Magnetic Aerogels for Room-Temperature Catalytic Production of Bis(indolyl)methane Derivatives.

The potential of aerogels as catalysts for the synthesis of a relevant class of bis-heterocyclic compounds such as bis(indolyl)methanes was investigated. In particular, the studied catalyst was a nanocomposite aerogel based on nanocrystalline nickel ferrite (NiFe2O4) dispersed on amorphous porous silica aerogel obtained by two-step sol-gel synthesis followed by gel drying under supercritical conditions and calcination treatments. It was found that the NiFe2O4/SiO2 aerogel is an active catalyst for the selected reaction, enabling high conversions at room temperature, and it proved to be active for three repeated runs. The catalytic activity can be ascribed to both the textural and acidic features of the silica matrix and of the nanocrystalline ferrite. In addition, ferrite nanocrystals provide functionality for magnetic recovery of the catalyst from the crude mixture, enabling time-effective separation from the reaction environment. Evidence of the retention of species involved in the reaction into the catalyst is also pointed out, likely due to the porosity of the aerogel together with the affinity of some species towards the silica matrix. Our work contributes to the study of aerogels as catalysts for organic reactions by demonstrating their potential as well as limitations for the room-temperature synthesis of bis(indolyl)methanes.

Seroepidemiological and biomolecular survey on Toxoplasma gondii in Sardinian wild boar (Sus scrofa).

Toxoplasma gondii is a zoonotic parasite able of infecting all warm-blooded animals. Toxoplasmosis is one of the major foodborne diseases globally. The consumption of wild boar (Sus scrofa) meat from recreational hunting has been linked to outbreaks of human toxoplasmosis. The island of Sardinia (Italy) contains a large wild boar population, thus providing an opportunity to assess the distribution of Toxoplasma in this species and the associated risks of transmission to humans. A total of 562 wild boars were screened: heart and meat juice samples were tested for T. gondii DNA via nested-PCR and IgG anti-Toxoplasma by commercial ELISA. Anti-Toxoplasma IgG were detected in 24.6% (138/562) of animals, while 37.2% (209/562) of the heart samples were PCR positive. The prevalence of T. gondii antibodies and DNA highlights the potential role of wild boar as an important reservoir for this parasite. The study suggests that wild boar could play a significant role in spreading the parasite to humans. As wild boar numbers are increasing throughout their range, their potential role in transmitting toxoplasmosis should be communicated to stakeholders, and the impact of different population control methods on disease transmission should be thoroughly assessed to mitigate potential threats effectively.

The groundbreaking impact of digitalization and artificial intelligence in sheep farming.

The integration of digitalization and Artificial Intelligence (AI) has marked the onset of a new era of efficient sheep farming in multiple aspects ranging from the general well-being of sheep to advanced web-based management applications. The resultant improvement in sheep health and consequently better farming yield has already started to benefit both farmers and veterinarians. The predictive analytical models embedded with machine learning (giving sense to machines) has helped better decision-making and has enabled farmers to derive most out of their farms. This is evident in the ability of farmers to remotely monitor livestock health by wearable devices that keep track of animal vital signs and behaviour. Additionally, veterinarians now employ advanced AI-based diagnostics for efficient parasite detection and control. Overall, digitalization and AI have completely transformed traditional farming practices in livestock animals. However, there is a pressing need to optimize digital sheep farming, allowing sheep farmers to appreciate and adopt these innovative systems. To fill this gap, this review aims to provide available digital and AI-based systems designed to aid precision farming of sheep, offering an up-to-date understanding on the subject. Various contemporary techniques, such as sky shepherding, virtual fencing, advanced parasite detection, automated counting and behaviour tracking, anomaly detection, precision nutrition, breeding support, and several mobile-based management applications are currently being utilized in sheep farms and appear to be promising. Although artificial intelligence and machine learning may represent key features in the sustainable development of sheep farming, they present numerous challenges in application.

