Gut microbiota and metabolomics profiles in patients with chronic stable angina and acute coronary syndrome.

Cardiovascular disease (CVD) is the leading cause of death worldwide. The gut microbiota and its associated metabolites may be involved in the development and progression of CVD, although the mechanisms and impact on clinical outcomes are not fully understood. This study investigated the gut microbiome profile and associated metabolites in patients with chronic stable angina (CSA) and acute coronary syndrome (ACS) compared with healthy controls. Bacterial alpha diversity in stool from patients with ACS or CSA was comparable to healthy controls at both baseline and follow-up visits. Differential abundance analysis identified operational taxonomic units (OTUs) assigned to commensal taxa differentiating patients with ACS from healthy controls at both baseline and follow-up. Patients with CSA and ACS had significantly higher levels of trimethylamine N-oxide compared with healthy controls (CSA: 0.032 ± 0.023 mmol/L, P < 0.01 vs. healthy, and ACS: 0.032 ± 0.023 mmol/L, P = 0.02 vs. healthy, respectively). Patients with ACS had reduced levels of propionate and butyrate (119 ± 4 vs. 139 ± 5.1 µM, P = 0.001, and 14 ± 4.3 vs. 23.5 ± 8.1 µM, P < 0.001, respectively), as well as elevated serum sCD14 (2245 ± 75.1 vs. 1834 ± 45.8 ng/mL, P < 0.0001) and sCD163 levels (457.3 ± 31.8 vs. 326.8 ± 20.7 ng/mL, P = 0.001), compared with healthy controls at baseline. Furthermore, a modified small molecule metabolomic and lipidomic signature was observed in patients with CSA and ACS compared with healthy controls. These findings provide evidence of a link between gut microbiome composition and gut bacterial metabolites with CVD. Future time course studies in patients to observe temporal changes and subsequent associations with gut microbiome composition are required to provide insight into how these are affected by transient changes following an acute coronary event.NEW & NOTEWORTHY The study found discriminative microorganisms differentiating patients with acute coronary syndrome (ACS) from healthy controls. In addition, reduced levels of certain bacterial metabolites and elevated sCD14 and sCD163 were observed in patients with ACS compared with healthy controls. Furthermore, modified small molecule metabolomic and lipidomic signatures were found in both patient groups. Although it is not known whether these differences in profiles are associated with disease development and/or progression, the findings provide exciting options for potential new disease-related mechanism(s) and associated therapeutic target(s).

From hype to hope: Considerations in conducting robust microbiome science.

Microbiome science has been one of the most exciting and rapidly evolving research fields in the past two decades. Breakthroughs in technologies including DNA sequencing have meant that the trillions of microbes (particularly bacteria) inhabiting human biological niches (particularly the gut) can be profiled and analysed in exquisite detail. This microbiome profiling has profound impacts across many fields of research, especially biomedical science, with implications for how we understand and ultimately treat a wide range of human disorders. However, like many great scientific frontiers in human history, the pioneering nature of microbiome research comes with a multitude of challenges and potential pitfalls. These include the reproducibility and robustness of microbiome science, especially in its applications to human health outcomes. In this article, we address the enormous promise of microbiome science and its many challenges, proposing constructive solutions to enhance the reproducibility and robustness of research in this nascent field. The optimisation of microbiome science spans research design, implementation and analysis, and we discuss specific aspects such as the importance of ecological principals and functionality, challenges with microbiome-modulating therapies and the consideration of confounding, alternative options for microbiome sequencing, and the potential of machine learning and computational science to advance the field. The power of microbiome science promises to revolutionise our understanding of many diseases and provide new approaches to prevention, early diagnosis, and treatment.

Effect of Christmas Holidays on Type 1 Diabetes Mellitus in Users of Glucose Flash Systems.

OBJECTIVE: Christmas holidays can impact weight and glycemic control in type 2 diabetes, but their effect on type 1 diabetes (T1D) remains understudied. This study assessed how Christmas holidays affect individuals with T1D who use flash continuous glucose monitoring systems. METHODS: This retrospective study involved 812 adults diagnosed with T1D recruited from 3 hospitals. Clinical, anthropometric, and socioeconomic data were collected. Glucose metrics from 14 days before January 1st, and before December 1st and February 1st as control periods, were recorded. Analyses adjusted for multiple variables were conducted to assess the holiday season's impact on glycemic control. RESULTS: The average time in range during the holidays (60.0 ± 17.2%) was lower compared to December (61.9 ± 17.2%, P < .001) and February (61.7 ± 17.7%, P < .001). Time above range (TAR > 180 mg/dL) was higher during Christmas (35.8 ± 18.2%) compared to December (34.1 ± 18.3%, P < .001) and February (34.2 ± 18.4%, P < .001). Differences were also observed in TAR >250 mg/dL, coefficient of variation, and average glucose (P < .05). No differences were found in time below range or other metrics. Linear regression models showed that the holidays reduced time in range by 1.9% (ß = -1.92, P = .005) and increased TAR >180 mg/dL by 1.8% (ß = 1.75, P = .016). CONCLUSION: Christmas holidays are associated with a mild and reversible deterioration in glucose metrics among individuals with T1D using flash continuous glucose monitoring, irrespective of additional influencing factors. These discoveries can be useful to advise individuals with diabetes during the festive season and to recognize potential biases within studies conducted during this timeframe.

