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In chronic lymphocytic leukemia (CLL), survival of neoplastic cells depends on microenvironmental signals at lymphoid sites where the crosstalk between the integrin VLA-4 (CD49d/CD29), expressed in ~40% of CLL, and the B-cell receptor (BCR) occurs. Here, BCR engagement inside-out activates VLA-4, thus enhancing VLA-4-mediated adhesion of CLL cells, which in turn obtain pro-survival signals from the surrounding microenvironment. We report that the BCR is also able to effectively inside-out activate the VLA-4 integrin in circulating CD49d-expressing CLL cells through an autonomous antigen-independent BCR signaling. As a consequence, circulating CLL cells exhibiting activated VLA-4 express markers of BCR pathway activation (phospho-BTK and phospho-PLC-γ2) along with higher levels of phospho-ERK and phospho-AKT indicating parallel activation of downstream pathways. Moreover, circulating CLL cells expressing activated VLA-4 bind soluble blood-borne VCAM-1 leading to increased VLA-4-dependent actin polymerization/re-organization and ERK phosphorylation. Finally, evidence is provided that ibrutinib treatment, by affecting autonomous BCR signaling, impairs the constitutive VLA-4 activation eventually decreasing soluble VCAM-1 binding and reducing downstream ERK phosphorylation by circulating CLL cells. This study describes a novel anchor-independent mechanism occurring in circulating CLL cells involving the BCR and the VLA-4 integrin, which help to unravel the peculiar biological and clinical features of CD49d+ CLL., (© 2024. The Author(s).)

Loss-of-function mutations in NFKBIE, which encodes for the NF-κB inhibitor IκBε, are frequent in chronic lymphocytic leukemia (CLL) and certain other B-cell malignancies and have been associated with accelerated disease progression and inferior responses to chemotherapy. Using in vitro and in vivo murine models and primary patient samples, we now show that NFKBIE-mutated CLL cells are selected by microenvironmental signals that activate the NF-κB pathway and induce alterations within the tumor microenvironment that can allow for immune escape, including expansion of CD8+ T-cells with an exhausted phenotype and increased PD-L1 expression on the malignant B-cells. Consistent with the latter observations, we find increased expression of exhaustion markers on T-cells from patients with NFKBIE-mutated CLL. In addition, we show that NFKBIE-mutated murine CLL cells display selective resistance to ibrutinib and report inferior outcomes to ibrutinib treatment in NFKBIE-mutated CLL patients. These findings suggest that NFKBIE mutations can contribute to CLL progression through multiple mechanisms, including a bidirectional crosstalk with the microenvironment and reduced sensitivity to BTK inhibitor treatment., (© 2024. The Author(s).)

Lymphoproliferative diseases are a heterogeneous set of malignant clonal proliferations of lymphocytes. Despite well-established diagnostic criteria, the diagnosis remains difficult due to their variety in clinical presentation and immunophenotypic profile. Lymphoid T-cell disorders are less common than B-cell entities, and the lack of a clear immunophenotypic characteristic makes their identification hard. Flow cytometry turned out to be a useful tool in diagnosing T-cell disorders and to resolve complicated cases, especially if the number of analyzable neoplastic cells is small. We present a case of a 55-year-old man with simultaneous lymphoproliferative neoplastic T-cell clones, one αβ and the other γδ, identified and characterized by flow cytometry (FC), exploiting the variable expression intensity of specific markers. However, the patient's rapid decline made it impossible to define a differential diagnosis in order to confirm the identity of the γδ clone, which remains uncertain. This case is added to the few other cases already documented in the literature, characterized by the co-existence of T-large granular lymphocytic leukemia (T-LGLL)-αβ and T-LGLL-γδ/Hepatosplenic T-cell lymphoma (HSTCL). Our case underlines the key role of sensitive diagnostic tools in the assessment of potential relationship between the diagnosis, prognosis, and treatment in the two pathologies.

Atypical chronic lymphocytic leukemia (CLL) is still defined according to morphological criteria. However, deviance from the typical surface immunological profile suggests an atypical immunological-based CLL. A large cohort of patients with CLL was retrospectively evaluated aiming at assessing morphological (FAB criteria), immunophenotypical (two or more discordances from the typical profile), and clinical-biological features of atypical CLL. Compared to typical cases, morphologically atypical CLL showed a greater percentage of unmutated IgVH and CD38 positivity, and a higher expression of CD20. Immunophenotypically atypical CLL was characterized by more advanced clinical stages, higher expression of CD20, higher rate of FMC7, CD79b and CD49d positivity, and by an intermediate-high expression of membrane surface immunoglobulin, compared to typical cases. When patients were categorized based on immunophenotypic and morphologic concordance or discordance, no difference emerged. Finally, morphological features better discriminated patients' prognosis in terms of time-to-first treatment, while concordant atypical cases showed overall a worse prognosis. Discordant cases by immunophenotype and/or morphology did not identify specific prognostic groups. Whether-in the era of molecular markers used as prognostic indicators-it does make sense to focus on morphology and immunophenotype features in CLL is still matter of debate needing further research.

