A gut microbiota rheostat forecasts responsiveness to PD-L1 and VEGF blockade in mesothelioma.

Malignant mesothelioma is a rare tumour caused by asbestos exposure that originates mainly from the pleural lining or the peritoneum. Treatment options are limited, and the prognosis is dismal. Although immune checkpoint blockade (ICB) can improve survival outcomes, the determinants of responsiveness remain elusive. Here, we report the outcomes of a multi-centre phase II clinical trial (MiST4, NCT03654833) evaluating atezolizumab and bevacizumab (AtzBev) in patients with relapsed mesothelioma. We also use tumour tissue and gut microbiome sequencing, as well as tumour spatial immunophenotyping to identify factors associated with treatment response. MIST4 met its primary endpoint with 50% 12-week disease control, and the treatment was tolerable. Aneuploidy, notably uniparental disomy (UPD), homologous recombination deficiency (HRD), epithelial-mesenchymal transition and inflammation with CD68+ monocytes were identified as tumour-intrinsic resistance factors. The log-ratio of gut-resident microbial genera positively correlated with radiological response to AtzBev and CD8+ T cell infiltration, but was inversely correlated with UPD, HRD and tumour infiltration by CD68+ monocytes. In summary, a model is proposed in which both intrinsic and extrinsic determinants in mesothelioma cooperate to modify the tumour microenvironment and confer clinical sensitivity to AtzBev. Gut microbiota represent a potentially modifiable factor with potential to improve immunotherapy outcomes for individuals with this cancer of unmet need.

Correction: Alves et al. WNK2 Inhibits Autophagic Flux in Human Glioblastoma Cell Line. Cells 2020, 9, 485.

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Holocene deglaciation drove rapid genetic diversification of Atlantic walrus.

Rapid global warming is severely impacting Arctic ecosystems and is predicted to transform the abundance, distribution and genetic diversity of Arctic species, though these linkages are poorly understood. We address this gap in knowledge using palaeogenomics to examine how earlier periods of global warming influenced the genetic diversity of Atlantic walrus (Odobenus rosmarus rosmarus), a species closely associated with sea ice and shallow-water habitats. We analysed 82 ancient and historical Atlantic walrus mitochondrial genomes (mitogenomes), including now-extinct populations in Iceland and the Canadian Maritimes, to reconstruct the Atlantic walrus' response to Arctic deglaciation. Our results demonstrate that the phylogeography and genetic diversity of Atlantic walrus populations was initially shaped by the last glacial maximum (LGM), surviving in distinct glacial refugia, and subsequently expanding rapidly in multiple migration waves during the late Pleistocene and early Holocene. The timing of diversification and establishment of distinct populations corresponds closely with the chronology of the glacial retreat, pointing to a strong link between walrus phylogeography and sea ice. Our results indicate that accelerated ice loss in the modern Arctic may trigger further dispersal events, likely increasing the connectivity of northern stocks while isolating more southerly stocks putatively caught in small pockets of suitable habitat.

Medical student deployment to a single metropolitan hospital during the COVID-19 pandemic: A mixed methods analysis.

OBJECTIVES: To explore student and staff satisfaction with the use of medical students as a surge workforce during the COVID-19 pandemic. METHOD: We conducted a mixed methods analysis of staff and student experiences with the medical student workforce at a single metropolitan ED over an 8-month period between December 2021 and July 2022, using an online survey tool. Students were invited to complete the survey fortnightly, whereas senior medical and nursing staff were invited weekly. RESULTS: There was a 32% response rate for surveys sent to medical student assistants (MSAs) and 18% and 15% for medical and nursing staff, respectively. Most students felt well prepared and supported in the role and would recommend it to other students. They reported that the role allowed them to gain experience and confidence within the ED, especially after much of their learning had moved online throughout the pandemic. Senior nurses and doctors found MSAs to be useful members of the team, largely through their assistance with task completion. Both staff and students recommended a more comprehensive orientation, changes to the supervision model and increased clarity in the students' scope of practice. CONCLUSIONS: The results of the present study provide insight into the use of medical students as an emergency surge workforce. Responses from medical students and staff suggested that the project was beneficial for both groups as well as for overall departmental performance. These findings are likely to be translatable beyond the COVID-19 pandemic setting.