Efficacy of T-Cell Receptor-Based Adoptive Cell Therapy in Cutaneous Melanoma: A Meta-Analysis.

BACKGROUND: T-cell receptor (TCR-T) therapies are based on the expression of an introduced TCR targeting a tumor associated antigen (TAA) which has been studied in several trials in cutaneous melanoma. We conducted a systematic review and meta-analysis aiming to assess the primary efficacy of TCR-based adoptive cell therapy in cutaneous melanoma. METHODS: We searched through PubMed electronic database from its inception until May 21, 2022. Primary endpoints were pooled objective response rate (ORR) and disease control rate (DCR). We conducted logistic regression analyses to identify potential predictive factors for tumor response. RESULTS: From 187 patients, 50 showed an objective response (pooled ORR 28%; 95% CI, 20%-37%) and a pooled DCR of 38% (95% CI, 27%-50%). Median PFS was 2, 9 months (95% CI, 1.4-3.1). A trend toward higher PFS was demonstrated for patients treated with cancer/testis antigens targeting TCR-T cells (HR 0.91 95% CI, 0.64-1.3, P = .61) among whom, patients treated with NYESO-1 targeting TCR-T showed a significantly higher PFS (HR 0.63 95% CI, 0.64-0.98, P = .03). In addition, the number of infused cells was associated with a significantly higher likelihood of tumor response (OR 6.61; 95% CI, 1.68-21.6; P = .007). CONCLUSION: TCR-T therapy shows promising results in terms of antitumor activity and survival similar to those reported for TILs with a significantly higher benefit for cancer/testis antigens targeting cells. Since TCR-based therapy shows advantages of great potential over classic ACT strategies, further research in solid cancers is warranted (PROSPERO ID CRD42022328011).

Insights into the associations between the gut microbiome, its metabolites, and heart failure.

Heart failure (HF) is the end stage of most cardiovascular diseases and remains a significant health problem globally. We aimed to assess whether patients with left ventricular ejection fraction ≤45% had alterations in both the gut microbiome profile and production of associated metabolites when compared with a healthy cohort. We also examined the associated inflammatory, metabolomic, and lipidomic profiles of patients with HF. This single center, observational study, recruited 73 patients with HF and 59 healthy volunteers. Blood and stool samples were collected at baseline and 6-mo follow-up, along with anthropometric and clinical data. When compared with healthy controls, patients with HF had reduced gut bacterial alpha diversity at follow-up (P = 0.004) but not at baseline. The stool microbiota of patients with HF was characterized by a depletion of operational taxonomic units representing commensal Clostridia at both baseline and follow-up. Patients with HF also had significantly elevated baseline plasma acetate (P = 0.007), plasma trimethylamine-N-oxide (TMAO) (P = 0.003), serum soluble CD14 (sCD14; P = 0.005), and soluble CD163 (sCD163; P = 0.004) levels compared with healthy controls. Furthermore, patients with HF had a distinct metabolomic and lipidomic profile at baseline when compared with healthy controls. Differences in the composition of the gut microbiome and the levels of associated metabolites were observed in patients with HF when compared with a healthy cohort. This was also associated with an altered metabolomic and lipidomic profile. Our study identifies microorganisms and metabolites that could represent new therapeutic targets and diagnostic tools in the pathogenesis of HF.NEW & NOTEWORTHY We found a reduction in gut bacterial alpha diversity in patients with heart failure (HF) and that the stool microbiota of patients with HF was characterized by depletion of operational taxonomic units representing commensal Clostridia at both baseline and follow-up. Patients with HF also had altered bacterial metabolites and increased inflammatory profiles compared with healthy controls. A distinct metabolomic and lipidomic profile was present in patients with HF at baseline when compared with healthy controls.

Tryptophan Modulation in Cancer-Associated Cachexia Mouse Models.

