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The communication of scientific knowledge to patients and society as a whole has never been more central than in modern times. Thanks to the recent pandemic, it has become evident how Scientific Communication (SC) has evolved over time, increasingly diverging from common language. However, it is also clear that it must be properly used by healthcare professionals to avoid comprehension issues that could be severe for the audience. Presently, science and technology are at the heart of progress and innovation; therefore, the proper dissemination of accurate yet accessible information to the population is vital to ensure that no one is left behind and to promote cohesive social advancement. This review aims to analyze the notions of SC and Scientific Method (SM), examining the relationships between them and providing suggestions on how to integrate them properly in both a broader context and the specific field of communication with oncology patients., Competing Interests: Declaration of Competing Interest No conflict, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Homologous recombination (HR) and mismatch repair (MMR) defects are driver mutational imprints and actionable biomarkers in DNA repair-defective tumors. Although usually thought as mutually exclusive pathways, recent preclinical and clinical research provide preliminary evidence of a functional crosslink and crosstalk between HRR and MMR. Shared core proteins are identified as key players in both pathways, broadening the concept of DNA repair mechanism exclusivity in specific tumor types. These observations may result in unexplored forms of synthetic lethality or hypermutable tumor phenotypes, potentially impacting the cancer risk management, and considerably expanding in the future the therapeutic window for DNA repair-defective tumors., (© 2024. The Author(s).)

Dravet syndrome, a developmental and epileptic encephalopathy, manifests with varying degrees of cognitive and communication impairment, postural and movement disorders (such as ataxia, coordination issues, and crouch gait) and behavioural challenges (including attention deficit/hyperactivity, oppositional/defiant behaviour, and autistic traits). Rehabilitation is a valuable tool for most patients, typically prescribed to address the most pressing issues. However, current practices often fall short in proactively preventing and treating known challenges associated with the syndrome, as indicated by the latest literature, at different life stages. Furthermore, there is a notable lack of evidence regarding treatment types and efficacy specific to people with Dravet Syndrome. Conducted in collaboration with one of the Italian Patient associations, this national survey provides a comprehensive view of the rehabilitation landscape in Dravet Syndrome, as perceived by caregivers. It outlines the types of treatments for 51 patients, based on age and relevant clinical features. The findings reveal a heterogenous rehabilitation approach, only partly tailored to the presence of specific comorbidities, and underline numerous unmet needs. Compared to the past there is indirect evidence that more patients are offered early rehabilitation. Nonetheless, while nowadays speech therapy and neuropsychomotor therapy are nearly universal for children up to the age of 10, some begin physiotherapy and psychotherapy thereafter, with a majority discontinuing treatments. Therefore, families of adolescent and adult patients often face a lack of comprehensive support, predominantly offered when epilepsy is more challenging to control affecting rehabilitation adherence and effectiveness. Finally, a negligible minority is offered treatments such as neurovisual training, augmentative and alternative communication, and occupational therapy. Many of these considerations could apply to other developmental and epileptic encephalopathy with lifelong disability. This survey calls for more data collection on this important topic for more efficient allocation of rehabilitation resources., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Although hereditary male neoplasms are quite rare, individuals harbouring germline BRCA1/2 pathogenic variants (PVs) may have a risk of developing tumours associated with Hereditary Breast and Ovarian Cancer (HBOC) syndrome, including male breast (MBC), prostate (PCa) and pancreatic (PC) cancers, and melanoma. Women and men showed a comparable genetic architecture of cancer susceptibility, but there are some gender-specific features. Since little is known about cancer genetic susceptibility in male population, our study was aimed at investigating the frequency of BRCA1/2 PVs in men with HBOC syndrome-associated tumors, in order to understand whether differences in gender may reflect in the prevalence and spectrum of germline alterations., Patients and Methods: We retrospectively collected and analysed clinical information of 352 HBOC-associated male cancer patients genetically tested for germline BRCA1/2 PVs by Next-Generation Sequencing analysis, enrolled, from February 2018 to January 2024, at the "Regional Center for the prevention, diagnosis and treatment of rare and heredo-familial tumors of adults" of the University-Hospital Policlinico "P. Giaccone" of Palermo (Italy)., Results: Our investigation revealed that 7.4% of patients was carrier of a germline BRCA PV, with an almost total prevalence of BRCA2 alterations. In particular, 65.4% of BRCA -positive patients developed MBC, 19.2% had PC, 11.6% developed PCa, and only 3.8% had melanoma. Specifically, MBC individuals showed a BRCA -associated genetic predisposition in 17% of cases, whereas patients with PCa or PC exhibited a lower frequency of BRCA2 PVs, taking into account the current national criteria for access to germline genetic testing., Discussion: Our study showed a high heterogeneity in prevalence of germline BRCA2 PVs among men which could reflect a potential gender-specific genetic heterogeneity. Therefore, BRCA -associated male tumours could be due to BRCA2 PVs different from those usually detected in women. In the event that it is demonstrated, in future, that male cancers are genetically distinct entities from those female this could improve personalized risk evaluation and guide therapeutic choices for patients of both sexes, in order to obtain a gender equality in cancer care., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Fanale, Corsini, Brando, Randazzo, Bono, Pedone, Perez, Sciacchitano, Cancelliere, Piraino, Giurintano, Bazan Russo, Ferraro, Rinaldi, Spinnato, Gennusa, Pernice, Vieni, Pantuso, Russo and Bazan.)

This dataset provides longitudinal survey data from a European project, ySKILLS, which was focused on the role of digital skills in youths' development. It contains data from 10,821 participants from Grades 6-10 (in Wave 1) in Estonia, Finland, Germany, Italy, Poland, and Portugal. The data was collected between Spring 2021 and Spring 2023, the participants were recruited through schools, where the data collection also took place, except for online data collections due to restrictions caused by COVID-19. The dataset is novel in its multidimensional approach to the construct of digital literacy. It provides insight into the development of digital skills in youth and the role of digital skills and internet usage in youths' positive and negative online experiences and wellbeing. It also contains data that allows for the analysis of the role of digital skills in class networks. The data are beneficial for researchers interested in the examination of youths' online skills, internet usage, online experiences, and wellbeing from a longitudinal perspective., (© 2024 The Authors.)

Background: The patient selection for optimal adjuvant therapy in gastrointestinal stromal tumors (GISTs) is provided by nomogram based on tumor size, mitotic index, tumor location, and tumor rupture. Although mutational status is not currently used to risk assessment, tumor genotype showed a prognostic influence on natural history and tumor relapse. Innovative measures, such as KIT/PDGFRA-mutant-specific variant allele frequency (VAF) levels detection from next-generation sequencing (NGS), may act as a surrogate of tumor burden and correlate with prognosis and overall survival of patients with GIST, helping the choice for adjuvant treatment., Patients and Methods: This was a multicenter, hospital-based, retrospective/prospective cohort study to investigate the prognostic role of KIT or PDGFRA-VAF of GIST in patients with radically resected localized disease. In the current manuscript, we present the results from the retrospective phase of the study., Results: Two-hundred (200) patients with GIST between 2015 and 2022 afferent to 6 Italian Oncologic Centers in the EURACAN Network were included in the study. The receiver operating characteristic (ROC) curves analysis was used to classify "low" vs. "high" VAF values, further normalized on neoplastic cellularity (nVAF). When RFS between the low and high nVAF groups were compared, patients with GIST with KIT/PDGFRA nVAF > 50% showed less favorable RFS than patients in the group of nVAF ≤ 50% (2-year RFS, 72.6% vs. 93%, respectively; P = .003). The multivariable Cox regression model confirmed these results. In the homogeneous sub-population of intermediate-risk, patients with KIT-mutated GIST, the presence of nVAF >50% was statistically associated with higher disease recurrence., Conclusion: In our study, we demonstrated that higher nVAF levels were independent predictors of GIST prognosis and survival in localized GIST patients with tumors harboring KIT or PDGFRA mutations. In the cohort of intermediate-risk patients, nVAF could be helpful to improve prognostication and the use of adjuvant imatinib., (© The Author(s) 2023. Published by Oxford University Press.)

