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Cigarette smoking is associated with impairment of repair mechanisms necessary for vascular endothelium homeostasis. Reducing the exposure to smoke toxicants may result in the mitigation of the harmful effect on the endothelium and cardiovascular disease development. Previous investigations evaluated in vitro the effect of electronic cigarette (EC) compared with cigarette smoke demonstrating a significant reduction in human umbilical vein endothelial cells (HUVECs) migration inhibition following EC aerosol exposure. In the present study, we replicated one of these studies, evaluating the effects of cigarette smoke on endothelial cell migration compared with aerosol from EC and heated tobacco products (HTPs). We performed an in vitro scratch wound assay on endothelial cells with a multi-center approach (ring-study) to verify the robustness and reliability of the results obtained in the replicated study, also testing the effect of aerosol from two HTPs on endothelial cells. Consistently with the original study, we observed a substantial reduction of the effects of aerosol from EC and HTPs on endothelial cell migration compared with cigarette smoke. While cigarette smoke reduced endothelial wound healing ability already at low concentrations (12.5%) and in a concentration-dependent manner, EC and HTPs aerosol showed no effect on endothelial cells until 80%–100% concentrations. In conclusion, our study further confirms the importance of EC and tobacco heated products as a possible harm reduction strategy for cardiovascular diseases development in smokers.

Inter-laboratory comparison is widely used to ensure quality control among laboratories. In in vitro toxicology studies for tobacco harm reduction (THR), exposure system performance and laboratory proficiency along with product smoke and aerosol stream are tested for variability to assess accuracy. Here we aim to test a novel inter-laboratory setup created in a new collaborative research group using identical and small footprint systems- in order to minimize variability factors and increase reproducibility.Seven independent laboratories from different geographical areas tested the aerosol and smoke stream and exposure system performance (LM1 and LM4E) using Cambridge Filter Pad (CFP) trapping techniques. We tested 1R6F reference cigarettes, two electronic cigarettes (Vype e-Pen and Vype e-Stick Maxx), and two tobacco heating products (IQOS and Glo™) under the appropriate ISO and/or HCI regimes. Nicotine quantification was performed by GC-FID at the laboratory of the leading center. The performance of participant laboratories was assessed by z-score values obtained from results either in relation to the mean and standard deviation of total participants or in relation to the reference leading center. Z-Scores were satisfactory when |z| ≤ 2, questionable when 2 < |z| < 3 and, unsatisfactory when |z| ≥ 3. In the first evaluation, for all the tested devices, Z- scores values generated by dosimetry data ranged from -2 to +2. However, high intra-laboratory variability (RSD> 10%) was observed for almost all laboratories. In the second, data showed borderline and unsatisfactory exposure performances versus LAB-A. Particularly, Z-scores ≥ 3 were observed once for LAB-B (e-Stick exposure) and LAB-G (e-Pen exposure), twice for LAB-C (1R6F-ISO and e-Stick exposures) and LAB-E (e-Pen and e-Stick exposures), and three times for LAB-F (1R6F-HCI, e-Pen, and Glo exposures).This study demonstrates that nicotine dosimetry is a fundamental method for quality assurance of smoke/vapor run exposure in the early stage of an interlaboratory study, allowing the identification and possibly the resolution of gaps. Extended practice sessions on exposure runs and several rounds of nicotine dosimetry testing should be planned to keep in check overall equipment and operator performance.

Cigarette smoking is associated with impairment of repair mechanisms necessary for vascular endothelium homeostasis. Reducing the exposure to smoke toxicants may result in the mitigation of the harmful effect on the endothelium and cardiovascular disease development. Previous investigations performed by the tobacco industries evaluated in vitro the effect of electronic cigarette (e-cig) compared to cigarette smoke demonstrating a significant reduction in endothelial cell migration inhibition following e-cig aerosol exposure. In the present study, we replicated one of these studies, evaluating the effects of cigarette smoke on endothelial cell migration compared to e-cig and heated tobacco products. We used a multi-center approach (ring-study) to verify the robustness and reliability of the results obtained in the replicated study. Consistently with the original study, we observed a substantial reduction of the effects of e-cig and tobacco heated products on endothelial cell migration compared to cigarette smoke. In conclusion, our study further confirms the importance of e-cig and tobacco heated products as a possible harm reduction strategy for cardiovascular diseases development in smokers.