Combination of mutations in genes controlling DNA repair and high mutational load plays a prognostic role in pancreatic ductal adenocarcinoma (PDAC): a retrospective real-life study in Sardinian population.

BACKGROUND: Patients with pancreatic ductal adenocarcinoma (PDCA) carrying impaired mismatch repair mechanisms seem to have an outcome advantage under treatment with conventional chemotherapy, whereas the role for the tumor mutation burden on prognosis is controversial. In this study, we evaluated the prognostic role of the mutated genes involved in genome damage repair in a real-life series of PDAC patients in a hospital-based manner from the main Institution deputed to surgically treat such a disease in North Sardinia. METHODS: A cohort of fifty-five consecutive PDAC patients with potentially resectable/border line resectable PDAC (stage IIB-III) or oligometastatic disease (stage IV) and tumor tissue availability underwent next-generation sequencing (NGS)-based analysis using a panel containing driver oncogenes and tumor suppressor genes as well as genes controlling DNA repair mechanisms. RESULTS: Genes involved in the both genome damage repair (DR) and DNA mismatch repair (MMR) were found mutated in 17 (31%) and 15 (27%) cases, respectively. One fourth of PDAC cases (14/55; 25.5%) carried tumors presenting a combination of mutations in repair genes (DR and MMR) and the highest mutation load rates (MLR-H). After correction for confounders (surgery, adjuvant therapy, stage T, and metastasis), multivariate Cox regression analysis indicated that mutations in DR genes (HR = 3.0126, 95% CI 1.0707 to 8.4764, p = 0.0367) and the MLR (HR = 1.0018, 95%CI 1.0005 to 1.0032, p = 0.009) were significantly related to worse survival. CONCLUSIONS: The combination of mutated repair genes and MLR-H, which is associated with a worse survival in our series of PDAC patients treated with conventional chemotherapy protocols, might become a predictive biomarker of response to immunotherapy in addition to its prognostic role in predicting survival.

Individual Differences on Wellbeing Indices during the COVID-19 Quarantine in Greece: A National Study.

The impact of COVID-19 and the associated lockdown measures on people's physical and mental wellbeing, as well as their daily lives and functioning, has been extensively studied. This study takes the approach of investigating the consequences of COVID-19 on a national scale, considering sociodemographic factors. The main objective is to make a contribution to ongoing research by specifically examining how age, gender, and marital status influence the overall impact of COVID-19 and wellbeing indicators during the second lockdown period that was implemented in response to the COVID-19 pandemic in the Greek population. The study involved a sample of 16,906 individuals of all age groups in Greece who completed an online questionnaire encompassing measurements related to personal wellbeing, the presence and search for meaning in life, positive relationships, as well as symptoms of depression, anxiety, and stress. Additionally, to gauge the levels of the perceived COVID-19-related impact, a valid and reliable scale was developed. The results reveal that a higher perception of COVID-19 consequences is positively associated with psychological symptoms and the search for meaning in life, while being negatively correlated with personal wellbeing and the sense of meaning in life. In terms of individual differences, the findings indicate that unmarried individuals, young adults, and females tend to report higher levels of psychological symptoms, a greater search for meaning in life, and a heightened perception of COVID-19-related impact. These findings are analyzed in depth, and suggestions for potential directions for future research are put forth.

Cumulative Negative Impacts of Invasive Alien Species on Marine Ecosystems of the Aegean Sea.

Biological invasions are a human-induced environmental disturbance that can cause major changes in ecosystem structure and functioning. Located in the northeastern Mediterranean basin, the Aegean Sea is a hotspot of biological invasions. Although the presence of alien species in the Aegean has been studied and monitored, no assessment has been conducted on their cumulative impacts on native biodiversity. To address this gap, we applied the CIMPAL index, a framework developed for mapping the cumulative impacts of invasive species, to identify the most affected areas and habitat types and determine the most invasive species in the region. Coastal areas showed stronger impacts than the open sea. The highest CIMPAL scores were four times more frequent in the South than in the North Aegean. Shallow (0-60 m) hard substrates were the most heavily impacted habitat type, followed by shallow soft substrates and seagrass meadows. We identified Caulerpa cylindracea, Lophocladia lallemandii, Siganus luridus, Siganus rivulatus, and Womersleyella setacea as the most impactful species across their range of occurrence in the Aegean but rankings varied depending on the habitat type and impact indicator applied. Our assessment can support marine managers in prioritizing decisions and actions to control biological invasions and mitigate their impacts.

