ALBERT

Description: The transport sector is commonly subordinate to several issues, such as traffic congestion and accidents. Despite this, in recent years, it is also evolving with regard to cooperation between vehicles. The fundamental objective of this trend is to increase road safety, attempting to anticipate the circumstances of potential danger. Vehicle-to-Vehicle (V2V), Vehicle-to-Infrastructure (V2I) and Vehicle-to-Everything (V2X) technologies strive to give communication models that can be employed by vehicles in different application contexts. The resulting infrastructure is an ad-hoc mesh network whose nodes are not only vehicles but also all mobile devices equipped with wireless modules. The interaction between the multiple connected entities consists of information exchange through the adoption of suitable communication protocols. The main aim of the review carried out in this paper is to examine and assess the most relevant systems, applications, and communication protocols that will distinguish the future road infrastructures used by vehicles. The results of the investigation reveal the real benefits that technological cooperation can involve in road safety.

Description: Introduction. Elderly patients with chronic lymphocytic leukemia (CLL) and younger patients with comorbidities are often treated with chlorambucil (Chl), despite the relatively low response rates. The addition of anti-CD20 monoclonal antibodies to Chl substantially increases the response rates, without negatively affecting tolerability. Overall response rates (ORR) between 66% to 84% have been reported with these combinations, with complete responses (CR)ranging from 8% to 26%. Methods. We conducted a retrospective analysis on the use of the Chl-rituximab (R) combination as frontline treatment for elderly (≥65 years) and/or unfit (CIRS 〉6) CLL patients treated at 15 different Italian hematologic centers. The main aim of the study was to further establish the safety and efficacy of the Chl-R protocol and investigate whether certain CLL patients for whom this protocol could be particularly effective could be identified. To this end, we performed a subgroup analysis stratifying patients according to FISH and IGHV results: high risk group (HR) included patients with 17p deletion, intermediate risk group (IR) patients with 11q deletion and/or unmutated IGHV, low risk group (LR) patients without 11q or 17p deletion and/or unmutated IGHV. Results. One hundred and two patients who underwent treatment between 2009 and 2011 were enrolled in the study. Patients' clinical and biologic characteristics are summarized in Table 1. Three patients discontinued treatment earlier than planned: 1 for an episode of autoimmune hemolytic anemia (AIHA) that developed after the 2nd cycle of Chl and before starting R treatment and 2 patients for disease progression after the 3rd and 5th cycle of Chl-R, respectively. The median number of Chl and R cycles administered in the 102 patients was 8 (range 2-12) and 6 (range 1-9), respectively. The planned treatment schedule was different among centers: the two main schedules used were Chl administered at 1 mg/kg for each cycle every 28 days, given at a fixed daily dose of 10 mg starting from day 1 and repeated for 8 cycles, and Chl administered at 8 mg/m2/day for seven days of each of eight 28-day-cycles. R was added to Chl from the 3rd cycle onwards and was administered on day 1 of each cycle at a dose of 375 mg/m2 during the first administration and 500 mg/m2 for the subsequent 5 cycles. On an intention to treat basis, the ORR was 87.1%. Thirty-two patients (31.7%) obtained a CR and 56 patients (55.4%) obtained a partial response (PR). Nostatistically significant differences were noted in terms of ORR for age above or below 70 years, fitness status, ECOG, bulky disease, cytogenetic risk abnormalities, IGHV mutational status, ZAP-70 or CD38 expression.Median progression-free survival (PFS) and time to retreatment (TTR) were reached at 43.7 and 72.3 months, respectively. Median overall survival (OS) was not reached; 86.1% and 81.2% of patients were alive at 48 and 60 months, respectively. The most frequent serious adverse event was grade 3-4 neutropenia, occurring in 13.7% of patients. Grade 3-4 extra-hematologic side effects were uncommon (9.8%). Subgroup analysis of the LR and IR patients (no HR patients were enrolled) showed that LR patients had a significantly better PFS than IR patients (65.8 months vs 35.2 months, p=0.001; Fig. 1),with 54.9% of patients remaining free from progression 60 months after treatment. Conclusions. Treatment of elderly and/or unfit CLL patients with the Chl-R regimen is associated with low toxicity, a high ORR and durable PFS. Particularly good results are achieved in CLL patients with a mutated IGHV profile and not carrying both 17p and 11q deletion, suggesting that in this low-risk subset of unfit patients Chl-R could represent the optimal therapeutic option, in consideration of safety, efficacy and costs. Disclosures D'Arena: Janssen-Cilag: Honoraria. Coscia:Gilead: Honoraria; ROCHE: Honoraria, Other: Advisory board; Janssen: Honoraria; Mundipharma: Honoraria; Karyopharm: Research Funding. Molica:Jansen: Membership on an entity's Board of Directors or advisory committees; Roche Italy: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Speakers Bureau. Efremov:Gilead: Honoraria.

