeutic target in HNSCC.

EP36 Vitamin D and interferon signature gene expression in systemic lupus erythematosus: a cross-sectional cohort study

Abstract Background Vitamin D deficiency is more prevalent in patients with systemic lupus eythematosus (SLE) as a result of sun avoidance. The potential negative impact of vitamin D deficiency on the disease activity of SLE has been shown in a number of studies. The expression of the interferon signature genes in SLE correlates positively with disease activity, and these genes are thought to mediate the clinical manifestations of the disease. The aim of this study was to establish whether a relationship exists between serum 25-hydroxyvitamin D level and the interferon signature gene expression in whole blood of SLE patients. Methods Informed consent was obtained from 92 SLE patients who were over the age of 18 and who fulfilled the SLICC classification criteria for SLE. The patients were interviewed and blood tests including full blood count, renal profile, calcium, 25-hydroxyvitamin D, complement 3 (C3), complement 4 (C4) and anti-double standed DNA (anti-dsDNA) titre were carried out. SLE disease activity was measured by SLE disease activity index-2K (SLEDAI-2K). RNA extraction was performed from whole blood. QuantiGene Plex technology was used to measure the expression of 12 interferon signature genes in the extracted RNA. The study was approved by the University Research Ethics Committee. Results 92.4% of the cohort studied were female. 58.7% were receiving vitamin D3 supplementation at a mean dose of 1031IU daily. 27.2% had vitamin D insufficiency (25-hydroxyvitamin D 21-29ng/ml) and 15.2% were vitamin D deficient (25-hydroxyvitamin D &amp;lt; 20ng/ml). Mean serum 25-hydroxyvitamin D was 30.75ng/ml (standard deviation 9.53 ng/ml). Median SLEDAI-2K was 4 (range 0-12). Serum 25-hydroxyvitamin D had a significant negative correlation with body mass index (BMI) (R=-0.258, p = 0.006) but there was no significant negative correlation with SLEDAI-2K or with the expression of the interferon signature genes. The expression of most interferon signatures genes measured (IFI35, OAS1, MX1, IFITM1, STAT2, IFIT3, IFIT1, STAT1, SOCS1) had a significant positive correlation with SLEDAI-2K. Conclusion This study did not show a significant relationship between serum vitamin D level and disease activity. In keeping with this, there was no significant negative correlation between serum 25-hydroxyvitamin D and interferon signature gene expression. Further prospective studies and randomised controlled trials are required to study this relationship in greater depth. Disclosures R. Magro None. C. Saliba None. L. Camilleri None. C. Scerri None. A.A. Borg None.

Hypoxia and Hypercapnia Diminish Intestinal Barrier Integrity in a Human Enteroid Model System

Warfighters are commonly exposed to unique physiologic stressors that affect their physical and mental performance, as well as their overall health. The adverse impact of these stressors may be in part due to effects on the microorganisms residing in the human gut, collectively known as the gut microbiome. Numerous studies have revealed the gut microbiome can influence multiple physiological process, including immunity, nutrient metabolism, and cognition. Hypoxia refers to a lack of oxygen in a given system and exists as a gradient in intestinal tissue, however, the intestine is sensitive to changes in oxygenation and can alter normal cellular process, such as inflammation and barrier function. Similarly, an excess of carbon dioxide (CO2), or hypercapnia, also affects intestinal tissue, as levels of CO2 are inversely coupled with the level of oxygen and are found to be elevated in hypoxic tissue. The GI tract is thought to have a low‐grade, albeit constant, level of inflammation and is home to multiple immune cell types. In addition, both epithelial and immune cells in the intestine can sense and respond to various stimuli including pathogens, and compromises to intestinal barrier integrity due to injury or stress can result. In this study, we sought to determine the extent to which hypoxia and hypercapnia degrade intestinal cellular integrity through inflammation‐mediated mechanisms. Intestinal epithelial organoids (enteroids) derived from the duodenum were exposed to hypoxic (0.5% O2) or hypercapnic (10%, 20% CO2) conditions for a period of 72 hours. Initial results indicated intestinal barrier integrity was compromised in hypoxia and 20% CO2 conditions as evidenced by transepithelial electrical resistance (TEER), with hypoxia (0.7 Ω) and 20% CO2 (0.85 Ω) being significantly lower than control (1.19 Ω; P &lt; 0.05). The Quantigene Plex array system was used to interrogate a panel of intestinal and inflammatory markers. Preliminary results suggest alterations in tight‐junction markers as well as in genes involved in inflammatory response pathways. In addition, IL‐8, a macrophage‐mediated chemokine produced in intestinal epithelial cells and involved in the innate immune response, was found to be significantly downregulated in hypoxia (58.7 pg/mL) and hypercapnia (42.5 pg in 10% CO2, 49.7 pg/mL in 20% CO2) compared to control (190.7 pg/mL; P &lt; 0.05). In sum, our data indicate hypoxic and hypercapnic conditions lead to alterations in molecules involved in the innate immune response pathway, tight‐junction markers, as well as compromised epithelial barrier integrity. Future experiments will be aimed at identifying the mechanism(s) responsible for diminished intestinal barrier function as well as identify potential preventative strategies.Support or Funding InformationFunding support provided by the Office of Naval Research award #N0001419MP00144 to SP FY18‐FY20.