Laboratory associated zoonotic parasitic infections: a review of main agents and biosecurity measures.

Laboratory workers are exposed to the risk of acquiring infections due to the manipulation of infectious materials. The biological hazard for researchers is seven times higher when compared with hospital and public health laboratory workers. Despite the implementation of standardized practices to control infections, multiple cases of Laboratory Associated Infections (LAIs) usually go unreported. There has been a lack of comprehensive epidemiological data regarding the situation of LAIs for parasitic zoonosis and besides, the available sources are not completely updated. Since most accounts of laboratory infections are organism-specific, this study has focused on common pathogenic/zoonotic species handled at parasitological laboratories and summarising the standard biosecurity protocols for the infectious agents. The main characteristics of Cryptosporidium spp., Entamoeba spp, Giardia duodenalis, Toxoplasma gondii, Leishmania spp., Echinococcus spp., Schistosoma spp., Toxocara canis, Ancylostoma caninum, Strongyloides stercoralis are considered in this review in order to assess the potential risk of developing occupational infections in the workplace along with stating prevention and prophylactic measures for each species. It was concluded that the LAIs from these agents can be prevented by using personal protective measures and good laboratory practices. However, further studies are necessary to better understand the environmental resistance of cysts, oocysts and eggs, with a view to select the most suitable disinfection methods. Furthermore, it is fundamental to constantly update epidemiological data of infection acquired by laboratory workers, to develop accurate risk indicators.

BRAFV600 variant allele frequency predicts outcome in metastatic melanoma patients treated with BRAF and MEK inhibitors.

BACKGROUND: The prognostic impact of variant allele frequency (VAF) on clinical outcome in BRAFV600 mutated metastatic melanoma patients (MMPs) receiving BRAF (BRAFi) and MEK inhibitors (MEKi) is unclear. MATERIALS AND METHODS: A cohort of MMPs receiving first line BRAFi and MEKi was identified by inspecting dedicated databases of three Italian Melanoma Intergroup centres. VAF was determined by next generation sequencing in pre-treatment baseline tissue samples. Correlation between VAF and BRAF copy number variation was analysed in an ancillary study by using a training and a validation cohort of melanoma tissue samples and cell lines. RESULTS: Overall, 107 MMPs were included in the study. The VAF cut-off determined by ROC curve was 41.3%. At multivariate analysis, progression-free survival (PFS) was significantly shorter in patients with M1c/M1d [HR 2.25 (95% CI 1.41-3.6, p < 0.01)], in those with VAF >41.3% [HR 1.62 (95% CI 1.04-2.54, p < 0.05)] and in those with ECOG PS ≥1 [HR 1.82 (95% CI 1.15-2.88, p < 0.05)]. Overall survival (OS) was significantly shorter in patients with M1c/M1d [HR 2.01 (95% CI 1.25-3.25, p < 0.01)]. Furthermore, OS was shorter in patients with VAF >41.3% [HR 1.46 (95% CI 0.93-2.29, p = 0.06)] and in patients with ECOG PS ≥1 [HR 1.52 (95% CI 0.94-2.87, p = 0.14)]. BRAF gene amplification was found in 11% and 7% of samples in the training and validation cohort, respectively. CONCLUSIONS: High VAF is an independent poor prognostic factor in MMP receiving BRAFi and MEKi. High VAF and BRAF amplification coexist in 7%-11% of patients.

ECOfast - An integrative ecological evaluation index for an ecosystem-based assessment of shallow rocky reefs.