Description: Introduction. Patients with CLL and FISH positive for trisomy 12 (+12) have unique clinical and biological features. We, therefore, performed an analysis of the association between demographic, clinical, laboratoristic and biological features and outcomes in treatment-naive patients with +12 CLL. Methods. This study included 312 treatment-naive patients with +12 CLL from 9 centers. These patients, diagnosed between January 2000 and July 2016, were compared to a control group of 580 treatment-naive patients with FISH negative CLL, matched by age and gender and followed in the same centers. An additional cohort of 250 patients with +12 CLL followed at a single US institution was used as external validation. Results. Patients' baseline characteristics are shown in Table 1. As compared to patients with negative FISH, patients with +12 had a significant higher prevalence of elevated LDH (p

Description: Background. TP53 mutations (TP53mut) along with 17p13 deletion (del17p) are strong predictors of poor survival and refractoriness to chemo-immunotherapies (CIT) in chronic lymphocytic leukemia (CLL), and their analyses should be always performed before treatment. Studies based upon ultra-deep-NGS have shown that TP53mut can be present at very low level in CLL cell populations, although their detrimental clinical impact in this setting is still matter of debate (Rossi, Blood 2014), and ERIC recomendations discourage to report TP53 mutations if subclonal Malcikova, Leukemia 2018). Aim. To investigated the presence and clinical relevance of clonal/subclonal TP53 aberrations in a large CLL cohort. Methods. The study includes 1,058 out of 1,613 CLL patients (509 treated with standard CIT) diagnosed between 1991 and 2018, and consecutively referred to a single institution for del17p analyses by FISH (167-kb 17p13 orange probe, MetaSystems), and TP53mut by ultra-deep NGS (MiSeq Illumina; median coverage 〉2,000X with an amplicon-based strategy covering exons 2-11 using 40ng DNA/test) in CD19-purified (〉85% pure) CLL samples, collected before treatment (as per ERIC recommendations). For TP53mut analyses, FASTQ files were aligned to the Hg19 reference with Burrows-Wheeler Aligner-MEM algorithm, and allele variants called by FreeBayes (Garrison & Marth, arXiv 2102) with non-stringent parameters. To calculate random/systematic errors we generated a specific database with all the variant allele frequencies (VAF) observed in a subset of TP53 wild type (wt) subjects (n=362). TP53mut were accepted if: i) validated by Fisher exact test after Bonferroni correction (p 12.5%) and 67 subclonal (all clones 0.4% for the clinical impact of TP53mut (Fig.D), and the c-index of combined clonal/subclonal TP53mut (0.645) was significantly higher than the c-index of clonal TP53mut alone (0.602; P10% of nuclei had significantly shorter OS than cases with del17p in 0.5% as the best cutoff. In keeping with this cutoff, TP53 mutated patients experienced a significantly shorter OS than wt patients. Again cases with clonal and subclonal mutation experienced the same OS (Fig.I). Importantly, the cutoff found in the training cohort was able to reproduce the very same results also in the validation cohort both in term of mutation per se and in terms of clonal and subclonal TP53 mutations (Fig.J). Conclusion. i) By applying ERIC recommendations and a rigorous pipeline of analysis, TP53mut impacted on OS also with VAF 10% of nuclei. These cutoffs may be employed for the clinical management of CLL patients. Figure Disclosures Di Raimondo: Takeda: Consultancy; Amgen: Consultancy, Honoraria, Research Funding. Rossi:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board.

Description: Our interest in chronic lymphocytic leukemia (CLL) derives primarily from the exploitation of human diseases as strategic models for defining the in vivo biological roles of CD38. Using this model, we showed that CD38 triggers robust proliferation/survival signals modulated through the interactions with the CD31 ligand expressed by nurselike cells and by the stromal/endothelial components. By analyzing a cohort of 56 patients with clinically and molecularly characterized CLL, we show that (1) patients with CD38+/ZAP-70+ are characterized by enhanced migration toward Stromal derived factor-1α (SDF-1α)/CXCL12; (2) CD38 ligation leads to tyrosine phosphorylation of ZAP-70, showing that these markers are functionally linked; (3) ZAP-70 represents a limiting factor for the CD38 pathway in the CLL context, as shown by studying CD38-mediated signal transduction in 26 molecularly characterized patients; and (4) the CLL subgroup of patients defined on the basis of migratory potential is marked by a specific genetic signature, with a significant number of differentially expressed genes being involved in cell-cell interactions and movement. Altogether, the results of this work provide biological evidence for why the combined analysis of CD38 and ZAP-70 expression as determined in several clinical trials results in more dependable identification of patients with CLL who have aggressive disease.

