Safety and immunogenicity of three doses of non-typeable Haemophilus influenzae-Moraxella catarrhalis (NTHi-Mcat) vaccine when administered according to two different schedules: a phase 2, randomised, observer-blind study.

BACKGROUND: Non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat) infections are frequently associated with exacerbations of chronic obstructive pulmonary disease (COPD). Results were reported with a two-dose (0-2 months) schedule of an investigational AS01E-adjuvanted NTHi-Mcat vaccine containing three surface proteins from NTHi and one from Mcat. We evaluated the safety and immunogenicity of three NTHi-Mcat vaccine doses administered in two different schedules to adults with a smoking history (≥ 10 pack-years), immunologically representing the COPD population. METHODS: In this 18-month, randomised (1:1), observer-blind study with 6-month open follow-up, 200 healthy adults aged 40-80 years received NTHi-Mcat vaccine at 0-2-6 months and placebo at 12 months (0-2-6 group), or vaccine at 0-2-12 months and placebo at 6 months (0-2-12 group). Solicited and unsolicited adverse events (AEs) were recorded for 7 and 30 days, respectively, post-vaccination, and potential immune-mediated diseases (pIMDs) and serious AEs (SAEs) throughout the study. Immune responses were assessed. RESULTS: No safety concerns were identified with the third vaccine dose or overall. Most solicited AEs were mild/moderate. Unsolicited AEs were reported in 16%, 16.1% and 14.4% of participants in the 0-2-6 group post-dose 1, 2 and 3, respectively, and 20%, 20.4% and 9.7%, respectively, in the 0-2-12 group. In 24 months, SAEs were reported in 12 participants in the 0-2-6 group and 9 in the 0-2-12 group (18 events in each group). There were three deaths (unknown cause, 0-2-6 group; myocardial infarction, lung cancer in 0-2-12 group). pIMDs were reported in three participants in the 0-2-6 group (non-serious inflammatory bowel disease, gout, psoriasis) and three in the 0-2-12 group (serious ulcerative colitis, two with non-serious gout). The SAEs, deaths and pIMDs were considered not causally related to vaccination. Antigen-specific antibody concentrations were higher at 12 months post-dose 1 with the 0-2-6 schedule than with the 0-2-12 schedule and at 12 months post-dose 3 were similar between schedules, remaining higher than baseline. CONCLUSIONS: No safety concerns were identified when the investigational NTHi-Mcat vaccine was administered via a 0-2-6 months or 0-2-12 months schedule to older adults with a smoking history. Persistent immune responses were observed after the third vaccine dose. Trial registration https://clinicaltrials.gov/ ; NCT03443427, registered February 23, 2018.

Anterior gradient protein 2 promotes survival, migration and invasion of papillary thyroid carcinoma cells.

BACKGROUND: Through a transcriptome microarray analysis, we have isolated Anterior gradient protein 2 (AGR2) as a gene up-regulated in papillary thyroid carcinoma (PTC). AGR2 is a disulfide isomerase over-expressed in several human carcinomas and recently linked to endoplasmic reticulum (ER) stress. Here, we analyzed the expression of AGR2 in PTC and its functional role. METHODS: Expression of AGR2 was studied by immunohistochemistry and real time PCR in normal thyroids and in PTC samples. The function of AGR2 was studied by knockdown in PTC cells and by ectopic expression in non-transformed thyroid cells. The role of AGR2 in the ER stress was analyzed upon treatment of cells, expressing or not AGR2, with Bortezomib and analyzing by Western blot the expression levels of GADD153. RESULTS: PTC over-expressed AGR2 at mRNA and protein levels. Knockdown of AGR2 in PTC cells induced apoptosis and decreased migration and invasion. Ectopic expression of AGR2 in non-transformed human thyroid cells increased migration and invasion and protected cells from ER stress induced by Bortezomib. CONCLUSIONS: AGR2 is a novel marker of PTC and plays a role in thyroid cancer cell survival, migration, invasion and protection from ER stress.

Imaging of thyroid tumor angiogenesis with microbubbles targeted to vascular endothelial growth factor receptor type 2 in mice.