The degradation of marine ecosystems is a growing concern worldwide, emphasizing the need for efficient tools to assess their ecological status. Herein, a novel ecosystem-based ecological evaluation index of shallow rocky reefs is introduced and tested in the Aegean and Ionian Seas (NE Mediterranean). The index focuses on a specific set of pre-selected species, including habitat-forming, key, commercially important, and non-indigenous species, across a wide range of trophic levels (1.00-4.53). Data acquisition is conducted through rapid non-destructive SCUBA diving surveys to assess all macroscopic food web components (macroalgae, invertebrates, and fish). Two versions of the index, ECOfast and ECOfast-NIS, were developed, each applying a different approach to account for the impact of non-indigenous species. In our case study, the correlations between the two versions of the index and sea surface temperature, protection status, occurrence of carnivorous fish, and non-indigenous herbivores were assessed through generalized additive models (GAMs). The assessment assigned 93% (ECOfast) or 96% (ECOfast-NIS) of the sites to a moderate to bad ecological status, indicating an alarming situation in the shallow rocky reefs of the NE Mediterranean. Sites evaluated as poor or bad were characterized by extensive coverage of ephemeral macroalgae, absence or minimal presence of large indigenous carnivorous fish, and complete absence of one to three out of five invertebrate functional trophic groups. The community composition of macroalgae, herbivorous species, and carnivorous fishes differed between the 5 m and 15 m depth zones. Surface temperature and carnivorous fish occurrence were the most important tested predictors of the ecological status of shallow rocky reefs. The best GAMs showed that the ECOfast score declined with sea surface temperature and increased with the occurrence of carnivorous fish; ECOfast-NIS declined with sea surface temperature and the occurrence of non-indigenous fish and increased with the occurrence of carnivorous fish. The non-destructive and integrative nature of this approach, its speed of data acquisition and analysis, and its capacity to account for highly mobile predatory fish and non-indigenous species render the ECOfast index a novel, robust, and valuable tool for assessing the ecological status of shallow rocky reefs.

First report of Echinococcus ortleppi and genotype G6 of E. canadensis cluster from southern Punjab, Pakistan and a global overview on genetic structure and host adaptation of E. ortleppi.

Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato, is a neglected tropical disease known mainly for its zoonotic nature. CE is endemic to Pakistan, however, the disease is not given due consideration and millions of people remain at health risk. This study was undertaken to assess the species and genotypes of E. granulosus sensu lato in sheep, buffaloes and cattle, brought to slaughterhouses of two major cities (Multan and Bahawalpur) of south Punjab, Pakistan. A total of 26 hydatid cyst specimens were characterized through complete cox1 mitochondrial gene (1609 bp) sequencing. Species and genotypes of E. granulosus sensu lato discovered in the southern Punjab consisted of E. granulosus sensu stricto (n =21), E. ortleppi (n=4) and genotype G6 of the E. canadensis cluster (n=1). Of E. granulosus s.s. isolates, the genotype G3 was predominantly involved in causing infections to the livestock of this region. Since all of these species are zoonotic, wide and effective surveillance studies are required to ascertain the risks to human population in Pakistan. Additionally, a global overview on cox1 phylogenetic structure of E. ortleppi was carried out. Despite widespread occurrence, the species is mostly limited to the southern hemisphere. The highest burden has been reported in South America (62.15%) and Africa (28.44%) and by far the most common host is cattle, accounting for >90% of cases.

Mammary-like adenocarcinoma of the vulva: a rare case report with next generation sequencing.