YES1 Kinase Mediates the Membrane Removal of Rescued F508del-CFTR in Airway Cells by Promoting MAPK Pathway Activation via SHC1.

Recent developments in CFTR modulator drugs have had a significant transformational effect on the treatment of individuals with Cystic Fibrosis (CF) who carry the most frequent F508del-CFTR mutation in at least one allele. However, the clinical effects of these revolutionary drugs remain limited by their inability to fully restore the plasma membrane (PM) stability of the rescued mutant channels. Here, we shed new light on the molecular mechanisms behind the reduced half-life of rescued F508del-CFTR at the PM of airway cells. We describe that YES1 protein kinase is enriched in F508del-CFTR protein PM complexes, and that its interaction with rescued channels is mediated and dependent on the adaptor protein YAP1. Moreover, we show that interference with this complex, either by depletion of one of these components or inhibiting YES1 activity, is sufficient to significantly improve the abundance and stability of modulator-rescued F508del-CFTR at the surface of airway cells. In addition, we found that this effect was mediated by a decreased phosphorylation of the scaffold protein SHC1, a key regulator of MAPK pathway activity. In fact, we showed that depletion of SHC1 or inhibition of MAPK pathway signaling was sufficient to improve rescued F508del-CFTR surface levels, whereas an ectopic increase in pathway activation downstream of SHC1, through the use of a constitutively active H-RAS protein, abrogated the stabilizing effect of YES1 inhibition on rescued F508del-CFTR. Taken together, our findings not only provide new mechanistic insights into the regulation of modulator-rescued F508del-CFTR membrane stability, but also open exciting new avenues to be further explored in CF research and treatment.

Worth the risk? Contemporary indications, yield and complications of lumbar punctures in a metropolitan Australian health service.

BACKGROUND: Performing lumbar punctures carries a risk of harm to the patient, but the information cerebrospinal fluid provides often makes this procedure necessary. Clinicians in the Australian setting would benefit from having more information on these procedures, in order to help them in a risk versus benefit analysis. AIMS: To describe the contemporary indications, cerebrospinal fluid findings and complications of lumbar punctures in a metropolitan Australian health service. METHODS: Retrospective electronic medical records audit of lumbar punctures performed on 525 adults within three acute hospitals between 1 July 2018 and 30 June 2019. Main outcome measures include frequency of indication for lumbar puncture by category, normal versus abnormal cerebrospinal fluid for each category, and frequency, severity and type of complications of lumbar punctures. RESULTS: Of 525 adult lumbar punctures that were assessed in this study, 466 were performed for a diagnostic indication. The most common diagnostic indications were acute severe headache (156 procedures; 33.5%) and encephalopathy (128 procedures; 27.5%). The yield of abnormal results varied by indication category, with the above indications yielding abnormal results in 85 (54.5%) and 72 (56.3%) cases respectively. A complication was recorded in 54 (10.3% of total) procedures. The majority (45; 8.6%) of complications were minor in severity and most frequently consisted of post-dural puncture headache (PDPH). CONCLUSIONS: In the era of an increased reliance on high quality neuroimaging, lumbar puncture has a high diagnostic yield with a low rate of major complications. The most common complication is PDPH, which is mild and self-limiting in most cases.

Adverse Outcome Pathways Associated with the Ingestion of Titanium Dioxide Nanoparticles-A Systematic Review.