Here we evaluated the epigenomic and transcriptomic profile of XPO1 mutant chronic lymphocytic leukaemia (CLL) and their clinical phenotype. By ATAC-seq, chromatin regions that were more accessible in XPO1 mutated CLL were enriched of binding sites for transcription factors regulated by pathways emanating from the B-cell receptor (BCR), including NF-κB signalling, p38-JNK and RAS-RAF-MEK-ERK. XPO1 mutant CLL, consistent with the chromatin accessibility changes, were enriched with transcriptomic features associated with BCR and cytokine signalling. By combining epigenomic and transcriptomic data, MIR155HG, the host gene of miR-155, and MYB, the transcription factor that positively regulates MIR155HG, were upregulated by RNA-seq and their promoters were more accessible by ATAC-seq. To evaluate the clinical impact of XPO1 mutations, we investigated a total of 957 early-stage CLL subdivided into 3 independent cohorts (N = 276, N = 286 and N = 395). Next-generation sequencing analysis identified XPO1 mutations as a novel predictor of shorter time to first treatment (TTFT) in all cohorts. Notably, XPO1 mutations maintained their prognostic value independent of the immunoglobulin heavy chain variable status and early-stage prognostic models. These data suggest that XPO1 mutations, conceivably through increased miR-155 levels, may enhance BCR signalling leading to higher proliferation and shorter TTFT in early-stage CLL., (© 2023 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Lanthanide-doped nanoparticles, featuring sharp emission peaks with narrow bandwidth, exhibit high downconversion luminescence intensity, making them highly valuable in the fields of bioimaging and drug delivery. High-crystallinity Y 2 O 3 nanoparticles (NPs) doped with Er 3+ ions were functionalized by using a pegylation procedure to confer water solubility and biocompatibility. The NPs were thoroughly characterized using transmission electron microscopy (TEM), inductively coupled plasma mass spectrometry (ICP-MS), and photoluminescence measurements. The pegylated nanoparticles were studied both from a toxicological perspective and to demonstrate their internalization within HCT-116 cancer cells. Cell viability tests allowed for the identification of the "optimal" concentration, which yields a detectable fluorescence signal without being toxic to the cells. The internalization process was investigated using a combined approach involving confocal microscopy and ICP-MS. The obtained data clearly indicate the efficient internalization of NPs into the cells with emission intensity showing a strong correlation with the concentrations of nanoparticles delivered to the cells. Overall, this research contributes significantly to the fields of nanotechnology and biomedical research, with noteworthy implications for imaging and drug delivery applications., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory checkpoint receptor that negatively regulates Tcell responses. CD226 competes with TIGIT for binding to the CD155 ligand, delivering a positive signal to the T cell. Here we studied the expression of TIGIT and CD226 in a cohort of 115 patients with chronic lymphocytic leukemia (CLL) and report expression of TIGIT and CD226 by leukemic cells. By devising a TIGIT/CD226 ratio, we showed that CLL cells favoring TIGIT over CD226 are typical of a more indolent disease, while those favoring CD226 are characterized by a shorter time to first treatment and shorter progression-free survival after first treatment. TIGIT expression was inversely correlated to the B-cell receptor (BCR) signaling capacity, as determined by studying BTK phosphorylation, cell proliferation and interleukin- 10 production. In CLL cells treated with ibrutinib, in which surface IgM and BCR signaling capacity are temporarily increased, TIGIT expression was downmodulated, in line with data indicating transient recovery from anergy. Lastly, cells from patients with Richter syndrome were characterized by high levels of CD226, with low to undetectable TIGIT, in keeping with their high proliferative drive. Together, these data suggest that TIGIT contributes to CLL anergy by downregulating BCR signaling, identifying novel and actionable molecular circuits regulating anergy and modulating CLL cell functions.

Background: B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the expansion of CD5 + malignant B lymphocytes. Recent discoveries have shown that double-negative T (DNT) cells, double-positive T (DPT) cells, and natural killer T (NKT)-cells may be involved in tumor surveillance., Methods: A detailed immunophenotypic analysis of the peripheral blood T-cell compartment of 50 patients with B-CLL (classified in three prognostic groups) and 38 healthy donors (as controls) matched for age was performed. The samples were analyzed by flow cytometry using a stain-lyse-no wash technique and a comprehensive six-color antibody panels., Results: Our data confirmed a reduction in percentage values and an increase in absolute values of T lymphocytes in patients with B-CLL, as already reported. In particular, DNT, DPT, and NKT-like percentages were significantly lower than in the controls, except for NKT-like in the low-risk prognostic group. Moreover, a significant rise in the absolute counts of DNT cells in each prognostic group and in the low-risk prognostic group of NKT-like cells was found. A significant correlation of the absolute values of NKT-like cells in the intermediate-risk prognostic group versus B cells was observed. Furthermore, we analyzed whether the increase in T cells was related to the subpopulations of interest. Only DNT cells were positively correlated with the increase in CD3 + T lymphocytes, regardless of the stage of the disease, supporting the hypothesis that this T-cell subset plays a key role in the immune T response in B-CLL., Conclusion: These early results supported that DNT, DPT, and NKT-like subsets may be related to disease progression and should encourage further studies aimed at identifying the potential immune surveillance role of these minority T subpopulations., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

This study aims to provide information on the behaviour and biopersistence rate (BP) of metallic nanoparticles (Ag-NPs, TiO 2 -NPs, ZnO-NPs) naturally occurring in canned seafood and subjected to static in vitro digestion. Single particle ICP-MS analysis was performed to determine NPs distribution and concentrations in oral, gastric, and intestinal digests. Depending on the conditions of the digestive phase and the sample matrix, the phenomena of agglomeration and dispersion were highlighted and confirmed by Dynamic Light Scattering (DLS) technique. In standard suspensions, Ag-NPs had lower biopersistence (BP) than ZnO and TiO 2 -NPs (BP 34%, 89% and >100%, respectively). Among Ag-NPs and TiO 2 -NPs naturally present in the food matrix, those in canned tuna were more degradable than those in canned clam (BP Ag-NPs 36% vs. > 100%; BP TiO 2 -NPs 96% vs. > 100%), while BP ZnO-NPs showed high biopersistence in both seafood matrix (>100%). The biopersistence rates were higher than the recommended limit set by European Food Safety Authority (EFSA) (12%), referred to nanotechnologies to be applied in the food and feed chain, thus the investigated naturally occurring NPs cannot be considered readily degradable., 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 Elsevier Ltd. All rights reserved.)

