Search

During the last ten years, the worldwide interest in using insects as food and feed has surged. Edible insects fall within the category of novel foods, i.e., the category of food not consumed in significant amounts in the European Union before 15 May 1997 (the date of entry into force of Regulation (EC) No. 258/1997, later repealed by Regulation (EU) No. 2283/2015). One of the most promising insect species to be raised for food is the house cricket (Acheta domesticus). In this study, the rearing of a stock of house crickets was studied over a period of four months. The microbiological quality of the farm was studied using swabs on the surface of the rearing boxes to analyze the trend over time of different populations of microorganisms (total aerobic mesophilic microbiota, Lactobacillus spp., enterococci, Staphylococcus spp., Enterobacteriaceae, total coliforms, Pseudomonas spp. and molds). The presence of four antimicrobial resistance genes (aph, blaZ, sul1, and tetM) was investigated by polymerase chain reaction. A production scheme was also developed in order to obtain a cricket-based flour, which was analyzed for its microbiological and chemical-centesimal profile. The results obtained in this study demonstrate that the contamination increases with time and that a proper management of the farming system for insects is of the utmost importance, as it is for conventional farm animals such as ungulates, poultry, and rabbits. The old-fashioned adage “all full, all empty” for the farming system summarizes the need for proper cleaning and disinfection of the structures at the end of each production cycle.

Abstract This study describes a simple method for the large‐scale isolation of pure Toxoplasma gondii tachyzoites and bradyzoites. T. gondii tachyzoites were obtained from infected human foreskin fibroblasts (HFFs) and peritoneal exudates of mice, while tissue cysts containing bradyzoites were collected from chronically infected mice. Harvested cells and brain tissues were incubated in Hanks balanced salt solution (HBSS), containing 0.25% trypsin and 0.5% taurodeoxycholic acid (TDC) for 5 min. Subsequent washes in phosphate buffered saline (PBS) were conducted, and the cell viability of the preparations was good, as determined by flow cytometry and ability to reinfect HFF cells and propagate in mice. The purification procedure allowed for a rapid preparation of pure T. gondii tachyzoites and bradyzoites in sufficient quantity that can be used for downstream procedures. The advantage of the new method is that it is convenient and inexpensive.

Staphylococcus aureus is a gram-positive bacterium, commonly found in the nostrils, on the skin and on the hair of warm-blooded animals, including humans. It can produce a wide variety of virulence factors, including staphylococcal enterotoxins (SEs). In literature, 24 different SEs and many variants have been identified; among these, only five (the so-called classic enterotoxins) have been well-defined. Due to their emetic activity, SEs are frequently responsible for staphylococcal food poisoning, when consumers ingest contaminated food. SEs are proteins with a high tolerance of denaturing and can maintain their activity, even when the vegetative form of the bacteria is inactivated during food processing. The enterotoxin encoding genes are found in a variety of different genetic elements and, as a result, enterotoxin production varies widely between different populations of S. aureus. SEs production is modulated by multiple, and often overlapping, regulatory pathways, which are influenced by environmental factors. Furthermore, complex food matrices possess many characteristics (storage temperature, pH, sugar or salt concentration, presence of competitive microorganisms, etc.) that have a high impact on S. aureus behaviour. The multiple factors influencing S. aureus growth in food matrices and the production of SE complicates risk assessment procedures. In this review, we focus on enterotoxin production by S. aureus in food of animal origin, its regulation and detection and on the most recent developments in predictive microbiology and risk assessment models.Highlights Staphylococcus aureus produces several virulence factors that contribute to the pathogenesis of several serious human diseases: among these staphylococcal enterotoxins (SEs) have emetic activity, and are responsible for staphylococcal food poisoning (SFP). SEs are proteins that maintain their activity even though the vegetative form of the bacteria is inactivated during food processing. Enterotoxin encoding genes are found in a variety of genetic elements and enterotoxin production varies and is regulated by multiple regulatory pathways, which are influenced by environmental factors.

California is the largest agricultural producer in the United States and local dairy industry produces 21.5% of the national milk supply. There are 1470 dairies, 1789 million dairy cows and a total annual milk production of 18 million metric tons. The amount of dead cows to be disposed of is remarkable in intensive farming and it increases in periods of extreme weather events, such as drought in California. Composting of bovine mortalities is prohibited in California as a means of disposal of carcases, and can only be done under an emergency declaration. Composting is an effective disposal method that can aid in carcase disposal, especially during an emergency. The objective of this study was to use screened dairy manure solids (SDMS) as the composting amendment for carcase decomposition. Our hypothesis was that temperatures would be sufficiently high and of sufficient duration to destroy most bacteria within the carcases and that the leachate from the carcases would penetrate less than one foot into the underlying soil. No significant amounts of leachate were noted in the collection pipes buried beneath either soil type. Total bacterial counts exceeded 1 × 106 CFU/ml in approximately 19% of the swab samples from the sample collection pipes. The sandy soil had higher bacterial counts than the clay soil. Results of these trials indicate that adult dairy cows can be successfully composted without significant impact on the nearby surrounding environment. The basic hypotheses have been verified by the simple and multiple regression and chi-square non-parametric test.

This study evaluated the effect of a ‘formulation of selected dairy starter cultures and commercial probiotics’, associated with ‘specific ripening conditions’ in the production of nitrite and nitrate-free, low-acid, fermented pork sausage, produced in a small-scale plant in Umbria (Italy), and their effect on microbiological, physico-chemical and sensorial properties of the products. The following conditions were investigated throughout the experiment: (i) the use of the ‘formulation of selected dairy starter cultures and commercial probiotics’ vs no starter; (ii) the use of ‘specific ripening conditions’ vs ‘classic ripening conditions’; (iii) the use of nitrate vs nitrite and nitrate-free sausages. Sensory properties were improved and pathogen control was greater in salami to which a ‘formulation of selected dairy starter cultures and commercial probiotics’ had been added, especially under ‘specific ripening conditions’. The proposed formulation, together with the specific ripening conditions, greatly inhibited the growth of undesirable microbiota, reduced the isolation rate of pathogens and preserved the acceptability of the salami.

The early diagnosis of mastitis is an essential factor for the prompt detection of the animal for further actions. In fact, if not culled, infected cows must be segregated from the milking herd and milked last, or milked with separate milking units. Besides microbiological analysis, the somatic cell count (SCC) commonly used as predictor of intramammary infection, frequently lead to a misclassification of milk samples. To overcome these limitations, more specific biomarkers are continuously evaluated. The total amino acid content increases significantly in mastitic milk compared to normal milk. S. aureus requires branched-chain amino acids (BCAAs—isoleucine, leucine, and valine) for protein synthesis, branched-chain fatty acids synthesis, and environmental adaptation by responding to their availability via transcriptional regulators. The increase of BCAAs in composite milk has been postulated to be linked to mammary infection by S. aureus. The aim of this work is to demonstrate, by a direct ion-pairing reversed-phase method, based on the use of the evaporative light-scattering detector (IP-RP-HPLC-ELSD), applied to 65 composite cow milk samples, a correlation between the concentration of isoleucine and leucine, and S. aureus load. The correlation coefficient, r, was found to be 0.102 for SCC (p = 0.096), 0.622 for isoleucine (p < 0.0001), 0.586 for leucine (p < 0.0001), 0.013 for valine (p = 0.381), and 0.07 for tyrosine (p = 0.034), standing for a positive correlation between S. aureus and isoleucine and leucine concentration. The link between the content of BCAAs, isoleucine and leucine, and udder infection by S. aureus demonstrated with our study has an important clinical value for the rapid diagnosis of S. aureus mastitis in cows.

Aging rooms of dry-cured ham and cheese are subject to the development of mold and arthropods, above all mites, which cause the erosion of the external part of these food products, reducing their surface to dust and determining their progressive degradation. This work is meant to give the results of an experiment carried out to test the level of protection supplied by AlistagTM, a coating agent based on Gluconodeltalactone, on a typical Sicilian cheese cosacavaddu ibleo. AlistagTM was applied by nebulization and by immersion. The two parameters took in consideration to evaluate their efficacy were the presence/absence of cracks and the level of dustiness on the surface of the cheese. AlistagTM turned out to be very efficient in protecting cheese both from the development of dust and from cracks; between the two kinds of application tested, immersion resulted to be the best one, for the protecting action lasts longer.