Cancer cachexia is a multifactorial syndrome that interferes with treatment and reduces the quality of life and survival of patients. Currently, there is no effective treatment or biomarkers, and pathophysiology is not clear. Our group reported alterations on tryptophan metabolites in cachectic patients, so we aim to investigate the role of tryptophan using two cancer-associated cachexia syngeneic murine models, melanoma B16F10, and pancreatic adenocarcinoma that is KPC-based. Injected mice showed signs of cancer-associated cachexia as reduction in body weight and raised spleen weight, MCP1, and carbonilated proteins in plasma. CRP and Myostatin also increased in B16F10 mice. Skeletal muscle showed a decrease in quadriceps weight and cross-sectional area (especially in B16F10). Higher expression of atrophy genes, mainly Atrogin1, was also observed. Plasmatic tryptophan levels in B16F10 tumor-bearing mice decreased even at early steps of tumorigenesis. In KPC-injected mice, tryptophan fluctuated but were also reduced and in cachectic patients were significantly lower. Treatment with 1-methyl-tryptophan, an inhibitor of tryptophan degradation, in the murine models resulted in the restoration of plasmatic tryptophan levels and an improvement on splenomegaly and carbonilated proteins levels, while changes in plasmatic inflammatory markers were mild. After the treatment, CCR2 expression in monocytes diminished and lymphocytes, Tregs, and CD8+, were activated (seen by increased in CD127 and CD25 expression, respectively). These immune cell changes pointed to an improvement in systemic inflammation. While treatment with 1-MT did not show benefits in terms of muscle wasting and atrophy in our experimental setting, muscle functionality was not affected and central nuclei fibers appeared, being a feature of regeneration. Therefore, tryptophan metabolism pathway is a promising target for inflammation modulation in cancer-associated cachexia.

Dabrafenib plus trametinib in patients with BRAFV600E-mutant low-grade and high-grade glioma (ROAR): a multicentre, open-label, single-arm, phase 2, basket trial.

BACKGROUND: Effective treatments are needed to improve outcomes for high-grade glioma and low-grade glioma. The activity and safety of dabrafenib plus trametinib were evaluated in adult patients with recurrent or progressive BRAFV600E mutation-positive high-grade glioma and low-grade glioma. METHODS: This study is part of an ongoing open-label, single-arm, phase 2 Rare Oncology Agnostic Research (ROAR) basket trial at 27 community and academic cancer centres in 13 countries (Austria, Belgium, Canada, France, Germany, Italy, Japan, the Netherlands, Norway, South Korea, Spain, Sweden, and the USA). The study enrolled patients aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 0, 1, or 2. Patients with BRAFV600E mutation-positive high-grade glioma and low-grade glioma received dabrafenib 150 mg twice daily plus trametinib 2 mg once daily orally until unacceptable toxicity, disease progression, or death. In the high-grade glioma cohort, patients were required to have measurable disease at baseline using the Response Assessment in Neuro-Oncology high-grade glioma response criteria and have been treated previously with radiotherapy and first-line chemotherapy or concurrent chemoradiotherapy. Patients with low-grade glioma were required to have measurable non-enhancing disease (except pilocytic astrocytoma) at baseline using the Response Assessment in Neuro-Oncology low-grade glioma criteria. The primary endpoint, in the evaluable intention-to-treat population, was investigator-assessed objective response rate (complete response plus partial response for high-grade glioma and complete response plus partial response plus minor response for low-grade glioma). This trial is ongoing, but is closed for enrolment, NCT02034110. FINDINGS: Between April 17, 2014, and July 25, 2018, 45 patients (31 with glioblastoma) were enrolled into the high-grade glioma cohort and 13 patients were enrolled into the low-grade glioma cohort. The results presented here are based on interim analysis 16 (data cutoff Sept 14, 2020). In the high-grade glioma cohort, median follow-up was 12·7 months (IQR 5·4-32·3) and 15 (33%; 95% CI 20-49) of 45 patients had an objective response by investigator assessment, including three complete responses and 12 partial responses. In the low-grade glioma cohort, median follow-up was 32·2 months (IQR 25·1-47·8). Nine (69%; 95% CI 39-91) of 13 patients had an objective response by investigator assessment, including one complete response, six partial responses, and two minor responses. Grade 3 or worse adverse events were reported in 31 (53%) patients, the most common being fatigue (five [9%]), decreased neutrophil count (five [9%]), headache (three [5%]), and neutropenia (three [5%]). INTERPRETATION: Dabrafenib plus trametinib showed clinically meaningful activity in patients with BRAFV600E mutation-positive recurrent or refractory high-grade glioma and low-grade glioma, with a safety profile consistent with that in other indications. BRAFV600E testing could potentially be adopted in clinical practice for patients with glioma. FUNDING: Novartis.

Avadomide monotherapy in relapsed/refractory DLBCL: safety, efficacy, and a predictive gene classifier.

Treatment options for relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) are limited, with no standard of care; prognosis is poor, with 4- to 6-month median survival. Avadomide (CC-122) is a cereblon-modulating agent with immunomodulatory and direct antitumor activities. This phase 1 dose-expansion study assessed safety and clinical activity of avadomide monotherapy in patients with de novo R/R DLBCL and transformed lymphoma. Additionally, a novel gene expression classifier, which identifies tumors with a high immune cell infiltration, was shown to enrich for response to avadomide in R/R DLBCL. Ninety-seven patients with R/R DLBCL, including 12 patients with transformed lymphoma, received 3 to 5 mg avadomide administered on continuous or intermittent schedules until unacceptable toxicity, disease progression, or withdrawal. Eighty-two patients (85%) experienced ≥1 grade 3/4 treatment-emergent adverse events (AEs), most commonly neutropenia (51%), infections (24%), anemia (12%), and febrile neutropenia (10%). Discontinuations because of AEs occurred in 10% of patients. Introduction of an intermittent 5/7-day schedule improved tolerability and reduced frequency and severity of neutropenia, febrile neutropenia, and infections. Among 84 patients with de novo R/R DLBCL, overall response rate (ORR) was 29%, including 11% complete response (CR). Responses were cell-of-origin independent. Classifier-positive DLBCL patients (de novo) had an ORR of 44%, median progression-free survival (mPFS) of 6 months, and 16% CR vs an ORR of 19%, mPFS of 1.5 months, and 5% CR in classifier-negative patients (P = .0096). Avadomide is being evaluated in combination with other antilymphoma agents. This trial was registered at www.clinicaltrials.gov as #NCT01421524.