Vulvar adenocarcinomas are rare tumors, representing approximately 5% of vulvar cancers. Mammary-like adenocarcinomas of the vulva (MLAV) are extremely rare, and their molecular features are poorly described in the scientific literature. We report a case of an 88-year-old woman affected by MLAV with comedo-like features, with a detailed description of the pathological, immunohistochemical and molecular features. Immunohistochemistry (IHC) showed strong staining for cytokeratin 7, GATA3, androgen receptor, GCFPD15, and weak staining for mammaglobin; no staining for Her-2 was found. The proliferation index (Ki-67) was 15%. Molecular testing detected a pathogenic mutation of the AKT1 gene, a likely pathogenic frameshift insertion of the JAK1 gene, and two likely pathogenic frameshift deletions of the KMT2C gene; in addition, two variants of unknown significance (VUS) involving the ARID1A and OR2T4 genes were detected. Finally, two CNVs of the BRCA1 gene were identified.

Comparison between Three Different Techniques for the Detection of EGFR Mutations in Liquid Biopsies of Patients with Advanced Stage Lung Adenocarcinoma.

Oncogenic mutations in the EGFR gene are targets of tyrosine kinase inhibitors (TKIs) in lung adenocarcinoma (LC) patients, and their search is mandatory to make decisions on treatment strategies. Liquid biopsy of circulating tumour DNA (ctDNA) is increasingly used to detect EGFR mutations, including main activating alterations (exon 19 deletions and exon 21 L858R mutation) and T790M mutation, which is the most common mechanism of acquired resistance to first- and second-generation TKIs. In this study, we prospectively compared three different techniques for EGFR mutation detection in liquid biopsies of such patients. Fifty-four ctDNA samples from 48 consecutive advanced LC patients treated with TKIs were tested for relevant EGFR mutations with Therascreen® EGFR Plasma RGQ-PCR Kit (Qiagen). Samples were subsequently tested with two different technologies, with the aim to compare the EGFR detection rates: real-time PCR based Idylla™ ctEGFR mutation assay (Biocartis) and next-generation sequencing (NGS) system with Ion AmpliSeq Cancer Hotspot panel (ThermoFisher). A high concordance rate for main druggable EGFR alterations was observed with the two real-time PCR-based assays, ranging from 100% for T790M mutation to 94% for L858R variant and 85% for exon 19 deletions. Conversely, lower concordance rates were found between real-time PCR approaches and the NGS method (L858R: 88%; exon19-dels: 74%; T790M: 37.5%). Our results evidenced an equivalent detection ability between PCR-based techniques for circulating EGFR mutations. The NGS assay allowed detection of a wider range of EGFR mutations but showed a poor ability to detect T790M.

'Fight the parasite': raising awareness of cystic echinococcosis in primary school children in endemic countries.

BACKGROUND: Cystic echinococcosis (CE) is a widespread zoonosis and a significant economic concern and cause of morbidity in humans. A scarcity of education on the sources of CE infection and containment measures is considered to be a key factor responsible for persistent transmission within communities. Recently, edutainment approaches have captured the attention of health education (HE) professionals due to the benefits of integrating cognitive and emotional learning processes. METHODS: A study was carried out in Sardinia, Italy, between 2020 and 2022, amid the SARS-Covid-19 pandemic. The project, designed to involve primary school children (via remote or face-to-face learning depending on the evolving Covid-19 containment measures) consisted of four distinct phases: (i) creation of material for school children and teachers focused on cystic echinococcosis; (ii) pre-intervention evaluation of CE knowledge (i.e. True False Don't Know [TFD] pre-intervention questionnaire based on CE-related knowledge and practices); (iii) edutainment activity (e.g. interactive lessons enhanced by the comic booklet and the "Fight the parasite" cartoon video, hands-on educational activities and drawing activities on CE); and (iv) post-intervention evaluation of CE knowledge (via TFD post-intervention questionnaire [same questionnaire as used for the pre-intervention assessment] on CE-related knowledge and practices) and on-site edutainment tour in primary schools taking part to the project. RESULTS: The percentage of correct answers increased from 65% for the questionnaire administered pre-intervention to 87.9% for the same questionnaire administered post-intervention (χ2 = 648.12, df = 1, P < 0.0001), while the percentage of uncertain answers (i.e. 'I don't know') decreased from 23% pre-intervention to 5% post-intervention (χ2 = 603.44, df = 1, P < 0.0001). These differences indicate a significantly enhanced understanding of CE among participating school children after the intervention. CONCLUSIONS: The results of the present survey indicate that the use of digital educational tools, the use of video animations as a model for science communication, as well as other participatory teaching methods, enabled children to retain key knowledge of the routes of CE transmission and ways to prevent it.