Measurable residual disease (MRD) is defined as the presence of residual cancer cells after treatment in patients with clinically undetectable disease, who would otherwise be considered in complete remission. It is a highly sensitive parameter which indicates the disease burden and predicts survival in this setting of patients. In recent years, MRD has gained a role in many hematological malignancies as a surrogate endpoint for clinical trials: undetectable MRD has been correlated to longer progression free survival (PFS) and overall survival (OS). New drugs and combinations have been developed with the aim to achieve MRD negativity, which would indicate favorable prognosis. Different methods to measure MRD have also been devised, which include flow cytometry, polymerase chain reaction (PCR) and next generation sequencing (NGS), with different sensitivity and accuracy in evaluating deep remission after treatment. In this review, we will analyze the current recommendations for the detection of MRD, with particular focus on its role in Chronic Lymphocytic Leukemia (CLL), as well as the different detection methods. Moreover, we will discuss the results of clinical trials and the role of MRD in new therapeutic schemes with inhibitors and monoclonal antibodies. MRD is not currently used in the clinical practice to evaluate response to treatment, due to technical and economical limitations, but it's gaining more and more interest in trials settings, especially since the introduction of venetoclax. The use of MRD in trials will likely be followed by a broader practical application in the future. The aim of this work is to provide a reader-friendly summary of the state of art in the field, as MRD will soon become an accessible tool to evaluate our patients, predict their survival and guide physician's therapeutic choices and preferences., 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 Benintende, Pozzo, Innocenti, Autore, Fresa, D’Arena, Gattei and Lurenti.)

Measurable residual disease (MRD) has emerged as a relevant parameter of response to therapy in chronic lymphocytic leukemia (CLL). Although several methods have been developed, flow cytometry has emerged as the most useful and standardized approach to measure and quantify MRD. The improved sensitivity of MRD measurements has been paralleled by the development of more effective therapeutic strategies for CLL, increasing the applicability of MRD detection in this setting. Chemotherapy and chemoimmunotherapy have firstly demonstrated their ability to obtain a deep MRD. Combined targeted therapies are also demonstrating a high molecular response rate and prospective trials are exploring the role of MRD to guide the duration of treatment in this setting. In this review we briefly summarize what we have learned about MRD with emphasis on its flow cytometric detection., (© 2022 John Wiley & Sons Ltd.)

Breast cancer is the most common malignancy among women worldwide. Several studies indicate that, in addition to established risk factors for breast cancer, other factors such as cortisol release related to psychological stress and drug treatment with high levels of glucocorticoids may also contribute significantly to the initiation of breast cancer. There are several possible mechanisms by which glucocorticoids might promote neoplastic transformation of breast tissue. Among these, the least known and studied is the inhibition of the nuclear erythroid factor 2-related (Nrf2)-antioxidant/electrophile response element (ARE/EpRE) pathway by high levels of glucocorticoids. Specifically, Nrf2 is a potent transcriptional activator that plays a central role in the basal and inducible expression of many cytoprotective genes that effectively protect mammalian cells from various forms of stress and reduce the propensity of tissues and organisms to develop disease or malignancy including breast cancer. Consequently, a loss of Nrf2 in response to high levels of gluco-corticoids may lead to a decrease in cellular defense against oxidative stress, which plays an important role in the initiation of human mammary carcinogenesis. In the present review, we provide a comprehensive overview of the current state of knowledge of the cellular mechanisms by which both glucocorticoid pharmacotherapy and endogenous GCs (cortisol in humans and corticosterone in rodents) may contribute to breast cancer development through inhibition of the Nrf2-ARE/EpRE pathway and the protective role of melatonin against glucocorticoid-induced apoptosis in the immune system., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s). Published by IMR Press.)