Efficacy and safety of pembrolizumab for patients with previously treated advanced vulvar squamous cell carcinoma: Results from the phase 2 KEYNOTE-158 study.

OBJECTIVE: Treatment options for advanced vulvar cancer are limited. We evaluated pembrolizumab monotherapy in patients with advanced vulvar squamous cell carcinoma (SCC) enrolled in the phase 2 multicohort, open-label KEYNOTE-158 study (NCT02628067). METHODS: Eligible patients had histologically or cytologically documented advanced vulvar SCC with prior treatment failure, measurable disease per RECIST v1.1, ECOG performance status 0-1, and a tumor sample available for biomarker analysis. Pembrolizumab 200 mg was administered intravenously Q3W for up to 35 cycles (approximately 2 years). The primary endpoint was objective response rate (ORR) per RECIST v1.1 by independent central radiologic review in all patients and subgroups based on PD-L1 combined positive score (≥1 [PD-L1-positive] versus <1 [PD-L1-negative]). RESULTS: 101 patients were enrolled. Median time from first dose to data cutoff was 36.0 months. The ORR (95% CI) was 10.9% (5.6%-18.7%) among all patients, 9.5% (4.2%-17.9%) among the 84 patients with PD-L1-positive tumors, and 28.6% (3.7%-71.0%) among the 7 patients with PD-L1-negative tumors. Among patients with a response, median DOR was 20.4 (range, 2.1+ to 28.0) months. Median (95% CI) PFS and OS were 2.1 (2.0-2.1) and 6.2 (4.9-9.4) months, respectively. Treatment-related AEs occurred in 50.5% of patients (grade 3-5, 11.9%) and led to discontinuation of treatment in 5.0% of patients. Two deaths were considered treatment-related (hepatitis, n = 2). CONCLUSIONS: Pembrolizumab monotherapy was associated with durable responses in a subset of patients with vulvar SCC. Responses occurred regardless of tumor PD-L1 status. No new safety signals emerged; overall, pembrolizumab was well tolerated.

Radiation and Dust Sensor for Mars Environmental Dynamic Analyzer Onboard M2020 Rover.

The Radiation and Dust Sensor is one of six sensors of the Mars Environmental Dynamics Analyzer onboard the Perseverance rover from the Mars 2020 NASA mission. Its primary goal is to characterize the airbone dust in the Mars atmosphere, inferring its concentration, shape and optical properties. Thanks to its geometry, the sensor will be capable of studying dust-lifting processes with a high temporal resolution and high spatial coverage. Thanks to its multiwavelength design, it will characterize the solar spectrum from Mars' surface. The present work describes the sensor design from the scientific and technical requirements, the qualification processes to demonstrate its endurance on Mars' surface, the calibration activities to demonstrate its performance, and its validation campaign in a representative Mars analog. As a result of this process, we obtained a very compact sensor, fully digital, with a mass below 1 kg and exceptional power consumption and data budget features.

First-in-Human, First-in-Child Trial of Autologous MSCs Carrying the Oncolytic Virus Icovir-5 in Patients with Advanced Tumors.

We present here the results of a first-in-human, first-in-child trial for patients with relapsed/refractory solid tumors using Celyvir, an advanced therapy medicine that combines autologous mesenchymal stem cells (MSCs) carrying an oncolytic adenovirus. Celyvir was manufactured from a bone marrow aspirate and then given intravenously. Patients received weekly infusions for 6 weeks at a dose of 2 × 106 cells/kg (children) or 0.5-1 × 106 cells/kg (adults), 2 × 104 viral particles per cell. Fifteen pediatric and 19 adult patients were recruited, but 18 were screen failures, mainly because rapid disease progression before Celyvir was available. No grade 2-5 toxicities were reported. Adenoviral replication detected by PCR was found in all but 2 pediatric patient and in none of the adult ones. Absolute numbers of circulating leukocytes suffered minor changes along therapy, but some subsets showed differences comparing the pediatric versus the adult cohorts. Two patients with neuroblastoma showed disease stabilization, and one of them continued on treatment for up to 6 additional weeks. Celyvir, the combination of MSCs and oncolytic adenovirus, is safe and warrants further evaluation in a phase 2 setting. The use of MSCs may be a strategy to increase the amount of oncolytic virus administered to patients, minimizing toxicities and avoiding direct tumor injections.