Landscape of immune-related signatures induced by targeting of different epigenetic regulators in melanoma: implications for immunotherapy.

BACKGROUND: Improvement of efficacy of immune checkpoint blockade (ICB) remains a major clinical goal. Association of ICB with immunomodulatory epigenetic drugs is an option. However, epigenetic inhibitors show a heterogeneous landscape of activities. Analysis of transcriptional programs induced in neoplastic cells by distinct classes of epigenetic drugs may foster identification of the most promising agents. METHODS: Melanoma cell lines, characterized for mutational and differentiation profile, were treated with inhibitors of DNA methyltransferases (guadecitabine), histone deacetylases (givinostat), BET proteins (JQ1 and OTX-015), and enhancer of zeste homolog 2 (GSK126). Modulatory effects of epigenetic drugs were evaluated at the gene and protein levels. Master molecules explaining changes in gene expression were identified by Upstream Regulator (UR) analysis. Gene set enrichment and IPA were used respectively to test modulation of guadecitabine-specific gene and UR signatures in baseline and on-treatment tumor biopsies from melanoma patients in the Phase Ib NIBIT-M4 Guadecitabine + Ipilimumab Trial. Prognostic significance of drug-specific immune-related genes was tested with Timer 2.0 in TCGA tumor datasets. RESULTS: Epigenetic drugs induced different profiles of gene expression in melanoma cell lines. Immune-related genes were frequently upregulated by guadecitabine, irrespective of the mutational and differentiation profiles of the melanoma cell lines, to a lesser extent by givinostat, but mostly downregulated by JQ1 and OTX-015. GSK126 was the least active drug. Quantitative western blot analysis confirmed drug-specific modulatory profiles. Most of the guadecitabine-specific signature genes were upregulated in on-treatment NIBIT-M4 tumor biopsies, but not in on-treatment lesions of patients treated only with ipilimumab. A guadecitabine-specific UR signature, containing activated molecules of the TLR, NF-kB, and IFN innate immunity pathways, was induced in drug-treated melanoma, mesothelioma and hepatocarcinoma cell lines and in a human melanoma xenograft model. Activation of guadecitabine-specific UR signature molecules in on-treatment tumor biopsies discriminated responding from non-responding NIBIT-M4 patients. Sixty-five % of the immune-related genes upregulated by guadecitabine were prognostically significant and conferred a reduced risk in the TCGA cutaneous melanoma dataset. CONCLUSIONS: The DNMT inhibitor guadecitabine emerged as the most promising immunomodulatory agent among those tested, supporting the rationale for usage of this class of epigenetic drugs in combinatorial immunotherapy approaches.

Marine heatwaves drive recurrent mass mortalities in the Mediterranean Sea.

Climate change is causing an increase in the frequency and intensity of marine heatwaves (MHWs) and mass mortality events (MMEs) of marine organisms are one of their main ecological impacts. Here, we show that during the 2015-2019 period, the Mediterranean Sea has experienced exceptional thermal conditions resulting in the onset of five consecutive years of widespread MMEs across the basin. These MMEs affected thousands of kilometers of coastline from the surface to 45 m, across a range of marine habitats and taxa (50 taxa across 8 phyla). Significant relationships were found between the incidence of MMEs and the heat exposure associated with MHWs observed both at the surface and across depths. Our findings reveal that the Mediterranean Sea is experiencing an acceleration of the ecological impacts of MHWs which poses an unprecedented threat to its ecosystems' health and functioning. Overall, we show that increasing the resolution of empirical observation is critical to enhancing our ability to more effectively understand and manage the consequences of climate change.