The present study aims to give information regarding the quantification of ZnO-NPs in canned seafood, which may be intentionally or unintentionally added, and to provide a first esteem of dietary exposure. Samples were subjected to an alkaline digestion and assessment of ZnO-NPs was performed by the single particle ICP-MS technique. ZnO-NPs were found with concentrations range from 0.003 to 0.010 mg/kg and a size mean range from 61.3 and 78.6 nm. It was not observed a clear bioaccumulation trend according to trophic level and size of seafood species, although the mollusk species has slightly higher concentrations and larger size. The number of ZnO-NPs/g does not differ significantly among food samples, observing an average range of 5.51 × 10 6 - 9.97 × 10 6 . Dissolved Zn determined with spICP-MS revealed comparable concentration to total Zn determined with ICP-MS in standard mode, confirming the efficiency of alkaline digestion on the extraction of the Zn. The same accumulation trend found for ZnO-NPs was observed more clearly for dissolved Zn. The ZnO-NPs intake derived from a meal does not differ significantly among seafood products and it ranges from 0.010 to 0.031 µg/kg b.w. in adult, and from 0.022 to 0.067 µg/kg b.w. in child. Conversely, the intake of dissolved Zn is significantly higher if it is assumed a meal of mollusks versus the fish products, with values of 109.3 µg/kg b.w. for adult and 240.1 µg/kg b.w. for child. Our findings revealed that ZnO-NPs have the potential to bioaccumulate in marine organisms, and seafood could be an important uptake route of ZnO-NPs. These results could be a first important step to understand the ZnO-NPs human dietary exposure, but the characterization and quantification of ZnO-NPs is necessary for a large number of food items., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Chronic lymphocytic leukemia (CLL) is characterized by a low CD20 expression, in part explained by an epigenetic-driven downregulation triggered by mutations of the NOTCH1 gene. In the present study, by taking advantage of a wide and well-characterized CLL cohort (n=537), we demonstrate that CD20 expression is downregulated in SF3B1-mutated CLL in an extent similar to NOTCH1-mutated CLL. In fact, SF3B1-mutated CLL cells show common features with NOTCH1-mutated CLL cells, including a gene expression profile enriched of NOTCH1-related gene sets and elevated expression of the active intracytoplasmic NOTCH1. Activation of the NOTCH1 signaling and down-regulation of surface CD20 in SF3B1-mutated CLL cells correlate with over-expression of an alternatively spliced form of DVL2, a component of the Wnt pathway and negative regulator of the NOTCH1 pathway. These findings are confirmed by separately analyzing the CD20-dim and CD20-bright cell fractions from SF3B1-mutated cases as well as by DVL2 knock-out experiments in CLL-like cell models. Altogether, the clinical and biological features that characterize NOTCH1-mutated CLL may also be recapitulated in SF3B1-mutated CLL, contributing to explain the poor prognosis of this CLL subset and providing the rationale for expanding novel agents-based therapies to SF3B1-mutated CLL.

Purpose: In chronic lymphocytic leukemia (CLL), TP53 mutations are associated with reduced survival and resistance to standard chemoimmunotherapy (CIT). Nevertheless, the clinical impact of subclonal TP53 mutations below 10% to 15% variant allele frequency (VAF) remains unclear., Experimental Design: Using a training/validation approach, we retrospectively analyzed the clinical and biological features of TP53 mutations above (high-VAF) or below (low-VAF) the previously reported 10.0% VAF threshold, as determined by deep next-generation sequencing. Clinical impact of low-VAF TP53 mutations was also confirmed in a cohort ( n = 251) of CLL treated with fludarabine-cyclophosphamide-rituximab (FCR) or FCR-like regimens from two UK trials., Results: In the training cohort, 97 of 684 patients bore 152 TP53 mutations, while in the validation cohort, 71 of 536 patients had 109 TP53 mutations. In both cohorts, patients with the TP53 mutation experienced significantly shorter overall survival (OS) than TP53 wild-type patients, regardless of the TP53 mutation VAF. By combining TP53 mutation and 17p13.1 deletion (del17p) data in the total cohort ( n = 1,220), 113 cases were TP53 mutated only (73/113 with low-VAF mutations), 55 del17p/ TP53 mutated (3/55 with low-VAF mutations), 20 del17p only, and 1,032 (84.6%) TP53 wild-type. A model including low-VAF cases outperformed the canonical model, which considered only high-VAF cases (c-indices 0.643 vs. 0.603, P < 0.0001), and improved the prognostic risk stratification of CLL International Prognostic Index. Clinical results were confirmed in CIT-treated cases ( n = 552) from the retrospective cohort, and the UK trials cohort., Conclusions: TP53 mutations affected OS regardless of VAF. This finding can be used to update the definition of TP53 mutated CLL for clinical purposes., (©2021 American Association for Cancer Research.)

Mediterranean diet protects from both cardiovascular disease (CVD) and cancer. In the 1960s, Ancel Keys defined the concept of Mediterranean diet in the South Italian region of Cilento and proposed it as a key factor for healthy ageing in the region. The aim of the current study was to compare the prevalence of CVD and cancer between a middle-aged population from Cilento and those of a Northern European population from Malmö, Sweden. We clinically characterized two middle-aged (50-67 years of age) population-based samples from Cilento (n = 809) and Malmö (n = 1025), Sweden, respectively. Logistic regression was used to calculate odds ratios (95% confidence interval) for disease prevalence in Malmö versus Cilento inhabitants adjusted for age and sex (model 1) and adjusted for all cardiometabolic risk factors (model 2). The prevalence of hypertension, current smoking, diabetes mellitus and levels of body mass index and triglycerides were lower, whereas HDL-cholesterol was higher in Malmö than in Cilento. LDL-cholesterol was higher and estimated glomerular filtration rate was lower in Malmö than in Cilento. The odds ratio for cardiovascular disease in Malmö versus Cilento inhabitants was 1.13 (0.69-1.87) (P = 0.62) in model 1, whereas it was significantly elevated in model 2 [2.03 (1.14-3.60) (P = 0.016)]. Moreover, the odds ratio for cancer in Malmö versus Cilento was 2.78 (1.81-4.27) (P < 0.001) in model 1 and 3.11 (1.97-4.92) (P < 0.001) in model 2. The higher odds of CVD and cancer in Malmö versus Cilento, when risk factors were accounted for, suggests the existence of unknown protective factors in Cilento., (© 2021. The Author(s).)

Membrane-bound CD200 is overexpressed in chronic lymphocytic leukemia (CLL), and there is some evidence that its soluble ectodomain (sCD200) could also be involved in the pathophysiology and the disease. However, very little is known about sCD200's prognostic significance. sCD200 was tested at diagnosis in 272 patients with CLL and in 78 age- and sex-matched healthy subjects using a specific human CD200 (OX-2 membrane glycoprotein) ELISA kit. A significantly higher concentration of sCD200 was found in CLL patients compared to controls. In our cohort, sCD200 was significantly higher in patients who were older than 66 years, with Binet stage C, unmutated IgVH and unfavorable (del11q or del17p) FISH. Time-to-first treatment and overall survival were significantly shorter in patients with higher sCD200 concentration, using as a cut-off 1281 pg/mL, the median value for sCD200 concentration in the whole CLL cohort. However, the prognostic impact of sCD200 was not confirmed in multivariate analysis. Baseline sCD200 values appeared to have an impact on the response to chemotherapy or chemo-immunotherapy, but not to targeted agents. Collectively, our data show that sCD200 serum levels correlate with more aggressive clinical and biological features and are able to predict a worse prognosis. This work supports the relevant role of CD200 not only as a diagnostic tool but also as a prognostic indicator and a potential therapeutic target in CLL.

This study provides a first insight on the chemical characterization and quantification of silver nanoparticles (AgNPs) and dissolved Ag in processed canned seafood products, where food-grade edible silver (E174) is not intentionally added nor is the nanoparticle contained in the food contact material. The aim was to evaluate the bioaccumulation potential of AgNPs and to contribute to the assessment of AgNPs and ionic Ag human dietary intake from processed seafood. It is known how seafood, and in particular pelagic fish, is a precious nutritional source of unsaturated fatty acids, protein, and different micronutrients. Nevertheless, it may cause possible health problems due to the intake of toxic compounds coming from environmental pollution. Among emerging contaminants, AgNPs are widely applied in several fields such as biomedicine, pharmaceutical, food industry, health care, drug-gene delivery, environmental study, water treatments, and many others, although its primary application is in accordance with its antimicrobial property. As a consequence, AgNPs are discharged into the aquatic environment, where the colloidal stability of these NPs is altered by chemical and physical environmental parameters. Its toxicity was demonstrated in in-vitro and in-vivo studies, although some findings are controversial because toxicity depends by several factors such as size, concentration, chemical composition, surface charge, Ag + ions released, and hydrophobicity. The new emerging technique called single-particle inductively coupled plasma mass spectrometry (spICP-MS) was applied, which allows the determination of nanoparticle number-based concentration and size distribution, as well as the dissolved element. Our findings highlighted comparable mean sizes across all species analysed, although AgNPs concentrations partly follow a trophic level-dependent trend. The low mean size detected could be of human health concern, since, smaller is the diameter higher is the toxicity. Dietary intake from a meal calculated for adults and children seems to be very low. Although seafood consumption represents only a small part of the human total diet, our findings represent a first important step to understand the AgNPs dietary exposure of the human population. Further studies are needed to characterize and quantify AgNPs in a large number of food items, both processing and not, and where AgNPs are added at the industrial level. They will provide a realistic exposure assessment, useful to understand if AgNPs toxicity levels observed in literature are close to those estimable through food consumption and implement data useful for risk assessors in developing AgNPs provisional tolerable daily intake.

Competing Interests: Competing interests: None declared.

FOXP3-expressing regulatory T-cells (Tregs), which suppress aberrant immune response against self-antigens, also suppress anti-tumor immune response. It has been shown that there is an increased proportion of Tregs in several different human malignancies, although the actual mechanism remains unclear. The research aims to explore the relationship between the number of Tregs and a predict prognosis in particular hematological diseases as monoclonal gammopathies of uncertain significance (MGUS). Tregs were evaluated by means of flow cytometry (CD4+CD25high/+ CD127low/-) in whole peripheral blood of 56 patients with MGUS to predict progression to overt multiple myeloma (MM). In two groups of patients, MGUS versus MGUS evolved to MM, we found a significative difference for the number of white blood cells, but not in terms of clinical and laboratory features evaluated at diagnosis. The study demonstrated the absence of a prognostic relevance of Tregs in MGUS. Nevertheless, their role in these disorders is still to be defined.

The significant increase in the production and variety of nanoparticles (NPs) has led to their release into the environment, especially into the marine environment. Titanium dioxide nanoparticles (TiO 2 -NPs) are used in different industrial sectors, from the food industry to several consumer and household products. Since the aquatic environment is highly sensitive to contamination by TiO 2 -NPs, this work aimed to give a preliminary assessment of the contamination of packaged seafood, where the food additive TiO 2 (E171) is not to be intentionally added. This allowed providing a chemical characterization and quantification of TiO 2 -NPs in processed canned fish products belonging to different trophic positions of the pelagic compartment and in canned clam. The new emerging technique called single-particle inductively coupled plasma mass spectrometry ( sp ICP-MS) was applied, which allows the determination of nanoparticle number-based concentration, as well as the dissolved titanium. This study highlights how processed food, where the pigment E171 was not intentionally added, contains TiO 2 in its nanoparticle form, as well as dissolved titanium. Processed clam represented the seafood with the highest content of TiO 2 -NPs. In pelagic fish species, we found progressively higher levels and smaller sizes of TiO 2 -NPs from smaller to larger fish. Our results highlight the importance of planning the characterization and quantification of TiO 2 -NPs in food both processed and not, as well as where the pigment E171 is intentionally added and not, as it is not the only source of TiO 2 -NPs. This result represents a solid step toward being able to estimate the real level of dietary exposure to TiO 2 -NPs for the general population and the related health risks.