Background: The BRCA proteins play a key role in the homologous recombination (HR) pathway. Beyond BRCA1/2, other genes are involved in the HR repair (HRR). Due to the prominent role in the cellular repair process, pathogenic or likely pathogenic variants (PV/LPVs) in HRR genes may cause inadequate DNA damage repair in cardiomyocytes., Patients and Methods: This was a multicenter, hospital-based, retrospective cohort study to investigate the heart toxicity from anthracycline-containing regimens (ACRs) in the adjuvant setting of breast cancer (BC) patients carrying germline BRCA PV/LPVs and no-BRCA HRR pathway genes. The left ventricular ejection fraction (LVEF) was assessed using cardiac ultrasound before starting ACR therapy and at subsequent time points according to clinical indications., Results: Five hundred and three BC patients were included in the study. We predefined three groups: (i) BRCA cohort; (ii) no-BRCA cohort; (iii) variant of uncertain significance (VUS)/wild-type (WT) cohort. When baseline (T0) and post-ACR (T1) LVEFs between the three cohorts were compared, pre-treatment LVEF values were not different (BRCA1/2 versus HRR-no-BRCA versus VUS/WT cohort). Notably, during monitoring (T1, median 3.4 months), patients carrying BRCA or HRR no-BRCA germline pathogenic or likely pathogenic variants showed a statistically significant reduction of LVEF compared to baseline (T0). To assess the relevance of HRR on the results, we included the analysis of the subgroup of 20 BC patients carrying PV/LPVs in other genes not involved in HRR, such as mismatch repair genes (MUTYH, PMS2, MSH6). Unlike HRR genes, no significant differences in T0-T1 were found in this subgroup of patients., Conclusion: Our data suggest that deleterious variants in HRR genes, leading to impaired HR, could increase the sensitivity of cardiomyocytes to ACR in early BC patients. In this subgroup of patients, other measurements, such as the global longitudinal strain, and a more in-depth assessment of risk factors may be proposed in the future to optimize cardiovascular risk management and improve long-term survival., (Copyright © 2023. Published by Elsevier Ltd.)

Specific tumor-derived extracellular vesicles, called exosomes, are considered as potential key players in cross-talk between immune system and tumor microenvironment in several solid tumors. Different studies highlighted the clinical relevance of exosomes in ovarian cancer (OC) for their role in early diagnosis, prognosis, chemoresistance, targeted therapy. The exosomes are nanosize vesicles carrying lipids, proteins, and nucleic acids. In particular, exosomes shuttle a wide spectrum of microRNAs (miRNAs) able to induce phenotypic reprogramming of target cells, contributing to tumor progression. In this review, we will discuss the promising role of miRNAs shuttled by exosomes, called exosomal miRNAs (exo-miRNAs), as potential biomarkers for early detection, tumour progression and metastasis, prognosis, and response to therapy in OC women, in order to search for new potential biological fingerprints able to better characterize the evolution of this malignancy and provide a clinically relevant non-invasive approach useful for adopting, in future, personalized therapeutic strategies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Breast cancer susceptibility gene 1 (BRCA1) and breast cancer susceptibility gene 2 (BRCA2) deleterious variants were the first and, still today, the main biomarkers of poly(ADP)ribose polymerase (PARP)-inhibitors (PARPis) benefit. The recent, increased, numbers of individuals referred for counseling and multigene panel testing, and the remarkable expansion of approved PARPis, not restricted to BRCA1/BRCA2-Pathogenic Variants (PVs), produced a strong clinical need for non-BRCA biomarkers. Significant limitations of the current testing and assays exist. The different approaches that identify the causes of Homologous Recombination Deficiency (HRD), such as the germline and somatic Homologous Recombination Repair (HRR) gene PVs, the testing showing its consequences, such as the genomic scars, or the novel functional assays such as the RAD51 foci testing, are not interchangeable, and should not be considered as substitutes for each other in clinical practice for guiding use of PARPi in non-BRCA, HRD-associated tumors. Today, the deeper knowledge on the significant relationship among all proteins involved in the HRR, not limited to BRCA, expands the possibility of a successful non-BRCA, HRD-PARPi synthetic lethality and, at the same time, reinforces the need for enhanced definition of HRD biomarkers predicting the magnitude of PARPi benefit., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Germline BRCA1/2 alterations in the Homologous Recombination (HR) pathway are considered as main susceptibility biomarkers to Hereditary Breast and Ovarian Cancers (HBOC). The modern molecular biology technologies allowed to characterize germline and somatic BRCA1/2 alterations in several malignancies, broadening the landscape of BRCA1/2-alterated tumors. In the last years, BRCA genetic testing, beyond the preventive value, also assumed a predictive and prognostic significance for patient management. The approval of molecules with agnostic indication is leading to a new clinical model, defined "mutational". Among these drugs, the Poly (ADP)-Ribose Polymerase inhibitors (PARPi) for BRCA1/2-deficient tumors were widely studied leading to increasing therapeutic implications. In this Review we provided an overview of the main clinical studies describing the association between BRCA-mutated tumors and PARPi response, focusing on the controversial evidence about the potential agnostic indication based on BRCA1/2 alterations in several solid tumors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The most common subtype of ovarian cancer (OC) showing immunogenic potential is represented by the high-grade serous ovarian cancer (HGSOC), which is characterized by the presence of tumor-infiltrating immune cells able to modulate immune response. Because several studies showed a close correlation between OC patient's clinical outcome and expression of programmed cell death protein-1 or its ligand (PD-1/PD-L1), the aim of our study was to investigate if plasma levels of immunomodulatory proteins may predict prognosis of advanced HGSOC women., Patients and Methods: Through specific ELISA tests, we analyzed plasma concentrations of PD-L1, PD-1, butyrophilin sub-family 3A/CD277 receptor (BTN3A1), pan-BTN3As, butyrophilin sub-family 2 member A1 (BTN2A1), and B- and T-lymphocyte attenuator (BTLA) in one hundred patients affected by advanced HGSOC, before surgery and therapy. The Kaplan-Meier method was used to generate the survival curves, while univariate and multivariate analysis were performed using Cox proportional hazard regression models., Results: For each analyzed circulating biomarker, advanced HGSOC women were discriminated based on long (≥ 30 months) versus short progression-free survival (PFS < 30 months). The concentration cut-offs, obtained by receiver operating characteristic (ROC) analysis, allowed to observe that poor clinical outcome and median PFS ranging between 6 and 16 months were associated with higher baseline levels of PD-L1 (> 0.42 ng/mL), PD-1 (> 2.48 ng/mL), BTN3A1 (> 4.75 ng/mL), pan-BTN3As (> 13.06 ng/mL), BTN2A1 (> 5.59 ng/mL) and BTLA (> 2.78 ng/mL). Furthermore, a lower median PFS was associated with peritoneal carcinomatosis, age at diagnosis > 60 years or Body Mass Index (BMI) > 25. A multivariate analysis also suggested that plasma concentrations of PD-L1 ≤ 0.42 ng/mL (HR: 2.23; 95% CI: 1.34 to 3.73; p = 0.002), age at diagnosis ≤ 60 years (HR: 1.70; 95% CI: 1.07 to 2.70; p = 0.024) and absence of peritoneal carcinomatosis (HR: 1.87; 95% CI: 1.23 to 2.85; p = 0.003) were significant prognostic marker for a longer PFS in advanced HGSOC patients., Conclusions: The identification of high-risk HGSOC women could be improved through determination of the plasma PD-L1, PD-1, BTN3A1, pan-BTN3As, BTN2A1 and BTLA levels., (© 2023. The Author(s).)

Background: Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer, associated with a worse prognosis. The Immune Checkpoint Inhibitors (ICIs) avelumab and pembrolizumab have been recently approved as first-line treatment in metastatic MCC (mMCC). The clinical observation of improved outcomes in obese patients following treatment with ICIs, known as the "obesity paradox", has been studied across many types of tumors. Probably due to the rarity of this tumor, data on mMMC patients are lacking., Patients and Methods: This is an observational, hospital-based, study to investigate the role of Body Mass Index (BMI) as predictive biomarker of ICI response in mMCC patients treated with avelumab as first-line treatment. The study population included the patients treated from February 2019 to October 2022 in an Italian referral center for rare tumors. Clinico-pathological characteristics, BMI, laboratory parameters (NLR and platelet count), and response to avelumab were analyzed from a MCC System database prospectively collected., Results: Thirty-two (32) patients were included. Notably, the presence of pre-treatment BMI ≥ 30 was significantly associated with longer PFS [BMI < 30 Group: median PFS, 4 months (95% CI: 2.5-5.4); BMI ≥ 30 Group: median PFS, not reached; p<0.001)[. Additionally, the median PFS was significantly higher in patients with higher PLT (median PFS: 10 months in the "low PLT" Group (95% CI: 4.9, 16.1) vs 33 months (95% CI: 24.3, 43.2) in the "high PLT" Group (p=0.006). The multivariable Cox regression model confirmed these results., Conclusion: To our knowledge, this is the first study that investigates the predictive role of BMI in MCC patients. Our data were consistent with the clinical observation of improved outcomes in obese patients across other tumor types. Thus, advanced age, a weakened immune system, and the obesity-associated "inflammaging", are key factors that could impact the cancer immune responses of mMCC patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Incorvaia, Dimino, Algeri, Brando, Magrin, De Luca, Pedone, Perez, Sciacchitano, Bonasera, Bazan Russo, Li Pomi, Peri, Gristina, Galvano, Giuffrida, Fazio, Toia, Cordova, Florena, Giordano, Bazan, Russo and Badalamenti.)