Description: Human SLAMF1 (signaling-lymphocytic-activation-molecule-family1, CD150) is a self-ligand adhesion/co-stimulatory molecule wich belongs to a family of 9 receptors. SLAMF1 is also a microbial sensor, as it regulates Gram- bacterial phagosome functions through an ubiquitous cellular autophagic machinery and serves as a receptor for Measles virus. In this work, we investigated expression and function of SLAMF1 in chronic lymphocytic leukemia (CLL) cells. Results indicate that expression of SLAMF1 is lost in a subset of patients with chronic lymphocytic leukemia characterized by an aggressive form of the disease, with shorter time to first treatment (median 2.2 years in SLAMF1- vs 7.6 in SLAMF1+ patients, P=.001) and overall survival (77.5% survival rate at 10 years in SLAMF1- vs 94.7% years in SLAMF1+ patients, P=.036). Consistently, SLAMF1low CLL patients are characterized by clinical or molecular markers of a more aggressive disease. Stable silencing of SLAMF1 in the CLL-like Mec-1 cell line (constitutively SLAMF1+) modulated pathways related to cell migration, cytoskeletal organization and intracellular vesicle formation/recirculation. Decreased expression of CXCR3 and an increased expression of CXCR4, CD38 and CD44 were maintained at the molecular level, likely explaining why SLAMF1- cells show enhanced chemotactic responses to CXCL12. This phenotype was confirmed in primary cells, by comparing a cohorts of SLAMF1high to one of SLAMF1low patients. Gene expression profiling also indicates profound modulation of pathways connected with vesicle formation and recirculation. Consistently, cross-linking of SLAMF1 with an agonisic mAb in primary cells and in the Mec-1 cell line enhanced the generation of autophagic vesicles and their fusion with the lysosomes. Ligation of SLAMF1 with this agonistic monoclonal antibody promoted the autophagic flux, by increasing accumulation of reactive oxygen species (ROS) and inducing phosphorylation of p38, JNK1/2 and bcl-2. The direct consequence was the formation of the autophagy macro-complex containing SLAMF1, the scaffold protein beclin1 and the enzyme Vps34. In agreement with the observation that many drugs used in CLL have autophagy-mediated effects, including fludarabine and the BH3 mimetic ABT-737, SLAMF1-silenced Mec-1 cells or SLAMF1low primary CLL cells were resistant to treatment with both agents. These results indicate that SLAMF1 plays as a critical role in CLL homeostasis. Loss of SLAMF1 expression modulates genetic pathways that regulate chemotaxis and autophagy and that potentially affect drug responses, thus providing a likely explanation for the unfavorable clinical outcome experienced by this patient subset. Restoring SLAMF1 expression in CLL cells would therefore be of therapeutic value for patients with aggressive CLL. Disclosures Gaidano: Morphosys, Roche, Novartis, GlaxoSmith Kline, Amgen, Janssen, Karyopharm: Honoraria, Other: Advisory boards; Celgene: Research Funding.