CD200, a transmembrane type Ia glycoprotein belonging to the immunoglobulin protein superfamily, is broadly expressed on a wide variety of cell types, such as B lymphocytes, a subset of T lymphocytes, dendritic cells, endothelial and neuronal cells. It delivers immunosuppressive signals through its receptor CD200R, which is expressed on monocytes/myeloid cells and T lymphocytes. Moreover, interaction of CD200 with CD200R has also been reported to play a role in the regulation of tumor immunity. Overexpression of CD200 has been reported in chronic lymphocytic leukemia (CLL) and hairy cell leukemia but not in mantle cell lymphoma, thus helping to better discriminate between these different B cell malignancies with different prognosis. In this review, we focus on the role of CD200 expression in the differential diagnosis of mature B-cell neoplasms and on the prognostic significance of CD200 expression in CLL, where conflicting results have been published so far. Of interest, increasing evidences indicate that anti-CD200 treatment might be therapeutically beneficial for treating CD200-expressing malignancies, such as CLL., 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 © 2020 D’Arena, De Feo, Pietrantuono, Seneca, Mansueto, Villani, La Rocca, D’Auria, Statuto, Valvano, Arruga, Deaglio, Efremov, Sgambato and Laurenti.)

Competing Interests: Competing interests: None declared.

We present a laboratory-based prognostic calculator (designated CRO score) to risk stratify treatment-free survival in early stage (Rai 0) chronic lymphocytic leukemia (CLL) developed using a training-validation model in a series of 1,879 cases from Italy, the United Kingdom and the United States. By means of regression analysis, we identified five prognostic variables with weighting as follows: deletion of the short arm of chromosome 17 and unmutated immunoglobulin heavy chain gene status, 2 points; deletion of the long arm of chromosome 11, trisomy of chromosome 12, and white blood cell count >32.0x10 3 /microliter, 1 point. Low-, intermediate- and high-risk categories were established by recursive partitioning in a training cohort of 478 cases, and then validated in four independent cohorts of 144 / 395 / 540 / 322 cases, as well as in the composite validation cohort. Concordance indices were 0.75 in the training cohort and ranged from 0.63 to 0.74 in the four validation cohorts (0.69 in the composite validation cohort). These findings advocate potential application of our novel prognostic calculator to better stratify early-stage CLL, and aid case selection in risk-adapted treatment for early disease. Furthermore, they support immunocytogenetic analysis in Rai 0 CLL being performed at the time of diagnosis to aid prognosis and treatment, particularly in today's chemofree era., (Copyright© 2020 Ferrata Storti Foundation.)

Background : Nonagenarians and centenarians (NCs) are an extremely fragile population, particularly in regard to their physical and cognitive function. The aim of this study was to define the neurocognitive profiles among 29 NCs and their 49 younger cohabitants aged 50-75 years from The Cilento Initiative on Aging Outcomes (CIAO) Pilot study in the South of Italy that had provided initial hypotheses regarding positive psychological traits related to exceptional longevity. Methods : During the home visits, lifestyle information with specific questionnaires, functional autonomy and the neuropsychological Mini Mental Scale Examination (MMSE), and the Alzheimer's Disease Assessment Scale-Cognitive (ADAS-Cog) scale were obtained by qualified study personnel. The total blood oxidative capacity was also determined by testing the reactive derivative of oxygen metabolites (d-ROM) and by the Biological Antioxidant Potential (BAP). In all individuals, the APOE genotype determination was also performed. Results : All the subjects in both groups showed high adherence to the Mediterranean Diet. None of the NCs had severe cognitive impairment, and a very low incidence of dementia was found. The data obtained on the Activities ed Instrumental Activities of Daily Living (ADL-IADL) scale showed that the majority of NCs (16/29) were autonomous in daily life activities. The comparative assessment of NCs and cohabitants showed no significant differences in the laboratory assessment of oxidative stress and APOE genotype. Conclusion: In the Cilento Region of Southern Italy, NCs seemed to have good cognitive status when compared to younger cohabitants aging 50-65 years without significant differences in oxidative stress markers or APOE genotype. These results might be related to optimal adherence to the Mediterranean diet, although other lifestyle factors and positive personality traits may also contribute to their healthy aging. Further studies on a larger population should be performed to confirm the results of this pilot study., Competing Interests: The authors declare no conflict of interest.

CD49d is a remarkable prognostic biomarker of chronic lymphocytic leukemia (CLL). The cutoff value for the extensively validated 30% of positive CLL cells is able to separate CLL patients into 2 subgroups with different prognoses, but it does not consider the pattern of CD49d expression. In the present study, we analyzed a cohort of 1630 CLL samples and identified the presence of ∼20% of CLL cases (n = 313) characterized by a bimodal expression of CD49d, that is, concomitant presence of a CD49d+ subpopulation and a CD49d- subpopulation. At variance with the highly stable CD49d expression observed in CLL patients with a homogeneous pattern of CD49d expression, CD49d bimodal CLL showed a higher level of variability in sequential samples, and an increase in the CD49d+ subpopulation over time after therapy. The CD49d+ subpopulation from CD49d bimodal CLL displayed higher levels of proliferation compared with the CD49d- cells; and was more highly represented in the bone marrow compared with peripheral blood (PB), and in PB CLL subsets expressing the CXCR4dim/CD5bright phenotype, known to be enriched in proliferative cells. From a clinical standpoint, CLL patients with CD49d bimodal expression, regardless of whether the CD49d+ subpopulation exceeded the 30% cutoff or not, experienced clinical behavior similar to CD49d+ CLL, both in chemoimmunotherapy (n = 1522) and in ibrutinib (n = 158) settings. Altogether, these results suggest that CD49d can drive disease progression in CLL, and that the pattern of CD49d expression should also be considered to improve the prognostic impact of this biomarker in CLL., (© 2020 by The American Society of Hematology.)