Individual response to immune checkpoint inhibitors (ICIs) is currently unpredictable in patients with melanoma. Recent findings highlight a striking improvement in the clinical outcomes of overweight/obese patients treated with ICIs, which seems driven, at least in part, by programmed cell death protein 1 (PD-1)-mediated T-cell dysfunction. A putative role of butyrophilins (BTNs) is under investigation as a novel mechanism of cancer immune evasion and obesity-associated inflammation. This study investigates the role of baseline plasma levels of soluble PD-1 (sPD-1), soluble programmed cell death ligand 1 (sPD-L1), BTN2A1 (sBTN2A1), BTN3A1 (sBTN3A1), along with body mass index (BMI), as predictive biomarkers of immunotherapy response in metastatic melanoma patients treated with nivolumab or pembrolizumab as first-line treatment. In all, 41 patients were included in the study. The baseline plasma level of sPD-1 was significantly lower, and the sBTN2A1 was significantly higher, in long-responder patients to nivolumab or pembrolizumab (median sPD-1: 10.3 ng/ml versus 16.6 ng/ml, p  = 0.001; median sBTN2A1: 4.4 ng/ml versus 3.77 ng/ml, p  = 0.004). Lower levels of sPD-1 and higher levels of sBTN2A1 were also significantly associated with better overall response rate. Notably, when we further stratified the study cohort using BMI along with sPD-1, patients with BMI ⩾ 25 and sPD-1 < 11.24 ng/ml had longer time to treatment failure after PD-1 inhibitor than other subgroups of patients ( p  < 0.001). Circulating sPD-1 and sBTN2A1 detection, along with BMI, could give more insights into the immune-metabolic interactions underlying the benefit observed in overweight/obese patients, improving the use of dynamic, noninvasive, biomarkers for patient selection., Competing Interests: The authors declare that there is no conflict of interest., (© The Author(s), 2023.)

Gastrointestinal Stromal Tumors (GISTs) represent a paradigmatic model of oncogene addiction. Despite the well-known impact of the mutational status on clinical outcomes, we need to expand our knowledge to other factors that influence behavior heterogeneity in GIST patients. A growing body of studies has revealed that the tumor microenvironment (TME), mostly populated by tumor-associated macrophages (TAMs) and lymphocytes (TILs), and stromal differentiation (SD) have a significant impact on prognosis and response to treatment. Interestingly, even though the current knowledge of the role of immune response in this setting is still limited, recent pre-clinical and clinical data have highlighted the relevance of the TME in GISTs, with possible implications for clinical practice in the near future. Moreover, the expression of immune checkpoints, such as PD-L1, PD-1, and CTLA-4, and their relationship to the clinical phenotype in GIST are emerging as potential prognostic biomarkers. Looking forward, these variables related to the underlying tumoral microenvironment in GIST, though limited to still-ongoing trials, might lead to the potential use of immunotherapy, alone or in combination with targeted therapy, in advanced TKI-refractory GISTs. This review aims to deepen understanding of the potential link between mutational status and the immune microenvironment in GIST.

The most common subtype of ovarian cancer (OC) is the high-grade serous ovarian carcinoma (HGSOC), accounting for 70%-80% of all OC deaths. Although HGSOC is a potentially immunogenic tumor, clinical studies assessing the effectiveness of inhibitors of programmed death protein and its ligand (PD-1/PD-L1) in OC patients so far showed only response rates <15%. However, recent studies revealed an interesting prognostic role of plasma PD-1/PD-L1 and other circulating immunoregulatory molecules, such as the B- and T-lymphocyte attenuator (BTLA), butyrophilin sub-family 3A/CD277 receptors (BTN3A), and butyrophilin sub-family 2 member A1 (BTN2A1), in several solid tumors. Since evidence showed the prognostic relevance of pretreatment serum CA125 levels in OC, the aim of our study was to investigate if soluble forms of inhibitory immune checkpoints can enhance prognostic power of CA125 in advanced HGSOC women. Using specific ELISA tests, we examined the circulating PD-1, PD-L1, pan-BTN3As, BTN3A1, BTN2A1, and BTLA levels in 100 advanced HGSOC patients before treatment, correlating them with baseline serum CA125, age at diagnosis, body mass index (BMI), and peritoneal carcinomatosis. A multivariate analysis revealed that plasma BTN3A1 ≤4.75 ng/ml (HR, 1.94; 95% CI, 1.23-3.07; p=0.004), age at diagnosis ≤60 years (HR, 1.65; 95% CI, 1.05-2.59; p=0.03) and absence of peritoneal carcinomatosis (HR, 2.65; 95% CI, 1.66-4.22; p<0.0001) were independent prognostic factors for a longer progression-free survival (PFS) (≥30 months) in advanced HGSOC women. However, further two-factor multivariate analyses highlighted that baseline serum CA125 levels >401 U/ml and each soluble protein above respective concentration cutoff were covariates associated with shorter PFS (<30 months) and unfavorable clinical outcome, suggesting that contemporary measurement of both biomarkers than CA125 only could strengthen prognostic power of serum CA125 in predicting PFS of advanced HGSOC women. Plasma PD-L1, PD-1, BTN3A1, pan-sBTN3As, BTN2A1, or BTLA levels could be helpful biomarkers to increase prognostic value of CA125., Competing Interests: JLI is cofounder of PanCa Therapeutics and PredicitngMed. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fanale, Corsini, Brando, Cutaia, Di Donna, Filorizzo, Lisanti, Randazzo, Magrin, Romano, Bazan Russo, Olive, Vieni, Pantuso, Chiantera, Russo, Bazan and Iovanna.)

In the last decade, tumor-infiltrating lymphocytes (TILs) have been recognized as clinically relevant prognostic markers for improved survival, providing the immunological basis for the development of new therapeutic strategies and showing a significant prognostic and predictive role in several malignancies, including ovarian cancer (OC). In fact, many OCs show TILs whose typology and degree of infiltration have been shown to be strongly correlated with prognosis and survival. The OC histological subtype with the higher presence of TILs is the high-grade serous carcinoma (HGSC) followed by the endometrioid subtype, whereas mucinous and clear cell OCs seem to contain a lower percentage of TILs. The abundant presence of TILs in OC suggests an immunogenic potential for this tumor. Despite the high immunogenic potential, OC has been described as a highly immunosuppressive tumor with a high expression of PD1 by TILs. Although further studies are needed to better define their role in prognostic stratification and the therapeutic implication, intraepithelial TILs represent a relevant prognostic factor to take into account in OC. In this review, we will discuss the promising role of TILs as markers which are able to reflect the anticancer immune response, describing their potential capability to predict prognosis and therapy response in OC.

MUTYH gene is involved in the base excision repair (BER) mechanism and its pathogenic alterations are associated with colorectal polyposis and cancer. MUTYH-associated polyposis (MAP) is a condition which is inherited in an autosomal recessive manner. MAP patients, beyond colorectal cancer (CRC), may develop other types of tumors, including duodenal, breast, ovarian, pancreatic, bladder and skin cancers. Carriers of biallelic MUTYH likely pathogenic/pathogenic variants exhibit a high lifetime risk of CRC, though cancer risk evidence becomes less clear when monoallelic carriers and extraintestinal tumors are considered. However, several studies recently reported an increased genetic susceptibility to cancer also for carriers of germline monoallelic MUTYH mutations. Moreover, experimental evidence highlighted the MUTYH involvement in many other biological functions. In future, MUTYH mutation carriers might benefit from new target therapies involving the use of PD-1 or KRAS inhibitors. Therefore, "MUTYH-associated tumor syndrome" might be the most appropriate term, due to the multiplicity of tumors observed in MAP patients and different biological contexts in which MUTYH acts as a "playmaker". In this Review, we will investigate the impact of germline mono- and biallelic MUTYH mutations on cancer risk, providing a proposal for clinical surveillance of mutation carriers., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Hereditary breast and ovarian cancer syndrome is caused by germline mutations in BRCA1/2 genes. These genes are very large and their mutations are heterogeneous and scattered throughout the coding sequence. In addition to the above-mentioned mutations, variants of uncertain/unknown significance (VUSs) have been identified in BRCA genes, which make more difficult the clinical management of the patient and risk assessment. In the last decades, several laboratories have developed different databases that contain more than 2000 variants for the two genes and integrated strategies which include multifactorial prediction models based on direct and indirect genetic evidence, to classify the VUSs and attribute them a clinical significance associated with a deleterious, high/low or neutral risk. This review provides a comprehensive overview of literature studies concerning the VUSs, in order to assess their impact on the population and provide new insight useful for the appropriate patient management in clinical practice., (Copyright © 2022. Published by Elsevier B.V.)

Lynch syndrome (LS) is an inherited genetic condition associated with increased predisposition to colorectal cancer (CRC) and other tumors and is caused by germline mutations in Mismatch Repair ( MMR ) or EPCAM genes. The identification of LS carriers is currently based on germline testing of subjects with MMR-deficient (dMMR) tumors or fulfilling clinical criteria, but the most efficient strategies to select patients who should be offered genetic testing are yet not well defined. In order to assess the most suitable selection mode to identify LS-related CRC patients, we retrospectively collected and analyzed all clinical and molecular information of 854 CRC patients, recruited from 2013 to 2021 at the University Hospital Policlinico "P. Giaccone" of Palermo (Italy), 100 of which were selected based on revised Bethesda guidelines, Amsterdam criteria II, or tissue MMR deficiency, and genetically tested for germline variants in LS-susceptibility genes. Our study showed that 32 out of 100 CRC patients harbored germline likely pathogenic/pathogenic variants in MMR genes. The analysis of tissue microsatellite instability (MSI) status according to the revised Bethesda guidelines has been to be the best selection approach. However, using different selection approaches as complementary strategies is useful to identify LS carriers, reducing underdiagnosis of this syndrome., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fanale, Corsini, Brando, Dimino, Filorizzo, Magrin, Sciacchitano, Fiorino, Bazan Russo, Calò, Iovanna, Francini, Russo and Bazan.)

Alterations in short-repetitive DNA sequences, known as microsatellite instability (MSI), can reflect deficiencies in Mismatch Repair (MMR) system which represents a major player in DNA integrity maintenance. The incidence of MSI-H/dMMR has been shown to be variable depending on the tumor type. Several studies confirmed that dMMR/MSI status, although less frequent than PD-L1 expression, may better predict response to immune-checkpoint inhibitors (ICIs) in patients with solid tumors. In October 2016, the FDA granted pembrolizumab as breakthrough therapy for the treatment of non-CRC, MSI-H/dMMR tumors, providing, for the first time, a tumor-agnostic indication. In the next future, the tissue-agnostic evaluation of MSI-H/dMMR could become the common denominator for the immunotherapy treatment of patients with different advanced solid tumors, in order to select patient subgroups which may benefit from this therapy. In this Review we provided an overview of the main clinical studies describing the association between MSI-H/dMMR tumors and immunotherapy response., (Copyright © 2022 Elsevier B.V. All rights reserved.)

This paper provides a taxonomic revision of the Neotropical ant genus Hylomyrma Forel (1912) (Myrmicinae: Pogonomyrmecini). Morphological traits combined with geographical data and natural history information led to the recognition of 30 species, fourteen of them described here as new: Hylomyrma adelae sp. n., Hylomyrma dandarae sp. n., Hylomyrma jeronimae sp. n., Hylomyrma lispectorae sp. n., Hylomyrma lopesi sp. n., Hylomyrma macielae sp. n., Hylomyrma margaridae sp. n., Hylomyrma mariae sp. n., Hylomyrma marielleae sp. n., Hylomyrma mitiae sp. n., Hylomyrma peetersi sp. n., Hylomyrma primavesi sp. n., Hylomyrma virginiae sp. n. and Hylomyrma wachiperi sp. n. Lectotypes for H. speciosa (junior synonym of H. balzani) and H. reitteri are here designated from syntypes to improve nomenclatural stability. Except for the three species most recently described (H. montana, H. plumosa, and H. villemantae), the external morphology of workers is described or redescribed, as well as for the known males and queens, most described here for the first time. Of the 30 recognized species herein, 11 present intercastes; at least three of them present female specimens with queen-like traits that may be understood as ergatoids. An updated identification key for Hylomyrma workers is provided, as well as high resolution photographs of all known sexes and castes, species distribution maps, and a summary of what is known from the biology of all species.

POLE, POLD1, and NTHL1 are involved in DNA replication and have recently been recognized as hereditary cancer-predisposing genes, because their alterations are associated with colorectal cancer and other tumors. POLE/POLD1-associated syndrome shows an autosomal dominant inheritance, whereas NTHL1-associated syndrome follows an autosomal recessive pattern. Although the prevalence of germline monoallelic POLE/POLD1 and biallelic NTHL1 pathogenic variants is low, they determine different phenotypes with a broad tumor spectrum overlapping that of other hereditary conditions like Lynch Syndrome or Familial Adenomatous Polyposis. Endometrial and breast cancers, and probably ovarian and brain tumors are also associated with POLE/POLD1 alterations, while breast cancer and other unusual tumors are correlated with NTHL1 pathogenic variants. POLE-mutated colorectal and endometrial cancers are associated with better prognosis and may show favorable responses to immunotherapy. Since POLE/POLD1-mutated tumors show a high tumor mutational burden producing an increase in neoantigens, the identification of POLE/POLD1 alterations could help select patients suitable for immunotherapy treatment. In this review, we will investigate the role of POLE, POLD1, and NTHL1 genetic variants in cancer predisposition, discussing the potential future therapeutic applications and assessing the utility of performing a routine genetic testing for these genes, in order to implement prevention and surveillance strategies in mutation carriers., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Although the gastrointestinal stromal tumor (GIST) genotype is not currently included in risk-stratification systems, a growing body of evidence shows that the pathogenic variant (PV) type and codon location hold a strong prognostic influence on recurrence-free survival (RFS). This information has particular relevance in the adjuvant setting, where an accurate prognostication could help to better identify high-risk tumors and guide clinical decision-making., Materials and Methods: Between January 2005 and December 2020, 96 patients with completely resected GISTs harboring a KIT proto-oncogene receptor tyrosine kinase ( KIT ) exon 11 PV were included in the study. We analyzed the type and codon location of the PV according to clinicopathological characteristics and clinical outcome; the metastatic sites in relapsed patients were also investigated., Results: Tumors harboring a KIT exon 11 deletion or deletion/insertion involving the 557 and/or 558 codons, showed a more aggressive clinical behavior compared with tumors carrying deletion/deletion/insertion in other codons, or tumors with duplication/insertion/single-nucleotide variant (SNV) (7-year RFS: 50% versus 73.1% versus 88.2%, respectively; p  < 0.001). Notably, among 18 relapsed patients with 557 and/or 558 deletion or deletion/insertion, 14 patients (77.8%) harbored deletions simultaneously involving 557 and 558 codons, while only 4 patients (22.2%) harbored deletions involving only 1 of the 557/558 codons. Thus, when 557 or 558 deletions occurred separately, the tumor showed a prognostic behavior similar to the GIST carrying deletions outside the 557/558 position. Remarkably, patients with GISTs stratified as intermediate risk, but carrying the 557/558 deletion, showed a similar outcome to the high-risk patients with tumors harboring deletions in codons other than 557/558, or duplication/insertion/SNV., Conclusion: Our data support the inclusion of the PV type and codon location in routine risk prediction models, and suggest that intermediate-risk patients whose GISTs harbor 557/558 deletions may also need to be treated with adjuvant imatinib like the high-risk patients., Competing Interests: The authors declare that there is no conflict of interest., (© The Author(s), 2021.)

Background: Hereditary breast cancer (BC), ovarian cancer (OC), and pancreatic cancer (PC) are the major BRCA-associated tumours. However, some BRCA1/2-wild-type (wt) patients with a strong personal and/or family history of cancer need a further genetic testing through a multi-gene panel containing other high- and moderate-risk susceptibility genes., Patients and Methods: Our study was aimed to assess if some BC, OC, or PC patients should be offered multi-gene panel testing, based on well-defined criteria concerning their personal and/or family history of cancer, such as earliness of cancer onset, occurrence of multiple tumours, or presence of at least two or more affected first-degree relatives. For this purpose, 205 out of 915 BC, OC, or PC patients, resulted negative for BRCA1/2 and with significant personal and/or family history of cancer, were genetically tested for germline pathogenic or likely pathogenic variants (PVs/LPVs) in genes different from BRCA1/2., Results: Our investigation revealed that 31 (15.1%) out of 205 patients harboured germline PVs/LPVs in no-BRCA genes, including PALB2, CHEK2, ATM, MUTYH, MSH2, and RAD51C. Interestingly, in the absence of an analysis conducted through multi-gene panel, a considerable percentage (15.1%) of PVs/LPVs would have been lost., Conclusions: Providing a multi-gene panel testing to BRCA1/2-wt BC/OC/PC patients with a strong personal and/or family history of cancer could significantly increase the detection rates of germline PVs/LPVs in other cancer predisposition genes beyond BRCA1/2. The use of a multi-gene panel testing could improve the inherited cancer risk estimation and clinical management of patients and unaffected family members., Competing Interests: Disclosure The authors have declared no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The introduction of checkpoint inhibitors (ICIs) in renal cell carcinoma (RCC) treatment landscape, resulted in improvements in overall survival (OS) in metastatic patients. Brain metastases (BMs) are a specific metastatic site of interest representing a predictive factor of poor prognosis. Patients with BMs were usually excluded from prospective clinical trials in the past. Despite recent evidence suggest the efficacy and safety of ICIs, the BMs treatment remains a challenge; the immunotherapy responsiveness seems to be multifactorial and dependent on several factors, such as the genetic intratumor heterogeneity and the immunosuppressive role of the brain tumor microenvironment. This review, starting from the immunological background in RCC BMs, provide an overview of the upcoming evidence from clinical trials, address the issues related to the neuroradiological immunotherapy response evaluation and, with a look to the future, describes how the epigenetic modulation of immune evasion could represent a background for new therapeutic strategies., (Copyright © 2021. Published by Elsevier B.V.)