Association of tumour mutational burden with outcomes in patients with advanced solid tumours treated with pembrolizumab: prospective biomarker analysis of the multicohort, open-label, phase 2 KEYNOTE-158 study.

BACKGROUND: Tumour mutational burden (TMB) has been retrospectively correlated with response to immune checkpoint blockade. We prospectively explored the association of high tissue TMB (tTMB-high) with outcomes in ten tumour-type-specific cohorts from the phase 2 KEYNOTE-158 study, which assessed the anti-PD-1 monoclonal antibody pembrolizumab in patients with selected, previously treated, advanced solid tumours. METHODS: In the multi-cohort, open-label, non-randomised, phase 2 KEYNOTE-158 study, patients were enrolled from 81 academic facilities and community-based institutions across 21 countries in Africa, the Americas, Asia, and Europe. Eligible patients were aged 18 years or older, had a histologically or cytologically confirmed advanced (ie, unresectable or metastatic, or both) incurable solid tumour (eligible tumour types were anal, biliary, cervical, endometrial, mesothelioma, neuroendocrine, salivary, small-cell lung, thyroid, and vulvar), progression on or intolerance to one or more lines of standard therapy, had measurable disease per Response Evaluation Criteria in Solid Tumors (RECIST; version 1.1) assessed by independent central radiological review, Eastern Cooperative Oncology Group performance status of 0 or 1, life expectancy of at least 3 months, adequate organ function, and a tumour sample for biomarker analysis. Participants were given pembrolizumab 200 mg intravenously every 3 weeks for up to 35 cycles. Tissue TMB (tTMB) was assessed in formalin-fixed paraffin-embedded tumour samples using the FoundationOne CDx assay (Foundation Medicine, Cambridge, MA, USA). The prespecified definition of tTMB-high status was at least 10 mutations per megabase. The primary endpoint was the proportion of patients with an objective response (complete or partial response) as per Response Evaluation Criteria in Solid Tumours (version 1.1) by independent central review. This prespecified analysis assessed the association between antitumour activity and tTMB in treated patients with evaluable tTMB data. Efficacy was assessed in all participants who received at least one dose of pembrolizumab, had evaluable tTMB data, and were enrolled at least 26 weeks before data cutoff (June 27, 2019), and safety was assessed in all participants who received at least one dose of pembrolizumab and had tTMB-high status. KEYNOTE-158 is registered at ClinicalTrials.gov, NCT02628067, and is ongoing. FINDINGS: Between Jan 15, 2016, and June 25, 2019, 1073 patients were enrolled. 1066 participants were treated as of data cutoff (June 27, 2019), of whom 805 (76%) were evaluable for TMB, and 105 (13%) of 805 had tTMB-high status and were assessed for safety. 1050 (98%) of 1066 patients enrolled by at least 26 weeks before data cutoff, of whom 790 (75%) were evaluable for TMB and included in efficacy analyses. 102 (13%) of these 790 patients had tTMB-high status (≥10 mutations per megabase), and 688 (87%) patients had non-tTMB-high status (<10 mutations per megabase). Median study follow-up was 37·1 months (IQR 35·0-38·3). Objective responses were observed in 30 (29%; 95% CI 21-39) of 102 patients in the tTMB-high group and 43 (6%; 5-8) of 688 in the non-tTMB-high group. 11 (10%) of 105 patients had treatment-related serious adverse events. 16 (15%) participants had a grade 3-5 treatment-related adverse event, of which colitis was the only such adverse event that occurred in more than one patient (n=2). One patient had fatal pneumonia that was assessed by the investigator to be treatment related. INTERPRETATION: tTMB-high status identifies a subgroup of patients who could have a robust tumour response to pembrolizumab monotherapy. tTMB could be a novel and useful predictive biomarker for response to pembrolizumab monotherapy in patients with previously treated recurrent or metastatic advanced solid tumours. FUNDING: Merck Sharp & Dohme Corp, a subsidiary of Merck & Co, Inc.

Common atrium/atrioventricular canal defect and postaxial polydactyly: A mild clinical subtype of Ellis-van Creveld syndrome caused by hypomorphic mutations in the EVC gene.

Clinical expression of Ellis-van Creveld syndrome (EvC) is variable and mild phenotypes have been described, including patients with mostly cardiac and limb involvement. Whether these cases are part of the EvC phenotypic spectrum or separate conditions is disputed. Herein, we describe a family with vertical transmission of atrioventricular canal defect (AVCD), common atrium, and postaxial polydactyly. Targeted sequencing of EVC, EVC2, WDR35, DYNC2LI1, and DYNC2H1 identified different compound heterozygosity in EVC genotypes in the two affected members, consisting of a nonsense (p.Arg622Ter) and a missense (p.Arg663Pro) variant in the father, and the same nonsense variant and a noncanonical splice-site in-frame change (c.1316-7A>G) in the daughter. Complementary DNA sequencing, immunoblot, and immunofluorescence experiments using patient-derived fibroblasts and Evc-/- mouse embryonic fibroblasts showed that p.Arg622Ter is a loss-of-function mutation, whereas p.Arg663Pro and the splice-site change c.1316-7A>G are hypomorphic variants resulting in proteins that retain, in part, the ability to complex with EVC2. Our molecular and functional data demonstrate that at least in some cases the condition characterized as "common atrium/AVCD with postaxial polydactyly" is a mild form of EvC due to hypomorphic EVC mutations, further supporting the occurrence of genotype-phenotype correlations in this syndrome.

Mechanistic added value of a trans-Sulfonamide-Platinum-Complex in human melanoma cell lines and synergism with cis-Platin.

BACKGROUND: Cisplatin is a potent antitumor agent. However, toxicity and primary and secondary resistance are major limitations of cisplatin-based chemotherapy, leading to therapeutic failure. We have previously reported that mono-sulfonamide platinum complexes have good antitumor activity against different tumoral cell lines and with a different and better cytotoxic profile than cisplatin. Besides, N-sulfonamides have been used extensively in medicinal chemistry as bactericides, anticonvulsant, inhibitors of the carbonic anhydrase, inhibitors of histone deacetylases, and inhibitors of microtubule polymerization, among others. METHODS: We aimed to compare the cytotoxic effects of cisplatin and a trans-sulfonamide-platinum-complex (TSPC), in two human melanoma cell lines that differ in their TP53 status: SK-MEL-5, TP53 wild type, and SK-MEL-28, TP53 mutated. We performed cytotoxicity assays with both drugs, alone and in combination, cell cycle analyses, western blotting and immunoprecipitation, and fluorescence immunocytochemistry. RESULTS: TSPC had similar antiproliferative activity than cisplatin against SK-MEL-5 (3.24 ± 1.08 vs 2.89 ± 1.12 µM) and higher against SK-MEL-28 cells (5.83 ± 1.06 vs 10.17 ± 1.29 µM). Combination of both drugs inhibited proliferation in both cell lines, being especially important in SK-MEL-28, and showing a synergistic effect. In contrast to cisplatin, TSPC caused G1 instead G2/M arrest in both cell lines. Our present findings indicate that the G1 arrest is associated with the induction of CDKN1A and CDKN1B proteins, and that this response is also present in melanoma cells containing TP53 mutated. Also, strong accumulation of CDKN1A and CDKN1B in cells nuclei was seen upon TSPC treatment in both cell lines. CONCLUSIONS: Overall, these findings provide a new promising TSPC compound with in vitro antitumor activity against melanoma cell lines, and with a different mechanism of action from that of cisplatin. Besides, TSPC synergism with cisplatin facilitates its potential use for co-treatment to reduce toxicity and resistance against cisplatin. TSPC remains a promising lead compound for the generation of novel antineoplastic agent and to explore its synergism with other DNA damaging agents.

Specific variants in WDR35 cause a distinctive form of Ellis-van Creveld syndrome by disrupting the recruitment of the EvC complex and SMO into the cilium.

Most patients with Ellis-van Creveld syndrome (EvC) are identified with pathogenic changes in EVC or EVC2, however further genetic heterogeneity has been suggested. In this report we describe pathogenic splicing variants in WDR35, encoding retrograde intraflagellar transport protein 121 (IFT121), in three families with a clinical diagnosis of EvC but having a distinctive phenotype. To understand why WDR35 variants result in EvC, we analysed EVC, EVC2 and Smoothened (SMO) in IFT-A deficient cells. We found that the three proteins failed to localize to Wdr35(-/-) cilia, but not to the cilium of the IFT retrograde motor mutant Dync2h1(-/-), indicating that IFT121 is specifically required for their entry into the ciliary compartment. Furthermore expression of Wdr35 disease cDNAs in Wdr35(-/-) fibroblasts revealed that the newly identified variants lead to Hedgehog signalling defects resembling those of Evc(-/-) and Evc2(-/-) mutants. Together our data indicate that splicing variants in WDR35, and possibly in other IFT-A components, underlie a number of EvC cases by disrupting targeting of both the EvC complex and SMO to cilia.

Nivolumab alone and nivolumab plus ipilimumab in recurrent small-cell lung cancer (CheckMate 032): a multicentre, open-label, phase 1/2 trial.