BACKGROUND: To evaluate whether Contrast Enhanced Ultrasund (CEUS) with microbubbles (MBs) targeted to VEGFR-2 is able to characterize in vivo the VEGFR-2 expression in the tumor vasculature of a mouse model of thyroid cancer (Tg-TRK-T1). METHODS: Animal protocol was approved by Institutional committee on Laboratory Animal Care. Contrast-enhanced ultrasound imaging with MBs targeted with an anti-VEGFR-2 monoclonal antibody (UCAVEGFR-2) and isotype control antibody (UCAIgG) was performed in 7 mice with thyroid carcinoma, 5 mice with hyperplasia or benign thyroid nodules and 4 mice with normal thyroid. After ultrasonography, the tumor samples were harvested for histological examination and VEGFR-2 expression was tested by immunohistochemistry. Data were reported as median and range. Paired non parametric Wilcoxon's test and ANOVA of Kruskal-Wallis were used. The correlation between the contrast signal and the VEGFR-2 expression was assessed by the Spearman coefficient. RESULTS: The Video intensity difference (VID) caused by backscatter of the retained UCAVEGFR-2 was significantly higher in mice harboring thyroid tumors compared to mice with normal thyroids (P < 0.01) and to mice harboring benign nodules (P < 0.01). No statistically significant differences of VID were observed in the group of mice carrying benign nodules compared to mice with normal thyroids. Moreover in thyroid tumors VID of retained VEGFR-2-targeted UCA was significantly higher than that of control UCAIgG (P <0.05). Results of immunohistochemical analysis confirmed VEGFR-2 overexpression. The magnitude of the molecular ultrasonographic signal from a VEGFR-2-targeted UCA retained by tissue correlates with VEGFR-2 expression determined by immunohistochemistry (rho 0.793, P=0.0003). CONCLUSIONS: We demonstrated that CEUS with UCAVEGFR-2 might be used for in vivo non invasive detection and quantification of VEGFR-2 expression in thyroid cancer in mice, and to differentiate benign from malignant thyroid nodules.

Non-typeable Haemophilus influenzae-Moraxella catarrhalis vaccine for the prevention of exacerbations in chronic obstructive pulmonary disease: a multicentre, randomised, placebo-controlled, observer-blinded, proof-of-concept, phase 2b trial.

BACKGROUND: Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are associated with changes in the sputum microbiome, including an increased prevalence of pathogenic bacteria. Vaccination against the most frequent bacteria identified in AECOPD might reduce exacerbation frequency. We assessed the efficacy, safety, and immunogenicity of a candidate vaccine containing surface proteins from non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat) in patients with COPD. METHODS: This multicentre, randomised, observer-blinded, placebo-controlled, proof-of-concept, phase 2b trial recruited patients with stable COPD, moderate-to-very severe airflow limitation (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 2, 3, or 4), at 67 clinical sites in Belgium, Canada, France, Germany, Italy, Spain, UK, and USA. Eligible patients were aged 40-80 years and had a history of at least one moderate or severe exacerbation in the previous year. Patients were allocated (1:1) using a minimisation algorithm to receive two intramuscular injections of NTHi-Mcat vaccine or placebo 60 days apart, in addition to standard care. The allocation algorithm considered age category, number of previous exacerbations, COPD severity at study entry, and country as minimisation factors, to guarantee treatment balance within each factor. Vaccine recipients and those responsible for evaluating study endpoints were masked to group allocation. In the analysis of efficacy, the primary outcome was the rate of any moderate or severe AECOPD occurring within a 1-year period, starting 1 month after the second dose in patients who received two vaccine doses (modified total vaccinated cohort). Safety was assessed in the total vaccinated cohort. The trial is registered with ClinicalTrials.gov, number NCT03281876, and is complete. FINDINGS: Between Nov 27, 2017, and Nov 30, 2018, 606 adults were enrolled and included in the total vaccinated cohort (304 in the NTHi-Mcat vaccine group, 302 in the placebo group); 571 received two doses and were included in the primary efficacy analysis (279 in the NTHi-Mcat vaccine group, 292 in the placebo group). 23 participants dropped-out in the NTHi-Mcat vaccine group and 39 in the placebo group; this included 4 patients in the NTHi-Mcat vaccine group and 15 in the placebo group who withdrew from the study because of an adverse event. The primary analysis included 340 exacerbations (in follow-up time 102 123 days) in the NTHi-Mcat vaccine group and 333 (in 104 443 days) in the placebo group, with a yearly rate of moderate or severe AECOPD of 1·22 in the NTHi-Mcat vaccine group and 1·17 in the placebo group, with vaccine efficacy in reducing the yearly rate of moderate or severe AECOPD estimated to be zero (vaccine efficacy point estimate 2·26% [87% CI -18·27 to 11·58]; p=0·82). Solicited local adverse events were more frequent in the NTHi-Mcat vaccine group (216 [72%] of 301 patients) than with placebo (34 [11%] of 299 patients), and the frequency of solicited general adverse events was similar between groups (239 [79%] of 301 vs 235 [79%] of 299 patients). There was one death in the NTHi-Mcat vaccine group (acute respiratory failure, not related to vaccination) and ten in the placebo group (seven due in part to COPD or respiratory failure). There were 158 serious adverse events (89 [29%] of 304 patients) in the NTHi-Mcat vaccine group, not related to vaccination, and 214 (99 [33%] of 302 patients) in the placebo group. INTERPRETATION: NTHi-Mcat vaccine administered to patients with COPD did not show efficacy in reducing the yearly rate of moderate or severe exacerbations. No safety concerns were identified. FUNDING: GlaxoSmithKline Biologicals SA.