About 10-20% of breast/ovarian (BC/OC) cancer patients undergoing germline BRCA1/2 genetic testing have been shown to harbor Variants of Uncertain Significance (VUSs). Since little is known about the prevalence of germline BRCA1/2 VUS in Southern Italy, our study aimed at describing the spectrum of these variants detected in BC/OC patients in order to improve the identification of potentially high-risk BRCA variants helpful in patient clinical management. Eight hundred and seventy-four BC or OC patients, enrolled from October 2016 to December 2020 at the "Sicilian Regional Center for the Prevention, Diagnosis and Treatment of Rare and Heredo-Familial Tumors" of University Hospital Policlinico "P. Giaccone" of Palermo, were genetically tested for germline BRCA1/2 variants through Next-Generation Sequencing analysis. The mutational screening showed that 639 (73.1%) out of 874 patients were BRCA - w.t. , whereas 67 (7.7%) were carriers of germline BRCA1/2 VUSs, and 168 (19.2%) harbored germline BRCA1/2 pathogenic/likely pathogenic variants. Our analysis revealed the presence of 59 different VUSs detected in 67 patients, 46 of which were affected by BC and 21 by OC. Twenty-one (35.6%) out of 59 variants were located on BRCA1 gene, whereas 38 (64.4%) on BRCA2 . We detected six alterations in BRCA1 and two in BRCA2 with unclear interpretation of clinical significance. Familial anamnesis of a patient harboring the BRCA1 -c.3367G>T suggests for this variant a potential of pathogenicity, therefore it should be carefully investigated. Understanding clinical significance of germline BRCA1/2 VUS could improve, in future, the identification of potentially high-risk variants useful for clinical management of BC or OC patients and family members., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fanale, Fiorino, Incorvaia, Dimino, Filorizzo, Bono, Cancelliere, Calò, Brando, Corsini, Sciacchitano, Magrin, Pivetti, Pedone, Madonia, Cucinella, Badalamenti, Russo and Bazan.)

Gastrointestinal stromal tumors (GISTs) represent 1% of all primary gastrointestinal tumors. Immune surveillance is often overcome by cancer cells due to the activation of immunoregulatory molecules such as programmed death protein (PD-1) and its ligand PD-L1, and butyrophilin sub-family 3A/CD277 receptors (BTN3A). Because several studies demonstrated that tumor PD-1 and PD-L1 expression may have a prominent prognostic function, this investigation aimed to discover if soluble forms of these molecules may be useful in predicting survival of metastatic GIST (mGIST) patients. Through specific ad hoc developed ELISA assays not yet available on the market, the circulating PD-1, PD-L1, BTN3A1, and pan-BTN3As levels were examined in 30 c-KIT exon 11-mutated mGIST patients, prior to imatinib therapy. Using specific thresholds derived by ROC analysis, we found that high baseline levels of sPD-1 (>8.1 ng/mL), sPD-L1 (>0.7 ng/mL), sBTN3A1 (>7.0 ng/mL), and pan-BTN3As (>5.0 ng/mL) were correlated with shorter progression-free survival (PFS) and poor prognosis. Contrariwise, subjects with lower plasma concentrations exhibited a median PFS about 20 months longer than to the earlier. Finally, an additional multivariate analysis revealed that circulating levels of sPD-L1 ≤ 0.7 ng/mL and pan-sBTN3As ≤ 5.0 ng/mL, and the absence of KIT exon 11 deletions or delins at codons 557 and/or 558 were associated with a longer PFS in mGIST patients. Our investigation, for the first time, revealed that evaluating the plasma concentration of some immune checkpoints may help prognosticate survival in mGIST patients, suggesting their potential use as prognostic biomarkers beyond the presence of KIT exon 11 Del or Delins at codons 557/558.

The HIPPO pathway (HP) is a highly conserved kinase cascade that affects organ size by regulating proliferation, cell survival and differentiation. Discovered in Drosophila melanogaster to early 2000, it immediately opened wide frontiers in the field of research. Over the last years the field of knowledge on HP is quickly expanding and it is thought will offer many answers on complex pathologies. Here, we summarized the results of several studies that have investigated HP signaling both in oncology than in cardiology field, with an overview on future perspectives in cardiology research., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Several available data suggest the association between specific molecular subtypes and BRCA1/2 mutational status. Previous investigations showed the association between BRCA1/2 pathogenic variants (PVs) in specific genomic regions and phenotypic variations of cancer relative risk, while the role of PV type and location in determining the breast cancer (BC) phenotypic features remains still unclear. The aim of this research was to describe the germline BRCA1/2 PVs in triple-negative breast cancer (TNBC) versus luminal-like BC and their potential leverage on BC phenotype., Patients & Methods: We retrospectively collected and analyzed all clinical information of 531 patients with BC genetically tested for germline BRCA1/2 PVs by Next-Generation Sequencing analysis at University Hospital Policlinico "P. Giaccone" of Palermo (Sicily) from January 2016 to February 2020., Results: Our results corroborate the evidence that BRCA1 -related tumors often have a profile which resembles the TNBC subtype, whereas BRCA2 -associated tumors have a profile that resembles luminal-like BC, especially the Luminal B subtype. Interestingly, our findings suggest that the PVs identified in TNBC were not largely overlapping with those in luminal-like tumors. Differences in the frequency of two PVs potentially associated with different molecular tumor subtypes were observed. BRCA1 -633delC was detected with relatively higher prevalence in patients with TNBC, whereas BRCA2 -1466delT was found mainly in Luminal B tumors, but in no TNBC patient., Conclusion: Future studies examining the type and location of BRCA1/2 PVs within different molecular subtypes are required to verify our hypothesis and could provide an interesting insight into the complex topic of genotype-phenotype correlations. Additionally, a more in-depth understanding of the potential correlations between BRCA PVs and clinical and phenotypic features of hereditary BC syndrome patients could be the key to develop better strategies of prevention and surveillance in BRCA -positive carriers without disease., Competing Interests: Conflict of interest statement: The authors declare that there is no conflict of interest., (© The Author(s), 2020.)

Introduction of checkpoint inhibitors resulted in durable responses and improvements in overall survival in advanced RCC patients, but the treatment efficacy is widely variable, and a considerable number of patients are resistant to PD-1/PD-L1 inhibition. This variability of clinical response makes necessary the discovery of predictive biomarkers for patient selection. Previous findings showed that the epigenetic modifications, including an extensive microRNA-mediated regulation of tumor suppressor genes, are key features of RCC. Based on this biological background, we hypothesized that a miRNA expression profile directly identified in the peripheral lymphocytes of the patients before and after the nivolumab administration could represent a step toward a real-time monitoring of the dynamic changes during cancer evolution and treatment. Interestingly, we found a specific subset of miRNAs, called "lymphocyte miRNA signature", specifically induced in long-responder patients (CR, PR, or SD to nivolumab >18 months). Focusing on the clinical translational potential of miRNAs in controlling the expression of immune checkpoints, we identified the association between the plasma levels of soluble PD-1/PD-L1 and expression of some lymphocyte miRNAs. These findings could help the development of novel dynamic predictive biomarkers urgently needed to predict the potential response to immunotherapy and to guide clinical decision-making in RCC patients.

Despite a proportion of renal cancer patients can experiment marked and durable responses to immune-checkpoint inhibitors, the treatment efficacy is widely variable and identifying the patient who will benefit from immunotherapy remains an issue. We performed a prospective study to investigate if soluble forms of the immune-checkpoints PD-1 (sPD-1), PD-L1 (sPD-L1), pan-BTN3As, BTN3A1, and BTN2A1, could be candidate to predict the response to immune-checkpoint blockade therapy. We evaluated the plasma levels in a learning cohort of metastatic clear cell renal carcinoma (mccRCC) patients treated with the anti-PD-1 agent nivolumab by ad hoc developed ELISA's. Using specific cut-offs determined through ROC curves, we showed that high baseline levels of sPD-1 (>2.11 ng/ml), sPD-L1 (>0.66 ng/ml), and sBTN3A1 (>6.84 ng/ml) were associated with a longer progression-free survival (PFS) to nivolumab treatment [median PFS, levels above thresholds: sPD-1, 20.7 months ( p < .0001); sPD-L1, 19 months ( p < .0001); sBTN3A1, 17.5 months ( p = .002)]. High sPD-1 and sBTN3A1 levels were also associated with best overall response by RECIST and objective response of >20%. The results were confirmed in a validation cohort of 20 mccRCC patients. The analysis of plasma dynamic changes after nivolumab showed a statistically significant decrease of sPD-1 after 2 cycles (Day 28) in the long-responder patients. Our study revealed that the plasma levels of sPD-1, sPD-L1, and sBTN3A1 can predict response to nivolumab, discriminating responders from non-responders already at therapy baseline, with the advantages of non-invasive sample collection and real-time monitoring that allow to evaluate the dynamic changes during cancer evolution and treatment., (© 2020 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Patients with unilateral breast cancer (UBC) have an increased risk of developing bilateral breast cancer (BBC). The annual risk of contralateral BC is about 0.5%, but increases by up to 3% in BRCA1 or BRCA2 pathogenic variant (PV) carriers. Our study was aimed to evaluate whether all BBC patients should be offered multi-gene panel testing, regardless their cancer family history and age at diagnosis. We retrospectively collected all clinical information of 139 BBC patients genetically tested for germline PVs in different cancer susceptibility genes by NGS-based multi-gene panel testing. Our investigation revealed that 52 (37.4%) out of 139 BBC patients harbored germline PVs in high- and intermediate-penetrance breast cancer (BC) susceptibility genes including BRCA1 , BRCA2 , PTEN , PALB2 , CHEK2 , ATM , RAD51C . Nineteen out of 53 positively tested patients harbored a PV in a known BC susceptibility gene (no- BRCA ). Interestingly, in the absence of an analysis performed via multi-gene panel, a significant proportion (14.4%) of PVs would have been lost. Therefore, offering a NGS-based multi-gene panel testing to all BBC patients may significantly increase the detection rates of germline PVs in other cancer susceptibility genes beyond BRCA1/2 , avoiding underestimation of the number of individuals affected by a hereditary tumor syndrome.

Pompe disease (PD) is a rare autosomal recessive disorder caused by mutations in the GAA gene, localized on chromosome 17 and encoding for acid alpha-1,4-glucosidase (GAA). Currently, more than 560 mutations spread throughout GAA gene have been reported. GAA catalyzes the hydrolysis of α-1,4 and α-1,6-glucosidic bonds of glycogen and its deficiency leads to lysosomal storage of glycogen in several tissues, particularly in muscle. PD is a chronic and progressive pathology usually characterized by limb-girdle muscle weakness and respiratory failure. PD is classified as infantile and childhood/adult forms. PD patients exhibit a multisystemic manifestation that depends on age of onset.Early diagnosis is essential to prevent or reduce the irreversible organ damage associated with PD progression. Here, we make an overview of PD focusing on pathogenesis, clinical phenotypes, molecular genetics, diagnosis, therapies, autophagy and the role of miRNAs as potential biomarkers for PD.

Introduction: A comprehensive smoking ban was recently enacted for acute-care hospital campuses in Spain. The aim of this study was to assess the prevalence and patterns of smoking among inpatients before and during hospitalization., Methods: Multi-center cross-sectional study was conducted in 13 hospitals in the province of Barcelona, Spain from May 2014 to May 2015. Participants were adults who provided informed consent. The sample size was calculated to be representative of each hospital (prevalence 29.4%, precision ± 5%, error 5%). We approached 1228 subjects, 888 accepted to participate and 170 were replaced (were not available or declined to participate). Final sample comprised 1047 subjects. We used a computer-assisted personal interview system to collect data, including sociodemographic variables and use of tobacco before and during hospitalization. Smoking status was validated with exhaled carbon monoxide. We calculated overall tobacco prevalence and investigated associations with participant and center characteristics. We performed multiple polytomous and multilevel logistic regression analyses to estimate odds ratios (ORs) and 95% confidence intervals (CIs), with adjustments for potential confounders., Results: In all, 20.5% (95% CI: 18.1-23.0) of hospitalized patients were smokers. Smoking was most common among men (aOR=7.47; 95% CI: 4.88-11.43), young age groups (18-64 years), and individuals with primary or less than primary education (aOR=2.76; 95% CI: 1.44-5.28). Of the smokers, 97.2% were daily consumers of whom 44.9% had medium nicotine dependence. Of all smokers, three-quarters expressed a wish to quit, and one-quarter admitted to consuming tobacco during hospitalization., Conclusions: Our findings indicate the need to offer smoking cessation interventions among hospitalized patients in all units and service areas, to avoid infringements and increase patient safety, hospital efficiency, and improve clinical outcomes. Hospitalization represents a promising window for initiating smoking interventions addressed to all patients admitted to smoke-free hospitals, specially after applying a smoke-free campus ban., Competing Interests: Authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none was reported.

The innate immune response is highly conserved across all eukaryotes and has been studied in great detail in several model organisms. Hemocytes, the primary immune cell population in mosquitoes, are important components of the mosquito innate immune response, yet critical aspects of their biology have remained uncharacterized. Using a novel method of enrichment, we isolated phagocytic granulocytes and quantified their proteomes by mass spectrometry. The data demonstrate that phagocytosis, blood-feeding, and Plasmodium falciparum infection promote dramatic shifts in the proteomic profiles of An. gambiae granulocyte populations. Of interest, large numbers of immune proteins were induced in response to blood feeding alone, suggesting that granulocytes have an integral role in priming the mosquito immune system for pathogen challenge. In addition, we identify several granulocyte proteins with putative roles as membrane receptors, cell signaling, or immune components that when silenced, have either positive or negative effects on malaria parasite survival. Integrating existing hemocyte transcriptional profiles, we also compare differences in hemocyte transcript and protein expression to provide new insight into hemocyte gene regulation and discuss the potential that post-transcriptional regulation may be an important component of hemocyte gene expression. These data represent a significant advancement in mosquito hemocyte biology, providing the first comprehensive proteomic profiling of mosquito phagocytic granulocytes during homeostasis blood-feeding, and pathogen challenge. Together, these findings extend current knowledge to further illustrate the importance of hemocytes in shaping mosquito innate immunity and their principal role in defining malaria parasite survival in the mosquito host., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: An effective malaria vaccine remains elusive. The most effective experimental vaccines confer only limited and short-lived protection despite production of protective antibodies. However, immunization with irradiated sporozoites, or with live sporozoites under chloroquine cover, has resulted in long-term protection apparently due to the generation of protective CD8+ T cells. The nature and function of these protective CD8+ T cells has not been elucidated. In the current study, the phenotype of CD8+ T cells generated after immunization of C57BL/6 mice with live Plasmodium berghei sporozoites under chloroquine cover was investigated., Methods: Female C57BL/6 mice, C57BL/6 mice B2 macroglobulin -/- [KO], or invariant chain-/- [Ic KO] [6-8 weeks old] were immunized with P. berghei sporozoites and treated daily with 800 μg/mouse of chloroquine for nine days. This procedure of immunization is referred to as "infection/cure". Mice were challenged by inoculating intravenously 1,000 infectious sporozoites. Appearance of parasitaemia was monitored by Giemsa-stained blood smears., Results: By use of MHC I and MHC II deficient animals, results indicate that CD8+ T cells are necessary for full protection and that production of protective antibodies is either CD4+ T helper cells dependent and/or lymphokines produced by CD4 cells contribute to the protection directly or by helping CD8+ T cells. Further, the phenotype of infection/cure P. berghei responsive CD8+ T cells was determined to be KLRG1high CD27low CD44high and CD62Llow., Conclusion: The KLRG1high CD27low CD44high and CD62Llow phenotype of CD8+ T cells is associated with protection and should be investigated further as a candidate correlate of protection.