The early diagnosis of cow subclinical mastitis represents a pivotal factor for a prompt and adequate animal treatment. Although several methods are available, the somatic cells count (SCC) still remains the elective test directly carried out on milk samples. In mastitis affected cows (even at subclinical stages), altered concentrations of specific metabolites, including free amino acids, is a well-known occurrence. In order to define the relationships between the variation of the unbound amino acids content with the SCC value, a direct ion-pairing reversed-phase method based on the use of the evaporative light-scattering detector (IP-RP-HPLC-ELSD) was applied to 65 cow milk samples. The statistical analysis of variance (ANOVA) was pursued in order to find a correlation between the SCC value and the concentration of isoleucine (Ile), leucine (Leu), valine (Val) and tyrosine (Tyr). Samples were divided in two groups according to their SCC value: Group I comprised all ones with SCC < 400,000 cells/mL; Group II encompassed those with a SCC > 400,000 cells/mL. Statistical analyses highlighted significant differences in the content of the branched-chain amino acids Ile and Leu, between the two groups (p < 0.02* and

The transfer of antibiotic resistance via the food chain is a global concern. Nevertheless, more attention is required to non-pathogenic strains, such as spoilage bacteria, which could transmit genes to pathogens. Although Lactic Acid Bacteria are microorganisms generally recognized as safe, Leuconostoc mesenteroides may reach and maintain high concentration levels on the surface of cooked products and ready-to-eat products throughout the entire shelf life. It is therefore important to consider the possibility for this species to carry antibiotic- resistance genes. The present research deals with the antibiotic susceptibility profile of strains of L. mesenteroides, isolated from vacuum packaged cooked meat products. In this study, the antimicrobial susceptibility of L.mesenteroides, previously isolated from cooked ham, was investigated through disk diffusion assay according to CLSI standards. Isolated strains from ready-to-eat food show high levels of resistance to ampicillin and methicillin and, according to a settled panel of 21 antibiotics, the antibiotic resistance was demonstrated for the 50% of the tested molecules.

This paper analyses data obtained from an online survey related to animal welfare and religious slaughter topics. The questionnaire was conducted with the purpose of examining the purchase behaviour of a group of consumers (with different religious orientation) and their views on animal protection and ritual slaughter. The main results of the consultation were two. The first evidenced the respondents’ great interest about the question on animal welfare, which is in accordance with the growing interest of European citizens concerning this issue. The second was the demand for a more transparent labelling of animal products, which would also reflect animal welfare and the slaughter method used. These results are in contrast with marketing analysis, which finds that consumers want to only receive positive information. Paradoxically, the more information is transmitted to reassure consumers, the higher is the risk to alarm them.

Several growth curves for selected pathogens and hygiene indicators alone and vs selected dairy starter cultures (LAB) and commercial probiotics have been performed. All strains for LAB and commercial probiotics were inoculated as pure cultures into skim milk to get an initial cocci:bacilli:enterocci ratio of 2:1:1 and a concentration of approximately 107 cfu mL–1 until challenge vs selected pathogens and hygiene indicators. Selected pathogens came from the collection of the Laboratorio di Ispezione degli Alimenti di O.A. or were reference strains (Escherichia coli, CSH26 K12, Staphylococcus aureus 27R, Salmonella Derby 27, Pseudomonas fluorescens ATCC 13525, Listeria innocua ATCC 33090). Each strain was inoculated into skim milk to get an initial concentration of approximately 106 cfu mL–1. Growth curves in skim milk for the following challenges were studied: i) sterility control; ii) association LAB; iii) association of LAB vs each selected pathogen or hygiene indicator; iv) selected pathogen or hygiene indicator alone. The challenges were carried out in BHI broth and in skim milk at 37°C. The highest reduction was observed in milk but in general the association of LAB and the probiotic was able to limit the growth of pathogens and hygiene indicators.

A total of 788 serum samples from dairy cattle in Umbria, Italy, were tested for the presence of antibodies to Mycobacterium avium subspecies paratuberculosis (Map) using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The sampled animals came from 19 herds representative of the central area of the Umbria county (Perugia and Assisi districts). Using the manufacturer suggested cut-off for a positive test, 44 animals (5.6%) were positive. Using the sensitivity and specificity claimed by the manufacturer of the ELISA kit, the true prevalence in Umbria dairy cattle overall was calculated as 9.7% (99% CI, 7.0%, 12.4%).

In the last years, direct sale of raw milk by vending machines has largely increased in several European Countries and Italy. As a consequence, adequate hygienic measures and correct consumer’s information is required in order to reduce any potential risk linked to this product. In the present study, the occurrence of pathogens (Salmonella spp., verocytotoxigenic E.coli, Campylobacter spp., Listeria monocytogenes, S.aureus) in raw milk and bovine faeces collected in 4 farms in the province of Pesaro-Urbino (Italy), between January 2007 and March 2009 has been investigated; 99.5% of milk samples resulted negative for the pathogens considered and complying with the regulation S. aureus limits. Campylobacter has been found in 0.44% of the samples, collected during summer, while only one sample resulted positive to a non-verocytotoxigenic E.coli O157. In respect to faeces, 62.6% of the samples resulted negative, 33.6% were contaminated by Campylobacter spp. (68% Campylobacter coli and 32% Campylobacter jejuni) and 3.8% by E.coli O157. No samples resulted positive for Salmonella spp. or Listeria monocytogenes. The results highlight the necessity of a strict plan of hygienic and sanitary controls, with particular attention to milking process hygiene and raw milk storage, to reduce the risk of contamination of the product.

Seminoma is the most common testicular cancer. Pituitary tumor-transforming gene 1 (PTTG1) is a securin showing oncogenic activity in several tumors. We previously demonstrated that nuclear PTTG1 promotes seminoma tumor invasion through its transcriptional activity on matrix metalloproteinase 2 ( MMP-2 ) and E-cadherin ( CDH1 ). We wondered if specific interactors could affect its subcellular distribution. To this aim, we investigated the PTTG1 interactome in seminoma cell lines showing different PTTG1 nuclear levels correlated with invasive properties. A proteomic approach upon PTTG1 immunoprecipitation uncovered new specific securin interactors. Western blot, confocal microscopy, cytoplasmic/nuclear fractionation, sphere-forming assay, and Atlas database interrogation were performed to validate the proteomic results and to investigate the interplay between PTTG1 and newly uncovered partners. We observed that spectrin beta-chain (SPTBN1) and PTTG1 were cofactors, with SPTBN1 anchoring the securin in the cytoplasm. SPTBN1 downregulation determined PTTG1 nuclear translocation, promoting its invasive capability. Moreover, a PTTG1 deletion mutant lacking SPTBN1 binding was strongly localized in the nucleus. The Atlas database revealed that seminomas that contained higher nuclear PTTG1 levels showed significantly lower SPTBN1 levels in comparison to non-seminomas. In human seminoma specimens, we found a strong PTTG1/SPTBN1 colocalization that decreases in areas with nuclear PTTG1 distribution. Overall, these results suggest that SPTBN1, along with PTTG1, is a potential prognostic factor useful in the clinical management of seminoma.

(1) Background: Sarcopenia lasting >1 year might be considered a chronic condition in many HNSCC patients. CT-scan-derived Skeletal Muscle Mass Index (SMI) is an established surrogate of sarcopenia; yet, the cut-off reported in the literature (literature-based, lb-SMI < 43.2) is mainly based on the risk of chemoradiotherapy-induced toxicity, and the optimal value to discriminate OS is under-investigated. (2) Methods: The effect on OS of the lb-SMI cutoff was compared with an untailored OS-oriented SMI cutoff obtained in a cohort of consecutive advanced HNSCC patients treated with primary chemoradiotherapy, bio-chemotherapy or chemo-immunotherapy (cohort-specific, cs-SMI cutoff). Gender- and BMI-tailored (gt-SMI and bt-SMI) cut-offs were also evaluated. Cutoff values were identified by using the maximally selected rank statistics for OS. (3) Results: In 115 HNSCC patients, the cs-SMI cutoff was 31.50, which was lower compared to the lb-SMI reported cut-off. The optimal cut-off separately determined in females, males, overweight and non-overweight patients were 46.02, 34.37, 27.32 and 34.73, respectively. gt-SMI categorization had the highest effect on survival ( p < 0.0001); its prognostic value was independent of the treatment setting or the primary location and was retained in a multivariate cox-regression analysis for OS including other HNSCC-specific prognostic factors ( p = 0.0004). (4) Conclusions: A tailored SMI assessment would improve clinical management of sarcopenia in chemoradiotherapy-, bio-chemotherapy- or chemo-immunotherapy-treated HNSCC patients. Gender-based SMI could be used for prognostication in HNSCC patients.

