Ferroptosis in Intrahepatic Cholangiocarcinoma: IDH1105GGT Single Nucleotide Polymorphism Is Associated With Its Activation and Better Prognosis.

Objectives: Intrahepatic cholangiocarcinoma (ICC) has a dismal prognosis and often demonstrates an anti-apoptotic landscape, which is a key step to chemotherapy resistance. Isocitrate dehydrogenase 1 or 2 (IDH1-2)-mutated ICCs have been described and associated with better prognosis. Ferroptosis is a regulated iron-mediated cell death induced by glutathione peroxidase 4 (GPX4) inhibition, and may be triggered pharmacologically. GPX4 is overexpressed in aggressive cancers, while its expression is inhibited by IDH1 R132C mutation in cell lines. We investigated tissue expression of ferroptosis activation markers in ICC and its correlation with clinical-pathological features and IDH1-2 status. Materials and Methods: We enrolled 112 patients who underwent hepatic resection or diagnostic liver biopsy for ICC. Immunostaining for transferrin-receptor 1 and GPX4, and Pearls' stain for iron deposits were performed to evaluate ferroptosis activation. Immunostaining for STAT3 was performed to study pro-inflammatory and anti-apoptotic landscape. Main IDH1-2 mutations were investigated in 90 cases by real-time polymerase chain reaction. Results: GPX4 overexpression was seen in 79.5% of cases and related to poor histological prognostic factors (grading and perineural and vascular invasion; p < 0.005 for all) and worse prognosis (OS p = 0.03; DFS p = 0.01). STAT3 was expressed in 95.5% of cases, confirming the inflammation-related anti-apoptotic milieu in ICC, and directly related to GPX4 expression (p < 0.0001). A high STAT3 expression correlated to a worse prognosis (OS p = 0.02; DFS p = 0.001). Nearly 12% of cases showed IDH1 105GGT single nucleotide polymorphism, which was never described in ICC up to now, and was related to lower tumor grade (p < 0.0001), longer overall survival (p = 0.04), and lower GPX4 levels (p = 0.001). Conclusion: Our study demonstrates for the first time that in most inflammatory ICCs ferroptosis is not active, and its triggering is related to IDH1-2 status. This supports the possible therapeutic role of ferroptosis-inducer drugs in ICC patients, especially in drug-resistant cases.

Association of CLDN18 Protein Expression with Clinicopathological Features and Prognosis in Advanced Gastric and Gastroesophageal Junction Adenocarcinomas.

The tight junction protein claudin-18 (CLDN18), is often expressed in various cancer types including gastric (GC) and gastroesophageal adenocarcinomas (GECs). In the last years, the isoform CLDN18.2 emerged as a potential drug target in metastatic GCs, leading to the development of monoclonal antibodies against this protein. CLDN18.2 is the dominant isoform of CLDN18 in normal gastric and gastric cancer tissues. In this work, we evaluated the immunohistochemical (IHC) profile of CLDN18 and its correlation with clinical and histopathological features including p53, E-cadherin, MSH2, MSH6, MLH1, PMS2, HER2, EBER and PD-L1 combined positive score, in a large real-world and mono-institutional series of advanced GCs (n = 280) and GECs (n = 70). The association of IHC results with survival outcomes was also investigated. High membranous CLDN18 expression (2+ and 3+ intensity ≥75%) was found in 117/350 (33.4%) samples analyzed. CLDN18 expression correlated with age <70 (p = 0.0035), positive EBV status (p = 0.002), high stage (III, IV) at diagnosis (p = 0.003), peritoneal involvement (p < 0.001) and lower incidence of liver metastases (p = 0.013). CLDN18 did not correlate with overall survival. The predictive value of response of CLDN18 to targeted agents is under investigation in several clinical trials and further studies will be needed to select patients who could benefit from these therapies.

Detection of HPV16/18 E6 Oncoproteins in Head and Neck Squamous Cell Carcinoma Using a Protein Immunochromatographic Assay.