A survey on Apicomplexa protozoa in sheep slaughtered for human consumption.

Infections with the Apicomplexa Toxoplasma gondii, Neospora caninum, and Sarcocystis spp. are common causes of reproductive disorders in sheep. However, few epidemiological studies regarding co-infections with these three protozoa are reported in sheep in Italy. For this reason, this study aims to evaluate possible co-infections with T. gondii, N. caninum, and Sarcocystis spp. in sheep slaughtered for human consumption. From April to July 2019, individual blood, brain, heart, and diaphragm samples were collected from 138 sheep after slaughtering. The presence of IgG anti-Toxoplasma in serum samples was evaluated through ELISA. DNA of the three protozoa was investigated using specific PCRs. Co-infection with T. gondii, N. caninum and Sarcocystis spp. was found in 66.7% of the examined sheep. Antibodies against T. gondii were found in the 36.2% of serum samples. The presence of T. gondii DNA was detected in the 67.4%, 77.5%, and 21.7% of the brain, heart, and diaphragm samples, respectively. Neospora caninum DNA was found in 72.5% of the examined brain samples. Sarcocystis spp. DNA was detected in 92% and 52.2% of the heart and diaphragm samples, respectively. Sequence analysis of the Sarcocystis spp. revealed the sole presence of Sarcocystis tenella. The present study demonstrates that sheep have a high risk of infection with the three Apicomplexa investigated, suggesting the need to adopt adequate measures to prevent the spread of these parasitic infections considering their clinical and economic impact on ovine production. Furthermore, the possible role sheep play in the zoonotic transmission of toxoplasmosis to humans was highlighted.

Global prognostic impact of driver genetic alterations in patients with lung adenocarcinoma: a real-life study.

BACKGROUND: Advanced lung adenocarcinoma (LAC) is one of the most lethal malignancies worldwide. The aim of this study was to evaluate the global survival in a real-life cohort of patients with LAC harboring driver genetic alterations. METHODS: A series of 1282 consecutive Sardinian LAC patients who underwent genetic testing from January 2011 through July 2016 was collected. Molecular tests were based on the clinical needs of each single case (EGFR-exon18/19/21, ALK, and, more recently, BRAF-exon15), and the availability of tissue (KRAS, MET, and presence of low-frequency EGFR-T790M mutated alleles at baseline). RESULTS: The mean follow-up time of the patients was 46 months. EGFR, KRAS, and BRAF mutations were detected in 13.7%, 21.3%, and 3% of tested cases, respectively; ALK rearrangements and MET amplifications were found respectively in 4.7% and 2% of tested cases. As expected, cases with mutations in exons 18-21 of EGFR, sensitizing to anti-EGFR tyrosine kinase inhibitors (TKIs) agents, had a significantly longer survival in comparison to those without (p < 0.0001); conversely, KRAS mutations were associated with a significantly lower survival (p = 0.0058). Among LAC patients with additional tissue section available for next-generation sequencing (NGS)-based analysis, 26/193 (13.5%) patients found positive for even low-rate EGFR-T790M mutated alleles at baseline were associated with a highly significant lower survival in comparison to those without (8.7 vs. 47.4 months, p < 0.0001). CONCLUSIONS: In addition to its predictive value for addressing targeted therapy approaches, the assessment of as more inclusive mutation analysis at baseline may provide clues about factors significantly impacting on global survival in advanced LAC patients.

An integrated assessment of the Good Environmental Status of Mediterranean Marine Protected Areas.