BACKGROUND: Treatments for small-cell lung cancer (SCLC) after failure of platinum-based chemotherapy are limited. We assessed safety and activity of nivolumab and nivolumab plus ipilimumab in patients with SCLC who progressed after one or more previous regimens. METHODS: The SCLC cohort of this phase 1/2 multicentre, multi-arm, open-label trial was conducted at 23 sites (academic centres and hospitals) in six countries. Eligible patients were 18 years of age or older, had limited-stage or extensive-stage SCLC, and had disease progression after at least one previous platinum-containing regimen. Patients received nivolumab (3 mg/kg bodyweight intravenously) every 2 weeks (given until disease progression or unacceptable toxicity), or nivolumab plus ipilimumab (1 mg/kg plus 1 mg/kg, 1 mg/kg plus 3 mg/kg, or 3 mg/kg plus 1 mg/kg, intravenously) every 3 weeks for four cycles, followed by nivolumab 3 mg/kg every 2 weeks. Patients were either assigned to nivolumab monotherapy or assessed in a dose-escalating safety phase for the nivolumab/ipilimumab combination beginning at nivolumab 1 mg/kg plus ipilimumab 1 mg/kg. Depending on tolerability, patients were then assigned to nivolumab 1 mg/kg plus ipilimumab 3 mg/kg or nivolumab 3 mg/kg plus ipilimumab 1 mg/kg. The primary endpoint was objective response by investigator assessment. All analyses included patients who were enrolled at least 90 days before database lock. This trial is ongoing; here, we report an interim analysis of the SCLC cohort. This study is registered with ClinicalTrials.gov, number NCT01928394. FINDINGS: Between Nov 18, 2013, and July 28, 2015, 216 patients were enrolled and treated (98 with nivolumab 3 mg/kg, three with nivolumab 1 mg/kg plus ipilimumab 1 mg/kg, 61 with nivolumab 1 mg/kg plus ipilimumab 3 mg/kg, and 54 with nivolumab 3 mg/kg plus ipilimumab 1 mg/kg). At database lock on Nov 6, 2015, median follow-up for patients continuing in the study (including those who had died or discontinued treatment) was 198·5 days (IQR 163·0-464·0) for nivolumab 3 mg/kg, 302 days (IQR not calculable) for nivolumab 1 mg/kg plus ipilimumab 1 mg/kg, 361·0 days (273·0-470·0) for nivolumab 1 mg/kg plus ipilimumab 3 mg/kg, and 260·5 days (248·0-288·0) for nivolumab 3 mg/kg plus ipilimumab 1 mg/kg. An objective response was achieved in ten (10%) of 98 patients receiving nivolumab 3 mg/kg, one (33%) of three patients receiving nivolumab 1 mg/kg plus ipilimumab 1 mg/kg, 14 (23%) of 61 receiving nivolumab 1 mg/kg plus ipilimumab 3 mg/kg, and ten (19%) of 54 receiving nivolumab 3 mg/kg plus ipilimumab 1 mg/kg. Grade 3 or 4 treatment-related adverse events occurred in 13 (13%) patients in the nivolumab 3 mg/kg cohort, 18 (30%) in the nivolumab 1 mg/kg plus ipilimumab 3 mg/kg cohort, and ten (19%) in the nivolumab 3 mg/kg plus ipilimumab 1 mg/kg cohort; the most commonly reported grade 3 or 4 treatment-related adverse events were increased lipase (none vs 5 [8%] vs none) and diarrhoea (none vs 3 [5%] vs 1 [2%]). No patients in the nivolumab 1 mg/kg plus ipilimumab 1 mg/kg cohort had a grade 3 or 4 treatment-related adverse event. Six (6%) patients in the nivolumab 3 mg/kg group, seven (11%) in the nivolumab 1 mg/kg plus ipilimumab 3 mg/kg group, and four (7%) in the nivolumab 3 mg/kg plus ipilimumab 1 mg/kg group discontinued treatment due to treatment-related adverse events. Two patients who received nivolumab 1 mg/kg plus ipilimumab 3 mg/kg died from treatment-related adverse events (myasthenia gravis and worsening of renal failure), and one patient who received nivolumab 3 mg/kg plus ipilimumab 1 mg/kg died from treatment-related pneumonitis. INTERPRETATION: Nivolumab monotherapy and nivolumab plus ipilimumab showed antitumour activity with durable responses and manageable safety profiles in previously treated patients with SCLC. These data suggest a potential new treatment approach for a population of patients with limited treatment options and support the evaluation of nivolumab and nivolumab plus ipilimumab in phase 3 randomised controlled trials in SCLC. FUNDING: Bristol-Myers Squibb.

Mutations in WNT1 cause different forms of bone fragility.

We report that hypofunctional alleles of WNT1 cause autosomal-recessive osteogenesis imperfecta, a congenital disorder characterized by reduced bone mass and recurrent fractures. In consanguineous families, we identified five homozygous mutations in WNT1: one frameshift mutation, two missense mutations, one splice-site mutation, and one nonsense mutation. In addition, in a family affected by dominantly inherited early-onset osteoporosis, a heterozygous WNT1 missense mutation was identified in affected individuals. Initial functional analysis revealed that altered WNT1 proteins fail to activate canonical LRP5-mediated WNT-regulated ß-catenin signaling. Furthermore, osteoblasts cultured in vitro showed enhanced Wnt1 expression with advancing differentiation, indicating a role of WNT1 in osteoblast function and bone development. Our finding that homozygous and heterozygous variants in WNT1 predispose to low-bone-mass phenotypes might advance the development of more effective therapeutic strategies for congenital forms of bone fragility, as well as for common forms of age-related osteoporosis.