OBJECTIVES/HYPOTHESIS: The accurate diagnostic assessment of clinically relevant human papillomavirus (HPV) infections in patients with head and neck squamous cell carcinoma represents an urgent unmet medical need. The aim of this study was to determine feasibility, accuracy, and clinical significance of HPV16/18 E6 oncoprotein detection on cytological specimens from oropharyngeal squamous cell carcinoma (OPSCC) and neck lymph node metastasis of SCC from unknown primary tumor (CUP) via a protein immunochromatographic assay. STUDY DESIGN: Cross-sectional study. METHODS: Cytological specimens from primary tumor and neck metastases were collected from 34 patients with OPSCC or CUP and applied to a lateral flow format test that detects HPV16 and HPV18 E6 oncoproteins. E6 oncoprotein positivity or negativity in these specimens was compared to the specimens' "HPV-driven" reference status, defined by presence of HPV-DNA in combination with p16INK4a overexpression and/or HPV E6 seropositivity. RESULTS: Eighteen of 29 OPSCC (62%) and three of five CUP (60%) were HPV-driven according to our reference method. The E6 oncoprotein lateral flow test had a sensitivity of 94% (95% CI: 70%-100%) and a specificity of 100% (95% CI: 66%-100%) on primary tumor, and a sensitivity of 88% (95% CI: 64%-99%) and a specificity of 100% (95% CI: 74%-100%) on neck metastases. Test agreement between the E6 lateral flow test and the clinical reference method, HPV-DNA plus p16INK4a was excellent, both for primary lesion and neck metastases. CONCLUSIONS: We found the detection of HPV16/18 E6 oncoproteins to be a feasible, highly reliable, and low-invasive method to assess "HPV-driven" status in OPSCC and CUP. LEVEL OF EVIDENCE: II Laryngoscope, 131:1042-1048, 2021.

TERT promoter hotspot mutations and their relationship with TERT levels and telomere erosion in patients with head and neck squamous cell carcinoma.

PURPOSE: To evaluate the prevalence of two recurrent somatic mutations (-124 C>T and -146 C>T) within the promoter of the gene encoding telomerase reverse transcriptase (TERT) as well as their relationship with TERT level, telomeres length, and outcome in patients with head and neck squamous cell carcinomas (HNSCCs). METHODS: We evaluate the prevalence of TERT promoter mutations, TERT levels, and telomere length in paired cancer tissue and adjacent mucosa (AM) in a series of HNSCCs. RESULTS: Cancer tissue and AM specimens from 105 patients were analyzed. Telomere length and TERT mRNA levels were estimated using real-time polymerase chain reaction. TERT promoter mutations were assessed using Sanger sequencing. Out of 105 cases, 101 were considered suitable for the analysis. TERT promoter harbored mutations in 12 tumors (11.9%), with -124 C>T and -146 C>T accounting for 83.3% and 16.7% of the alterations, respectively. No mutations were detected in AM samples. The prevalence of TERT promoter mutations was significantly higher in oral cavity SCCs (10 out of 27 tumors; 37%), and telomere length in AM was shorter in patients with tumors carrying TERT promoter mutations than in patients with unmutated TERT promoter cancers (p = 0.023). TERT levels in tumor did not significantly differ according to the mutational status of TERT promoter. No significant association was found between TERT promoter status and overall survival. CONCLUSION: TERT promoter mutations are most likely a late event in tumor development, occurring in a context of critically short telomeres, mostly in patients with oral cavity SCC. TERT levels, but not TERT promoter mutational status impact clinical outcome.

Absence of disruptive TP53 mutations in high-risk human papillomavirus-driven neck squamous cell carcinoma of unknown primary.

