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•Rapid targeted genotyping of CSF prospectively detected hallmark single-nucleotide variants in 42% of patients with new CNS tumors.•Integrating this approach in a CLIA environment accelerated times to diagnosis and treatment for patients with newly diagnosed CNS lymphoma.

Coral reef metabolism is dominated by benthic photoautotrophic communities that comprise varying combinations of algae, coral, and sand. Rates of daily gross primary production (GPP) for these benthic functional types (BFTs) are remarkably consistent across biogeographical regions, supporting the idea that reefs exhibit modal metabolism. Most variability in reported rates likely arises from differences in light availability. In fact, GPP is a linear function of incident photosynthetically active radiation (PAR), the fraction of PAR absorbed (fAPAR) by photoautotrophic organisms or communities, and light‐use efficiency (ε), which parameterizes photosynthesizers' biochemical capacity for CO2fixation: GPP = ε× fAPAR × PAR. On time scales of days to weeks, fAPAR and εare far more stable than PAR. εis a critical parameter, because it represents productive response integrated across all environmental conditions, other than light. If BFTs exhibit consistent GPP across wide geographic ranges, then their εs must also be consistent. The aim of this study was to estimate εfor algae, coral, and sand. Using data collected during NASA's CORAL mission in 2016–2017, εwas calculated for 32 mixed communities at Lizard Island, Australia (10); Kāne'ohe Bay, Hawai'i (8); Guam (6); and Palau (8). Nonnegative least squares was used to solve for εof each BFT, producing values of 0.038, 0.060, and 0.016 C photon−1for algae, coral, and sand, respectively. These values can be used in light‐driven models of reef metabolism. Further work is necessary to refine these estimates and, importantly, to explore how εis affected by environmental conditions.

Introduction: Biomarker testing is mandatory for the clinical management of patients with advanced non-small cell lung cancer (NSCLC). Myriads of technical platforms are now available for biomarker analysis with differences in terms of multiplexing capability, analytical sensitivity, and turnaround time (TAT). We evaluated the technical performance of the diagnostic workflows of 24 representative Italian institutions performing molecular tests on a series of artificial reference specimens built to mimic routine diagnostic samples. Methods: Sample sets of eight slides from cell blocks of artificial reference specimens harboring exon 19 EGFR(epidermal growth factor receptor) p.E746_AT50del, exon 2 KRAS(Kirsten rat sarcoma viral oncogene homologue) p.G12C, ROS1(c-ros oncogene 1)-unknown gene fusion, and MET(MET proto-oncogene, receptor tyrosine kinase) Δ exon 14 skipping were distributed to each participating institution. Two independent cell block specimens were validated by the University of Naples Federico II before shipment. Methodological and molecular data from reference specimens were annotated. Results: Overall, a median DNA concentration of 3.3 ng/µL (range 0.1–10.0 ng/µL) and 13.4 ng/µL (range 2.0–45.8 ng/µL) were obtained with automated and manual technical procedures, respectively. RNA concentrations of 5.7 ng/µL (range 0.2–11.9 ng/µL) and 9.3 ng/µL (range 0.5–18.0 ng/µL) were also detected. KRASexon 2 p.G12C, EGFRexon 19 p.E736_A750del hotspot mutations, and ROS1aberrant transcripts were identified in all tested cases, whereas 15 out of 16 (93.7%) centers detected METexon 14 skipping mutation. Conclusions: Optimized technical workflows are crucial in the decision-making strategy of patients with NSCLC. Artificial reference specimens enable optimization of diagnostic workflows for predictive molecular analysis in routine clinical practice.

Introduction:PIK3CAgene mutations occur in approximately 40% of hormone receptor-positive/HER2-negative (HR+/HER2−) metastatic breast cancers (MBCs), electing them to targeted therapy. Testing PIK3CAstatus is complex due to selection of biological specimen and testing method. Materials & methods:This work investigates real-life experience on PIK3CAtesting in HR+/HER2−MBC. Clinical, technical and molecular data on PIK3CAtesting were collected from two referral laboratories. Additionally, the results of a nationwide PIK3CAsurvey involving 116 institutions were assessed. Results:Overall, n = 35 MBCs were PIK3CA-mutated, with mutations mostly occurring in exons 9 (n = 19; 51.4%) and 20 (n = 15; 40.5%). The nationwide survey revealed significant variability across laboratories in terms of sampling methodology, technical assessment and clinical report signing healthcare figures for PIK3CAmolecular testing in diagnostic routine practice. Conclusion:This study provides insights into the real-world routine of PIK3CAtesting in HR+/HER2−MBC and highlights the need for standardization and networking in predictive pathology.

Craniosynostosis is a group of disorders of premature calvarial suture fusion. The identity of the calvarial stem cells (CSCs) that produce fusion-driving osteoblasts in craniosynostosis remains poorly understood. Here we show that both physiologic calvarial mineralization and pathologic calvarial fusion in craniosynostosis reflect the interaction of two separate stem cell lineages; a previously identified cathepsin K (CTSK) lineage CSC1(CTSK+CSC) and a separate discoidin domain-containing receptor 2 (DDR2) lineage stem cell (DDR2+CSC) that we identified in this study. Deletion of Twist1, a gene associated with craniosynostosis in humans2,3, solely in CTSK+CSCs is sufficient to drive craniosynostosis in mice, but the sites that are destined to fuse exhibit an unexpected depletion of CTSK+CSCs and a corresponding expansion of DDR2+CSCs, with DDR2+CSC expansion being a direct maladaptive response to CTSK+CSC depletion. DDR2+CSCs display full stemness features, and our results establish the presence of two distinct stem cell lineages in the sutures, with both populations contributing to physiologic calvarial mineralization. DDR2+CSCs mediate a distinct form of endochondral ossification without the typical haematopoietic marrow formation. Implantation of DDR2+CSCs into suture sites is sufficient to induce fusion, and this phenotype was prevented by co-transplantation of CTSK+CSCs. Finally, the human counterparts of DDR2+CSCs and CTSK+CSCs display conserved functional properties in xenograft assays. The interaction between these two stem cell populations provides a new biologic interface for the modulation of calvarial mineralization and suture patency.

ABSTRACTIntroductionCollaborative computing has attracted great interest, enabling researchers worldwide to come together and work seamlessly. Its relevance has never been so apparent as during the recent pandemic given that it allows for the strengthening of scientific collaborations whilst avoiding physical interaction.This technology evaluation reviews the MEDIATE initiative, launched by the Exscalate4Cov consortium, which invites researchers to contribute their virtual screening simulations that are then combined with AI-based consensus approaches to provide robust and method-independent predictions. The best compounds are subsequently tested, and the biological results are then shared with the scientific community.Areas coveredIn this paper, the MEDIATE initiative is described. This shares compounds’ libraries and protein structures prepared to perform standardized virtual screenings. Preliminary analyses are also reported which provide encouraging results emphasizing the MEDIATE initiative’s capacity to identify active compounds.Expert opinionStructure-based virtual screening is well-suited for collaborative projects provided that the participating researchers work on the same input file. Until now, such a strategy was rarely pursued and most initiatives in the field were organized as challenges. The MEDIATE platform is focused on SARS-CoV-2 targets but can be seen as a prototype which can be utilized to perform collaborative virtual screening campaigns in any therapeutic field by sharing the appropriate input files.

In the era of personalised medicine, testing for an increasing number of predictive biomarkers is becoming a priority. However, tissue biopsies from these patients are oftentimes insufficient for conventional approaches, a common issue that deprives them of the clinical benefits of biomarker-directed treatments. To tackle this problem, many clinical laboratories are resorting to circulating tumour DNA (ctDNA), which is becoming increasingly appreciated as a valuable source for biomarker testing. In this context, next-generation sequencing (NGS) has become essential. Indeed, different NGS systems are able to detect several clinically relevant low-frequency hot-spot mutations simultaneously in a single run. However, their reproducibility in the analysis of ctDNA has not yet been investigated. The purpose of this study was to evaluate the reproducibility of using Illumina MiSeq and Thermo Fisher Ion S5 Plus platforms to assess pathogenic alterations in non-small cell lung cancer (NSCLC) liquid biopsy specimens. Using the in vitro diagnostic (IVD) NGS panel Myriapod NGS Cancer panel DNA (Diatech Pharmacogenetics) on MiSeq platform (Illumina), we reanalysed ctDNA extracted from a retrospective series of n=40 patients with advanced NSCLC previously tested with a custom NGS panel (SiRe) on Thermo Fisher Ion S5 Plus system. Overall, 13 out of 40 (32.5%) ctDNA samples displayed pathogenic alterations in at least two genes, namely, EGFRand KRAS. A concordance rate of 100% was identified between the two methodologies in terms of sample mutational status and total number of detected variables. All NGS platforms featured a high degree of concordance.

DNA mismatch repair complex is involved in the maintenance of DNA stability. In the recent years, a plethora of technical approaches for microsatellite instability (MSI) analysis emerged. Here, we review the results of our MSI status evaluation by adopting a customised workflow on microfluidic system obtained in 4 years of diagnostic routine practice. Data from MSI status were retrieved from our institutional archive covering the period from January 2017 to December 2021. Microfluidic analysis was carried out on microfluidic platform. Results were inspected with a proprietary software. Overall, microsatellite stability (MSS) and MSI-high (MSI-H) profile was detected in n=423/458 (92.36%) and n=35/458 (7.64%) patients with metastatic CRC (mCRC), respectively. In addition, n=78/86 (90.70%) and n=8/86 (9.30%) patients without CRC showed an MSS and MSI-H profile. This review highlights the suitability of microfluidic approach in patients with cancer for MSI testing.

AimsGene fusions assays are key for personalised treatments of advanced human cancers. Their implementation on cytological material requires a preliminary validation that may make use of cell line slides mimicking cytological samples. In this international multi-institutional study, gene fusion reference standards were developed and validated.MethodsCell lines harbouring EML4(13)–ALK(20) and SLC34A2(4)–ROS1(32) gene fusions were adopted to prepare reference standards. Eight laboratories (five adopting amplicon-based and three hybridisation-based platforms) received, at different dilution points two sets of slides (slide A 50.0%, slide B 25.0%, slide C 12.5% and slide D wild type) stained by Papanicolaou (Pap) and May Grunwald Giemsa (MGG). Analysis was carried out on a total of 64 slides.ResultsFour (50.0%) out of eight laboratories reported results on all slides and dilution points. While 12 (37.5%) out of 32 MGG slides were inadequate, 27 (84.4%) out of 32 Pap slides produced libraries adequate for variant calling. The laboratories using hybridisation-based platforms showed the highest rate of inadequate results (13/24 slides, 54.2%). Conversely, only 10.0% (4/40 slides) of inadequate results were reported by laboratories adopting amplicon-based platforms.ConclusionsReference standards in cytological format yield better results when Pap staining and processed by amplicon-based assays. Further investigation is required to optimise these standards for MGG stained cells and for hybridisation-based approaches.

Here, we present a protocol for investigating the non-genetic heterogeneity of membrane proteins expression within murine muscle stem cell (MuSC) population isolated from injured skeletal muscles. We describe a protocol that employs flow cytometry technology to detect variations in membrane CRIPTO protein levels and ensure measurements standardization. We detail steps for muscle digestion, bulk muscle cell staining, and phenotypic analysis. This approach allows for the identification of MuSC fractions with distinct phenotypic and functional properties.

Smaller operative exposures associated with suturectomy for craniosynostosis may result in difficulties visualizing the prematurely fused suture during surgery. The authors report cases of suturectomy for lambdoid and metopic craniosynostosis in which neuronavigation or frameless stereotaxy was used to assist with incision planning and intraoperative localization of the fused suture. In both cases, neuronavigation integrated easily and safely into established workflows and was associated with complete suture release. To our knowledge, this is the first report of applying this noninvasive technology, which does not require cranial pinning or rigid fixation, to suturectomy, and the authors demonstrate its use as an adjunct, especially for surgeons beginning in practice. Larger studies are needed to determine if neuronavigation in suturectomy is associated with a clinically significant reduction in blood loss or operative time or an increase in the rate of complete suturectomy.

AimsTo date, reverse transcriptase PCR (RT-PCR) on nasopharyngeal swabs is the ‘gold standard’ approach for the diagnosis of COVID-19. The need to develop easy to use, rapid, robust and with minimal hands-on time approaches are warranted. In this setting, the Idylla SARS-CoV-2 Test may be a valuable option. The aim of our study is to evaluate the analytical and clinical performance of this assay on previously tested SARS-CoV-2 people by conventional RT-PCR based approach in different settings, including initial diagnosis and clinical follow-up.MethodsTo evaluate the sensitivity and specificity of the Idylla SARS-CoV-2 Test, we retrieved 55 nasopharyngeal swabs, previously analysed by a fully validated assay, from symptomatic patients or from people who have been in close contact with COVID-19 positive cases. Discordant or high discrepant cases were further analysed by a third technique. In addition, a second subset of 14 nasopharyngeal swab samples with uncertain results (cycle threshold between 37 and 40), by using the fully validated assay, from patients with viral infection beyond day 21, were retrieved.ResultsOverall, Idylla showed a sensitivity of 93.9% and a specificity of 100.0%. In addition, in the additional 14 nasopharyngeal swab samples, only five (35.7%) featured a positive result by the Idylla SARS-CoV-2 Test.ConclusionsWe demonstrated that the Idylla SARS-CoV-2 Test may represent a valid, fast, highly sensitive and specific RT-PCR test for the identification of SARS-CoV-2 infection.

AimNext generation sequencing (NGS) represents a key diagnostic tool to identify clinically relevant gene alterations for treatment-decision making in cancer care. However, the complex manual workflow required for NGS has limited its implementation in routine clinical practice. In this worldwide study, we validated the clinical performance of the TargetPlex FFPE-Direct DNA Library Preparation Kit for NGS analysis. Impressively, this new assay obviates the need for separate, labour intensive and time-consuming pre-analytical steps of DNA extraction, purification and isolation from formalin-fixed paraffin embedded (FFPE) specimens in the NGS workflow.MethodsThe TargetPlex FFPE-Direct DNA Library Preparation Kit, which enables NGS analysis directly from FFPE, was specifically developed for this study by TargetPlex Genomics Pleasanton, California. Eleven institutions agreed to take part in the study coordinated by the Molecular Cytopathology Meeting Group (University of Naples Federico II, Naples, Italy). All participating institutions received a specific Library Preparation Kit to test eight FFPE samples previously assessed with standard protocols. The analytical parameters and mutations detected in each sample were then compared with those previously obtained with standard protocols.ResultsOverall, 92.8% of the samples were successfully analysed with the TargetPlex FFPE-Direct DNA Library Preparation Kit on Thermo Fisher Scientific and Illumina platforms. Altogether, in comparison with the standard workflow, the TargetPlex FFPE-Direct DNA Library Preparation Kit was able to detect 90.5% of the variants.ConclusionThe TargetPlex FFPE-Direct DNA Library Preparation Kit combined with the SiRe panel constitutes a convenient, practical and robust cost-saving solution for FFPE NGS analysis in routine practice.

ObjectiveAberrant Rho-associated protein kinase (ROCK) activity is implicated in several vascular and immunologic disorders. We previously demonstrated increased ROCK activity in histopathologically negative temporal artery biopsies (TABs) in subjects with clinical giant cell arteritis (GCA) compared to those without GCA. This current study aimed to examine ROCK activity in a larger cohort of biopsy-negative GCA subjects and to validate the prior findings.Methods.Based on clinical data 6 months after TAB, subjects were categorized into 2 groups: biopsy-negative GCA and controls without GCA. Paraffin-embedded TABs were stained for phosphorylated ezrin/radixin/ moesin (pERM), a surrogate of ROCK activity, and scored by 2 pathologists blinded to clinical diagnosis using a previously derived scoring system measuring staining intensity in 3 areas of the vessel.Results.Thirty-six subjects with biopsy-negative GCA and 43 controls were analyzed. The mean (SD) pERM intensity score in non-GCA subjects was 3.9 (1.4), compared to 5.0 (1.4) in those with GCA (P= 0.002). Using the predetermined cut-off of 4 to define high pERM intensity, subjects with GCA were significantly more likely to have a high pERM intensity score compared to non-GCA (odds ratio 3.67, 95% CI 1.19-11.36; P= 0.02. The sensitivity of high pERM intensity score for diagnosis of GCA in histologically negative TABs was 86% (95% CI 70-95).Conclusion.In this well-characterized cohort, those with biopsy-negative GCA had significantly higher pERM intensity scores compared to subjects without GCA. pERM staining has diagnostic significance in enhancing the sensitivity of TAB and may help to define the clinically important group of biopsy-negative GCA.

AimsIn thyroid cytopathology, the undetermined diagnostic categories still pose diagnostic challenges. Although next-generation sequencing (NGS) is a promising technique for the molecular testing of thyroid fine-needle aspiration (FNA) specimens, access to such technology can be difficult because of its prohibitive cost and lack of reimbursement in countries with universal health coverage. To overcome these issues, we developed and validated a novel custom NGS panel, Nexthyro,specifically designed to target 264 clinically relevant mutations involved in thyroid tumourigenesis. Moreover, in this study, we compared its analytical performance with that of our previous molecular testing strategy.MethodsThe panel, which includes 15 genes (BRAF, EIF1AX, GNAS, HRAS, IDH1, KRAS, NF2, NRAS, PIK3CA, PPM1D, PTEN, RET, DICER1, CHEK2, TERT promoter),was validated with a cell-line derived reference standard and 72 FNA archival samples previously tested with the 7-gene test.ResultsNexthyroyielded 100% specificity and detected mutant alleles at levels as low as 2%. Moreover, in 5/72 (7%) FNAs, it detected more clinically relevant mutations in BRAFand RASgenes compared with the 7-gene test. Nexthyroalso revealed better postsequencing metrics than the previously adopted commercial ‘generic’ NGS panel.ConclusionOur comparative analysis indicates that Nexthyrois a reliable NGS panel. The study also implies that a custom-based solution for routine thyroid FNA is sustainable at the local level, allowing patients with undetermined thyroid nodules affordable access to NGS.

AimsIn metastatic colorectal carcinomas (mCRC), RAS/RAFgenes mutations are first tested to determine the eligibility for anti-EGFR (Epidermal Growth Factor Receptor) therapy in combination with conventional cytotoxic agents. Recent advancements in next-generation sequencing (NGS) have highlighted the potential of multi-gene panels. This multi-gene analysis may provide useful information for the molecular characterisation of mCRC, other than the status of RAS/RAFgenes. Aim of this study was to evaluate the feasibility of two NGS custom multi-gene panels in the characterisation of CRC cases and evaluating the relevance of PIK3CAmutation in a routine cohort of consecutive CRC cases.MethodsA total of 961 formalin-fixed and paraffin-embedded specimens from two medical centres (Bologna and Naples) were analysed using two lab-developed NGS multi-gene panels.ResultsKRASmutations (56.2%) were the more frequent alterations observed in our cohort. Intriguingly, PIK3CAmutations were more frequent (16.8%) than variants observed in the other two genes nowadays analysed in CRC clinical practice (NRASand BRAF, 4.2% and 9.6%, respectively). Moreover, in more than 10% of samples, coexistent mutations were detected in our cohort of CRC.ConclusionsOur study demonstrates the feasibility and efficacy of lab-developed targeted multi-gene NGS panels in the clinical practice of CRC. Moreover, the data lead to hypothesise that PIK3CAmutations, together with those of RAS/BRAF, worth to be further investigated in clinical CRC specimens.

AimsHeterogeneous implementation of molecular tests in current diagnostic algorithm at a European and international level is emerging as a major issue for efficient lung cancer molecular profiling.MethodsFrom May 2017 until October 2017, N=1612 patients referring to 13 Italian institutions were selected, at advanced stage non-small cell lung cancer (NSCLC), and prospectively evaluated. Principal endpoints were: the percentage of diagnoses performed on cytological and histological material, the proportion of requests for epidermal growth factor receptor (EGFR) mutational status, and resistance mutations detected on tissue and/or liquid biopsy samples after first-generation or second-generation tyrosine kinase inhibitors, the proportion of requests for anaplastic lymphoma kinase (ALK) gene rearrangements, ROS proto-oncogene 1 (ROS1) and Kirsten Rat Sarcoma (KRAS) determinations, the proportion of requests for programmed death-ligand1 (PD-L1) evaluation and, finally, the different assays used for the detection of EGFR mutations, ALK and ROS1 gene rearrangements and PD-L1 expression.ResultsOf 1325 patients finally included, only 50.8% requests were related to driver mutations with target agents already available in first-line at that preplanned time, while 49.2% were associated with PD-L1, ROS1, KRAS and others. Multiplex genomic assays (such as next-generation sequencing) were considered by all participating centres.ConclusionsTo the best of our knowledge, this is the first study in a ‘real-life daily practice’ involving both pathologists and oncologists evaluating routinely workflow and trends towards improvements in molecular requests. Collected data aim to describe the applied algorithms and evolution of molecular screening for stage IV NSCLC in clinical practice.

Diagnosing primary central nervous system lymphoma (PCNSL) frequently requires neurosurgical biopsy due to nonspecific radiologic features and the low yield of cerebrospinal fluid (CSF) studies. We characterized the clinical evaluation of suspected PCNSL (N = 1007 patients) and designed a rapid multiplexed genotyping assay for MYD88, TERT promoter, IDH1/2, H3F3A, and BRAF mutations to facilitate the diagnosis of PCNSL from CSF and detect other neoplasms in the differential diagnosis. Among 159 patients with confirmed PCNSL, the median time to secure a diagnosis of PCNSL was 10 days, with a range of 0 to 617 days. Permanent histopathology confirmed PCNSL in 142 of 152 biopsies (93.4%), whereas CSF analyses were diagnostic in only 15/113 samplings (13.3%). Among 86 archived clinical specimens, our targeted genotyping assay accurately detected hematologic malignancies with 57.6% sensitivity and 100% specificity (95% confidence interval [CI]: 44.1% to 70.4% and 87.2% to 100%, respectively). MYD88 and TERT promoter mutations were prospectively identified in DNA extracts of CSF obtained from patients with PCNSL and glioblastoma, respectively, within 80 minutes. Across 132 specimens, hallmark mutations indicating the presence of malignancy were detected with 65.8% sensitivity and 100% specificity (95% CI: 56.2%-74.5% and 83.9%-100%, respectively). This targeted genotyping approach offers a rapid, scalable adjunct to reduce diagnostic and treatment delays in PCNSL.

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MRG15 is a transcription factor containing the methyl-lysine reader chromodomain. Despite its involvement in different physiological and pathological states, to date the role of this protein has not been fully elucidated due to the lack of a specific and potent chemical probe.

AimsIn the time of COVID-19, predictive molecular pathology laboratories must still timely select oncological patients for targeted treatments. However, the need to respect social distancing measures may delay results generated by laboratory-developed tests based on sequential steps a long hands-on time. Laboratory workflows should now be simplified.MethodsThe organisation of the University of Naples Federico II predictive pathology laboratory was assessed before (March–April 2019) and during (March–April 2020) the Italian lockdown.ResultsThe number of patients undergoing single or multiple biomarker testing was similar in 2019 (n=43) and in 2020 (n=45). Considering adequate samples for molecular testing, before the outbreak, next-generation sequencing was mostly used (35/42, 83.3%). Testing six genes had a reagent cost of €98/patient. Conversely, in 2020, almost all cases (38/41, 92.7%) were analysed by automated testing. This latter had for any single assay/gene a significant reagent cost (€95–€136) and a faster mean turnaround time (5.3 vs 7.9 working days).ConclusionIn the times of coronavirus, laboratory fully automated platforms simplify predictive molecular testing. Laboratory staff may be more safely and cost-effectively managed.

BackgroundAneurysmal ruptures typically cause subarachnoid bleeding with intraparenchymal and intraventricular extension. However, rare instances of acute aneurysmal ruptures present with concomitant, non-traumatic subdural hemorrhage (SDH). We explored the incidence and difference in outcomes of SDH with aneurysmal subarachnoid hemorrhage (aSAH) as compared with aSAH alone.MethodsRetrospective cohort study from 2012 to 2015 from the National (Nationwide) Inpatient Sample (NIS) (20% stratified sample of all hospitals in the United States). NIS database (2012 to September 2015) queried to identify all patients presenting with aSAH. From this population, the patients with concomitant SDH were identified.ResultsA total of 10 075 patients with both cerebral aneurysms and aSAH were included. Of these, 335 cases of concomitant SDH and aSAH were identified. There was no significant change in the rate of SDH in aSAH over time. SDH with aSAH patients had a mortality of 24% compared with 12% (p=0.003) in the SAH only group, and only 16% were discharged home vs 37% (p=0.003) in the SAH group.ConclusionsThere is a 3.5% incidence of acute SDH in patients presenting with non-traumatic aSAH. Patients with SDH and aSAH have nearly double the mortality, higher rate of discharge to nursing home and rehabilitation, and a significantly lower rate of discharge to home and return to routine functioning. This information is useful in counseling and prognostication of patients with concomitant SDH and aSAH.

AimsLung cancer predictive biomarker testing is essential to select advanced-stage patients for targeted treatments and should be carried out without delays even during health emergencies, such as the coronavirus (COVID-19) outbreak.MethodsFifteen molecular laboratories from seven different European countries compared 4 weeks of national lockdown to a corresponding period in 2019, in terms of tissue and/or plasma-based molecular test workload, analytical platforms adopted, number of cases undergoing programmed death-ligand1 (PD-L1) expression assessment and DNA-based molecular tests turnaround time.ResultsIn most laboratories (80.0%), tissue-based molecular test workload was reduced. In 40.0% of laboratories (6/15), the decrease was >25%, and in one, reduction was as high as 80.0%. In this instance, a concomitant increase in liquid biopsy was reported (60.0%). Remarkably, in 33.3% of the laboratories, real-time PCR (RT-PCR)-based methodologies increased, whereas highly multiplexing assays approaches decreased. Most laboratories (88.9%) did not report significant variations in PD-L1 volume testing.ConclusionsThe workload of molecular testing for patients with advanced-stage lung cancer during the lockdown showed little variations. Local strategies to overcome health emergency-related issues included the preference for RT-PCR tissue-based testing methodologies and, occasionally, for liquid biopsy.

V-Raf murine sarcoma viral oncogene homolog B (BRAF) gene mutations have recently been approved to select advanced stages non-small cell lung cancer (NSCLC) patients for tyrosine kinase inhibitors treatments. In this setting, liquid biopsy may represent a valuable option for BRAFmutational testing in patients without tissue availability. Here, we reviewed 196 plasma based liquid biopsies analysed by an in-house developed next generation sequencing panel, termed SiRe. On the overall, 6 (3.1%) out of 196 BRAFmutated cases were identified, with an overall median allelic frequency of 3.4%. Exon 15 p.V600E was the most common detected mutation (2/6, 33.3%). Our data highlighted that the SiRe panel is a robust tool for BRAFmutation assessment on circulating tumour DNA. Further investigation is required to develop a diagnostic algorithm to harmonise BRAFtesting on tissue and blood in advanced stages NSCLC patients.

ABSTRACTIntroduction: Driver molecular aberrations, such as EGFRand BRAFmutations, ALKand ROS1rearrangements, play an important role in the oncogenesis of non-small-cell lung cancer (NSCLC). Overall, these molecular targets select about 20% of advanced nonsquamous NSCLC Caucasian patients who can be treated with the corresponding tyrosine kinase inhibitors (TKIs). Many novel driver mutations are being or have been investigated in NSCLC, with varying degrees of success and failure. These emerging molecular targets are responsible for both primary involvement in cancer growth and have acquired resistance to previous TKIs, and for which potential therapeutic strategies are under investigation.Areas covered: This review will focus on the main emerging molecular targets in advanced stage of clinical development for the management of advanced NSCLC. A systematic review of bibliographic databases for peer-reviewed research literature and of key meetings was undertaken in order to discuss these topics.Expert opinion: Many emerging driver mutations are being investigated in metastatic NSCLC. Defining the most appropriate methods of detection of molecular aberrations, focusing on their role in the mechanisms of intrinsic and acquired resistance within appropriate preclinical and clinical trials, are needed in order to improve therapeutic benefit for NSCLC patients.

B7–H3 (CD276), a member of the B7 superfamily, is an important factor in downregulating immune responses against tumors. It is also aberrantly expressed in many human malignancies. Beyond immune regulatory roles, its overexpression has been linked to invasive metastatic potential and poor prognosis in patients with cancer. Antibody-dependent cell-mediated cytotoxicity strategies targeting B7–H3 are currently in development, and early-phase clinical trials have shown encouraging preliminary results.

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Viral hepatitis is a major global public health problem affecting hundreds of millions of people and is associated with significant morbidity and mortality. Five major biologically unrelated hepatotropic viruses cause most of the global burden of viral hepatitis. Hepatitis B and hepatitis C are associated with a significant number of chronic infections. Most deaths from viral hepatitis are due to hepatitis B and hepatitis C. An estimated 257 million people were living with HBV and 71 million people were living with HCV. Most people are asymptomatic. New diagnostics and highly effective, pangenotypic direct-acting antivirals provide opportunities to cure and eradicate chronic hepatitis C virus infection.

Background Limited literature exists on the morbidity and mortality of AVM associated intracerebral hemorrhage (ICH) compared with non-AVM ICH.Objective We examine morbidity and mortality in cAVM in a large nationwide inpatient sample to create a prognostic inpatient ruptured AVM mortality score.Methods This retrospective cohort study from 2008 to 2014 compares outcomes in cAVM related hemorrhages and ICH utilizing the National Inpatient Sample database. Diagnostic codes for ICH and AVM underlying ICH were identified. We compared case fatality according to medical complications. Multivariate analysis was used to derive hazard ratios and 95% confidence intervals to assess odds of mortality.Results We identified 6496 patients with ruptured AVMs comparing them to 627,185 admitted with ICH. Mortality was lower for ruptured AVMs (11%) compared to ICH (22%) [p< 0.01]. Mortality associated factors were liver disease (OR 2.64, CI 1.81–3.85, p< .001), diabetes mellitus (OR 2.42, CI 1.38–4.22, p= 0.002), alcohol abuse (OR 1.81, CI 1.31–2.49, p= 0.001), hydrocephalus (OR 3.35 CI 2.81–4.00, p< 0.001), cerebral edema (OR 1.5, 1.25–1.85, p< 0.001), cardiac arrest (OR 15, CI 7.9–30, p< 0.001), and pneumonia (OR 1.93, CI 1.51–2.47, p< 0.001). A 0–5 ruptured AVM mortality score was developed: Cardiac arrest (=3), age >60 (=1), Black race (=1), chronic liver failure (=1) diabetes mellitus (=1), pneumonia (=1), alcohol abuse (=1) and cerebral edema (=1). Mortality increased with score. No patient with 5 or more points survived.Conclusion The Ruptured AVM Mortality Score allows for risk stratification on patients with ICH due to ruptured AVM. This scale could prove useful in prognostication and patient education.

Cancer, a leading cause of global mortality, has ignited substantial interest in the advancement of chemotherapy drugs. Nonetheless, challenges such as limited blood circulation time, drug instability, and severe side effects have curtailed the effectiveness of conventional treatments. In recent times, the groundbreaking electrospinning technique has ushered in the era of nanofibers imbued with a diverse array of chemotherapeutic agents. This innovation harnesses the potential of natural, synthetic polymers, and composite materials. The resulting nanofibers exhibit a customizable interplay of mechanical, physical, and chemical attributes, thereby facilitating controlled drug release and targeted drug delivery. This, in turn, augments the therapeutic efficacy. This article undertakes a comprehensive journey through the recent strides achieved in the domain of cancer therapy, specifically concerning the utilisation of electrospinning. It delves into a myriad of electrospinning techniques, delving into the materials employed in the creation of nanofibers and the multifaceted properties they exhibit. Furthermore, this review encompasses an expansive exploration of the manifold applications of these nanofibers as potent drug delivery systems within the realm of cancer therapy. These applications span a wide spectrum, ranging from localized chemotherapy to the integration of multiple anticancer drugs, facilitating combinatorial therapies. Additionally, these nanofibers play an instrumental role in cancer cell detection, capture, and isolation, as well as the nuanced manipulation of cancer cell behavior. Furthermore, they are instrumental in constructing intricate cancer models, enabling in-depth studies of metastasis.

The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP+exosomes. Moreover, uptake of CEMIP+exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnfand Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.

Since the discovery of compound BIX01294 over 10 years ago, only a very limited number of nonquinazoline inhibitors of H3K9-specific methyltransferases G9a and G9a-like protein (GLP) have been reported. Herein, we report the identification of a novel chemotype for G9a/GLP inhibitors, based on the underinvestigated 2-alkyl-5-amino- and 2-aryl-5-amino-substituted 3H-benzo[e][1,4]diazepine scaffold. Our research efforts resulted in the identification 12a(EML741), which not only maintained the high in vitro and cellular potency of its quinazoline counterpart, but also displayed improved inhibitory potency against DNA methyltransferase 1, improved selectivity against other methyltransferases, low cell toxicity, and improved apparent permeability values in both parallel artificial membrane permeability assay (PAMPA) and blood–brain barrier-specific PAMPA, and therefore might potentially be a better candidate for animal studies. Finally, the co-crystal structure of GLP in complex with 12aprovides the basis for the further development of benzodiazepine-based G9a/GLP inhibitors.

AimsOur laboratory is a centralised centre receiving routine non-small cell lung cancer (NSCLC) samples for programmed death ligand-1 (PD-L1) immunohistochemical (IHC) evaluation. Since literature data are not concordant here we review our clinical records to assess the rate of PD-L1 positive and negative NSCLC cases in real-world practice.MethodsPD-L1 expression was evaluated by a validated 22C3 IHC laboratory developed test on 211 prospectively collected routine NSCLC samples, received from 10 outside institutions. PD-L1 expression was assessed by the tumour proportion score (TPS) and reported by using a three cut-point system: TPS<1, TPS 1%–49% and TPS≥50%.ResultsOverall, 193 out of 211 samples (91.5%) meet the criteria for adequacy (more than 100 viable neoplastic cells). In 62.7% (121/193) of samples TPS was <1%; 17.6% of samples (34/193) expressed PD-L1 with a TPS of 1%–49% and 19.7% (38/193) with a TPS of >50%. There was no significant difference in PD-L1 expression rates between different histotypes and site of sampling. Instead, a statistically significant difference was associated to the type of samples: in fact, cytological samples were more frequently negative for PD-L1 expression (TPS<1%) and less often displayed PD-L1 expression at high levels (TPS>50%) than surgical resections and biopsies.ConclusionsWe present a referral laboratory experience on IHC PD-L1 expression of prospectively collected routine NSCLC samples. Data from the real-world practice can better clarify the percentage of PD-L1 positive and negative cases, to establish benchmarks for good practice standards.

AimThe rapid and fully automated Idylla EGFRMutation Assay has been specifically designed to process formalin-fixed, paraffin-embedded sections without requiring preliminary DNA extraction. This study evaluates whether this approach can also process archival smears from patients with non–small cell lung cancer (NSCLC) by scraping the stained cellular material directly into the cartridge.MethodsThe study was divided into two parts. In the first part, we carried out Idylla EGFRMutation Assay on archival stained smears from 39 patients with NSCLC. Among these, 14 cases harboured a mutation in either exon 19 (n=11) or exon 21 (n=3), previously detected on DNA extracts by fragment length and TaqMan assays. In the second part, we evaluated whether de-staining of the smears could reduce background fluorescence.ResultsThe Idylla EGFRMutation Assay confirmed the presence of EGFRmutation in 11 instances (78.6%). However, concordance was higher for exon 19 deletions (10/11) than for exon 21 p.L858R assessments. Raw data showed a high background fluorescence in channel 2, where the EGFRexon 21 p.L858R mutation was detected. This interference, due to dye residues from the original staining, was partially reduced by de-staining the cytological material.ConclusionsOur data, although preliminary, show that the Idylla EGFRMutation Assay can reliably process most archival smears without requiring preliminary DNA extraction. Results may be further improved by de-staining the cellular material before insertion into the cartridge.

AimsFollowing the development for liquid biopsies of the SiRe next-generation sequencing (NGS) panel that covers 568 clinical relevant mutations in EGFR, KRAS, NRAS, BRAF, cKITand PDGFRagenes, in this current study, we apply this small NGS panel on tissue samples of lung cancer.MethodsA total of 322 specimens were prospectively tested. Technical parameters were analysed on both cytological and histological samples. In a subset of 75 samples, the EGFRSiRe results were compared with those generated by the European Community (CE)–IVD EGFRassay on Idylla platform. Clinical outcomes of 11 patients treated, on the basis of SiRe results, were also evaluated.ResultsOnly 28 (8.7%) specimens failed to produce a library; out of the 294 remaining samples, a total of 168 somatic mutations were found. In nearly all instances (74/75–99%), the EGFRSiRe results were confirmed by Idylla. In general, SiRe analytical parameters were excellent. However, histological and cytological specimens differed in relation to average reads for sample, mean number of mapped reads, median read length and average reads for amplicon. Treatment outcome evaluation in 11 patients showed a partial response in 82 % (9/11) patients with a median progression-free survival of 340 days.ConclusionsThe small gene panel SiRe is a clinically relevant tool useful to widespread the adoption of NGS in predictive molecular pathology laboratories.

Abiotic hydrocarbons and carboxylic acids are known to be formed on Earth, notably during the hydrothermal alteration of mantle rocks. Although the abiotic formation of amino acids has been predicted both from experimental studies and thermodynamic calculations, its occurrence has not been demonstrated in terrestrial settings. Here, using a multimodal approach that combines high-resolution imaging techniques, we obtain evidence for the occurrence of aromatic amino acids formed abiotically and subsequently preserved at depth beneath the Atlantis Massif (Mid-Atlantic Ridge). These aromatic amino acids may have been formed through Friedel–Crafts reactions catalysed by an iron-rich saponite clay during a late alteration stage of the massif serpentinites. Demonstrating the potential of fluid-rock interactions in the oceanic lithosphere to generate amino acids abiotically gives credence to the hydrothermal theory for the origin of life, and may shed light on ancient metabolisms and the functioning of the present-day deep biosphere.

In the present study, we analysed 44 formalin fixed paraffin embedded (FFPE) from different solid tumours by adopting two different next generation sequencing platforms: GeneReader (QIAGEN, Hilden, Germany) and Ion Torrent (Thermo Fisher Scientific, Waltham, Massachusetts, USA). We highlighted a 100% concordance between the platforms. In addition, focusing on variant detection, we evaluated a very good agreement between the two tests (Cohen’s kappa=0.84) and, when taking into account variant allele fraction value for each variant, a very high concordance was obtained (Pearson’s r=0.94). Our results underlined the high performance rate of GeneReader on FFPE samples and its suitability in routine molecular predictive practice.