NOTCH1 mutations in chronic lymphocytic leukemia (CLL) lead to accumulation of NOTCH1 intracellular domain (NICD) and prolong signaling. These mutations associate with a more aggressive disease compared to wild-type (WT) CLL. In this work we demonstrate a bidirectional functional relationship between NOTCH1 and the B cell receptor (BCR) pathways. By using highly homogeneous cohorts of primary CLL cells, activation of NOTCH1 is shown to increase expression of surface IgM, as well as LYN, BTK, and BLNK, ultimately enhancing BCR signaling responses, including global mRNA translation. Upon BCR cross-linking, NOTCH1 itself is actively translated and increased on cell surface. Furthermore, BCR ligation induces calcium mobilization that can facilitate ligand-independent NOTCH1 activation. These data suggest that the two pathways are functionally linked, providing a rationale for dual inhibition strategies. Consistently, addition of the γ-secretase inhibitor DAPT to ibrutinib significantly potentiates its effects, both in vitro and in a short-term patient-derived xenograft model. While this observation may find limited applications in the CLL field, it is more relevant for Richter's Syndrome (RS) management, where very few successful therapeutic options exist. Treatment of RS-patient-derived xenografts (RS-PDX) with the combination of ibrutinib and DAPT decreases disease burden and increases overall survival.

Morphological and immunohistochemical (IHC) analysis of bone marrow biopsies (BMB) is routinely performed during staging of patients with non-Hodgkin's lymphoma (NHL). Aiming to evaluate the possible diagnostic value of flow cytometry (FC) on bone marrow aspirates (BMA), as compared with BMB, we retrospectively reviewed BMA specimen of 354 NHL. In 305 cases (86.1 %), there was a concordance between the two investigations. A discordance was detected in 49 cases (14 %): in 33 of these (9.3 % of total population), FC analysis of BMA was positive, whereas BMB, supported by IHC, was negative; in 16 (4.5 % of total population), FC did not detected lymphoid infiltration, while BMB was positive. Although the clinical implications of such an observation remain unclear, we think our results may be useful in the context of current staging procedures, also opening a possible future perspective in the setting of minimal measurable disease in these patients., (Copyright © 2019. Published by Elsevier Ltd.)

Chronic Lymphocytic Leukemia (CLL) patients with +12 have been reported to have specific clinical and biologic features. We performed an analysis of the association between demographic; clinical; laboratory; biologic features and outcome in CLL patients with +12 to identify parameters predictive of disease progression; time to treatment; and survival. The study included 487 treatment-naive CLL patients with +12 from 15 academic centers; diagnosed between January 2000 and July 2016; and 816 treatment-naïve patients with absence of Fluorescence In Situ Hybridization (FISH) abnormalities. A cohort of 250 patients with +12 CLL followed at a single US institution was used for external validation. In patients with +12; parameters associated with worse prognosis in the multivariate model were high Lactate DeHydrogenase (LDH) and β-2-microglobulin and unmutated immunoglobulin heavy-chain variable region gene (IGHV). CLL patients with +12 and high LDH levels showed a shorter Progression-Free-Survival (PFS) (30 months vs. 65 months; p < 0.001), Treatment-Free-Survival (TFS) (33 months vs. 69 months; p < 0.001), Overall Survival (OS) (131 months vs. 181 months; p < 0.001) and greater CLL-related mortality (29% vs. 11% at 10 years; p < 0.001) when compared with +12 CLL patients with normal LDH levels. The same differences were observed in the validation cohort. These data suggest that serum LDH levels can predict PFS; TFS; OS and CLL-specific survival in CLL patients with +12.

Breast cancer is the most common malignancy among women worldwide. Various studies indicate that prolonged exposure to elevated levels of estrogens is associated with development of breast cancer. Both estrogen receptor-dependent and independent mechanisms can contribute to the carcinogenic effects of estrogens. Among them, the oxidative metabolism of estrogens plays a key role in the initiation of estradiol-induced breast cancer by generation of reactive estrogen quinones as well as the associated formation of oxygen free radicals. These genotoxic metabolites can react with DNA to form unstable DNA adducts which generate mutations leading to the initiation of breast cancer. A variety of endogenous and exogenous factors can alter estrogen homeostasis and generate genotoxic metabolites. The use of specific phytochemicals and dietary supplements can inhibit the risk of breast cancer not only by the modulation of several estrogen-activating enzymes (CYP19, CYP1B1) but also through the induction of various cytoprotective enzymes (eg, SOD3, NQO1, glutathione S-transferases, OGG-1, catechol- O -methyltransferases, CYP1B1A, etc.) that reestablish the homeostatic balance of estrogen metabolism via nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent and independent mechanisms., Competing Interests: The authors report no conflicts of interest in this work.