BACKGROUND: To enforce the evidence for causality between high-risk human papillomavirus (hrHPV) infections and neck squamous cell carcinoma from unknown primary (NSCCUP) and provide biological basis for treatment de-intensification, we searched for TP53 mutations in association with HPV status. METHODS: TP53 mutations were searched for by amplification of exons 4 to 10. RESULTS: Of the 70 NSCCUP, 27 (39%) harbored HPV infection. TP53 sequencing resulted in the identification of 19 patients harboring single mutations including 16 disruptive alterations (84%). The association of TP53 mutations and HPV could be evaluated in 48 NSCCUP including those with disruptive mutation in any exon (n = 16) and those without mutations but with complete sequence of exons 4 to 9 (n = 32): no disruptive mutations were found in the 17 HPV-driven NSCCUP but in 16 of the 31 non-HPV-driven NSCCUP (P = .0002). CONCLUSION: In a fraction of cases, NSCCUP is an HPV-driven entity harboring wild-type TP53 gene or nondisruptive TP53 mutations. HPV-driven NSCCUP might benefit from treatment de-intensification.

The influence of fertiliser and pesticide emissions model on life cycle assessment of agricultural products: The case of Danish and Italian barley.

Barley is an ancient crop and a great source of nutrients. It is the third largest agricultural commodity produced in Denmark and represents a relevant crop in Italy too. Due to the increasing customers awareness of sustainability issues, it has become essential to evaluate the environmental impact and the use of resources in food production and distribution systems. However, especially in agriculture, difficulties are encountered when emissions from fertilisers and pesticides need to be modelled, due to a variety of modelling options and their dependency on the availability of site-specific information. How to address these difficulties might affect the results reliability. Hence, this study aims to evaluate, using the life cycle assessment (LCA) methodology, the influence of different models for estimating emissions from fertilisers and pesticides on the environmental impacts of barley cultivation in Denmark and Italy. Two models for fertilisers and pesticides' emissions have been applied; these differ on the extent of data requirements and complexity of calculation algorithms, which might increase the results accuracy and robustness. The results show that the modelling options do affect the environmental impacts of barley production, in particular climate change, eutrophication categories, acidification and freshwater eco-toxicity. This study estimates that the variations for such categories range from 15% in the case of climate change to 89% in the case of marine eutrophication. These findings highlight the importance of the emission modelling options as well as the constraints of data requirements, critical aspects when a LCA study on agricultural products is carried out.

Concomitant KIT/BRAF and PDGFRA/BRAF mutations are rare events in gastrointestinal stromal tumors.

AIM: The BRAF mutation is a rare pathogenetic alternative to KIT/PDGFRA mutation in GIST and causes Imatinib resistance. A recent description of KIT and BRAF mutations co-occurring in an untreated GIST has challenged the concept of their being mutually exclusive and may account for ab initio resistance to Imatinib, even in the presence of Imatinib-sensitive KIT mutations. BRAF sequencing is generally limited to KIT/PDGFRA wild-type cases. Hence, the frequency of concomitant mutations may be underestimated. METHODS: We screened for KIT (exon 9, 11 ,13 ,17), PDGFRA (exon 12,14, 18) and BRAF (exon 15) mutations a series of 407 GIST. Additionally, we evaluated the BRAF V600E mutation-specific antibody, VE1, as a surrogate for V600E mutation, on a series of 313 GIST (24 on whole sections, 288 cases on tissue array), including 6 cases molecularly ascertained to carry the BRAF V600E mutation. RESULTS: No concomitant KIT/BRAF or PDGFRA/BRAF mutations were detected. BRAF mutation was detected only in one case, wild-type for KIT/PDGFRA. All the 6 BRAF-mutant cases stained positive with the VE1 antibody. A weak VE1 expression was observed in 14/287 (4.9%) BRAF wild-type cases, as observed also in 2/6 BRAF-mutant cases. Overall in our series, sensitivity and specificity of the VE1 antobody were 100% and 95.1%, respectively. CONCLUSIONS: The concomitance of BRAF mutation with either KIT or PDGFRA mutation is rare in GIST. In these tumors, moderate/strong VE1 immunoreactivity is a valuable surrogate for molecular analysis. Instead, genotyping is warranted in the presence of weak VE1 staining.

Prognostic significance of circulating and endothelial progenitor cell markers in type 2 diabetic foot.