Glioblastoma (GBM) is the most frequent adult malignant brain tumour and despite different therapeutic efforts, the median overall survival still ranges from 14 to 18 months. Thus, new therapeutic strategies are urgently needed. However, the identification of cancer-specific targets is particularly challenging in GBM, due to the high heterogeneity of this tumour in terms of histopathological, molecular, genetic and epigenetic features. Telomerase reactivation is a hallmark of malignant glioma. An activating mutation of the hTERT gene, encoding for the active subunit of telomerase, is one of the molecular criteria to establish a diagnosis of GBM, IDH-wildtype, in the 2021 WHO classification of central nervous system tumours. Telomerase inhibition therefore represents, at least theoretically, a promising strategy for GBM therapy: pharmacological compounds, as well as direct gene expression modulation therapies, have been successfully employed in in vitro and in vivo settings. Unfortunately, the clinical applications of telomerase inhibition in GBM are currently scarce. The aim of the present systematic review is to provide an up-to-date report on the studies investigating telomerase inhibition as a therapeutic strategy for malignant glioma in order to foster the future translational and clinical research on this topic.

Background: Recently, it was reported that the Bladder EpiCheck test is likely to represent a valid tool in the diagnostic process of patients who have suspected bladder carcinoma, with some controversial management decisions because of the technical limitations of cytology., Methods: Two hundred ninety patients with a diagnosis of nonmuscle-invasive bladder carcinoma who were admitted at the authors' department from March 2019 to December 2019 were treated and followed for 1 year. During follow-up, all patients were evaluated by voided urine cytology, white-light cystoscopy (according to European Association of Urology guidelines), and the Bladder EpiCheck test., Results: The cytologic diagnoses of high-grade urothelial carcinoma (HGUC) and suspicious for HGUC were histologically confirmed in 5 of 20 patients (25%) who had quantitative Bladder EpiCheck scores (EpiScores) from 60 to 69, in 23 of 36 patients (64%) who had EpiScores from 70 to 79, and in 42 of 56 patients (75%) and 57 of 63 patients (90%) who had EpiScores between 80 and 89 and EpiScores >90, respectively. Of 48 patients who had a cytologic diagnosis of HGUC or suspicious for HGUC with EpiScores ≥60 and negative histology, 20 (42%) had a recurrence of HGUC, which was cytologically and histologically confirmed, at 6-12 months during follow-up., Conclusions: To the best of the authors' knowledge, this is the first study in which patients at high risk for HGUC were stratified using the Bladder EpiCheck EpiScore. The results validate this methylation analysis tool as a useful method for predicting recurrent HGUC during the follow-up of patients with nonmuscle-invasive bladder carcinoma., (© 2022 The Authors. Cancer Cytopathology published by Wiley Periodicals LLC on behalf of American Cancer Society.)

Background: Failure of clinical trials with targeted therapies in glioblastoma (GBM) is probably related to the enrollment of molecularly unselected patients. In this study we report the results of a precision medicine protocol in recurrent GBM., Methods: We prospectively evaluated 34 patients with recurrent GBM. We determined the expression of vascular endothelial growth factor (VEGF), epidermal growth factor receptor variant III (EGFRvIII), and phosphatase and tensin homolog (PTEN). According to the molecular pattern we administered bevacizumab alone in patients with VEGF overexpression, absence of EGFRvIII, and normal PTEN (group A; N.=16); bevacizumab + erlotinib in patients with VEGF overexpression, expression of EGFRvIII, and normal PTEN (group B; N.=14); and bevacizumab + sirolimus in patients with VEGF overexpression and loss of PTEN, irrespective of the EGFRvIII status (group C; N.=4). We evaluated the response rate, the clinical benefit rate, the 6-month progression-free survival (PFS-6), the 12-month PFS (PFS-12) and the safety profile of the treatment. Moreover, we compared our results with the ones of EORTC 26101 trial., Results: Response rate was 50% in the whole cohort with the highest rate in group C (75%). Clinical benefit rate was 71% with the highest rate in group C (75%). PFS-6 was 56% in the whole cohort with the highest rate in group B (64%). PFS-12 was 21% in the whole cohort with the highest rate in group B (29%). When comparing our results with those from the combination arm of the EORTC 26101 trial we found a significantly higher PFS-6 and PFS-12 in our cohort., Conclusions: The precision medicine protocol for recurrent GBM is feasible and leads to improved results if compared with studies lacking molecular selection.

Grade 3 meningiomas are rare malignant tumors that can originate de novo or from the progression of lower grade meningiomas. The molecular bases of anaplasia and progression are poorly known. We aimed to report an institutional series of grade 3 anaplastic meningiomas and to investigate the evolution of molecular profile in progressive cases. Clinical data and pathologic samples were retrospectively collected. VEGF, EGFR, EGFRvIII, PD-L1; and Sox2 expression; MGMT methylation status; and TERT promoter mutation were assessed in paired meningioma samples collected from the same patient before and after progression using immunohistochemistry and PCR. Young age, de novo cases, origin from grade 2 in progressive cases, good clinical status, and unilateral side, were associated with more favorable outcomes. In ten progressive meningiomas, by comparing molecular profile before and after progression, we identified two subgroups of patients, one defined by Sox2 increase, suggesting a stem-like, mesenchymal phenotype, and another defined by EGFRvIII gain, suggesting a committed progenitor, epithelial phenotype. Interestingly, cases with Sox2 increase had a significantly shortened survival compared to those with EGFRvIII gain. PD-L1 increase at progression was also associated with worse prognosis, portending immune escape. We thus identified the key drivers of meningioma progression, which can be exploited for personalized treatments.

In January 2022, our institution launched a comprehensive cancer genome profiling program on 10 cancer types using a non-IVD solution named the TruSight Oncology 500 Assay provided by Illumina ® . The assay analyzes both DNA and RNA, identifying Single-Nucleotide Variants (SNV)s and Insertion-Deletion (InDel) in 523 genes, as well as known and unknown fusions and splicing variants in 55 genes and Copy Number Alterations (CNVs), Mutational Tumor Burden (MTB) and Microsatellite Instability (MSI). According to the current European IVD Directive 98/79/EC, an internal validation was performed before running the test. A dedicated open-source bioinformatics pipeline was developed for data postprocessing, panel assessment and embedding in high-performance computing framework using the container technology to ensure scalability and reproducibility. Our protocols, applied to 71 DNA and 64 RNA samples, showed full agreement between the TruSight Oncology 500 assay and standard approaches, with only minor limitations, allowing to routinely perform our protocol in patient screening.

(1) Background: PTTG1 sustains the EMT process and the invasiveness of several neoplasms. We previously showed the role of nuclear PTTG1 in promoting invasiveness, through its transcriptional target MMP2, in seminoma in vitro models. Here, we investigated the key players involved in PTTG1-mediated EMT in human seminoma. (2) Methods: Two seminoma cell lines and four human seminoma tumor specimens were used. E-Cadherin gene regulation was investigated using Western blot, real-time PCR, and luciferase assay. Immunoprecipitation, ChIP, RE-ChIP, and confocal microscopy analysis were performed to evaluate the interplay between PTTG1 and ZEB1. Matrigel invasion and spheroid formation assays were applied to functionally investigate PTTG1 involvement in the EMT of seminoma cell lines. RNA depletion and overexpression experiments were performed to verify the role of PTTG1/ZEB1 in E-Cadherin repression and seminoma invasiveness. E-Cadherin and ZEB1 levels were analyzed in human testicular tumors from the Atlas database. (3) Results: PTTG1 transcriptionally represses E-Cadherin in seminoma cell lines through ZEB1. The cooperation of PTTG1 with ZEB1 has a significant impact on cell growth/invasion properties involving the EMT process. Analysis of the Atlas database of testicular tumors showed significantly lower E-Cadherin levels in seminoma, where PTTG1 showed nuclear staining. Finally, PTTG1 and ZEB1 strongly localize together in the periphery of the tumors. (4) Conclusions: These results strengthen the evidence for a role of PTTG1 in the EMT process in human seminomas through its cooperation with the transcriptional repressor ZEB1 on the E-Cadherin gene. Our data enrich the molecular characterization of seminoma, suggesting that PTTG1 is a prognostic factor in seminoma clinical management.

Meningiomas are mostly benign tumors that, at times, can behave aggressively, displaying recurrence despite gross-total resection (GTR) and progression to overt malignancy. Such cases represent a clinical challenge, particularly because they are difficult to recognize at first diagnosis. SOX2 (Sex-determining region Y-box2) is a transcription factor with a key role in stem cell maintenance and has been associated with tumorigenesis in a variety of cancers. The purpose of the present work was to dissect the role of SOX2 in predicting the aggressiveness of meningioma. We analyzed progressive/recurrent WHO grade 1−2 meningiomas and WHO grade 3 meningiomas; as controls, non-recurring WHO grade 1 and grade 2 meningioma patients were enrolled. SOX2 expression was evaluated using both immunohistochemistry (IHC) and RT-PCR. The final analysis included 87 patients. IHC was able to reliably assess SOX2 expression, as shown by the good correlation with mRNA levels (Spearman R = 0.0398, p = 0.001, AUC 0.87). SOX2 expression was an intrinsic characteristic of any single tumor and did not change following recurrence or progression. Importantly, SOX2 expression at first surgery was strongly related to meningioma clinical behavior, histological grade and risk of recurrence. Finally, survival data suggest a prognostic role of SOX2 expression in the whole series, both for overall and for recurrence-free survival (p < 0.0001 and p = 0.0001, respectively). Thus, SOX2 assessment could be of great help to clinicians in informing adjuvant treatments during follow-up.

Predictive factors for response to regorafenib in recurrent glioblastoma, IDH-wildtype, are scarcely recognized. The objective of this study was to identify molecular predictive factors for response to regorafenib using a clinically available platform. We analyzed a prospective cohort of 30 patients harboring recurrent glioblastoma, IDH-wildtype, and treated with regorafenib. Next-generation sequencing (NGS) analysis was performed on DNA extracted from paraffin-embedded tissues using a clinically available platform. Moreover, MGMT methylation and EGFRvIII expression analyses were performed. Six-month progression-free survival (PFS) was 30% and median overall survival (OS) was 7.5 months, in line with literature data. NGS analysis revealed a mutation in the EGFR pathway in 18% of cases and a mutation in the mitogen-activated protein-kinase (MAPK) pathway in 18% of cases. In the remaining cases, no mutations were detected. Patients carrying MAPK pathway mutation had a poor response to regorafenib treatment, with a significantly shorter PFS and a nonsignificantly shorter OS compared to EGFR-mutated patients (for PFS, 2.5 vs 4.5 months, p = 0.0061; for OS, 7 vs 9 months, p = 0.1076). Multivariate analysis confirmed that MAPK pathway mutations independently predicted a shorter PFS after regorafenib treatment (p = 0.0188). The negative prognostic role of MAPK pathway alteration was reinforced when we combined EGFR-mutated with EGFRvIII-positive cases. Recurrent glioblastoma tumors with an alteration in MAPK pathway could belong to the mesenchymal subtype and respond poorly to regorafenib treatment, while EGFR-altered cases have a better response to regorafenib. We thus provide a molecular selection criterion easy to implement in the clinical practice., (© 2022. The Author(s).)

The prognostic role of epidermal growth factor receptor variant III (EGFRvIII), a constitutively activated oncogenic receptor, in glioblastoma is controversial. We performed a prospective study enrolling 355 patients operated on for de novo glioblastoma at a large academic center. The molecular profile, including EGFRvIII status, MGMT promoter methylation, and VEGF expression, was assessed. Standard parameters (age, clinical status and extent of surgical resection) were confirmed to hold prognostic value. MGMT promoter methylation portended a slightly improved survival. In the whole series, confirming previous results, EGFRvIII was not associated with worsened prognosis. Interestingly, female sex was associated with a better outcome. Such findings are of interest for the design of future trials.

Background: The management of non-muscle invasive bladder carcinoma (NMIBC) after transurethral resection of a bladder tumor consists of adjuvant intravesical therapy and strict and long surveillance with urine cytology and cystoscopy. The Bladder EpiCheck test (Nucleix Ltd) (BE) is a newly developed urinary markers based on DNA methylation changes in a panel of 15 genomic biomarkers, with a promising performance in term of non-invasive NMIBC detection., Methods: In this study we prospectively enrolled 151 consecutive patients with high grade NMIBC, treated with intravesical BCG and mitomycin C therapy and evaluated during the follow-up by voided urine cytology and white-light cystoscopy, according to the European Association of Urology Guidelines. The Bladder EpiCheck test was performed at the same time of urine cytology in voided specimen. In all cases with positive cytology the diagnosis was confirmed by histology and a diagnosis was made according to the 2017 tumor, node, metastasis (TNM) classification and graded using both the 1973 and the 2004 World Health Organization (WHO) classifications., Results: At three months of follow-up, we reported similar overall specificity rates for BE and urine cytology (85,1% vs 86,3%). In the group of patients with carcinoma in situ (CIS), we found the same specificity for BE and urine cytology (81,4%), while in the groups of patients with papillary high grade NMIBC, the specificity of BE was higher compared to cytology (96,3% vs 90,4%). The sensitivity of BE was always higher compared to cytology during all the follow-up both for papillary NMIBC and CIS., Conclusion: In the early follow-up of NMIBC the EpiCheck test might replace urinary cytology., Competing Interests: Conflict of interest The authors have no conflicts of interest to disclose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Numerous studies showed that bladder urothelial carcinoma and upper urothelial tract carcinoma (UTUC) display clinical and genomic similarities. In order to analyze that the same panel of biomarkers used in the diagnosis of bladder urothelial carcinoma could be suitable for early detection of UTUC, we performed a retrospective study in which we analyzed Bladder EpiCheck scores in the urinary samples obtained by selective ureteral catheterization in a high-grade UTUC cohort, correlating the results with urinary cytology and diagnostic urethral biopsies. The present study represents a retrospective analysis of 82 patients with clinically localized high-grade UTUC (60 renal pelvis UTUC, 22 ureter UTUC) who had undergone a radical nephroureterectomy (RNU) at our Urology department from June 2018 to November 2020. Before any surgical procedure, one sample of urine, obtained by selective ureteral catheterization, was collected for each patient for cytological examination, and the remaining material was stored for the Bladder EpiCheck test. Our results showed that the sensitivity of the methylation test for high-grade UTUC was about 97.4%, significantly higher than the sensitivity of urinary cytology either considering the HGUC cytological diagnosis or including in the positive cases the SHGUC cytological diagnosis (97.4% versus 59% or 70.5%). The methylation analysis of urinary samples may represent a valid tool in the diagnostic process of patients with suspected UTUC. In cases with a difficult clinical decision after upper urinary tract biopsy and cytology, the methylation test could assist in the clinical management of UTUC patients., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Mutational analysis contributes to the diagnosis and prognosis of thyroid nodules analyzed with fine-needle aspiration cytology (FNAC). Although several advanced molecular tests based on multiple molecular markers are available for clinical use and have increased their impact on clinical management of patients, they are not widely available. Among them is BRAF V600E, one of the most studied mutations. Other genetic alterations, such as TERT promoter mutations, may coexist in thyroid carcinomas. Previous studies have demonstrated that this duet might be involved in the aggressiveness of thyroid cancer, although its prognostic value related to mortality remains undefined. The detection of such genetic alterations in thyroid liquid-based cytology (LBC) thus may assist with patient management., Methods: From January 2013 to June 2014, 356 thyroid FNAC samples were processed by LBC, including 174 surgical follow-up samples. BRAF V600E and TERT mutation analyses were performed on both LBC and histopathology., Results: The study included 119 samples categorized as atypia of undetermined significance, 42 categorized as follicular neoplasms, 61 categorized as suspicious for malignancy, and 34 categorized as positive for malignancy. BRAF V600E mutation was detected in 10.4% of all cases, whereas TERT promoter mutations were identified in 1.1%. TERT-mutated cases belonged to the positive for malignancy category, with a histologic diagnosis of tall cell variant of papillary thyroid carcinoma. These genetic alterations correlated with lymph node metastases (P = .0349) and higher disease stage., Conclusions: BRAF V600E and TERT analysis can be performed on LBC. TERT mutations are rarely identified in well differentiated thyroid carcinoma but are associated with higher stage. Although a larger molecular panel may offer more information, analyzing these few point mutations is still likely to be useful for managing potentially more aggressive thyroid carcinomas., (© 2021 American Cancer Society.)

If KRAS mutation status of primary colorectal tumor is representative of corresponding colorectal liver metastases (CRLM) mutational pattern, is controversial. Several studies have reported different rates of KRAS discordance, ranging from 4 to 32%. Aim of this study is to assess the incidence of discordance and its impact on overall survival (OS) in a homogenous group of patients. KRAS mutation status was evaluated in 107 patients resected for both primary colorectal tumor and corresponding CRLM at the same institution, between 2007 and 2018. Discordance rate was 15.9%. Its incidence varied according to the time interval between the two mutation analyses ( p = 0.025; Pearson correlation = 0.2) and it was significantly higher during the first 6 months from the time of primary tumor evaluation. On multivariable analysis, type of discordance (wild-type in primary tumor, mutation in CRLM) was the strongest predictor of poor OS ( p < 0.001). At multivariable logistic regression analysis, the number of CRLM >3 was an independent risk factor for the risk of KRAS discordance associated with the worst prognosis (OR = 4.600; p = 0.047). Results of our study suggested that, in the era of precision medicine, possibility of KRAS discordance should be taken into account within multidisciplinary management of patients with metastatic colorectal cancer.