Titanium dioxide nanoparticles (TiO2-NPs) are widely used, and humans are exposed through food (E171), cosmetics (e.g., toothpaste), and pharmaceuticals. The oral and gastrointestinal (GIT) tract are the first contact sites, but it may be systemically distributed. However, a robust adverse outcome pathway (AOP) has not been developed upon GIT exposure to TiO2-NPs. The aim of this review was to provide an integrative analysis of the published data on cellular and molecular mechanisms triggered after the ingestion of TiO2-NPs, proposing plausible AOPs that may drive policy decisions. A systematic review according to Prisma Methodology was performed in three databases of peer-reviewed literature: Pubmed, Scopus, and Web of Science. A total of 787 records were identified, screened in title/abstract, being 185 used for data extraction. The main endpoints identified were oxidative stress, cytotoxicity/apoptosis/cell death, inflammation, cellular and systemic uptake, genotoxicity, and carcinogenicity. From the results, AOPs were proposed where colorectal cancer, liver injury, reproductive toxicity, cardiac and kidney damage, as well as hematological effects stand out as possible adverse outcomes. The recent transgenerational studies also point to concerns with regard to population effects. Overall, the findings further support a limitation of the use of TiO2-NPs in food, announced by the European Food Safety Authority (EFSA).

Species-specific reservoir effect estimates: A case study of archaeological marine samples from the Bering Strait.

Due to the marine reservoir effect, radiocarbon dates of marine samples require a correction. Marine reservoir effects, however, may vary among different marine species within a given body of water. Factors such as diet, feeding depth and migratory behaviour all affect the 14C date of a marine organism. Moreover, there is often significant variation within single marine species. Whilst the careful consideration of the ΔR values of a single marine species in a given location is important, so too is the full range of ΔR values within an ecosystem. This paper illustrates this point, using a sample pairing method to estimate the reservoir effects in 17 marine samples, of eight different species, from the archaeological site of Ekven (Eastern Chukotka, Siberia). An OxCal model is used to assess the strength of these estimates. The marine reservoir effects of samples passing the model range from ΔR (Marine20) = 136 ± 41-ΔR = 460 ± 40. Marine reservoir effect estimates of these samples and other published samples are used to explore variability in the wider Bering Strait region. The archaeological implications of this variability are also discussed. The calibrating of 14C dates from human bone collagen, for example, could be improved by applying a dietary relevant marine reservoir effect correction. For humans from the site of Ekven, a ΔR (Marine20) correction of 289 ± 124 years or reservoir age correction of 842 ± 123 years is suggested.

Active symptom control with or without oral vinorelbine in patients with relapsed malignant pleural mesothelioma (VIM): A randomised, phase 2 trial.

Background: Currently, there is no US Food and Drug Administration approved therapy for patients with pleural mesothelioma who have relapsed following platinum-doublet based chemotherapy. Vinorelbine has demonstrated useful clinical activity in mesothelioma, however its efficacy has not been formally evaluated in a randomised setting. BRCA1 expression is required for vinorelbine induced apoptosis in preclinical models. Loss of expression may therefore correlate with vinorelbine resistance. Methods: In this randomised, phase 2 trial, patients were eligible if they met the following criteria: age ≥ 18 years, Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1, histologically confirmed pleural mesothelioma, post platinum-based chemotherapy, and radiological evidence of disease progression. Consented patients were randomised 2:1 to either active symptom control with oral vinorelbine versus active symptom control (ASC) every 3 weeks until disease progression, unacceptable toxicity or withdrawal at an initial dose of 60 mg/m2 increasing to 80 mg/m2 post-cycle 1. Randomisation was stratified by histological subtype, white cell count, gender, ECOG performance status and best response during first-line therapy. The study was open label. The primary endpoint was progression-free survival (PFS), measured from randomisation to time of event (or censoring). Analyses were carried out according to intention-to-treat (ITT) principles. Recruitment and trial follow-up are complete. This trial is registered with ClinicalTrials.gov, number NCT02139904. Findings: Between June 1, 2016 and Oct 31, 2018, we performed a randomised phase 2 trial in 14 hospitals in the United Kingdom. 225 patients were screened for eligibility, of whom 154 were randomly assigned to receive either ASC + vinorelbine (n = 98) or ASC (n = 56). PFS was significantly longer for ASC+vinorelbine compared with ASC alone; 4.2 months (interquartile range (IQR) 2.2-8.0) versus 2.8 months (IQR 1.4-4.1) for ASC, giving an unadjusted hazard ratio (HR) of 0·60 (80% CI upper limit 0.7, one-sided unadjusted log rank test p = 0.002); adjusted HR 0.6 (80% CI upper limit 0.7, one-sided adjusted log rank test p < 0.001). BRCA1 did not predict resistance to ASC+vinorelbine. Neutropenia was the most common grades 3, 4 adverse events in the ASC +vinorelbine arm. Interpretation: Vinorelbine plus ASC confers clinical benefit to patients with relapsed pleural mesothelioma who have progressed following platinum-based doublet chemotherapy. Funding: This study was funded by Cancer Research UK (grant CRUK A15569).