Circumsporozoite protein (CSP) of Plasmodium falciparum is a protective human malaria vaccine candidate. There is an urgent need for models that can rapidly down-select novel CSP-based vaccine candidates. In the present study, the mouse-mosquito transmission cycle of a transgenic Plasmodium berghei malaria parasite stably expressing a functional full-length P. falciparum CSP was optimized to consistently produce infective sporozoites for protection studies. A minimal sporozoite challenge dose was established, and protection was defined as the absence of blood-stage parasites 14 days after intravenous challenge. The specificity of protection was confirmed by vaccinating mice with multiple CSP constructs of differing lengths and compositions. Constructs that induced high NANP repeat-specific antibody titers in enzyme-linked immunosorbent assays were protective, and the degree of protection was dependent on the antigen dose. There was a positive correlation between antibody avidity and protection. The antibodies in the protected mice recognized the native CSP on the parasites and showed sporozoite invasion inhibitory activity. Passive transfer of anti-CSP antibodies into naive mice also induced protection. Thus, we have demonstrated the utility of a mouse efficacy model to down-select human CSP-based vaccine formulations.

Background: The worldwide burden of malaria remains a major public health problem due, in part, to the lack of an effective vaccine against the Plasmodium falciparum parasite. An effective vaccine will most likely require the induction of antigen specific CD8(+) and CD4(+) T-cells as well as long-lasting antibody responses all working in concert to eliminate the infection. We report here the effective modification of a self-assembling protein nanoparticle (SAPN) vaccine previously proven effective in control of a P. berghei infection in a rodent model to now present B- and T-cell epitopes of the human malaria parasite P. falciparum in a platform capable of being used in human subjects., Methodology/principal Findings: To establish the basis for a SAPN-based vaccine, B- and CD8(+) T-cell epitopes from the P. falciparum circumsporozoite protein (PfCSP) and the universal CD4 T-helper epitope PADRE were engineered into a versatile small protein (∼125 amino acids) that self-assembles into a spherical nanoparticle repetitively displaying the selected epitopes. P. falciparum epitope specific immune responses were evaluated in mice using a transgenic P. berghei malaria parasite of mice expressing the human malaria full-length P. falciparum circumsporozoite protein (Tg-Pb/PfCSP). We show that SAPN constructs, delivered in saline, can induce high-titer, long-lasting (1 year) protective antibody and poly-functional (IFNγ(+), IL-2(+)) long-lived central memory CD8(+) T-cells. Furthermore, we demonstrated that these Ab or CD8(+) T-cells can independently provide sterile protection against a lethal challenge of the transgenic parasites., Conclusion: The SAPN construct induces long-lasting antibody and cellular immune responses to epitope specific sequences of the P. falciparum circumsporozoite protein (PfCSP) and prevents infection in mice by a transgenic P. berghei parasite displaying the full length PfCSP.

We have designed and produced a prototypic malaria vaccine based on a highly versatile self-assembling polypeptide nanoparticle (SAPN) platform that can repetitively display antigenic epitopes. We used this platform to display a tandem repeat of the B cell immunodominant repeat epitope (DPPPPNPN)(2)D of the malaria parasite Plasmodium berghei circumsporozoite protein. Administered in saline, without the need for a heterologous adjuvant, the SAPN construct P4c-Mal conferred a long-lived, protective immune response to mice with a broad range of genetically distinct immune backgrounds including the H-2(b), H-2(d), and H-2(k) alleles. Immunized mice produced a CD4(+) T cell-dependent, high-titer, long-lasting, high-avidity Ab response against the B cell epitope. Mice were protected against an initial challenge of parasites up to 6 mo after the last immunization or for up to 15 mo against a second challenge after an initial challenge of parasites had successfully been cleared. Furthermore, we demonstrate that the SAPN platform not only functions to deliver an ordered repetitive array of B cell peptide epitopes but operates as a classical immunological carrier to provide cognate help to the P4c-Mal-specific B cells.

We report the first safety and immunogenicity trial of the Plasmodium falciparum vaccine candidate FMP2.1/AS02A, a recombinant E. coli-expressed protein based upon the apical membrane antigen-1 (AMA-1) of the 3D7 clone formulated with the AS02A adjuvant. We conducted an open-label, staggered-start, dose-escalating Phase I trial in 23 malaria-naïve volunteers who received 8, 20 or 40microg of FMP2.1 in a fixed volume of 0.5mL of AS02A on a 0, 1, and 2 month schedule. Nineteen of 23 volunteers received all three scheduled immunizations. The most frequent solicited local and systemic adverse events associated with immunization were injection site pain (68%) and headache (29%). There were no significant laboratory abnormalities or vaccine-related serious adverse events. All volunteers seroconverted after second immunization as determined by ELISA. Immune sera recognized sporozoites and merozoites by immunofluorescence assay (IFA), and exhibited both growth inhibition and processing inhibition activity against homologous (3D7) asexual stage parasites. Post-immunization, peripheral blood mononuculear cells exhibited FMP2.1-specific lymphoproliferation and IFN-gamma and IL-5 ELISPOT assay responses. This is the first PfAMA-1-based vaccine shown to elicit both potent humoral and cellular immunity in humans. Encouraged by the potential of FMP1/AS02A to target host immunity against PfAMA-1 that is known to be expressed by sporozoite, hepatic and erythrocytic stages, we have initiated field trials of FMP2.1/AS02A in an endemic population in the Republic of Mali.

Liver-stage antigen 1 (LSA1) is expressed by Plasmodium falciparum only during the intrahepatic cell stage of the parasite's development. Immunoepidemiological studies in regions where malaria is endemic suggested an association between the level of LSA1-specific humoral and cell-mediated immune responses and susceptibility to clinical malaria. A recombinant LSA1 protein, FMP011, has been manufactured as a preerythrocytic vaccine to induce an immune response that would have the effect of controlling parasitemia and disease in humans. To evaluate the immunogenicity of FMP011, we analyzed the immune response of three inbred strains of mice to antigen immunization using two different adjuvant formulations, AS01B and AS02A. We report here the ability of BALB/c and A/J mice, but not C57BL/6J mice, to mount FMP011-specific humoral (antibody titer) and cellular (gamma interferon [IFN-gamma] production) responses following immunization with FMP011 formulated in AS01B or AS02A. Immunization of BALB/c and A/J mice with FMP011/AS01B induced more antigen-specific IFN-gamma-producing splenocytes than immunization with FMP011/AS02A. A slightly higher titer of antibody was induced using AS02A than AS01B in both strains. C57BL/6J mice did not respond with any detectable FMP011-specific IFN-gamma splenocytes or antibody when immunized with FMP011 in AS01B or AS02A. Intracellular staining of cells isolated from FMP011/AS01B-immunized BALB/c mice indicated that CD4(+) cells, but not CD8(+) cells, were the main IFN-gamma-producing splenocyte. However, inclusion of blocking anti-CD4(+) antibody during the in vitro restimulation ELISpot analysis failed to completely abolish IFN-gamma production, indicating that while CD4(+) T cells were the major source of IFN-gamma, other cell types also were involved.

Background: In the current study, we determined whether bovine dialyzable leukocyte extract (bDLE) modulates lipopolysaccharide (LPS)-induced nitric oxide and cytokine overproduction., Methods: Human whole blood cells were treated with LPS (50 ng) + bDLE (1 U)., Results: The bDLE treatment decreased nitric oxide as well as TNF-alpha, IL-6 and IL-10 (P <0.01) cytokine production. In addition, it decreased TNF-alpha, IL-1beta and IL-6 mRNA expression and suppressed IL-10 and IL-12p40 mRNA expression, but did not modulate IL-8 mRNA expression in LPS-stimulated human blood cells., Discussion: Our results suggest that bDLE may effectively modulate the fatal symptoms of hypotensive shock associated with endotoxin (LPS)-induced nitric oxide and cytokine production, and this may offer therapeutic potential for the treatment of endotoxic shock.

Background: Bovine dialyzable leukocyte extract (bDLE) is a dialyzate of a heterogeneous mixture of low molecular weight substances released from disintegrated blood leukocytes or lymphoid tissue obtained from homogenized bovine spleen. The purpose of this study was to determine if bDLE had cytotoxic effects and modulated apoptosis gene expression in breast cancer cells., Methods: The MCF-7, BT-474, MDA-MB-453, A-427, Calu-1, U937 and L5178Y cancer cell lines and PBMC human cells were treated with bDLE (0-0.66 U/mL) for 72 h. The bDLE effect on cell growth proliferation was evaluated by MTT assay, and the MCF-7 was evaluated by ethidium bromide-acridine orange staining; total DNA was evaluated for DNA fragmentation, and total RNA was isolated for p53, bag-1, c-myc, bim, bax, bcl-2 and bad mRNA expression., Results: The bDLE had dose-dependent cytotoxic effects and demonstrated an IC50 at a dosage of 0.06 U/mL (P<0.05). The bDLE did not affect the viability of normal human PBMC. The bDLE induced DNA fragmentation at doses of 0.06 and 0.13 U/mL in MCF-7 breast cancer cells. The bDLE induced cytotoxic effects and suppressed the p53, bag-1, c-myc, bax, bcl-2, and bad mRNA expression that influences apoptosis in MCF-7 breast cancer cells. Bim mRNA expression was not detected., Discussion: This may open up interesting prospects for the treatment of human breast cancer.