The ciliary Evc/Evc2 complex interacts with Smo and controls Hedgehog pathway activity in chondrocytes by regulating Sufu/Gli3 dissociation and Gli3 trafficking in primary cilia.

Hedgehog (Hh) signaling is involved in patterning and morphogenesis of most organs in the developing mammalian embryo. Despite many advances in understanding core components of the pathway, little is known about how the activity of the Hh pathway is adjusted in organ- and tissue-specific developmental processes. Mutations in EVC or EVC2 disrupt Hh signaling in tooth and bone development. Using mouse models, we show here that Evc and Evc2 are mutually required for localizing to primary cilia and also for maintaining their normal protein levels. Consistent with Evc and Evc2 functioning as a complex, the skeletal phenotypes in either single or double homozygous mutant mice are virtually indistinguishable. Smo translocation to the cilium was normal in Evc2-deficient chondrocytes following Hh activation with the Smo-agonist SAG. However, Gli3 recruitment to cilia tips was reduced and Sufu/Gli3 dissociation was impaired. Interestingly, we found Smo to co-precipitate with Evc/Evc2, indicating that in some cells Hh signaling requires direct interaction of Smo with the Evc/Evc2 complex. Expression of a dominantly acting Evc2 mutation previously identified in Weyer's acrodental dysostosis (Evc2Δ43) caused mislocalization of Evc/Evc2Δ43 within the cilium and also reproduced the Gli3-related molecular defects observed in Evc2(-/-) chondrocytes. Moreover, Evc silencing in Sufu(-/-) cells attenuated the output of the Hh pathway, suggesting that Evc/Evc2 also promote Hh signaling in the absence of Sufu. Together our data reveal that the Hh pathway involves Evc/Evc2-dependent modulations that are necessary for normal endochondral bone formation.

Prognostic value of dual-specificity phosphatase 6 expression in non-small cell lung cancer.

Dual-specificity phosphatase 6 (DUSP6/MKP-3) is a mitogen-activated protein kinase phosphatase that regulates extracellular signal-regulated kinases (ERKs) activity via feedback mechanisms, with an increasingly recognized role in tumour biology. The aim of this study was to explore the role of DUSP6 expression in the prognosis of human non-small cell lung cancer (NSCLC). DUSP6 expression levels were evaluated by real-time quantitative reverse transcription polymerase chain reaction (PCR) in 60 NSCLC samples from patients who underwent pulmonary resection at 12 de Octubre University Hospital. We performed a statistical analysis to investigate the correlation of DUSP6 expression and the clinical outcomes. We found that 66.7% of the tumour samples show the downregulation of DUSP6 at the messenger RNA (mRNA) levels compared to benign epithelial lung tissues and 55% of them show at least twofold downregulation of DUSP6 gene expression. Patients were classified into three groups according to their DUSP6 expression levels and those with very low levels (at least twofold downregulation) had the worst outcomes. Using the value of twice below the mean value in benign epithelial lung tissue as a cutoff, the overall survival of patients with very low DUSP6 levels was significantly lower than that in the rest of patients (31.9 ± 18.8 months vs. not reached, P = 0.049). This was most pronounced in adenocarcinoma histology and high-stage tumour samples. Our results suggest that DUSP6 gene expression in tumour samples may be a prognostic marker in NSCLC.

The kinetic analysis of the substrate specificity of motif 5 in a HAD hydrolase-type phosphosugar phosphatase of Arabidopsis thaliana.

The Arabidopsis thaliana gene AtSgpp (locus tag At2g38740), encodes a protein whose sequence motifs and expected structure reveal that it belongs to the HAD hydrolases subfamily I, with the C1-type cap domain (Caparrós-Martín et al. in Planta 237:943-954, 2013). In the presence of Mg(2+) ions, the enzyme has a phosphatase activity over a wide range of phosphosugar substrates. AtSgpp promiscuity is preferentially detectable on D-ribose-5-phosphate, 2-deoxy-D-ribose-5-phosphate, 2-deoxy-D-glucose-6-phosphate, D-mannose-6-phosphate, D-fructose-1-phosphate, D-glucose-6-phosphate, DL-glycerol-3-phosphate, and D-fructose-6-phosphate. Site-directed mutagenesis analysis of the putative signature sequence motif-5 (IAGKH), which defines its specific chemistry, brings to light the active-site residues Ala-69 and His-72. Mutation A69M, changes the pH dependence of AtSgpp catalysis, and mutant protein AtSgpp-H72K was inactive in phosphomonoester dephosphorylation. It was also observed that substitutions I68M and K71R slightly affect the substrate specificity, while the replacement of the entire motif for that of homologous DL-glycerol-3-phosphatase AtGpp (MMGRK) does not switch AtSgpp activity to the specific targeting for DL-glycerol-3-phosphate.