Local, regional and global targets have been set to halt marine biodiversity loss. Europe has set its own policy targets to achieve Good Environmental Status (GES) of marine ecosystems by implementing the Marine Strategy Framework Directive (MSFD) across member states. We combined an extensive dataset across five Mediterranean ecoregions including 26 Marine Protected Areas (MPAs), their reference unprotected areas, and a no-trawl case study. Our aim was to assess if MPAs reach GES, if their effects are local or can be detected at ecoregion level or up to a Mediterranean scale, and which are the ecosystem components driving GES achievement. This was undertaken by using the analytical tool NEAT (Nested Environmental status Assessment Tool), which allows an integrated assessment of the status of marine systems. We adopted an ecosystem approach by integrating data from several ecosystem components: the seagrass Posidonia oceanica, macroalgae, sea urchins and fish. Thresholds to define the GES were set by dedicated workshops and literature review. In the Western Mediterranean, most MPAs are in good/high status, with P. oceanica and fish driving this result within MPAs. However, GES is achieved only at a local level, and the Mediterranean Sea, as a whole, results in a moderate environmental status. Macroalgal forests are overall in bad condition, confirming their status at risk. The results are significantly affected by the assumption that discrete observations over small spatial scales are representative of the total extension investigated. This calls for large-scale, dedicated assessments to realistically detect environmental status changes under different conditions. Understanding MPAs effectiveness in reaching GES is crucial to assess their role as sentinel observatories of marine systems. MPAs and trawling bans can locally contribute to the attainment of GES and to the fulfillment of the MSFD objectives. Building confidence in setting thresholds between GES and non-GES, investing in long-term monitoring, increasing the spatial extent of sampling areas, rethinking and broadening the scope of complementary tools of protection (e.g., Natura 2000 Sites), are indicated as solutions to ameliorate the status of the basin.

NGS-Based Analysis of Atypical Deep Penetrating Nevi.

Deep penetrating nevi (DPNs) are rare melanocytic neoplasms consisting of pigmented spindled or epithelioid melanocytes with a distinctive wedge-shaped configuration showing activation of the WNT pathway, with unusual cyto-architectural features. It is unclear whether they show a distinct genomic profile associated with a diverse metastatic potential. We describe herein a cohort of 21 atypical DPNs analyzed by next-generation sequencing using the Ion AmpliSeq™ Comprehensive Cancer Panel. We found that ß-catenin exon 3 was mutated in 95% and MAP kinase pathway genes in 71% of the cases. Less frequent mutations were observed in HRAS (19%) and MAP2K1 (24%). Isocitrate dehydrogenases 1 (IDH1) mutations, including R132C, V178I, and S278L, were identified in 38% of cases and co-existed with BRAF/HRAS mutations. The only case with progressive nodal disease carried alterations in the ß-catenin pathway and mutations in IDH1 and NRAS (codon 61). By a comprehensive mutation analysis, we found low genetic heterogeneity and a lack of significant associations between specific gene mutations and histopathological features, despite atypical features. Whether the acquisition of an NRAS or IDH1 mutation in an atypical DPN may represent a molecular evolution implying a pathway to melanoma progression should be confirmed in a larger series.

Are Molecular Alterations Linked to Genetic Instability Worth to Be Included as Biomarkers for Directing or Excluding Melanoma Patients to Immunotherapy?

The improvement of the immunotherapeutic potential in most human cancers, including melanoma, requires the identification of increasingly detailed molecular features underlying the tumor immune responsiveness and acting as disease-associated biomarkers. In recent past years, the complexity of the immune landscape in cancer tissues is being steadily unveiled with a progressive better understanding of the plethora of actors playing in such a scenario, resulting in histopathology diversification, distinct molecular subtypes, and biological heterogeneity. Actually, it is widely recognized that the intracellular patterns of alterations in driver genes and loci may also concur to interfere with the homeostasis of the tumor microenvironment components, deeply affecting the immune response against the tumor. Among others, the different events linked to genetic instability-aneuploidy/somatic copy number alteration (SCNA) or microsatellite instability (MSI)-may exhibit opposite behaviors in terms of immune exclusion or responsiveness. In this review, we focused on both prevalence and impact of such different types of genetic instability in melanoma in order to evaluate whether their use as biomarkers in an integrated analysis of the molecular profile of such a malignancy may allow defining any potential predictive value for response/resistance to immunotherapy.