Table S1 CDK inhibitors utilized in clinical trials for the treatment of various types of malignancies. (DOCX 21 kb)

Cancer patients do not always respond in the same way to the same drug treatment: the administration of the same dose of an antiblastic drug in a population of patients induces the manifestation of a wide range of toxicity, which sometimes can even be deadly. The intersubject variation, that is observed in efficacy and toxicity of anticancer drugs, can be determined by complex interactions among components of the physiological, environmental and genetic factors. The activities carried out in the framework of the PhD project have focused the attention on the influence of genetic factors on the outcome of drug treatment, identifying possible prognostic and predictive genetic biomarkers. To evaluate the correlation between genotype and phenotype of the patient, we considered a pharmacogenetics study which defined the relationship between a specific polymorphism (UGT1A1 * 28) and the effects of the drug. A phase Ib clinical trial was conducted at the CRO, Aviano (PN). The purpose of this study was to modulate the dosage of CPT-11 in the presence of the inhibitor angiogenetic BV for each individual patient, based on genotype of UGT1A1, in order to obtain an improvement of the therapeutic index . To do this, it was considered necessary to develop a specific method of pharmacokinetic analysis and we proceeded as follows : * Development of a quantitative method for the analysis of the drug of interest * Validation of the method in accordance with FDA guidelines * Measurement of plasma concentrations of the drug * Determination of the pharmacokinetics of the drug * Correlation of pharmacokinetics and pharmacogenetics We enrolled patients with a histological diagnosis of metastatic colorectal adenocarcinoma, naïve or treated with adjuvant chemotherapy (excluding irinotecan), and corresponding to the criteria for eligibility/exclusion described in the protocol. The patients were assigned to their treatment group based on genotype (*1/*1 or *1/*28) until the completion of recruitment for each dose level in each group of patients. Patients with genotype *28/*28 were excluded because of high risk of toxicity. The starting dose of irinotecan administered in patients carrying the *1 (wild-type and heterozygous) was 260mg/m2. The dose of BV was 5mg/kg and it was administered in infusion after two weeks. In the treatment group with lower dose, the irinotecan dose was increased to 310 and 370mg/m2 in case of lack of toxicity. The evaluation of pharmacokinetic and pharmacodynamic interactions between bevacizumab (BV) and irinotecan (CPT-11) was conducted describing the pharmacokinetic profile of the CPT -11 (and its metabolites) in the absence and in the presence of BV in the same patient. The obteined pharmacokinetic parameters exclude an effect of BV on the pharmacokinetics of CPT-11. However, the registered values ??of MTD were lower than those determined by previous works with FOLFIRI alone, and that might suggest that the addition of BV results in changes in the manifestation of toxicity in the high-dose regimens. The drugs that have been investigated in this protocol have been present in the clinical practice since several years, and the high variability of the response and toxicity makes it essential the definition of useful criteria for treatment personalization. Basic criteria for personalization of therapy are clinical parameters, like gender and age of the patients. Many of the elderly subjects, in fact, receive reduced treatments comparing standard clinical protocols, with a considerable reduction of the dosage of the drug or of the number of therapy cycles. Due to their physiological characteristics, the older cancer patients reported greater toxicity effects associated with the treatment. In clinical trials, elderly patients are under-represented because they don’t satisfy all the inclusion criteria, like comorbid conditions and baseline functional status of the patient. Similarly, women are often excluded from some protocols especially for the variability that characterizes the hormonal status. In a second part of the PhD project, we considered a very large series of patients with CRC treated with other medications associated with fluoropyrimidine (FOLFOX or FOLFIRI) at the CRO, Aviano (PN), and other centers participating in the program. We performed a pharmacogenetic study to see if there are genetic biomarkers useful to define a personalization of therapy for subjects considered elderly (age at diagnosis >70 years). In particular, the aim was to identify possible pharmacogenetic (PG) determinants which influence drug effects (toxicity and response) in a different way between young and elderly subjects, and between the two genders (male and female). We analyzed several polymorphisms of genes coding for proteins involved in the mechanisms of activation, metabolism and elimination of drugs mainly used in therapies against CRC and other polymorphisms in genes that regulate the cell cycle and involved in the process of carcinogenesis and tumor progression. Many allelic variations confirm an association with the risk of developing cancer risk, while others with the survival of patients. In particular, the variation in the copy number of GSTM1 and GSTT1 genes, showed a correlation with the age of diagnosis of the patients, as well as with the gender. The increase in copies of GSTM1 or GSTT1 reduction are associated with greater survival in male patients older than 70 years (PGSTM1=0.047, HR=3.937, 95% CI=0.89-0:31; PGSTT1=0.039, HR=4.246). Moreover, we further found a specific association of genotype GSTnull with an increased risk of tumor in young subjects, but not in the elderly. It can therefore be assumed that there is a deficiency in the enzymatic activity of glutathione S-transferase deficiency resulting in reduced cell detoxification mechanisms that involve an increase in damage to the DNA damnage. