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Background New treatments are currently offering new opportunities and challenges in clinical management and research in the migraine field. There is the need of homogenous criteria to identify candidates for treatment escalation as well as of reliable criteria to identify refractoriness to treatment. To overcome those issues, the European Headache Federation (EHF) issued a Consensus document to propose criteria to approach difficult-to-treat migraine patients in a standardized way. The Consensus proposed well-defined criteria for resistant migraine (i.e., patients who do not respond to some treatment but who have residual therapeutic opportunities) and refractory migraine (i.e., patients who still have debilitating migraine despite maximal treatment efforts). The aim of this study was to better understand the perceived impact of resistant and refractory migraine and the attitude of physicians involved in migraine care toward those conditions. Methods We conducted a web-questionnaire-based cross-sectional international study involving physicians with interest in headache care. Results There were 277 questionnaires available for analysis. A relevant proportion of participants reported that patients with resistant and refractory migraine were frequently seen in their clinical practice (49.5% for resistant and 28.9% for refractory migraine); percentages were higher when considering only those working in specialized headache centers (75% and 46% respectively). However, many physicians reported low or moderate confidence in managing resistant (8.1% and 43.3%, respectively) and refractory (20.7% and 48.4%, respectively) migraine patients; confidence in treating resistant and refractory migraine patients was different according to the level of care and to the number of patients visited per week. Patients with resistant and refractory migraine were infrequently referred to more specialized centers (12% and 19%, respectively); also in this case, figures were different according to the level of care. Conclusions This report highlights the clinical relevance of difficult-to-treat migraine and the presence of unmet needs in this field. There is the need of more evidence regarding the management of those patients and clear guidance referring to the organization of care and available opportunities.

In semiconductor manufacturing plants, monitoring of all wafers is fundamental in order to maintain good yield and high quality standards. However, this is a costly approach and in practice only few wafers in a lot are actually monitored. With a Virtual Metrology (VM) system it is possible to partly overcome the lack of physical metrology. In a VM scheme, tool data are used to predict, for every wafer, metrology measurements. In this paper, we present a VM system for a Chemical Vapor Deposition (CVD) process. Various data mining techniques are proposed. Due to the huge fragmentation of data derived from CVD's mixed production, several kind of data clustering have been adopted. The proposed models have been tested on real productive industrial data sets.

A multicentre, randomised, open trial with a 2 x 2 factorial design was conducted to compare the benefits and risks of two thrombolytic agents, streptokinase (SK, 1·5 MU infused intravenously over 30-60 min) and alteplase (tPA, 100 mg infused intravenously over 3 h) in patients with acute myocardial infarction admitted to coronary care units within 6 h from onset of symptoms. The patients were also randomised to receive heparin (12 500 U subcutaneously twice daily until discharge from hospital, starting 12 h after beginning the tPA or SK infusion) or usual therapy. All patients without specific contraindications were given atenolol (5-10 mg iv) and aspirin (300-325 mg a day). The end-point of the study was the combined estimate of death plus severe left ventricular damage. 12 490 patients were randomised to four treatment groups (SK alone, SK plus heparin, tPA alone, tPA plus heparin). No specific differences between the two thrombolytic agents were detected as regards the combined end-point (tPA 23·1%; SK 22·5%; relative risk 1·04, 95% Cl 0·95-1·13), nor after the addition of heparin to the aspirin treatment (hep 22·7%, no hep 22·9%; RR 0·99, 95% Cl 0·91-1·08). The outcome of patients allocated to the four treatment groups was similar with respect to baseline risk factors such as age, Killip class, hours from onset of symptoms, and site and type of infarct. The rates of major in-hospital cardiac complications (reinfarction, post-infarction angina) were also similar. The incidence of major bleeds was significantly higher in SK and heparin treated patients (respectively, tPA 0·5%, SK 1·0%, RR 0·57, 95% Cl 0·38-0·85; hep 1·0%, no hep 0·6%, RR 1·64, 95% Cl 1·09-2·45), whereas the overall incidence of stroke was similar in all groups. SK and tPA appear equally effective and safe for use in routine conditions of care, in all infarct patients who have no contraindications, with or without post-thrombolytic heparin treatment. The 8·8% hospital mortality of the study population (compared with approximately 13% in the control cohort of the GISSI-1 trial) indicates the beneficial impact of the proven acute treatments for AMI.

In advanced stage non-small cell lung cancer (NSCLC) patients, Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) testing may soon acquire a predictive significance to select patients for AMG510 treatment. Since tissue samples are not always available, liquid biopsy may represent a viable option for KRAS testing. Here, we review the last three years clinical practice performed on 194 plasma based liquid biopsies by next generation sequencing (NGS) SiRe(®) panel. In particular, 36 (18.6%) KRAS mutated cases were identified, with an overall median allelic frequency of 5.0% (ranging between 0.2% and 46.8%). No concomitant mutations were observed in the other NSCLC clinical relevant genes included in the SiRe(®) panel, such as epidermal growth factor receptor (EGFR) and v-Raf murine sarcoma viral oncogene homolog B (BRAF). Exon 2 p.G12C was the most common detected mutation (13/36, 36.1%). In conclusion, our data update and confirm that SiRe(®) NGS panel represents a robust analytical tool to assess KRAS mutational status on circulating tumor DNA. Further investigation is required to design more cost-effective diagnostic algorithms to harmonize clinical relevant biomarker testing on tissue and blood in advanced stage NSCLC clinical practice.

Stroke is a major challenge in modern medicine and understanding the role of the neuronal extracellular matrix (NECM) in its pathophysiology is fundamental for promoting brain repair. Currently, stroke research is focused on the neurovascular unit (NVU). Impairment of the NVU leads to neuronal loss through post-ischemic and reperfusion injuries, as well as coagulatory and inflammatory processes. The ictal core is produced in a few minutes by the high metabolic demand of the central nervous system. Uncontrolled or prolonged inflammatory response is characterized by leukocyte infiltration of the injured site that is limited by astroglial reaction. The metabolic failure reshapes the NECM through matrix metalloproteinases (MMPs) and novel deposition of structural proteins continues within months of the acute event. These maladaptive reparative processes are responsible for the neurological clinical phenotype. In this review, we aim to provide a systems biology approach to stroke pathophysiology, relating the injury to the NVU with the pervasive metabolic failure, inflammatory response and modifications of the NECM. The available data will be used to build a protein–protein interaction (PPI) map starting with 38 proteins involved in stroke pathophysiology, taking into account the timeline of damage and the co-expression scores of their RNA patterns The application of the proposed network could lead to a more accurate design of translational experiments aiming at improving both the therapy and the rehabilitation processes.

Over the past decade, immunotherapy has emerged as one of the most promising cancer treatments. Several monoclonal antibodies targeting the programmed death 1 (PD‐1)/ programmed death ligand‐1 (PD‐L1) pathway have been integrated into standard‐of‐care treatments for a wide range of cancer types. Although all the available PD‐L1 immunohistochemistry (IHC) assays have been developed on formalin‐fixed histological specimens, a growing body of research has recently suggested the feasibility of PD‐L1 testing on cytological samples. Although promising results have been reported, several important issues still need to be addressed. Among these are pre‐analytical issues, cyto‐hystological correlation, and inter‐observer agreement. This review will briefly summarise the knowledge gaps and future directions of cytopathology in the immuno‐oncology scenario., Programmed death ligand‐1 (PD‐L1) assays have been developed on formalin‐fixed histological specimens, and a growing body of research is being dedicated to confirming the feasibility of applying these assays to cytological samples. Promising results have been reported but several important issues still need to be addressed. This review summarizes the current status, knowledge gaps and future directions of cytopathology in the context of immuno‐oncology.

Whenever tissue sample is not available, non-small cell lung cancer (NSCLC) biomarker testing is performed with liquid biopsy. The Kirsten rat sarcoma viral oncogene homolog (KRAS) p.G12C mutation is a novel target in patients with NSCLC. In this study, 33 NSCLC frozen plasma samples, previously characterised for KRAS mutational status by next generation sequencing (NGS), were processed by the fully automated Idylla KRAS assay. In 30/33 cases, archival matched cell-free DNA (cfDNA) was also directly pipetted in the cartridge. Overall, 30/33 plasma and 28/30 cfDNA samples yielded valid results. In 29/30 of KRAS p.G12C mutant plasma samples and 26/28 of cfDNA, Idylla confirmed the NGS results. In conclusion, the Idylla NSCLC KRAS liquid biopsy assay may represent a reliable tool to assess KRAS p.G12C mutation.

Introduction In the selection of non-small cell lung cancer (NSCLC) patients for immunotherapy, specimen processed as cell blocks (CBs) may be the only available material to assess PD-L1 expression. Therefore, optimal CB preparation becomes paramount. In this context, here we assessed whether inadequate fixation time might be one of the pre-analytical factors affecting PD-L1 expression. Methods Ex vivo CBs from placental (n = 3) and NSCLC (n = 8) resection specimens were obtained. PD-L1 staining was performed on CBs prepared at increasing fixation times (12 hours, 48 hours, 72 hours, 96 hours, 168 hours and 504 hours) using the companion diagnostic SP263 Assay and a validated 22C3 laboratory developed test (LDT). Staining intensity and percentage of positive cells were evaluated. Results All placental CBs showed moderate to strong PD-L1 positivity in most cells, regardless of the fixation time. Likewise, the percentage of SP263-stained NSCLC cells was similar at all fixation times except for one case, which showed less intense SP263 staining at 168 hours. Conversely, in 5/8 cases, the 22C3 LDT percentage of positive cells and staining intensity decreased at 168 hours and 504 hours. Conclusions Our results show that fixation time influences the performance of 22C3 LDT on CBs. Thus, we recommend that the fixation time of cytological materials be carefully checked, especially when PD-L1 testing is delayed until the oncology request. Indeed, delays in tissue processing and paraffin embedding may lead to sub-optimal performance of PD-L1 staining on CBs.

Carcinomas do rarely metastasize to the finger. Non-small-cell lung cancer is most frequently involved. To date, only a few cases have been reported, on histology. Here we describe the cytological features of a metastatic lung adenocarcinoma case to the phalanx of the right forefinger as initial manifestation of the neoplastic disease in a 69-years-old woman. The cytological microscopic findings, complemented by a wide array of ancillary techniques, were confirmed by histology. Neoplastic cells were positive for cytokeratin 7 (CK7) and thyroid transcription factor 1 (TTF-1), harboring, in tissue and in bloodstream samples, an uncommon epidermal growth factor receptor (EGFR) exon 20 p.S768_D760DUP, detected by next generation sequencing (NGS) and liquid biopsy. Cytology corroborated by immunocytochemistry, NGS, and liquid biopsy is a modern approach to provide diagnostic and therapeutic information even in rare and challenging cases.

Background Thyroid fine-needle aspirates (FNAs) with undetermined morphology can be outsourced to centralized laboratories for comprehensive molecular profiling. When a local, rapid screening rules out easily detectable BRAF and NRAS mutations outsourcing is minimized, leading to cost savings. The fully automated Idylla technology, that does not require trained staff, is an emerging option. However, Idylla platform has only been validated to process formalin fixed paraffin embedded (FFPE) sections. Here we investigate whether also the FNA needle rinse could be genotyped by the same cytopathologist who performs the FNA, a procedure that can be termed rapid on site molecular evaluation (ROME). Methods To validate this approach, the Idylla BRAF and NRAS Test was performed on the rinses from 25 simulated (bench-top) FNAs, in a first part of the study. Genotyping data were compared with those obtained on matched histological FFPE blocks. The second part of the study was carried out on 25 prospectively collected routine FNAs to assess the performance of the Idylla BRAF and NRAS assay against a gold standard real time polymerase chain reaction method. Results Idylla NRAS-BRAF Mutation Test was performed on needle rinse as well as histological FFPE blocks. A sensitivity of 88.9%, a specificity of 100.0% were obtained comparing the Idylla NRAS-BRAF Mutation Test on needle rinse to the reference method. Conclusions The FNA needle rinse can be directly genotyped. This obviates the need of cell block preparation, making possible a rapid combined morphological and molecular evaluation. Since DNA extraction is no longer necessary, the cytopathologist can perform ROME him/herself.

Patients with high-frequency resistant migraine and medication-overuse headache are still the main clinical challenge in tertiary headache centers. The approval of targeted antibodies against the calcitonin gene-related peptide (CGRP) and its receptor represents a powerful instrument. In this study, we observed how biological and clinical features of resistant migraineurs responded to erenumab, fremanezumab, or galcanezumab. We found a reduction in advanced oxidation protein products (AOPP) as a biomarker of improved redox state after six months of treatment. We also found that treatment efficacy was precocious and maintained with high individual responder rates. In particular, seven out of ten patients achieved a reduction of 50% from the baseline at three months, which was maintained at six months, while about one out of our patients experienced a 75% reduction in headache frequency from the first month of treatment. The migraine disability assessment (MIDAS) and the associated fatigue, anxiety, and sleep quality also significantly improved. The allodynia symptom dropped from moderate/severe to mild/absent as a sign of central sensitization reduction. Our study confirmed the safety and efficacy of CGRP inhibition in real-life, high-challenging patients. Additional evidence is needed to understand the role of oxidative stress as a migraine biomarker.

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. The neuropathological hallmark of the disease is the loss of dopamine neurons of the substantia nigra pars compacta. The clinical manifestations of PD are bradykinesia, rigidity, resting tremors and postural instability. PD patients often display non-motor symptoms such as depression, anxiety, weakness, sleep disturbances and cognitive disorders. Although, in 90% of cases, PD has a sporadic onset of unknown etiology, highly penetrant rare genetic mutations in many genes have been linked with typical familial PD. Understanding the mechanisms behind the DA neuron death in these Mendelian forms may help to illuminate the pathogenesis of DA neuron degeneration in the more common forms of PD. A key step in the identification of the molecular pathways underlying DA neuron death, and in the development of therapeutic strategies, is the creation and characterization of animal models that faithfully recapitulate the human disease. In this review, we outline the current status of PD modeling using mouse, rat and non-mammalian models, focusing on animal models for autosomal recessive PD.

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 BRAF mutational 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 BRAF mutated 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 BRAF mutation assessment on circulating tumour DNA. Further investigation is required to develop a diagnostic algorithm to harmonise BRAF testing on tissue and blood in advanced stages NSCLC patients.

Migraine is a primary headache with high prevalence among the general population, characterized by functional hypersensitivity to both exogenous and endogenous stimuli particularly affecting the nociceptive system. The hyperresponsivity of cortical neurons could be due to a disequilibrium in the excitatory/inhibitory signaling. This study aimed to investigate the anatomo-functional pathway from the retina to the primary visual cortex using visual evoked potentials (VEP). Contrast gain protocol was used in 15 patients diagnosed with migraine without aura (at baseline and after 3 months of topiramate therapy) and 13 controls. A saturation (S) index was assessed to monitor the response of VEP’s amplitude to contrast gain. Non-linear nor monotone growth of VEP (S &lt, 0.95) was defined as supersaturation. A greater percentage of migraine patients (53%) relative to controls (7%) showed this characteristic. A strong inverse correlation was found between the S index and the number of days separating the registration of VEP from the next migraine attack. Moreover, allodynia measured through the Allodynia Symptoms Check-list (ASC-12) correlates with the S index both at baseline and after 3 months of topiramate treatment. Other clinical characteristics were not related to supersaturation. Topiramate therapy, although effective, did not influence electrophysiological parameters suggesting a non-intracortical nor retinal origin of the supersaturation (with possible involvement of relay cells from the lateral geniculate nucleus). In conclusion, the elaboration of visual stimuli and visual cortex activity is different in migraine patients compared to controls. More data are necessary to confirm the potential use of the S index as a biomarker for the migraine cycle (association with the pain-phase) and cortical sensitization (allodynia).

Despite the recognized potential of nature-based solutions (NBSs) to support climate adaptation, there are still wide barriers for a wider uptake of such NBS in urban areas. While tailored NBS tools could facilitate and accelerate this process, a comprehensive mapping of their availability and capacity to respond to cities’ challenges is missing. This research aims to provide an overview of tools that intend to facilitate the uptake of NBS for urban climate adaptation supporting cities in overcoming their challenges. To do so, this paper (i) presents the results of interviews and workshops with municipal officers and decision-makers from different European cities that identified the challenges they experience with NBS uptake, (ii) selects and reviews NBS tools and (iii) analyzes them on their capacity to address these implementation challenges. Our research revealed four key challenges that municipal officers experience: resources availability, level of expertise, know-how or competence, the institutional setting, and collaborative governance and planning. The results from the tools’ review show that existing tools can support overcoming a lack of expertise (31), but, to a smaller extent, can also be of use when experiencing the institutional setting (13), availability of resources (11), and collaborative governance and planning (10) as a challenge. This work provides researchers and tool developers with insights into potential market saturation as well as scarcity of certain types of tools that would match cities’ challenges, highlighting needs and opportunities for new tool development.

Background Immune-checkpoint inhibitors (ICIs) represent an important treatment option for patients who have advanced stage non-small cell lung cancer (NSCLC). Currently, evaluation of the expression level of programmed death-ligand 1 (PD-L1) has proven highly successful as a positive predictive biomarker for ICIs. In addition to PD-L1, other promising predictive biomarkers are emerging, including high tumor mutational burden (TMB-H). However, measuring TMB-H remains challenging for several reasons, among which is the difficulty in obtaining adequate tissue material from NSCLC patients. There are no data in the current literature regarding the possibility of adopting cell blocks (CBs) for TMB evaluation; therefore, our goal was to evaluate the feasibility of analyzing TMB on CBs. Methods For evaluation of differences in run metric parameters, 8 pairs of histological and CB samples from patients with NSCLC were analyzed using the Oncomine Tumor Mutational Load Assay on Ion Torrent S5 GS next-generation sequencing (NGS) platform. Results Most CBs (6/8, 75.0%) were successfully analyzed by adopting the broad NGS panel approach. CBs provided results similar to those obtained on histological matched specimens in terms of median total reads (7207048.80 vs 7558817.80), median mapped reads (7075753.83 vs 7513822.00), median read lengths (115.50 vs. 113.00), median percentage of reads on-target (97.49% vs. 98.45%), median average reads per amplicon (454.67 vs 476.14), and median uniformity of amplicon coverage (83.52% vs 84.13%). Conclusion In this pilot study, we demonstrated the technical feasibility of assessing TMB on CBs.

Immunohistochemistry (IHC) and polymerase chain reaction (PCR) and fragment separation by capillary electrophoresis represent the current clinical laboratory standard for the evaluation of microsatellite instability (MSI) status. The importance of reporting MSI status in colorectal cancer is based on its potential for guiding treatment and as a prognostic indicator. It is also used to identify patients for Lynch syndrome testing. Our aim was to evaluate pre-analytical factors, such as age of formalin-fixed and paraffin-embedded (FFPE) block, neoplastic cell percentage, mucinous component, and DNA integrity, that may influence the accuracy of MSI testing and assess the concordance between three different MSI evaluation approaches. We selected the mucinous colorectal cancer (CRC) histotype for this study as it may possibly represent an intrinsic diagnostic issue due to its low tumor cellularity. Seventy-five cases of mucinous CRC and corresponding normal colon tissue samples were retrospectively selected. MMR proteins were evaluated by IHC. After DNA quality and quantity evaluation, the Idylla&trade, and TapeStation 4200 platforms were adopted for the evaluation of MSI status. Seventy-three (97.3%) cases were successfully analyzed by the three methodologies. Overall, the Idylla&trade, platform showed a concordance rate with IHC of 98.0% for microsatellite stable (MSS)/proficient MMR (pMMR) cases and 81.8% for MSI/deficient MMR (dMMR) cases. The TapeStation 4200 system showed a concordance rate with IHC of 96.0% for MSS/pMMR cases and 45.4% for MSI/dMMR cases. The concordance rates of the TapeStation 4200 system with respect to the Idylla&trade, platform were 98.1% for MSS profile and 57.8% for MSI profile. Discordant cases were analyzed using the Titano MSI kit. Considering pre-analytical factors, no significant variation in concordance rate among IHC analyses and molecular systems was observed by considering the presence of an acellular mucus cut-off &gt, 50% of the tumor area, FFPE year preparation, and DNA concentration. Conversely, the Idylla&trade, platform showed a significant variation in concordance rate with the IHC approach by considering a neoplastic cell percentage &gt, 50% (p-value = 0.002), and the TapeStation 4200 system showed a significant variation in concordance rate with the IHC approach by considering a DNA integrity number (DIN) &ge, 4 as cut-off (p-value = 0.009). Our data pinpoint a central role of the pre-analytical phase in the diagnostic outcome of MSI testing in CRC.

The fifth cranial nerve is the common denominator for many headaches and facial pain pathologies currently known. Projecting from the trigeminal ganglion, in a bipolar manner, it connects to the brainstem and supplies various parts of the head and face with sensory innervation. In this review, we describe the neuroanatomical structures and pathways implicated in the sensation of the trigeminal system. Furthermore, we present the current understanding of several primary headaches, painful neuropathies and their pharmacological treatments. We hope that this overview can elucidate the complex field of headache pathologies, and their link to the trigeminal nerve, to a broader field of young scientists.

Background: Next-generation sequencing (NGS) needs to be validated and standardized to ensure that cancer patients are reliably selected for target treatments. In Italy, NGS is performed in several institutions and harmonization of wet and dry procedures is needed. To this end, a consortium of five different laboratories, covering the most part of the Italian peninsula, was constituted. A narrow gene panel (SiRe®) covering 568 clinically relevant mutations in six different genes (EGFR, KRAS, NRAS, BRAF, cKIT, and PDGFRα) with a predictive role for therapy selection in non-small cell lung cancer (NSCLC), gastrointestinal stromal tumor, colorectal carcinoma (CRC), and melanoma was evaluated in each participating laboratory. Methods: To assess the NGS inter-laboratory concordance, the SiRe® panel, with a related kit and protocol for library preparation, was used in each center to analyze a common set of 20 NSCLC and CRC routine samples. Concordance rate, in terms of mutation detected and relative allelic frequencies, was assessed. Then, each institution prospectively analyzed an additional set of 40 routine samples (for a total of 160 specimens) to assess the reproducibility of the NGS run parameters in each institution. Results: An inter-laboratory agreement of 100% was reached in analyzing the data obtained from the 20 common sample sets; the concordance rate of allelic frequencies distribution was 0.989. The prospective analysis of the run metric parameters obtained by each center locally showed that the analytical performance of the SiRe® panel in the different institutions was highly reproducible. Conclusions: The SiRe® panel represents a robust diagnostic tool to harmonize the NGS procedure in different Italian laboratories.

In this review, we describe molecular pathology testing to predict response to targeted treatment of solid tumors, focusing on Italian routine clinical practice. The combination of the universal health care system organized at national, regional, and local levels has led a decentralized model, with a large number of local laboratories performing in-house molecular testing following guidelines issued and external quality assessment organized by the Italian Society of Pathology and Cytopathology–Italian Division of the International Academy of Pathology. In this framework, in the early days of predictive testing, sponsored informatics platforms support to set up national programs that aimed to integrate the activity of oncologists and pathologists to test cancer patients for druggable alterations. More recently, reimbursement for molecular testing is being covered completely by the Italian National Health Service. In the near future, considering the development of complex technologies, we expect that outsourcing samples to next-generation sequencing referral laboratories will take place.

Aberrant p53 immunohistochemical expression is used to identify the copy-number-high/TP53-mutant subgroup of endometrial cancer (EC). We aimed to determine the diagnostic accuracy of p53 immunohistochemistry as surrogate for TP53 sequencing through a systematic review and meta-analysis. Electronic databases were searched from their inception to June 2019. All studies assessing p53 expression and TP53 mutations in EC were included. Diagnostic accuracy was assessed based on area under the curve (AUC). Immunohistochemical criteria used to define aberrant p53 expression were “overexpression” and “overexpression or complete absence”. Subgroup analysis was based on the sequencing technique adopted (Polymerase Chain Reaction + sequencing, or next generation sequencing, NGS). Thirteen observational studies with 727 endometrial cancers were included. Both “overexpression” and “overexpression or complete absence” showed high diagnostic accuracy (AUC = 0.9088 and 0.9030, respectively). The subgroup with “overexpression” and NGS showed the best results, with very high diagnostic accuracy (AUC = 0.9927). In conclusion, immunohistochemistry for p53 is a highly accurate surrogate of TP53 sequencing. Overexpression of p53 in ≥70−80% showed the best accuracy in predicting TP53 mutations. Further studies in this field should adopt optimized immunohistochemical procedures and take into account less common p53 patterns (e.g. cytoplasmic expression).

AimWe aimed to study the prognostic value of KRAS, NRAS, BRAF mutations and microsatellite stable (MSS)/instable (MSI) in the field of colorectal cancer invading the submucosa (ie, pT1 colorectal cancer (CRC)).MethodsWe led a case-control study in tumour samples from 60 patients with pT1 CRC with (20 cases) and without (40 cases) metastatic evolution (5 years of follow-up) which were analysed for KRAS, NRAS, BRAF mutations (Idylla testing and next generation sequencing, NGS) and MSS/MSI status (Idylla testing and expression of mismatch repair (MMR) proteins using immunohistochemistry).ResultsKRAS mutations were encountered in 11/20 (55%) cases and 21/40 (52.5%) controls (OR=1.11 (0.38 to 3.25), p=0.8548), NRAS mutations in 1/20 (5%) cases and 3/40 (7.5%) controls (OR=3.08 (0.62 to 15.39), p=0.1698) and BRAF mutations in 3/20 (15%) cases and 6/40 (15%) controls (OR=1.00 (0.22 to 4.5), p=1.00). A MSI status was diagnosed in 3/20 (15%) cases and 5/40 (12.5%) controls (OR=1.2353 (0.26 to 5.79), p=0.7885). Beyond the absence of significant association between the metastatic evolution and any of the studied molecular parameters, we observed a very good agreement between methods analysing KRAS, NRAS and BRAF mutations (Kappa value of 0.849 (0.748 to 0.95) between Idylla and NGS) and MSS/MSI (Idylla)—proficient MMR/deficient MMR (immunohistochemistry) status (Kappa value of 1.00).ConclusionAlthough being feasible using the fully automated Idylla method as well as NGS, the molecular testing of KRAS, NRAS, BRAF and MSS/MSI status does not seem useful for prognostic purpose in the field of pT1 CRC.

The pathophysiological mechanisms of migraine transformation are debated. Modifications of plasma oxidative stress biomarkers have been described in chronic migraine. OnabotulintoxinA (BoNT/A) treatment, approved for chronic migraine prophylaxis, possibly reduces pain neurotransmitters release and oxidative stress products. Aims of our study were to investigate differences in the levels of selected plasmatic oxidative stress biomarkers (Advanced Oxidation Protein Products (AOPP), Ferric Reducing Antioxidant Power (FRAP), Thiolic Groups (SH)) comparing chronic migraineurs (CM) and healthy controls (HC). We also explored possible clinical and biochemical modifications in the CM group after six months of treatment with BoNT/A. At the baseline, we found higher values of AOPP (p &lt, 0.001), and lower values of SH (p &lt, 0.001) and FRAP (p = 0.005) in the CM group. At the six-month follow-up we found a reduction of AOPP (p &lt, 0.001) and an increase of FRAP (p &lt, 0.001) and SH (p = 0.023) within the CM group. BoNT/A treatment improved migraine symptoms in the CM group. We confirmed previous reports of imbalanced antioxidant mechanisms in chronic migraine showing lower antioxidant capacities in patients than controls. BoNT/A improved the levels of plasma oxidative stress biomarkers and confirmed its role as an effective prophylactic treatment for CM. Other studies should investigate the potential antioxidant properties of BoNT/A treatment.

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: TPSResultsOverall, 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 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 (TPS50%) 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.

This paper deals with a quantitative risk analysis, performed using the rockfall risk management (RO.MA.) method, for a road affected by rockfalls. This approach allows evaluating the rockfall risk, also considering existing protection devices. Rockfall hazard and risk were assessed for an important and very busy road stretch linking Positano to Amalfi in the Campania region of southern Italy. An estimate of the rockfall return periods for assigned volume classes was obtained using the magnitude– frequency curves (MFCs) computed through the analysis of a rockfall inventory covering a time span from 1996 to 2008. For rockfall volumes of 0.1, 1.0, and 10 m3, using a 2D trajectory simulating code, the involved kinetic energies and run-out distances of boulders that may hit the road were calculated. The risk, expressed as the annual probability of a fatal accident for the three rockfall hazard scenarios has been evaluated both without and with protection devices, respectively. The total final risk is given by the sum of partial risks related to the three scenarios. The analysis showed that regardless of whether there are any rockfall protection devices, the total risk remains almost constant. This is due to the wide spread of slopes protected with inadequate restraining metallic nets characterized by decreasing catching capacities, as possible rockfall magnitude increases. The individual risk is not acceptable, and some actions are requested in order to lower it.

The expanding therapeutic use of high-dose intravenous immunoglobulin (IVIg) in autoimmune diseases has raised important practical and conceptual issues over the last few years. These have prompted a number of research efforts aimed at characterizing aspects of the mechanism of action of current IVIg preparations, which might lead to the development of standardized, more cost-effective agents. Although polyclonal IgG in these preparations are mostly thought to act via direct interference with disease-specific, pathogenic autoantibodies, evidence from clinical and experimental work points to the involvement of crucial checkpoints upstream of self-reactive B-cell activation and autoantibody production. Reviewed herein are the results of the most recent studies documenting the crucial role of regulatory T cells (Treg) in the immunomodulatory activity of IVIg, and the molecular mechanisms mediating the effect of specific IgG fragments and glycoforms on Treg activity and the ensuing downregulation of T-cell effector responses of different sign and magnitude. Further progress in this area of translational research may lead to the development of innovative strategies aimed at restoring tolerance in autoimmune diseases. © 2014 Elsevier Ltd.

Metastatic colorectal cancer harbouring a mutation in codon 12 or 13 of the KRAS gene does not benefit from therapy with antibodies targeting the epidermal growth factor receptor (EGFR). The implementation of community KRAS testing is generating a rapid flow of new data that have implications for the pathologist and testing guidelines besides the physician. Therefore, it seems timely to draw together the threads of this large body of information in order that pathologists can be knowledgeable partners in the multidisciplinary process of targeted cancer therapy and to help refine current testing guidelines. This review addresses (1) the most relevant methodological and technical aspects of KRAS testing in terms of sample site (primary/metastatic), test specimens (resection/biopsy/cytology) and the diverse molecular methods available; (2) the issues related to daily practice, namely, the timing of the test, its turnaround time and the quality control procedures; and (3) the evidence related to the relationship between KRAS genetic intratumoural heterogeneity, clinical sensitivity of mutational detection tools and anti-EGFR treatment outcome. Hopefully, in the near future, elucidation of the potential of biomarker panels and of the mechanisms underlying primary and acquired resistance to anti-EGFR therapy will refine even further personalised treatment regimens for patients with metastatic colorectal cancer.

Background: Molecular techniques are relevant to modern cytopathology, but their implementation is difficult without molecular expertise and infrastructure. The assessment of KRAS mutational status on cytological preparations may be useful either to refine uncertain diagnoses on pancreatic aspirates or to yield predictive information to plan targeted treatment of metastatic colorectal cancer (mCRC). The novel test Idylla™ enables fully automated KRAS genotyping in approximately 2 h, even in less experienced hands. Materials and Methods: This study aims to validate this methodology to detect KRAS mutations on archival cytological preparations of pancreatic cancer (n = 9) and mCRC (n = 9) by comparing the Idylla™ performance to that of standard real-time polymerase chain reaction. Results: The same 11 mutations (n = 4: p.G12D; n = 2: p.G12V; n = 2: p.A59E/G/T; n = 1: p.G12R; n = 1: p.G13D; n = 1: p.Q61H) were detected by both techniques. Conclusion: Even in less experienced laboratories, a cytopathologist may easily integrate morphological diagnostic report with accurate KRAS mutation detection, which is relevant for diagnostic and treatment decisions.

Objective: Guidelines from the College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC) and the Association for Molecular Pathology (AMP) consider cytology suitable for testing epidermal growth factor receptor (EGFR) mutations in lung adenocarcinoma. The guidelines recommend that cytopathologists first discuss the possibility of testing squamous cell carcinomas (SqCC) in multidisciplinary meetings. Second, cell blocks should be analysed rather than smear preparations and, third, specimens should be sent to external molecular laboratories within three working days of receiving requests. This study monitored how these recommendations are met in practice. Methods: Our laboratory received 596 requests from cytologists from 13 different institutions. For each case, the cytological diagnosis, cytopreparation type, and time between the request and sample mailing were compared with the recommendations. Results: Of the 596 samples, 32 (5.4%) had been reported as SqCC. Three of these (9.4%) showed EGFR mutation. Cytological slides, either ThinPrep TM (51.2%) or direct smears (43.2%), weremore frequently received than cell blocks (5.7%). The mean time between the oncologist's request and specimen dispatching was 5.8 working days. Conclusions: The occurrence of mutations in samples reported as SqCC was higher than expected. This questions the reliability of the original diagnosis, which reinforced the recommendation to evaluate the opportunity for testing non-adenocarcinoma cytology on a case-by-case basis. In spite of CAP/IASLC/AMP recommendations, cell blocks were underutilized for EGFR testing, but cytological slides were suitable for DNA analyses. Significant efforts are needed to avoid delays in outsourcing cytological samples for EGFR testing.

Objectives: In this study we reviewed our practice of lung cancer epidermal growth factor receptor (EGFR) mutational testing in an academic centralized laboratory setting, where direct smears and liquid-based cytology (LBC) slides represent the most frequent cytological specimens received. The aim was to assess the differences, if any, between these sample types in terms of DNA yield, adequacy rates and overall EGFR testing performance. Study Design: A total of 362 cases were retrieved - received from January 2012 to January 2014 for EGFR testing - including 204 LBC specimens and 158 smears. Exon 19 deletions and the L858R point mutation in exon 21, detected by fragment assay and TaqMan assay, respectively, were confirmed by direct sequencing or by high-resolution melting. Results: Although the direct smears showed a higher DNA yield (60.94 vs. 23.07 ng/µl) and were more frequently cell-rich (54%) than the LBC slides (31.4%), the differences in adequacy (direct smears: 97.4%; LBCs: 94.1%) and in mutant rate (direct smears: 10.3%; LBCs: 14.0%) between the two sample types did not reach statistical significance. Conclusions: Not only direct smears but also LBC slides represent an effective preparation and storage medium for cytological material to be used for EGFR molecular testing.

Summary Aim To report an outbreak of extensively drug-resistant (XDR) Acinetobacter baumannii in the neonatal intensive care unit (NICU) of an Italian university hospital. Patient risk profiles for acquisition of A. baumannii and measures used to control the outbreak are described. Methods Antibiotic susceptibility of strains was evaluated by microdilution. Genotyping was performed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing. Carbapenemase genes were analysed by polymerase chain reaction and DNA sequencing. A case–control study was designed to identify risk factors for acquisition of A. baumannii . Findings A. baumannii was isolated from 22 neonates, six of whom were infected. One major PFGE type was identified, assigned to sequence type (ST) 2, corresponding to International Clone II; this was indistinguishable from isolates from the adult ICU in the same hospital. A. baumannii isolates were resistant to aminoglycosides, quinolones and classes of β-lactam antibiotics, but were susceptible to tigecycline and colistin. Carbapenem resistance was associated with the presence of transposon Tn 2006 carrying the bla OxA-23 gene. Length of NICU stay, length of exposure to A. baumannii , gestational age, use of invasive devices and length of exposure to invasive devices were significantly associated with acquisition of A. baumannii on univariate analysis, while length of exposure to central venous catheters and assisted ventilation were the only independent risk factors after multi-variate analysis. Conclusions This XDR A. baumannii outbreak in an NICU was probably caused by intrahospital transfer of bacteria via a colonized neonate whose mother was admitted to the adult ICU. Strengthened infection control measures were necessary to control the outbreak.

Transition to parenthood represents an important life event increasing vulnerability to psychological disorders. Postpartum depression and parenting distress are the most common psychological disturbances and a growing scientific evidence suggests that both mothers and fathers are involved in this developmental crisis. This paper aims to explore maternal and paternal experience of transition to parenthood in terms of parenting distress and risk of postpartum depression. Seventy-five couples of first-time parents were invited to compile the Edinburgh Postnatal Depression Scale and the Parenting Stress Index-Short Form in the first month of children life. Study sample reported very high levels of parenting distress and a risk of postpartum depression in 20.8% of mothers and 5.7% of fathers. No significant correlation between parenting distress and the risk of postpartum depression emerged, both in mothers than in fathers group while maternal distress levels are related to paternal one. The first month after partum represents a critical phase of parents life and it could be considered a developmental crisis characterized by anxiety, stress and mood alterations that could have important repercussions on the child psycho-physical development.

INTRODUCTIONGastric metastases from lung adenocarcinoma are rare and usually associated with disseminated disease. The great majority is asymptomatic and in few cases discovered during autopsy studies. Reports of single metachronous metastases during the lifetime are anecdotal. We describe a case of solitary gastric metastasis 5 years after lung surgery.PRESENTATION OF CASEA 68-year-old male submitted in 2006 to right lobectomy for lung adenocarcinoma was referred at Emergency Room department in 01/2011 because of chronic epigastric pain. Radiologic and endoscopic evaluation showed a bulky lesion inside the stomach, originating from the muscular layer, suspected for GIST. He underwent a subtotal gastrectomy and the pathologic examination revealed an undifferentiated adenocarcinoma, positive for Thyroid Transcriptional Factor-1, Cytokeratin 7, AE 1/3 and CEA, confirming the pulmonary origin.DISCUSSIONAt the time of diagnosis about 50% of lung cancer are metastatic, with survival rates of 1% at 5-year. Gastric metastasis is very rare; autopsy studies report an incidence of 0.2–0.5%. They develop in the submucosa, usually without any symptom and the diagnosis is incidental during the staging of primary cancer or the follow-up. There are no guidelines about surgical treatment; however few cases of long-term survival following the operation were reported. Pathologic diagnosis is difficult, but the immunohistochemical staining helps to recognize the primary origin.CONCLUSIONSolitary metachronous gastric metastasis from pulmonary adenocarcinoma is an exceptional event, but it could happen during the follow-up. It seems that a radical resection, in absence of systemic implants, might provide survival benefits in selected patients.