Long-term immunogenicity and safety of a non-typeable Haemophilus influenzae-Moraxella catarrhalis vaccine: 4-year follow-up of a phase 1 multicentre trial.

A multicomponent vaccine has been developed to reduce the frequency of acute exacerbations of COPD associated with non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat) infections, containing NTHi (PD and PE-PilA) and Mcat (UspA2) surface proteins. In a randomised, observer-blind, placebo-controlled study with two steps (NCT02547974), the investigational vaccine had good immunogenicity and no safety concerns were identified. In step 2, 90 adults aged 50-71 years with smoking history received two doses 60 days apart of one of two AS01E-adjuvanted formulations containing 10 µg of each antigen (10-10-AS01) or 10 µg NTHi antigens and 3.3 µg UspA2 (10-3-AS01), or placebo. Long-term persistence of antigen-specific humoral antibodies was assessed in 81 participants during 3 years of follow-up after the initial 14-month study (NCT03201211). Antigen-specific antibody concentrations were measured in blood samples taken every 6 months. Safety monitoring evaluated serious adverse events (SAEs) and potential immune-mediated disease (pIMD). Immune responses against NTHi antigens persisted up to 4 years post-vaccination. For PD, PE and PilA, at each follow-up time point, adjusted antibody geometric mean concentrations (GMCs) were higher (non-overlapping 95% confidence intervals [CIs]) in the vaccine groups versus placebo and versus pre-vaccination. Antibody GMC point estimates were higher with 10-3-AS01 than with 10-10-AS01. For UspA2, 95% CIs included 1 for GMC ratios of 10-10-AS01 or 10-3-AS01 to placebo at each time point. During follow-up, SAEs were reported in nine (11.1%) participants, one of which was fatal (lung cancer, 607 days after second 10-10-AS01 dose). One non-serious pIMD, trigeminal neuralgia, was reported 771 days after second 10-3-AS01 dose. The SAEs and pIMD were considered not related to vaccination. Immune responses against NTHi antigens persisted for 4 years after two-dose vaccination with the investigational NTHi-Mcat vaccine. There was no persistent response against the Mcat antigen. No safety concerns were identified during the long-term follow-up.

TWIST1/miR-584/TUSC2 pathway induces resistance to apoptosis in thyroid cancer cells.

TWIST1, a transcription factor, plays a pivotal role in cancer initiation and progression. Anaplastic thyroid carcinoma (ATC) is one of the deadliest human malignancies; TWIST1 is overexpressed in ATC and increases thyroid cancer cell survival, migration and invasion. The molecular mechanisms underlying the effects of TWIST1 are partially known. Here, using miRNome profiling of papillary thyroid cancer cells (TPC-1) ectopically expressing TWIST1, we identified miR-584. We showed that TWIST1 directly binds miR-584 using chromatin immunoprecipitation. Importantly, miR-584 was up-regulated in human ATC compared to papillary thyroid carcinoma (PTC) and normal thyroid samples. Overexpression of miR-584 in TPC cells induced resistance to apoptosis, whereas stable transfection of anti-miR-584 in TPC-TWIST1 and 8505C cells increased the sensitivity to apoptosis. Using bioinformatics programs, we identified TUSC2 (tumor suppressor candidate 2) as a novel target of miR-584. TUSC2 mRNA and protein levels were decreased in TPC miR-584 and increased in TPC-TWIST1 anti-miR-584 cells. Luciferase assays demonstrated direct targeting. Restored expression of TUSC2 rescued the inhibition of apoptosis induced by miR-584. Finally, qRT-PCR and immunohistochemical analysis showed that TUSC2 was down-regulated in ATC and PTC samples compared to normal thyroids. In conclusion, our study identified a novel TWIST1/miR-584/TUSC2 pathway that plays a role in resistance to apoptosis of thyroid cancer cells.

High-Frequency Ultrasound-Guided Injection for the Generation of a Novel Orthotopic Mouse Model of Human Thyroid Carcinoma.