Objective: To identify in which cases after cytological diagnosis, the Bladder EpiCheck test could represent an effective tool in non-muscle invasive bladder carcinoma or an useless expence., Materials and Methods: 375 patients diagnosed with non-muscle invasive bladder cancer, 269 with high grade urothelial carcinoma and 106 with carcinoma in situ, were treated and followed for 1 year. The treatment was an intravesical instillation of Bacillus Calmette-Guerin in 305 patients and Mitomycin-C in 70 patients. During the follow-up patients were evaluated by voided urine cytology and white-light cystoscopy, according to the European Association of Urology Guidelines. Bladder EpiCheck test was performed together with cytology in all cases., Results: Analyzing Bladder Epicheck results for each category defined by the Paris System for Reporting Urinary Cytology, we found that the Episcore >60 correlates with histological diagnosis of high grade urothelial carcinoma (HGUC) in atypical urothelial cells and Suspicious for High Grade Urothelial Carcinoma (P = 0.0002 Odds Ratio 0.05926 95% Confidence Interval from 0.01127 to 0.3116 and P = 0.0009 Odds Ratio 0.03155 95% Confidence Interval from 0.001683 to 0.5914, Fisher's exact test, respectively), while in Negative for high grade urothelial carcinoma and HGUC patients Episcore is not helpful to identify cases with histological diagnosis of HGUC (P = 0.101 and P = 0.58 Fisher's exact test, respectively). Considering an Episcore ≥ 90 in the HGUC cytological group, this seems not to be correlated with a histological diagnosis of HGUC (P = 0.090 Fisher's exact test)., Conclusions: Cytology and Bladder EpiCheck test in combination may have the potential to reduce cystoscopies in the follow-up of non-muscle invasive bladder cancer only for cytological diagnoses of atypical urothelial cells and Suspicious for High Grade Urothelial Carcinoma . Moreover, in patients with a cytological diagnosis of Negative for high grade urothelial carcinoma or HGUC, cytology alone seems to be safe and cost-effective., Competing Interests: Conflict of interest The authors have no conflicts of interest to disclose., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Aims: Bladder EpiCheck is one of several urinary tests studied to identify bladder tumours and analyses 15 methylation biomarkers determining bladder cancer presence on the basis of methylation profile., Methods: 374 patients diagnosed with high-grade non-muscle invasive bladder cancer were treated and followed for 1 year with voided urine cytology and white-light cystoscopy and biopsies according to European Association of Urology Guidelines. 268 cases were diagnosed with high-grade papillary carcinoma, while 106 cases were carcinoma in situ. Bladder EpiCheck test was performed together with cytology in all cases., Results: Comparing cytological categories of negative for high-grade urothelial carcinoma (NHGUC) and atypical urothelial cells (AUCs), we found that an EpiScore <60 correlates with NHGUC (p=0.0003, Fisher's exact test), while comparing AUC and suspicious for high-grade urothelial carcinoma (SHGUC) or SHGUC and high-grade urothelial carcinoma (HGUC) categories, an EpiScore ≥60 correlates with SHGUC and HGUC, respectively (p=0.0031 and p=0.0027, Fisher's exact test). In each TPS category, we found that sensitivity, specificity, Positive Predicitve Value (PPV) and Negative Predictive Value (NPV) of the Bladder EpiCheck test in HGUC category were higher than those observed in SHGUC group (sensitivity=98%, specificity=100%, NPV=85.7%, PPV=100% vs sensitivity=86.6%, specificity=52.3%, NPV=84.6%, PPV=56.5%)., Conclusions: Analysing methylation study results, we demonstrated that different TPS cytological categories also carry a distinct molecular signature. Moreover, our results confirm that cytological categories SHGUC and HGUC are different entities also from a molecular point of view and should continue to represent distinct groups in TPS., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Alterations in the transcriptional factor c-MYC could be involved in the anti-EGFR resistance in metastatic colorectal cancer (mCRC). The c-MYC expression was evaluated in 121 RAS and BRAF wild-type mCRC before treatment with anti-EGFR+Folfiri therapy and in 33 subsequent metastases collected during target therapy (TT) or in TT resistance phase. We analyzed the expression and the functional role of some c-MYC linked miRNAs (miR-31-3p, miR-143 and miR-145) in our patient group and in two CRC cell lines, also performing a c-MYC target PCR array. Patients with higher c-MYC expression (HME) showed a significant lower PFS and OS when compared to those with low c-MYC expression (LME). HME pattern was significantly more frequent in the metastases after TT and significantly associated to anti-EGFR molecular resistance alterations. We also found a significant correlation between the expression of the above-mentioned c-MYC linked miRNAs, c-MYC level and anti-EGFR resistance. Moreover, expression gene profiling pointed out the pivotal role of c-MYC in CRC-related cell-cycle, apoptosis, signal transduction and cell-growth pathways. c-MYC expression might distinguish patients with a lower PFS and OS in anti-EGFR treated mCRC. The individuation of some miRNAs involved in the c-MYC pathway regulation and the downstream c-MYC effector genes could provide a new possible target to overcome the anti-EGFR resistance in mCRC.

Seminomas are the most frequent kind of testicular germ cell tumors (TGCTs), accounting for 50% of tumor diagnosis in young men, whereas non-seminomas account for 40% and mixed forms for 10% of cases. It is currently supposed that TGCTs evolve from a pre-invasive stage of carcinoma in situ (CIS). Octamer-binding transcription factor 4 (OCT4) is essential for self-renewal of stem cells. It is considered as a major regulator of cell pluripotency. Prior studies have shown that seminoma expresses OCT4. Transcription factor Krüppel-like factor 4 (KLF4) has moreover associated with embryonic stem cell maintenance. Finally, we previously demonstrated the expression of PTTG1 in CIS and seminomas. In this pilot study, we compared the combined expression of PTTG1 with KLF4 and OCT4 in seminoma, in order to validate our hypotesis that PTTG1 marks a specific population of stem cells in neoplastic tissue, strictly related with tumor. Formalin-fixed and paraffin-embedded testicular tissues by 5 patients who underwent an orchidectomy for seminoma have been collected and immunofluorescence analysis was performed using antibody rabbit monoclonal PTTG-1 and mouse monoclonal OCT4 or mouse monoclonal KLF4 antibody. In seminoma we observed that tumor cells strongly express OCT-4 in all seminomas and in the intratubular areas of seminoma. Expression of KLF-4 was observed in many tumor cells. PTTG1 marks some specific OCT4- and KLF4-positive tumor cells, mainly localized at the periphery of the neoplasm. In the intertubular infiltration areas nests of cells expressing both OCT4/KLF4 and PTTG1 have been observed. This is the first identification of a cell population in seminoma characterized for being OCT4, KLF4, and PTTG1 positive cells in seminoma, associated with cancer invasiveness. Further investigation is needed to elucidate if a functional abrogation of PTTG1 might be used in order to offer new therapeutic approaches in the clinical workout of seminoma., (Copyright © 2019 Grande, Milardi, Martini, Cenci, Gulino, Mancini, Bianchi, Pontecorvi and Pierconti.)

Background: Succinate dehydrogenase (SDH)-deficient gastrointestinal stromal tumors (GISTs) constitute a small KIT/PDGFRA-WT GIST subgroup featuring DNA methylation which, although pervasive, appears nevertheless not randomly distributed. Although often indolent, these tumors are mostly chemorefractory in aggressive cases. Promoter methylation-induced O 6 -methylguanine DNA methyltransferase (MGMT) inactivation improves the efficacy of alkylating agents in gliomas, colorectal cancer and diffuse large B cell lymphoma. MGMT methylation has been found in some GISTs, without determining SDH status. Thirty-six GISTs were enrolled in past sarcoma trials testing alkylating agents, with negative results. Nevertheless, a possible effect on MGMT-methylated GISTs could have escaped detection, since tested GISTs were neither selected by genotype nor investigated for SDH; MGMT was studied in two cases only, revealing baseline activity; these trials were performed prior to the adoption of Choi criteria, the most sensitive for detecting GIST responses to therapy. Under these circumstances, we investigated whether MGMT methylation is preferentially found in SDH-deficient cases (identified by SDHB immunohistochemistry) by analyzing 48 pathogenetically heterogeneous GISTs by methylation-specific PCR, as a premise for possible investigations on the use of alkylating drugs in these tumors., Results: Nine GISTs of our series were SDH-deficient, revealing significantly enriched in MGMT-methylated cases (6/9-67%-, vs. 6/39-15%- of SDH-proficient GISTs; p = 0.004). The pathogenetically heterogeneous KIT/PDGFRA-WT GISTs were also significantly MGMT-methylated (11/24-46%-, vs. 1/24-4%- of KIT/PDGFRA-mutant cases, p = 0.002)., Conclusions: A subset of KIT/PDGFRA-WT GISTs, including their largest pathogenetically characterized subgroup (i.e., SDH-deficient ones), is preferentially MGMT-methylated. This finding could foster a reappraisal of alkylating agents for treating malignant cases occurring among these overall chemorefractory tumors.

Treatment options for recurrent glioblastoma are scarce; targeted therapy trials were disappointing, probably due to enrollment of patients without molecular selection. We treated with bevacizumab and erlotinib a 66-year-old male suffering from recurrent glioblastoma, IDH-wildtype and MGMT unmethylated, after three neurosurgeries. Treatment was tailored on molecular profile of recurrent tumor-namely, EGFRvIII positivity, VEGF overexpression, normal PTEN, low total VEGF and VEGF-121 mRNA-and resulted in complete, exceptionally durable response (51-month progression-free survival). Notably, histology of further recurrence after therapy was reminiscent of sarcoma. We suggest a thorough molecular screening for personalization of targeted therapy in recurrent glioblastoma.

Introduction: Several authors have underlined the limits of morphological analysis mostly in the diagnosis of follicular neoplasms (FN). The application of ancillary techniques, including immunocytochemistry (ICC) and molecular testing, contributes to a better definition of the risk of malignancy (ROM) and management of FN. According to literature, the application of models, including the evaluation of ICC, somatic mutations (ie, BRAF V 600E ), micro RNA analysis is proposed for FNs. This study discusses the validation of a diagnostic algorithm in FN with a special focus on the role of morphology then followed by ancillary techniques., Methods: From June 2014 to January 2016, we enrolled 37 FNs with histological follow-up. In the same reference period, 20 benign nodules and 20 positive for malignancy were selected as control. ICC, BRAF V 600E mutation and miR-375 were carried out on LBC., Results: The 37 FNs included 14 atypia of undetermined significance/follicular lesion of undetermined significance and 23 FN. Specifically, atypia of undetermined significance/follicular lesion of undetermined significance resulted in three goitres, 10 follicular adenomas and one NIFTP whereas FN/suspicious for FN by seven follicular adenomas and 16 malignancies (nine non-invasive follicular thyroid neoplasms with papillary-like nuclear features, two invasive follicular variant of papillary thyroid carcinoma [PTC] and five PTC). The 20 positive for malignancy samples included two invasive follicular variant of PTC, 16 PTCs and two medullary carcinomas. The morphological features of BRAF V 600E mutation (nuclear features of PTC and moderate/abundant eosinophilic cytoplasms) were associated with 100% ROM. In the wild type cases, ROM was 83.3% in presence of a concordant positive ICC panel whilst significantly lower (10.5%) in a negative concordant ICC. High expression values of MirR-375 provided 100% ROM., Conclusions: The adoption of an algorithm might represent the best choice for the correct diagnosis of FNs. The morphological detection of BRAF V 600E represents the first step for the identification of malignant FNs. A significant reduction of unnecessary thyroidectomies is the goal of this application., (© 2018 John Wiley & Sons Ltd.)

Aging rooms of dry-cured ham and cheese are subject to the development of mold and arthropods, above all mites, which cause the erosion of the external part of these food products, reducing their surface to dust and determining their progressive degradation. This work is meant to give the results of an experiment carried out to test the level of protection supplied by Alistag TM , a coating agent based on Gluconodeltalactone, on a typical Sicilian cheese cosacavaddu ibleo . Alistag TM was applied by nebulization and by immersion. The two parameters took in consideration to evaluate their efficacy were the presence/absence of cracks and the level of dustiness on the surface of the cheese. Alistag TM turned out to be very efficient in protecting cheese both from the development of dust and from cracks; between the two kinds of application tested, immersion resulted to be the best one, for the protecting action lasts longer., Competing Interests: Conflict of interest: the authors declare no potential conflict of interest.

Background: Recently, we demonstrated that hypermethylation of SOCS3 determines a significant reduction of its mRNA and protein expression and identifies a subgroup of prostate cancer with aggressive behavior. In this paper, our objective was to investigate whether the immunohistochemical expression of the SOCS3 protein could represent an alternative method to molecular analysis for the individualization of aggressive prostate carcinoma., Materials and Methods: We analyzed the SOCS3 immunohistochemical expression in 65 patients undergoing biopsies at the Institute of Urology of our hospital between September 2011 and October 2011 (median age, 66.4 y; range, 50 to 73 y), and in 35 cases, a subset of 65 cases originally used for the immunohistochemical study, we studied the methylation status of the SOCS3 promoter., Results: We found that the percentage of cases with SOCS3 negativity (-) or with SOCS3 weak staining in <50% of the neoplastic glands (+/-) correlated to the worst prognosis in terms of the Gleason score (P=0.0001; Fisher's exact test), the pT stage (P=0.012; Fisher's exact test), and progression-free survival (P=0.0334; hazard ratio, 0.34; and 95% confidence interval, from 0.1261 to 0.9188). Moreover, some cases with an SOCS3 unmethylated pattern showed SOCS3-negative immunostaining (-) or SOCS3-negative glands with weak cytoplasmatic staining in <50% of the neoplastic glands (+/-)., Conclusions: Our data suggest that in prostatic cancer biopsies, the immunohistochemical analysis of SOCS3 protein expression may provide a method that is less expensive and easier to apply than SOCS3 methylation analysis for the distinction of a subgroup of prostate cancer with a more aggressive behavior.

Context: - Fine-needle aspiration cytology has been increasingly used as the first tool in the evaluation of several diseases. Although cytology has a relevant role in the discrimination between benign and malignant lesions, conventional slides cannot lead to 100% conclusive results. It was hoped that the introduction of liquid-based cytology (LBC) would improve the efficacy of cytology through standardization, quality improvement, and the possibility of carrying out ancillary techniques on the residual stored material. In recent decades, the application of genomic alterations has been studied on cytologic samples with feasible and reliable results. The molecular analysis offers a powerful aid to define the best clinical or surgical approaches and follow-up for patients. In recent years, the application of different ancillary techniques has been carried out on conventional slides even though LBC represents a useful additional and alternative method for molecular testing., Objective: - To demonstrate the relevance of LBC as a valid aid to overcoming the difficulties encountered in the application of ancillary techniques on conventional slides., Data Sources: - We examined and reviewed our experience with the application of ancillary techniques on LBC performed on different body sites., Conclusions: - We emphasize that LBC achieves significant and accurate results. It represents a valid method for cytologic evaluation and it provides highly reproducible and informative molecular yields.

Background: Analysis of K- and N-RAS mutations is mandatory before planning treatment of metastatic colorectal cancer, because only RAS wild-type (WT) patients can benefit from treatment with anti-EGFR monoclonal antibodies (cetuximab and panitumumab)., Case Report: Here we report the case of a 69-year-old male patient affected by metastatic sigmoid cancer. He underwent left hemicolectomy, and histology diagnosed a well-differentiated, pT4, node-positive adenocarcinoma; KRAS analysis performed with direct sequencing identified a mutation in exon 2 of the KRAS gene (GGT->GTT). After first-line chemotherapy with FOLFOX6 plus bevacizumab, the patient underwent surgical resection of residual liver metastases. Histology showed metastatic deposits from colic adenocarcinoma with extensive coagulative necrosis. Mutational analysis of the KRAS gene was also performed on liver metastases by pyrosequencing assay, and no mutation was identified. Due to the discordant results (GGT->GTT exon 2 KRAS mutation in the primary tumor, and KRAS-WT in the liver metastases), mutational analysis on liver metastasis was repeated using next-generation sequencing and enriching the sample in tumor cells by manual microdissection; the same type of mutation of the primary tumor (GGT->GTT exon 2 KRAS gene) was confirmed., Conclusions: Accurate tissue sampling and adequately sensitive assays are essential to correctly identify colorectal cancer patients who can be treated with an anti-EGFR monoclonal antibody.

Background: Advances from glioma stemlike cell (GSC) research, though increasing our knowledge of glioblastoma (GBM) biology, do not influence clinical decisions yet. We explored the translational power of GSC-enriched cultures from patient-derived tumorspheres (TS) in predicting treatment response., Methods: The relationship between TS growth and clinical outcome was investigated in 52 GBMs treated with surgical resection followed by radiotherapy and temozolomide (TMZ). The effect on TS of radiation (6 to 60 Gy) and of TMZ (3.9 μM to 1 mM) was related with patients' survival., Results: Generation of TS was an independent factor for poor overall survival (OS) and poor progression-free survival (PFS) (P < .0001 and P = .0010, respectively). Growth rate and clonogenicity of TS predicted poor OS. In general, TS were highly resistant to both radiation and TMZ. Resistance to TMZ was stronger in TS with high clonogenicity and fast growth (P < .02). Shorter PFS was associated with radiation LD50 (lethal dose required to kill 50% of TS cells) >12 Gy of matched TS (P = .0484). A direct relationship was found between sensitivity of TS to TMZ and patients' survival (P = .0167 and P = .0436 for OS and PFS, respectively). Importantly, values for TMZ half-maximal inhibitory concentration <50 μM, which are in the range of plasma levels achieved in vivo, identified cases with longer OS and PFS (P = .0020 and P = .0016, respectively)., Conclusions: Analysis of TS holds translational relevance by predicting the response of parent tumors to radiation and, particularly, to TMZ. Dissecting the clonogenic population from proliferating progeny in TS can guide therapeutic strategies to a more effective drug selection and treatment duration., (© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

When one is dealing with pediatric thyroid lesions, fine-needle aspiration is the first diagnostic tool for the correct characterization of these nodules. Despite the apparent infrequency of thyroid cancers in children, recent data from the National Cancer Institute prove that the incidence has been increasing, especially in adolescents. With the same data, a higher prevalence of well-differentiated cancers can be estimated, with 90% diagnosed as papillary thyroid cancer. Nonetheless, some publications have demonstrated that some specific malignant variants are more frequent in children and have a more aggressive behavior that justifies the increased number of surgical procedures. For this reason, the American Thyroid Association recommends the performance of neck ultrasonography and fine-needle aspiration cytology (FNAC) for the evaluation of pediatric thyroid nodules. Accordingly, as reported in adult thyroid series, several authors have documented the high sensitivity and diagnostic accuracy of FNAC in pediatric series; they have also shared the same problematic issues encountered in adult populations, mostly in the diagnosis of indeterminate lesions. To provide precise clinical and/or surgical management, the correct cytological identification of specific malignant histotypes/entities should be mandatory because lymph nodes, distant metastases, and extrathyroidal infiltration are more frequent within specific histotypes. A perusal of the literature shows that their identification has not been extensively studied and investigated in cytological samples. This review focuses on the analysis of data from the literature on the evaluation of malignancies and specific morphological features in pediatric thyroid lesions. Cancer Cytopathol 2017;125:594-603. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

Background: Presently, few options are available for refractory colorectal cancer (CRC). O6-methyl-guanine-DNA-methyltransferase (MGMT) promoter methylation is a frequent and early event in CRC tumourigenesis. This epigenetic silencing is a predictor of response to the alkylating drug temozolomide in glioblastoma. Preclinical evidences and some case reports showed temozolomide activity in CRC with MGMT silencing, but the available data from clinical trials are inconsistent., Methods: This was a multicentre, phase 2 trial, planned according to a two-stage Simon's optimal design to investigate activity and safety of temozolomide in refractory CRC harbouring MGMT promoter methylation. The primary end point was overall response rate (ORR). Patients who failed two or more prior treatments received temozolomide at a dose of 150-200 mg m -2 per day on days 1-5 every 28 days., Results: From July 2012 to June 2016, 225 patients were screened, 80 showed MGMT promoter methylation and 41 were enrolled. Overall response rate was 10% and disease control rate was 32%. Median progression-free survival and overall survival were 1.9 and 5.1 months, respectively., Conclusions: Temozolomide showed a modest activity in this heavily pretreated population and the study did not meet its primary end point. The role of temozolomide in CRC remains still controversial and further research is warranted.

Background: In the 2014, The International Society of Urological Pathology (ISUP) consensus conference update the grading of prostate, last revised in 2005. In this study we evaluate the SOCS3 immunohistochemical protein expression in different Gleason prostatic adenocarcinoma: classical Gleason grade 3, classical Gleason grade 3 upgraded to Gleason grade 4 according to the ISUP modifications and classical and modified Gleason grade 4. The major conclusions were: (i) Cribriform glands should be assigned a Gleason pattern 4, regardless of morphology; (ii) Glomeruloid glands should be assigned a Gleason pattern 4, regardless of morphology; (iii) Grading of mucinous carcinoma of the prostate should be based on its underlying growth pattern rather than all as pattern 4; and (iv) Intraductal carcinoma of the prostate without invasive carcinoma should not assigned Gleason grade and a comment about aggressive carcinoma probably associated should be made. In a recent report we analyzed the methylathion status of cytokine signaling (SOCS) proteins 3 (SOCS3) gene and the consequences of promoter hypermethylation on mRNA and protein expression in a collection of prostate cancer and benign prostate hyperplasia (BPH) and for the first time we demonstrated that a hypermethylation of SOCS3 with a significant reduction of its mRNA and protein expression identifies a subgroup of prostate cancer with a more aggressive behavior. Moreover we demonstrated that the immunohystochemical analysis of SOCS3 protein expression in prostatic cancer biopsies may provide a useful and easier method than SOCS3 methylation analysis to individuate in cancer with intermediate-high grade Gleason score a subgroup of prostate cancer with a more aggressive behavior., Methods: A total of 148 radical prostatectomy with diagnosis of prostatic acinar adenocarcinoma were stratified into three different categories on the basis of Gleason grade: (i) Twenty-six prostatic adenocarcinoma with classical and modified Gleason grade 3; (ii) Fifty seven prostatic adenocarcinoma with classical Gleason grade 3 upgraded to Gleason grade 4 by 2005 and 2014 ISUP Consensus Conference; and (iii) Sixty five prostatic adenocarcinoma with classical and modified Gleason grade 4. Immunohistochemical analysis for SOCS3 was performed and SOCS3 staining intensity were evaluated by two pathologists in three different ways on the basis of the intensity of cytoplasmatic staining: positive (intense cytoplasmatic staining in more than 50% of neoplastic cells) (+), negative (absence of cytoplasmatic staining in more than 50% of neoplastic cells) (-), weakly positive (weak cytoplasmatic staining in more than 50% of neoplastic cells (+/-)., Results: In the group of prostatic adenocarcinoma Gleason grade 3 we found that SOCS3 positivity (+) were observed in 19 out of 26 cases (73.1%); in 5 out of 26 prostatic adenocarcinoma the neoplastic glands showed weak intensity SOCS3 staining (+/-) (19.2%), while in only two cases we found SOCS-3 negativity (-) (7.7%); in the group of cases with prostatic adenocarcinoma with Gleason grade 4, 16 out 65 cases (24.6%) showed SOCS3 positivity (+); 18 out 65 cases (27.7%) SOCS3 weakly positive (+/-), and in 31 cases (47.7%) SOCS3 negative staining (-) were observed. Interestingly, the group of prostatic adenocarcinoma with histological Gleason 3 pattern upgraded to Gleason 4 pattern according to the 2005 and 2014 ISUP modified grading system, showed SOCS3 positivity (+) in 16 out of 57 cases (28%), in 16 out 57 cases (28%) a weakly positive for SOCS3 (+/-) were observed, while 25 cases (44%) showed negative SOCS3 staining (-)., Conclusions: In this study we demonstrated a significant association of SOCS3 positivity (+) with prostatic carcinoma classical Gleason pattern 3 (P < 0.0001), while SOCS3 negative pattern (-) or SOCS3 weakly positive pattern (+/-) were associated to prostatic carcinomas with Gleason pattern 3 upgraded to Gleason pattern 4 (P = 0.0002) and with classical Gleason pattern 4. The significant difference of SOCS3 immunohistochemical expression between classical Gleason grade 3 and Gleason grade 4 upgraded to grade 4 seems to support the definitions and the modifications of Gleason grade 4 of the 2005 and the 2014 International Society of Urological Pathology (ISUP). The hypoexpression of SOCS3 protein in glomeruloid glands could support the hypothesis that from molecular point of view this growth pattern could be different from classical Gleason pattern 3 and biologically more closely to Gleason pattern 4, confirming the conclusions of the 2014 ISUP Conference assigning a Gleason pattern 4 to glomeruloid glands regardless of morphology. Prostate 77: 597-603, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