Targeting Cancer by Using Nanoparticles to Modulate RHO GTPase Signaling.

Functionalized nanomaterials have recently been introduced as efficient vehicles for targeted delivery of drugs and other tailored molecules to cancer cells. They emerge as new opportunities for addressing particular challenging targets such as RHO guanosine triphosphatases (GTPases), a group of signaling molecules involved in the progression of a variety of tumor types. RHO GTPases comprise a subfamily of the Ras superfamily of small GTPases. They are best known for their role in cell migration through the remodeling of the actin cytoskeleton. However, they are also key regulators of a broad number of cellular functions, ranging from proliferation to cell adhesion and differentiation. Not surprisingly, their dysregulation has been implicated in the development and progression of many types of cancer. The RHO GTPase subfamily includes 20 members that can be further separated into typical and atypical RHO GTPases. The typical RHO family members include the classical RHOA, RAC1 and CDC42 proteins, which cycle between an active GTP-bound and inactive GDP-bound conformation, under the coordinated action of three types of regulators: GEFs, GAPs and GDIs. Atypical RHO family members have small changes in key residues that alter their regulatory mechanisms. Nevertheless, both typical and atypical RHO GTPases contribute to cancer progression but, in contrast to Ras proteins, very few mutations have been found in tumors. In most cancers, it is the expression level and/or activity of RHO GTPases that is dysregulated. RHO GTPase signaling has thus long been seen as an attractive target for cancer treatment but their ubiquity and the lack of isoform-specific drugs have posed significant obstacles to the development of viable therapeutic strategies. Based on the success of recent nanomedicine approaches, this chapter reviews representative studies of how functionalized nanoparticles can be designed to target tumor-specific molecules and directly or indirectly modulate the expression and/or activity of particular RHO GTPases in cancer cells.

Pro-Inflammatory Cytokines Trigger the Overexpression of Tumour-Related Splice Variant RAC1B in Polarized Colorectal Cells.

An inflammatory microenvironment is a tumour-promoting condition that provides survival signals to which cancer cells respond with gene expression changes. One example is the alternative splicing variant Rat Sarcoma Viral Oncogene Homolog (Ras)-Related C3 Botulinum Toxin Substrate 1 (RAC1)B, which we previously identified in a subset of V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF)-mutated colorectal tumours. RAC1B was also increased in samples from inflammatory bowel disease patients or in an acute colitis mouse model. Here, we used an epithelial-like layer of polarized Caco-2 or T84 colorectal cancer (CRC) cells in co-culture with fibroblasts, monocytes or macrophages and analysed the effect on RAC1B expression in the CRC cells by RT-PCR, Western blot and confocal fluorescence microscopy. We found that the presence of cancer-associated fibroblasts and M1 macrophages induced the most significant increase in RAC1B levels in the polarized CRC cells, accompanied by a progressive loss of epithelial organization. Under these conditions, we identified interleukin (IL)-6 as the main trigger for the increase in RAC1B levels, associated with Signal Transducer and Activator of Transcription (STAT)3 activation. IL-6 neutralization by a specific antibody abrogated both RAC1B overexpression and STAT3 phosphorylation in polarized CRC cells. Our data identify that pro-inflammatory extracellular signals from stromal cells can trigger the overexpression of tumour-related RAC1B in polarized CRC cells. The results will help to understand the tumour-promoting effect of inflammation and identify novel therapeutic strategies.