Description: Background. Trisomy 12 (tris12) is a recurrent cytogenetic abnormality in chronic lymphocytic leukemia (CLL), occurring in approximately 15-20% of cases, often as the unique cytogenetic alteration, that is usually considered a clonal driver lesion occurring early in CLL evolution. In the Dohner hierarchical categorization, tris12 CLL are identified as having an intermediate prognostic risk, although recent reports suggest a more complex and heterogeneous clinical behavior. Compared to CLL lacking this cytogenetic abnormality, tris12 CLL show more atypical morphology and immunophenotype, more frequent expression of the negative prognostic markers CD49d and CD38, and presence of NOTCH1 mutations and an unmutated (UM) IGHV gene status. The increased fraction of tris12 CLL carrying adverse prognostic features is in contrast to the intermediate clinical behavior associated with most tris12 CLL cases. Aim. To perform a comprehensive evaluation of the clinical impact of the major genetic, immunogenetic and immunophenotypic prognostic markers in tris12 CLL. Methods. The study was based on a multicenter series of tris12 CLL defined according to Dohner (n=283, including 73 cases also bearing del13q), and a comparison group (control) of 553 cases with either del13q (n=308) or without any cytogenetic abnormality (no del17p, del11q, tris12, del13q, n=245). Median follow-up of patients in the tris12 and control groups were 4 years (range 0-22) and 7 years (range 0-28), with 54% and 57% treated patients, and 18% and 15% deaths, respectively. Patient characterization included modified Rai stage, CD49d (CD49dhigh, ≥30% positive cells by flow cytometry), CD38 (CD38high, ≥30% positive cells by flow cytometry) and ZAP-70 (ZAP-70high, ≥20% positive cells by flow cytometry) expression, and IGHV mutational status (mutated, M, or UM according to the 2% cutoff). TP53, BIRC3, NOTCH1 andSF3B1 mutations were screened either at diagnosis or before therapy by NGS with at least 1000X coverage and 1% of sensitivity. Groups were compared by chi-square test; overall survival (OS) was computed from diagnosis to death or censored at last observation, and analyzed by Cox regression analysis. Results. Comparing the tris12 and the control groups, median age was 64 years (range 30-92) vs 66 years (range 33-92), male gender 55% vs 56% (p=0.86), the modified Rai stage was early in 52% vs 54%, intermediate in 41% vs 42% and advanced in 7% vs 4% (p=0.20). As previously reported, tris12 CLL were characterized by a higher prevalence of cases expressing CD49d (85% vs 31%) and CD38 (62% vs 17%; all p

Description: Background: Thecross talk between neoplastic cells and microenvironment is mediated by direct cell-to-cell contacts, secretion of soluble factors and release of extracellular vesicles (EVs). EVs deriving from tumor cells in chronic lymphocytic leukemia (CLL) may affect the surrounding microenvironment, playing a key role on survival of neoplastic clone. Of note, EVs are increased in CLL patients compared to normal subjects. In this study, we performed a comprehensive characterization of serum EVs from previously untreated CLL patients, investigating, in particular, phenotype and absolute number, in order to test their possible prognostic significance. Patients and methods: Serum samples of 131 newly diagnosed CLL and from 28 healthy subjects were analyzed. One milliliter of serum was processed with serial ultracentrifugations. Each sample of EV-enriched pellet, from patients and controls, was freshly analyzed by FACS Calibur (Becton Dickinson BD) cytometer using Cell Quest software (BD). The system was calibrated using standard microbeads with a diameter of 0.3-0.9-3 μm to define the size limit for microvesicles (MV), a subtype of EVs. To determine the number of MV/μL serum, TruCOUNT beads (BD) were added immediately prior to analysis by flow cytometry. MV morphology was characterized by transmission electron microscope (TEM). MV were then labeled with fluorochrome-conjugated monoclonal antibodies (anti-CD19, CD3, CD94, CD20, CD2, CD56, CD52, CD37) and their specific isotypic controls. Finally, MV were correlated with the main clinical and biological disease's characteristics, including clinical outcome. Results: Flow cytometric analysis of MVs showed a size within 1mm, based on forward and side scatter evaluation and the use of standard beads. The analysis was carried out on MV population isolated by the gating strategy. MV were also visualized by TEM, showing a spheroid morphology. We found a significantly higher mean number of MV in CLL patients with respect to healthy subjects (p