MicroRNA (miRNA) deregulation has been frequently associated with different human cancers. Not only have miRNAs been involved in almost every cellular function but they have also been linked with a significant number of cancers including thyroid carcinomas. Specifically, thyroid tumors encompass several different miRNA profiles based on the histotypes. Furthermore, thyroid lesions with their broad spectrum of neoplasms (from benign to malignant entities) offer the possibility of studying and recognizing specific subsets of different up-and downregulated miRNAs in each different entity. To date, the majority of authors completed their evaluation mostly by including histologic samples of thyroid tumors. Nonetheless, in the last years, a few studies are focusing on the role of miRNA expression in thyroid fine-needle aspiration cytology (FNAC) regardless of the cytologic preparation, including liquid-based cytology. This growing interest is driven by the possible role of miRNAs in the malignant risk stratification, especially for the indeterminate categories of follicular neoplasms (FNs). In this review we overview the reliability of analyzing miRNAs on thyroid lesions, including those diagnosed as FNs, to identify whether their profiles are likely to distinguish benign from malignant lesions, providing a predictive molecular diagnosis on FNAC., (Copyright © 2017 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

Objective: BRAF V 600E represents the most common diagnostic marker in papillary thyroid carcinoma (PTC). A few papers have demonstrated the correlation between BRAF V 600E and specific morphological findings on PTCs in the adult population. This is the first reported series investigating cytological morphological parameters in paediatric thyroid carcinoma., Methods: One hundred and nineteen paediatric samples (56 male and 63 female patients), diagnosed in the period between April 2013 and July 2015, were enrolled in the study. Fifteen patients with inadequate results were excluded. Cytological cases were processed with liquid-based cytology (LBC). BRAF V 600E and immunocytochemistry for the VE1 antibody were performed on LBC., Results: The diagnostic series included 10 mutated and 94 wild-type (WT) cases. Twenty two percent surgical samples showed 96% cytohistological concordance. The morphological analysis revealed plump cells (abundant eosinophilic cytoplasm and PTC nuclei) in all 10 mutated cases with only four cases showing a focal (less than 20% of the cells) plump component. None of the WT showed plump cells. A sickle nuclear shape was seen only in the mutated cases. VE1 yielded 100% positivity on mutated cases with three cytohistological discrepancies., Conclusions: The BRAF V 600E mutation is also seen in paediatric cytology and the morphological features showed a high accuracy as both predictive mutational parameters and a helpful aid in management mainly of the aggressive BRAF V 600E mutated carcinomas., (© 2016 John Wiley & Sons Ltd.)

Fine needle aspiration cytology (FNAC) plays an essential role in the evaluation of thyroid nodules especially for the category of follicular neoplasms (FN) representing 25 % of all thyroid cases including different neoplastic entities. Hence, one of the most promising areas is the application of molecular tests to FNAC. Among them, microRNAs (miRNA),identified as negative (post-transcriptional) gene expression regulators involved in tumor development, are likely to discriminate among FNs. Limited data explored the use of miRNAs on FNAC as well as their role in the malignant risk stratification. We aimed to define whether liquid-based cytology (LBC) is a valid method for miRNA evaluation. From June 2014 to March 2015, we enrolled 27FNs with histological follow-up. In the same reference period, 13 benign nodules (BN) and 20 positive for malignancy (PM) were selected as controls. Histologically, FNs resulted in 14 malignancies (3 papillary thyroid carcinoma-PTC and 11 follicular variant of PTC-FVPC) and 13 follicular adenomas (FA). The 20 PMs included two FVPC, 16 PTC and two medullary thyroid carcinoma (MTC). Five miRNAs (10b, 92a, 221/222 cluster, and 375) were studied on LBC and quantified by real-time PCR. Only miR-375 was over-expressed in the FNs diagnosed as carcinomas and in the PMs. A cut-off of 12 miR-375/U6 relative ratio recognized all BNs and 95 % PMs. Specifically, in each category, FVPCs and PTCs did not show any difference while MTCs had the highest value. miR-375 shows 97.1 % sensitivity, 100 % specificity, 96.3 % negative predictive value (NPV), 100 % positive predictive value (PPV), and 98.3 % diagnostic accuracy. LBC is suitable for miRNAs evaluation. miR-375 resulted over-expressed in all malignant FNs and 95 % PMs. It may represent a valid aid in ruling out BNs and supporting PTCs and/or FVPCs.

The vast majority of gastrointestinal stromal tumors (GISTs) occur as sporadic tumors. Rarely, however, these neoplasms can arise in syndromic contexts. Under these circumstances, GISTs are often multiple and associated with accompanying signs peculiar of the hosting syndrome. Moreover, syndromic GISTs themselves tend to show heterogeneous features depending on the underlying condition. Multiple inflammatory fibroid polyps (IFPs) and a jejunal spindle-cell GIST were resected in a germline PDGFRA-mutant individual. Although the association of IFP and GIST is typical of this genetic setting (PDGFRA mutations can in fact trigger both these tumor types), PDGFRA-mutant GISTs are usually epithelioid and gastric. This discrepancy was settled evidencing a somatic KIT mutation in the GIST. The awareness of possible somatic mutations can be critical in the management of high-risk/malignant GISTs arising in syndromic settings. GIST features unusual for a given GIST-predisposing syndrome are a valuable tool in the hands of physicians for suspecting these "extra" triggers, which could not be sought for once a diagnosis of GIST-prone syndrome is well established, in a bona fide cost/benefit perspective., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Kirsten-Ras (KRAS) mutations are widely accepted negative predictive factors for anti-EGFR therapies in metastatic colorectal cancer (mCRC), while their prognostic significance is still under discussion., Objective: This mono-institutional retrospective study aims to investigate the real-life impact of exon 2 codon 12 and 13 mutations in mCRC., Methods: All mCRC patients treated at our institution between 2008 and 2014 carrying KRAS exon 2 mutations were included. The primary endpoint was to determine any significant difference in overall survival (OS) between codon 12 and 13 mutations. Secondary endpoints included progression-free survival (PFS), OS in both populations according to antiangiogenic treatment, and OS in liver-limited disease (LLD)., Results: Of 620 mCRC patients, 218 carried KRAS exon 2 mutations (35.1%): 162 (26.1%) at codon 12 and 56 (9.0 %) at codon 13. Median OS results were similar: 32.0 months (codon 12) and 31.0 months (codon 13). PFS was also comparable, reaching 10.8 months in both populations. The addition of bevacizumab to chemotherapy conferred a trend toward survival advantage in codon 12 but not codon 13 mutation (p = 0.058). A high proportion of LLD patients underwent hepatic surgery with radical purpose (62.3%): in these patients, median OS has not yet been reached, while OS in non-LLD patients was 30.2 months., Conclusions: No difference in OS between KRAS codon 12/13 mutated disease was found. This analysis showed a very prolonged OS for KRAS-mutated patients, even when LLD patients were excluded; OS of our real-life series favorably compares with OS of all-RAS wild-type patients in recent randomized studies.

Background: Mutational analysis is reshaping the practice of fine-needle aspiration cytology for the diagnosis of thyroid nodules. The v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) valine (V) to glutamic acid (E) substitution at codon 600 (BRAF(V600E)) is the most effective diagnostic/prognostic marker and is used mainly for papillary thyroid carcinomas (PTCs). Although BRAF(V600E) represents 95% of all BRAF mutations, uncommon BRAF mutations have been identified in thyroid carcinomas. For the current study, the authors evaluated morphologic (plump pink cells and sickle-shaped nuclei) anti-BRAF(V600E) antibody (VE1) immunocytochemical and molecular findings of BRAF mutations in PTCs and in the follicular variant of PTC (FVPC)., Methods: Between January 2013 and June 2014, there were 150 cytologic samples with surgical follow-up at the authors' institution. BRAF mutations, which were identified using liquid-based cytology, were classified into wild-type BRAF, BRAF(V600E), and uncommon BRAF mutations. All clinicopathologic correlations between BRAF and FVPCs were analyzed., Results: Forty-four of 150 samples were identified as benign histologic lesions, and the authors focused on the 106 cytologic samples from patients who had malignant outcomes (60 PTCs and 46 FVPCs). The series included 16 follicular neoplasms, 36 samples diagnosed as suspicious of malignancy, and 54 samples diagnosed as positive for malignancy. The BRAF(V600E) mutation was detected in 17.4% of FVPCs and in 66.6% of PTCs, whereas uncommon BRAF mutations were detected only in FVPCs. Plump pink cells and VE1 expression were not identified in samples that had uncommon BRAF mutations. VE1 immunocytochemistry yielded positive results in all 36 samples that had the BRAF(V600E) mutation., Conclusions: Uncommon BRAF mutations were observed only in FVPCs and were linked to less aggressive behavior. Negative/weak VE1 expression was observed in both wild-type and uncommon BRAF mutations. The current investigation did not reveal any plump cells or morphologic BRAF findings in samples that had uncommon BRAF mutations. In the authors' experience, BRAF mutations detected by DNA methods were more accurate in identifying FVPCs., (© 2015 American Cancer Society.)