Including acoustic modes in the vortex-sheet eigenbasis of a jet.

Vortex-sheet (V-S) models of jets are widely used to describe the dynamics of modes, such as the Kelvin-Helmholtz instability and guided acoustic waves. However, the absence of the free-stream acoustic modes in the V-S eigenspectrum is seldom pointed out in the literature. This family of modes is important if, for example, one is interested into the problems of sound emission or flow-acoustic interactions. In this work, it is shown how a distantly confined jet may be used as a surrogate problem for the free jet, in which the free-stream acoustic waves appear as a set of discrete modes. Comparing the modes observed in the free jet with those of the distantly confined jet, we show that other than the free-stream acoustic modes, the eigenvectors and eigenvalues converge exponentially with the wall distance. The proposed surrogate problem, thus, efficiently reproduces the dynamics of the original problem while accounting for the dynamics of the free-stream acoustic eigenmodes.

Abemaciclib in patients with p16ink4A-deficient mesothelioma (MiST2): a single-arm, open-label, phase 2 trial.

BACKGROUND: Genetically stratified therapy for malignant mesothelioma is unavailable. Mesotheliomas frequently harbour loss of the chromosome 9p21.3 locus (CDKN2A-MTAP), which is associated with shorter overall survival due to loss of the tumour suppressor p16ink4A, an endogenous suppressor of cyclin-dependent kinase (CDK)4 and CDK6. Genetic restoration of p16ink4A suppresses mesothelioma in preclinical models, underpinning the rationale for targeting CDK4 and CDK6 in p16ink4A-negative mesothelioma. We developed a multicentre, stratified, phase 2 trial to test this hypothesis. METHODS: The MiST2 study was a single-arm, open-label, phase 2 clinical trial done two UK centres. Patients older than 18 years with any histologically confirmed subtype of mesothelioma (pleural or peritoneal) with radiological progression after at least one course of platinum-based chemotherapy were molecularly screened by immunohistochemistry for p16ink4A. Patients with p16ink4A-negative mesothelioma were eligible for inclusion in the study. Patients were required to have measurable disease by modified Response Evaluation Criteria in Solid Tumours version 1.1 for malignant mesothelioma, a predicted life expectancy of at least 12 weeks, and an Eastern Cooperative Oncology Group performance status score of 0-1. Patients received oral abemaciclib 200 mg twice daily, administered in 28-day cycles for 24 weeks. The primary endpoint was the disease control rate (patients with complete responses, partial responses, or stable disease) at 12 weeks. The null hypothesis could be rejected if at least 11 patients had disease control. The efficacy and safety populations were defined as all patients who received at least one dose of the study drug. The study is registered with ClinicalTrials.gov, NCT03654833, and is ongoing (but MiST2 is now closed). FINDINGS: Between Sept 31, 2019, and March 2, 2020, 27 eligible patients consented to molecular screening. The median follow-up was 18·4 weeks (IQR 6·7-23·9). One patient was excluded before treatment because of a serious adverse event before study drug allocation. 26 (100%) of 26 treated patients were p16ink4A deficient and received at least one dose of abemaciclib. Disease control at 12 weeks was reported in 14 (54%) of 26 patients (95% CI 36-71). Grade 3 or worse treatment-related adverse events (of any cause) occurred in eight (27%) of 26 patients (diarrhoea, dyspnoea, thrombocytopenia, vomiting, urinary tract infection, increased alanine aminotransferase, ascites, chest infection or suspected chest infection, neutropenic sepsis, alopecia, blood clot left calf, fall [broken neck and collar bone], haemoptysis, lower respiratory tract infection, and pulmonary embolism). Grade 3 or worse treatment-related adverse events occurred in three (12%) of 26 patients (diarrhoea, thrombocytopenia, vomiting, increased alanine aminotransferase, and pulmonary embolism). Serious adverse events occurred in six (23%) of 26 patients, leading to treatment discontinuation in one (4%) patient (diarrhoea, urinary tract infection, chest infection, neutropenic sepsis, fall [broken neck and collar bone], haemoptysis, lower respiratory tract infection, and pulmonary embolism). One patient had a serious adverse event related to abemaciclib (diarrhoea). One (4%) of 26 patients died from an adverse event (neutropenic sepsis). INTERPRETATION: This study met its primary endpoint, showing promising clinical activity of abemaciclib in patients with p16ink4A-negative mesothelioma who were previously treated with chemotherapy, and warrants its further investigation in a randomised study as a targeted stratified therapy. FUNDING: University of Leicester, Asthma UK and British Lung Foundation Partnership, and the Victor Dahdaleh Foundation.

Neolithic farmers or Neolithic foragers? Organic residue analysis of early pottery from Rakushechny Yar on the Lower Don (Russia).

The emergence of pottery in Europe is associated with two distinct traditions: hunter-gatherers in the east of the continent during the early 6th millennium BC and early agricultural communities in the south-west in the late 7th millennium BC. Here we investigate the function of pottery from the site of Rakushechny Yar, located at the Southern fringe of Eastern Europe, in this putative contact zone between these two economic 'worlds'. To investigate, organic residue analysis was conducted on 120 samples from the Early Neolithic phase (ca. mid-6th millennium BC) along with microscopic and SEM analysis of associated foodcrusts. The results showed that the earliest phase of pottery use was predominantly used to process riverine resources. Many of the vessels have molecular and isotopic characteristics consistent with migratory fish, such as sturgeon, confirmed by the identification of sturgeon bony structures embedded in the charred surface deposits. There was no evidence of dairy products in any of the vessels, despite the fact these have been routinely identified in coeval sites to the south. Further analysis of some of the mammalian bones using ZooMS failed to demonstrate that domesticated animals were present in the Early Neolithic. Nevertheless, we argue that intensive exploitation of seasonally migratory fish, accompanied by large-scale pottery production, created storable surpluses that led to similar socio-economic outcomes as documented in early agricultural societies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12520-021-01412-2.

Resolvent-based modeling of turbulent jet noise.

Resolvent analysis has demonstrated encouraging results for modeling coherent structures in jets when compared against their data-educed counterparts from high-fidelity large-eddy simulations (LES). We formulate resolvent analysis as an acoustic analogy that relates the near-field resolvent forcing to the near- and far-field pressure. We use an LES database of round, isothermal, Mach 0.9 and 1.5 jets to produce an ensemble of realizations for the acoustic field that we project onto a limited set of resolvent modes. In the near-field, we perform projections on a restricted acoustic output domain, r/D=[5,6], while the far-field projections are performed on a Kirchhoff surface comprising a 100-diameter arc centered at the nozzle. This allows the LES realizations to be expressed in the resolvent basis via a data-deduced, low-rank, cross-spectral density matrix. We find that a single resolvent mode reconstructs the most energetic regions of the acoustic field across Strouhal numbers, St=[0-1], and azimuthal wavenumbers, m=[0,2]. Finally, we present a simple function that results in a rank-1 resolvent model agreeing within 2 dB of the peak noise for both jets.

Nivolumab versus placebo in patients with relapsed malignant mesothelioma (CONFIRM): a multicentre, double-blind, randomised, phase 3 trial.