Description: Chronic lymphocytic leukemia (CLL), is characterized by the expansion of mature B lymphocytes present in blood, bone marrow and lymphoid organs. Clinical behavior is highly heterogeneous, thus requiring timely identification of high-risk patients. NOTCH1 encodes a trans-membrane receptor acting as a ligand-activated transcription factor. NOTCH signaling initiates when the ligand, from either the Jagged or Delta families, binds to the receptor and induces successive proteolytic cleavages, resulting in the release and nuclear translocation of the NOTCH intra-cellular domain (NICD). Signaling is terminated by phosphorylation of the PEST domain of NOTCH1, triggering its ubiquitination and proteasomal degradation. Whole exome sequencing approaches have revealed NOTCH1 mutations in 5-10% newly diagnosed CLL cases, with their prevalence increasing to 15-20% in progressive or relapsed patients. The most frequent mutation is 7544-7545delCT frameshift deletion in exon 34, resulting in disruption of the C-terminal PEST domain. Truncation of the PEST domain is predicted to result in NOTCH1 impaired degradation, stabilization of the active NOTCH1, and deregulated signaling. The present study was undertaken with the aim to compare NOTCH1 expression and functional role in CLL patients harboring wild type (WT) or mutated (M) NOTCH1 gene. NOCTH1 mRNA and surface protein were expressed at comparable high levels in peripheral blood (PB) CLL cells obtained from NOTCH1 M and WT patients, consistent with a more general requirement of NOTCH1 signaling in this leukemia. However, at a variance of NOTCH1 WT cases, NOCTH1 M patients displayed remarkable accumulation of both the intermediate molecular species of the activated NOCTH1 receptor, as well as of the active NICD. Consistently, by gene expression profiling NOCTH1 M patients displayed significantly higher levels of HES1 and DTX1, the main NOTCH1 target genes. Overall, these data suggest a more active signaling pathway in NOTCH1 M CLL than in NOTCH1 WT cases. Expression of NOTCH1 and of its target gene (DTX1) varied across disease compartments, being higher in CLL cells obtained from the lymph nodes (LN), as compared to paired samples derived from the PB or the bone marrow (BM). By immunohistochemical analyses of primary LN tissue samples, NOTCH1 M CLL showed an intense nuclear staining as opposed to the more cytoplasmic distribution observed in NOTCH1 WT samples. These data suggest a more active NOTCH1 signaling in CLL residing in the LN microenvironment and confirm the functional effect of NOTCH1 mutations in vivo. When PB CLL cells were cultured in vitro in the absence of any supporting layer or stimulation, they showed a rapid down regulation of the NOTCH pathway, with complete loss of NICD after 24 hours paralleled by a sharp decrease in HES1 and DTX1 transcription. Consistently, levels of presenilin-1 (PSEN1), the catalytic subunit of the g-secretase complex, were also down-regulated offering a partial mechanistic explanation for the NICD loss. NOTCH1 mRNA levels remained unchanged, with accumulation of the receptor at the plasma membrane. These effects were independent of NOTCH1 mutation status and suggested the dependence of NOTCH1 signaling activation upon in vivo microenvironmental interaction, even in NOTCH1 M CLL. Within primary LN biopsies from CLL patients, the NOTCH1 ligand, was highly expressed on CD68+ elements of myeloid origin. This observation prompted the in vitro recreation of a lymphoid niche by co-culturing Jagged1+ nurse-like cells (NLC) with autologous CLL cells. Under these conditions, NOTCH1 activity in CLL cells was sustained over time, as shown by Q-PCR analyses of DTX1 and PSEN1. Moreover, NLCs protect NOTCH1 M CLL cells from fludarabine-induced apoptosis. This microenvironment-induced chemoresistance was prevented by pre-treatment of NOTCH1 M CLL cells with specific g-secretase inhibitors, to block NOTCH1 activation. Taken together, these results show that the 7544-7545delCT mutation in the PEST domain of NOTCH1 has a stabilizing effect on NOTCH1 signaling pathway. They also show that micro-environmental interactions are critical in activating NOTCH1 pathway both in the M and WT patients. Lastly, these results show that NOTCH1 signals micht create local conditions that favour drug resistance, thus making NOTCH1 a potential molecular target in CLL. Disclosures: No relevant conflicts of interest to declare.

Description: Background. The pivotal role of the Immunoglobulin (Ig) receptor and antigenic stimulation have been proven to be landmarks for the understanding of the ontogeny and evolution of chronic lymphocytic leukemia (CLL). In addition, the mutational status of the Immunoglobulin Heavy-Chain Variable region gene (IGHV) was confirmed to be a reliable prognostic factor, supporting an antigen-driven model of CLL development. To clarify aspects regarding an antigenic involvement in CLL evolution, studies focusing on intraclonal diversification (ID) of Ig genes have provided relevant information, although mainly conducted in a pre-Next Generation Sequencing (NGS) era. Aim. To apply a NGS approach to investigate ID in CLL. Methods. IGHV genes from 530 CLL patients with Royal Masden Hospital score 4-5 (Fig. 1A) was sequenced using NGS (Lymphotrack). The bio-informatic pipeline was based on the pRESTO/ChangeO packages. Specific pathological clones were selected based on the presence of same IGHV, junction genes and with similar HCDR3 sequence according to Hamming's distance. Through the R-Alakazam package, we generated rarefaction curves to evaluate the clonal diversity inside the pathological clone (Fig. 1B). Focusing on the Simpson index (represented by the Hill number of order q=2), which gives more weight to larger clones minimizing the smaller ones (Fig. 1B), we selected a Diversity Score (DS) of 4 for the definition of cases without ID (clonal; DS