BACKGROUND: Thyroid carcinoma is the most common endocrine malignancy and has an increasing incidence. High-frequency ultrasound (HFUS) has a spatial resolution of 30 µm, which is a property that has been exploited for thyroid visualization and analysis in mice. The aim of this study was to generate a novel orthotopic mouse model of human follicular thyroid carcinoma (FTC) using an HFUS-guided injection system. METHODS: Twenty Balb/C nude mice were injected in the right lobe of the thyroid with 2 × 10(6) FTC-133 cells using the microinjection HFUS-guided system, and 20 mice, used as a control, underwent surgical orthotopic implantation of 2 × 10(6) FTC-133 cells in the right lobe of the thyroid. All mice underwent HFUS imaging two weeks after cell injection; HFUS examinations and tumor volume (TV) measurements were repeated weekly. Micro-computed tomography was performed at different time points to determine whether lung metastasis had occurred. TVs were compared between the two models (surgical vs. HFUS-guided) using the Mann-Whitney U-test, and the Mantel-Cox log-rank test was applied to evaluate the death hazard. Hematoxylin and eosin analysis of formalin-fixed, paraffin-embedded mouse tissue was performed to validate the in vivo imaging results. RESULTS: Of the HFUS-guided injected mice, 9/18 survived up to 40 days after the injection of tumor cells. Mice injected surgically had 100% mortality at day 29. Of 38 mice, 29 (14/18 HFUS, 15/20 surgical) showed metastasis in the salivary glands and lymph nodes, and 13 (10/18 HFUS, 3/20 surgical) also showed metastasis in the lungs, which was confirmed by histological analysis. In the surgical group, there was an evident, frequent (12/20 mice) involvement of the contralateral lobe of the thyroid, whereas this feature was only detected in 1/18 mice in the HFUS group. Statistical analysis showed the same pattern of growth in the two groups, and a significant hazard in the mice in the surgical group (p = 0.03). CONCLUSIONS: This study demonstrated the technical feasibility of an HFUS-guided orthotopic mouse model of FTC. The HFUS-guided orthotopic model is easily reproducible and allows prolonged monitoring of the disease because the animals showed an increased survival rate.

Identification of targets of Twist1 transcription factor in thyroid cancer cells.

CONTEXT: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human tumors. Twist1 is a basic helix-loop-helix transcription factor involved in cancer development and progression. We showed that Twist1 affects thyroid cancer cell survival and motility. OBJECTIVE: We aimed to identify Twist1 targets in thyroid cancer cells. DESIGN: Transcriptional targets of Twist1 were identified by gene expression profiling the TPC-Twist1 cells in comparison with control cells. Functional studies were performed by silencing in TPC-Twist1 and in CAL62 cells the top 10 upregulated genes and by evaluating cell proliferation, survival, migration, and invasion. Chromatin immunoprecipitation was performed to verify direct binding of Twist1 to target genes. Quantitative RT-PCR was applied to study the expression level of Twist1 target genes in human thyroid carcinoma samples. RESULTS: According to the gene expression profile, the top functions enriched in TPC-Twist1 cells were cellular movement, cellular growth and proliferation, and cell death and survival. Silencing of the top 10 upregulated genes reduced viability of TPC-Twist1 and of CAL62 cells. Silencing of COL1A1, KRT7, and PDZK1 also induced cell death. Silencing of HS6ST2, THRB, ID4, RHOB, and PDZK1IP also impaired migration and invasion of TPC-Twist1 and of CAL62 cells. Chromatin immunoprecipitation showed that Twist1 directly binds the promoter of the top 10 upregulated genes. Quantitative RT-PCR showed that HS6ST2, COL1A1, F2RL1, LEPREL1, PDZK1, and PDZK1IP1 are overexpressed in thyroid carcinoma samples compared with normal thyroids. CONCLUSIONS: We identified a set of genes that mediates Twist1 biological effects in thyroid cancer cells.

TWIST1 plays a pleiotropic role in determining the anaplastic thyroid cancer phenotype.

CONTEXT: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human tumors; it is characterized by chemoresistance, local invasion, and distant metastases. ATC is invariably fatal. OBJECTIVE: The aim was to study the role of TWIST1, a basic helix-loop-helix transcription factor, in ATC. DESIGN: Expression of TWIST1 was studied by immunohistochemistry and real-time PCR in normal thyroids and well-differentiated, poorly differentiated, and ATC. The function of TWIST1 was studied by RNA interference in ATC cells and by ectopic expression in well-differentiated thyroid carcinoma cells. RESULTS: ATCs up-regulate TWIST1 with respect to normal thyroids as well as to poorly and well-differentiated thyroid carcinomas. Knockdown of TWIST1 by RNA interference in ATC cells reduced cell migration and invasion and increased sensitivity to apoptosis. The ectopic expression of TWIST1 in thyroid cells induced resistance to apoptosis and increased cell migration and invasion. CONCLUSIONS: TWIST1 plays a key role in determining malignant features of the anaplastic phenotype in vitro.