BACKGROUND: No phase 3 trial has yet shown improved survival for patients with pleural or peritoneal malignant mesothelioma who have progressed following platinum-based chemotherapy. The aim of this study was to assess the efficacy and safety of nivolumab, an anti-PD-1 antibody, in these patients. METHODS: This was a multicentre, placebo-controlled, double-blind, parallel group, randomised, phase 3 trial done in 24 hospitals in the UK. Adult patients (aged ≥18 years) with an Eastern Cooperative Oncology Group performance status of 0 or 1, with histologically confirmed pleural or peritoneal mesothelioma, who had received previous first-line platinum-based chemotherapy and had radiological evidence of disease progression, were randomly assigned (2:1) to receive nivolumab at a flat dose of 240 mg every 2 weeks over 30 min intravenously or placebo until disease progression or a maximum of 12 months. The randomisation sequence was generated within an interactive web response system (Alea); patients were stratified according to epithelioid versus non-epithelioid histology and were assigned in random block sizes of 3 and 6. Participants and treating clinicians were masked to group allocation. The co-primary endpoints were investigator-assessed progression-free survival and overall survival, analysed according to the treatment policy estimand (an equivalent of the intention-to-treat principle). All patients who were randomly assigned were included in the safety population, reported according to group allocation. This trial is registered with Clinicaltrials.gov, NCT03063450. FINDINGS: Between May 10, 2017, and March 30, 2020, 332 patients were recruited, of whom 221 (67%) were randomly assigned to the nivolumab group and 111 (33%) were assigned to the placebo group). Median follow-up was 11·6 months (IQR 7·2-16·8). Median progression-free survival was 3·0 months (95% CI 2·8-4·1) in the nivolumab group versus 1·8 months (1·4-2·6) in the placebo group (adjusted hazard ratio [HR] 0·67 [95% CI 0·53-0·85; p=0·0012). Median overall survival was 10·2 months (95% CI 8·5-12·1) in the nivolumab group versus 6·9 months (5·0-8·0) in the placebo group (adjusted HR 0·69 [95% CI 0·52-0·91]; p=0·0090). The most frequently reported grade 3 or worse treatment-related adverse events were diarrhoea (six [3%] of 221 in the nivolumab group vs two [2%] of 111 in the placebo group) and infusion-related reaction (six [3%] vs none). Serious adverse events occurred in 90 (41%) patients in the nivolumab group and 49 (44%) patients in the placebo group. There were no treatment-related deaths in either group. INTERPRETATION: Nivolumab represents a treatment that might be beneficial to patients with malignant mesothelioma who have progressed on first-line therapy. FUNDING: Stand up to Cancer-Cancer Research UK and Bristol Myers Squibb.

LDLR variants functional characterization: Contribution to variant classification.

BACKGROUND AND AIMS: Familial hypercholesterolaemia (FH) is an autosomal disorder of lipid metabolism presenting with increased cardiovascular risk. LDLR mutations are the cause of disease in 90% of the cases but functional studies have only been performed for about 15% of all LDLR variants. In the Portuguese Familial Hypercholesterolemia Study (PFHS), 142 unique LDLR alterations were identified and 44 (30%) lack functional characterization. The aim of the present work is to increase evidence for variant classification by performing functional characterization of 13 LDLR missense alterations found in Portugal and in 20 other countries. METHODS: Different LDLR mutants were generated by site-directed mutagenesis and expressed in CHO-ldlA7 cells lacking endogenous expression of LDLR. To determine the effects of alterations on LDLR function, cell surface expression, binding and uptake of FITC-LDL were assessed by flow cytometry and Western blot. RESULTS: Of the 13 variants studied 7 were shown to affect LDLR function - expression, binding or uptake, with rates lower than 60%: p.(Cys184Tyr), p.(Gly207_Ser213del); p.(His211Asp); p.(Asp221Tyr); p.(Glu288Lys); p.(Gly592Glu) and p.(Asp601Val)). The remaining 6 variants do not alter the LDLR function. CONCLUSIONS: These studies contributed to an update of these variants classification: from the 9 variants classified as variants of unknown significance, 7 have reached now a final classification and 3 variants have improved classification from likely pathogenic to pathogenic. In Portugal, an additional 55 patients received an FH definite diagnosis thanks to these studies. Since only likely pathogenic and pathogenic variants are clinically actionable, this work shows the importance of functional studies for variant classification.