There is a large body of evidence showing a strong correlation between carcinogenesis of several types of human tumors, including chronic lymphocytic leukemia (CLL), and oxidative stress (OS). The mechanisms by which OS may promote cancer pathogenesis have not been completely deciphered yet and, in CLL, as in other neoplasms, whether OS is a primary cause or simply a downstream effect of the disease is still an open question. It has been demonstrated that, in CLL, OS concomitantly results from increased reactive oxygen species (ROS) production, mainly ascribable to CLL cells mitochondrial activity, and impaired antioxidant defenses. Interestingly, OS evaluation in CLL patients, at diagnosis, seems to have a prognostic significance, thus getting new insights in the biological comprehension of the disease with potential therapeutic implications.

Monoclonal B-cells lymphocytosis (MBL) is a benign condition that may precede chronic lymphocytic leukemia (CLL), not rarely present in peripheral blood of healthy elderly people, among which there is also a male prevalence. Though CLL has been associated with various types of solid tumors, including prostate cancer (PC), no data exist about the relationship between PC and MBL. We studied the frequency of CLL-like MBL clones in a group of 48 patients affected by PC and followed them during and after whole-pelvis radiotherapy (WPRT) treatment. We found four MBL clones (8.3%), two of which (4.2%) had a B-cell clonal count >1000 cells/µL ('clinical MBL'). A single case (1.8%) of 'low-count' MBL occurred in a control group of 54 healthy males. Notably, normal B-lymphocytes were consistently affected by WPRT, while MBL clones were less radiosensitive. Our results suggest a possible association between 'clinical' MBL and PC and show a different impact of the radiation on monoclonal respect to normal B-cells, which could also imply a greater risk of clonal transformation., Competing Interests: Competing interests: None declared., (© American Federation for Medical Research 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Competing Interests: The authors declare that they have no conflict of interest.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Informed consent was obtained from the patient.

Competing Interests: Competing interests: None declared.

Therapeutic re-challenge is currently a debated issue in the field of multiple myeloma (MM), given the recent availability of several new drugs and combinations. However, very few specific evidences are available about bortezomib re-use at first relapse. This multicenter, observational, retrospective study enrolled 134 MM patients with significant response after bortezomib-based frontline regimens and who had received a first salvage treatment containing bortezomib at relapse. The overall response rate was 71%, including 40% partial responses, 24% very good partial responses, and 7% complete responses. Re-treatment was well-tolerated, with no significant new or unexpected toxicities observed. The median duration of second progression-free survival (PFS) was 15 months, while median PFS2 was 55 months. With a median follow-up of 56 months, overall survival was 94 months for the entire series, without significant differences between patients undergoing or not undergoing transplant procedures. This real-life survey indicates that re-treatment including bortezomib as a first salvage therapy could be still considered in MM patients achieving durable response after initial exposure to bortezomib.

Objective: The aim of this review is to analyze preclinical and clinical studies investigating the anxiety effects of Citrus aurantium or Citrus sinensis essential oils (EOs)., Design: The bibliographic research was made on the major scientific databases. Analysis included only articles written in English and published on peer-reviewed scientific journals describing preclinical experiments and clinical trials carried out to investigate the antianxiety effects of Citrus aurantium or Citrus sinensis EOs on anxiety disorders. Clinical studies reporting the antianxiety effects of products containing Citrus aurantium or Citrus sinensis EOs in combination with other active substances, including medicinal plants, were excluded. Nine clinical studies fulfilled the criteria adopted for analysis., Results: Data show that Citrus aurantium or Citrus sinensis EOs produce anxiolytic effects both in preclinical experiments and in different clinical conditions. Citrus aurantium EO aromatherapy reduced anxiety level in the great part of stress conditions studied (subjects affected by chronic myeloid leukemia and preoperative patients) except for a sample of patients subjected to colonoscopy. Exposition to Citrus sinensis EO in clinical studies shows to be positive in reducing anxiety level in patients waiting for dental treatment as well as in healthy volunteers submitted to an anxiogenic situation., Conclusions: Overview of clinical trials conducted with Citrus aurantium or Citrus sinensis on people with anxiety showed that inhalation or oral administration of Citrus aurantium and inhalation of Citrus sinensis can exert beneficial effects on anxiety; however, because of incomplete accuracy in the reporting of methodology, further more complete clinical studies are warranted.

Competing Interests: Compliance with Ethical StandardsAll Authors have no conflict of interest to disclose.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Informed consent was obtained from the patient.

Competing Interests: Competing interests: None declared.

In mammals, a master clock is located within the suprachiasmatic nucleus (SCN) of the hypothalamus, a region that receives input from the retina that is transmitted by the retinohypothalamic tract. The SCN controls the nocturnal synthesis of melatonin by the pineal gland that can influence the activity of the clock's genes and be involved in the inhibition of cancer development. On the other hand, in the literature, some papers highlight that artificial light exposure at night (LAN)-induced circadian disruptions promote cancer. In the present review, we summarize the potential mechanisms by which LAN-evoked disruption of the nocturnal increase in melatonin synthesis counteracts its preventive action on human cancer development and progression. In detail, we discuss: (i) the Warburg effect related to tumor metabolism modification; (ii) genomic instability associated with L1 activity; and (iii) regulation of immunity, including regulatory T cell (Treg) regulation and activity. A better understanding of these processes could significantly contribute to new treatment and prevention strategies against hormone-related cancer types.