The major histocompatibility complex nonrestricted cytotoxic leukemic T cell line T acute lymphoblastic leukemia (TALL)-104 is being pursued as a therapeutic agent for cancer. However, the receptors and effector mechanisms responsible for its broad tumoricidal function remain undefined. Here, we examined the roles played by natural cytotoxicity receptors (NCR), killer cell immunoglobulin-like receptors, cytolytic granule components, and tumor necrosis factor (TNF) family members in tumor recognition and lysis by TALL-104 cells. The perforin-granzyme pathway, TNF-related apoptosis-inducing ligand (TRAIL), and Fas were each involved in the lysis of particular tumor targets by TALL-104. Furthermore, phorbol 12-myristate 13-acetate/ionomycin treatment induced surface expression of Fas-L and TRAIL. In addition, supernatants from CD3-stimulated TALL-104 cultures exhibited antiproliferative activity, which was blocked 50-90% by anti-TNF-alpha monoclonal antibody (mAb). Although negative for the NCR natural killer (NK)p44, this cell line was found to express NKp46. An anti-NKp46 antibody strongly blocked TALL-104-mediated lysis of certain targets and directly induced cytokine production, granule release, and redirected lysis responses. Anti-NKG2D and anti-2B4 also stimulated redirected cytotoxicity by TALL-104. By contrast, anti-NKG2A mAb did not stain the cells or inhibit killing responses. Alternatively, KIR3DL2 was detected on TALL-104, and expression of its reported ligand, human leukocyte antigen (HLA)-A, on target cells provided protection from cytotoxicity. Thus, NKp46, NKG2D, and 2B4 are activating receptors, and KIR3DL2 is an inhibitory receptor on TALL-104. The data demonstrate the ability of TALL-104 cells to recognize a wide variety of tumors with NK cell receptors and kill them with a broad arsenal of cytolytic effector mechanisms, including cytolytic granules and TNF family ligands.

Viper venom disintegrins have been used frequently to study the cellular receptors which characterize various types of cells, including platelets, endothelial cells and cancer cells. While the majority of such analyses have pointed to involvement of integrin receptors alphavbeta3, alpha5beta1 or alphaIIbbeta3, this may not always be so. Eristostatin, from Eristocophis macmahoni, is a potent inhibitor of ADP-induced platelet aggregation as well as of human and murine melanoma metastases in mouse model systems. This disintegrin requires an RGDW motif, as well as an intact C-terminus, in order to interact with both platelets and four different types of melanoma cells. Eristostatin causes nonmetastatic SBc12 melanoma cells to show higher susceptibility to specific killing by NK-like TALL-104 cells. While it is known that eristostatin binds to alphaIIbbeta3 on platelets, the receptor with which eristostatin binds to the melanoma cells has not yet been identified., (Copyright 2002 S. Karger AG, Basel)

The venom of Echis carinatus suchoreki contains a monomeric disintegrin echistatin (Mr 5,500 Da) that strongly inhibits alphaIIbbeta3, alphavbeta3, and alpha5beta1 integrins and a heterodimeric disintegrin called EC3 (M(r) 14,762 Da). At nanomolar concentration, EC3 inhibits adhesion of human cell lines expressing alpha4beta1 and alpha4beta7 to immobilized VCAM-1; it has a lower inhibitory effect on alpha5beta1-mediated cell adhesion. In this study, we demonstrated that EC3, in contrast to echistatin, inhibited binding of monoclonal anti-alpha4 and anti-alpha5 antibodies to cells expressing alpha4beta7. In a dose-dependent manner and to the same extent, EC3 inhibited adhesion of Jurkat cells and murine splenic lymphocytes to immobilized VCAM-1, whereas echistatin was not active. EC3 injected intraperitoneally into nonobese diabetic (NOD mice) suppressed development of insulitis and sialoadenitis, whereas echistatin had no significant effect. We propose that the effect of EC3 is mediated, at least, in part, by blocking alpha4beta1 and alpha4beta7 on murine lymphocytes., (Copyright 2000 Academic Press.)

The "outside-in" signals produced by the interaction of integrin molecules with the extracellular matrix (ECM) trigger a multitude of cellular events. The vitronectin receptor (VNR), an alpha v beta 3 heterodimer, functions as a costimulatory molecule for the activation of a subset of V gamma 1.1/C gamma 4-bearing gamma/delta T cells, which have been postulated to recognize a ubiquitous self-antigen. We addressed the question of whether stimulation of these T cells requires both engagement of the VNR by ECM proteins and engagement of the TCR by its Ag. We introduced into a TCR- but VNR+ mutant T cell hybridoma, TG40 (derived from 2B4), a chimeric molecule that contains the cytoplasmic tail of the TCR zeta-chain fused to the cytoplasmic and transmembrane region of either human CD8 or human CD25. The transfectants expressing the chimeric molecules secreted IL-2 constitutively when the VNR was engaged with a ligand, e.g., provided by ECM proteins present in FCS. This constitutive cytokine secretion could be blocked with mAb directed against the VNR, with or the peptide RGD, or by growth in serum-free medium. VNR-mediated cell activation also induced the phosphorylation of the zeta-chain. Signaling through the zeta-chain was required, as cells transfected with a chimera containing only a 22 amino-acid long, truncated zeta-chain did not secrete IL-2 constitutively. Thus, we demonstrated that the binding of the VNR to ECM protein in the presence of the zeta-chain is sufficient to induce cytokine secretion by T cells and does not require the recognition of an Ag by the TCR. Such integrin-mediated, Ag-independent activation of T cells may play a critical role in the potentiation of inflammatory responses.

The murine vitronectin receptor (VNR, alpha V beta 3) is expressed on T cell hybridomas expressing both the alpha beta and the gamma delta TCR as well as on TCR-alpha beta cells activated for prolonged periods of time by mitogens or alloantigens. The VNR functions as a costimulatory molecule for the activation of a subset of gamma delta T cells that express the V gamma 1.1 C gamma 4 V delta 6 TCR and that may recognize a ubiquitously expressed autoantigen. To characterize further some of the signal transduction parameters observed after engagement of the VNR in stimulated T lymphocytes, we have examined the effect of ligation of the VNR by RGDS-containing proteins on the pattern of protein phosphorylation. We demonstrate the appearance of a 115-kDa, tyrosine-phosphorylated protein (pp115) after engagement of the VNR with its ligand, RGDS. pp115 was shown to be immunologically distinct from focal adhesion kinase, did not possess inherent kinase activity, and may represent an as yet unidentified substrate in the integrin signal transduction pathway. Although induction of pp115 was independent of TCR expression, because it was seen in the TG40 hybridoma, which expresses neither the alpha beta nor the gamma delta TCR, pp115 could also be induced by cross-linking of the TCR in a murine TCR gamma delta hybridoma in the absence of any extracellular matrix proteins. This result raises the possibility that induction of pp115 is a common biochemical step in signal transduction by both the TCR and the VNR.

Murine T helper cloned cells permeabilized with the bacterial lysin, tetanolysin, were used to investigate the role of intracellular Ca2+ in regulating myo-[2-3H] inositol phospholipid (InsPL) hydrolysis triggered upon perturbation of the T cell receptor-CD3 complex. [Ca2+] was controlled by a calcium/magnesium/EGTA buffer. Antibody (mAb) aggregation of CD3 induced InsPL hydrolysis in the absence of added Ca2+. However, stimulated InsPL hydrolysis increased with the free [Ca2+], reaching a maximum at 100-300 nM [Ca2+]. Ca2+ increased the overall efficiency of hydrolysis without changes in EC50 of the anti-CD3 mAb. The response diminished at > 300 nM [Ca2+] due to a mixed type inhibition. Ca2+ alone had no effect on inositol phosphate levels. Polyphosphoinositides were preferentially cleaved, since no accumulation of Ins(1)P/Ins(3)P was detected, indicating that direct hydrolysis of phosphatidylinositol did not occur, irrespective of the Ca2+ concentration. [Ca2+] above 300 nM shifted the relative amounts of CD3-induced inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) in favor of the latter. Unlabeled permeabilized cells exposed to > or = 100 nM [Ca2+] showed enhanced conversion of [3H]Ins(1,4,5)P3 to [3H]Ins(1,3,4,5)P4. In conclusion, InsPL hydrolysis is optimally triggered by CD3 perturbation at intracellular Ca2+ levels approximating those observed in intact resting lymphocytes (100 nM). Ca2+ concentrations similar to those triggered by InsPL-derived metabolites may inhibit InsPL hydrolysis and promote Ins(1,3,4,5)P4 production, thus controlling the amounts of Ins(1,4,5)P3.