Objective. We studied circulating precursor cells (CPC) in type 2 diabetes mellitus (T2DM) with neuropathic foot lesions with or without critical limb ischemia and relationships between endothelial precursor cells (EPC) and peripheral neuropathy. Methods and Subjects. We measured peripheral blood CD34, CD133, and CD45 markers for CPC and KDR, CD31 markers for EPC by citofluorimetry and systemic neural nociceptor CGRP (calcitonin gene related protein) by ELISA in 8 healthy controls (C) and 62 T2DM patients: 14 with neuropathy (N), 20 with neuropathic foot lesions (N1), and 28 with neuroischemic recent revascularized (N2) foot lesions. Timing of lesions was: acute (until 6 weeks), healed, and not healed. Results. CD34+ and CD133+ were reduced in N, N1, and N2 versus C, and CD34+ were lower in N2 versus N1 (P = 0.03). In N2 CD34+KDR+ remain elevated in healed versus chronic lesions and, in N1 CD133+31+ were elevated in acute lesions. CGRP was reduced in N2 and N1 versus C (P < 0.04 versus C 26 ± 2 pg/mL). CD34+KDR+ correlated in N2 with oximetry and negatively in N1 with CGRP. Conclusions. CD34+ CPC are reduced in diabetes with advanced complications and diabetic foot. CD34+KDR+ and CD31+133+ EPC differentiation could have a prognostic and therapeutic significance in the healing process of neuropathic and neuroischemic lesions.

Review of LCA studies of solid waste management systems--part II: methodological guidance for a better practice.

Life cycle assessment (LCA) is increasingly used in waste management to identify strategies that prevent or minimise negative impacts on ecosystems, human health or natural resources. However, the quality of the provided support to decision- and policy-makers is strongly dependent on a proper conduct of the LCA. How has LCA been applied until now? Are there any inconsistencies in the past practice? To answer these questions, we draw on a critical review of 222 published LCA studies of solid waste management systems. We analyse the past practice against the ISO standard requirements and the ILCD Handbook guidelines for each major step within the goal definition, scope definition, inventory analysis, impact assessment, and interpretation phases of the methodology. Results show that malpractices exist in several aspects of the LCA with large differences across studies. Examples are a frequent neglect of the goal definition, a frequent lack of transparency and precision in the definition of the scope of the study, e.g. an unclear delimitation of the system boundaries, a truncated impact coverage, difficulties in capturing influential local specificities such as representative waste compositions into the inventory, and a frequent lack of essential sensitivity and uncertainty analyses. Many of these aspects are important for the reliability of the results. For each of them, we therefore provide detailed recommendations to practitioners of waste management LCAs.

Review of LCA studies of solid waste management systems--part I: lessons learned and perspectives.

The continuously increasing solid waste generation worldwide calls for management strategies that integrate concerns for environmental sustainability. By quantifying environmental impacts of systems, life cycle assessment (LCA) is a tool, which can contribute to answer that call. But how, where and to which extent has it been applied to solid waste management systems (SWMSs) until now, and which lessons can be learnt from the findings of these LCA applications? To address these questions, we performed a critical review of 222 published LCA studies of SWMS. We first analysed the geographic distribution and found that the published studies have primarily been concentrated in Europe with little application in developing countries. In terms of technological coverage, they have largely overlooked application of LCA to waste prevention activities and to relevant waste types apart from household waste, e.g. construction and demolition waste. Waste management practitioners are thus encouraged to abridge these gaps in future applications of LCA. In addition to this contextual analysis, we also evaluated the findings of selected studies of good quality and found that there is little agreement in the conclusions among them. The strong dependence of each SWMS on local conditions, such as waste composition or energy system, prevents a meaningful generalisation of the LCA results as we find it in the waste hierarchy. We therefore recommend stakeholders in solid waste management to regard LCA as a tool, which, by its ability of capturing the local specific conditions in the modelling of environmental impacts and benefits of a SWMS, allows identifying critical problems and proposing improvement options adapted to the local specificities.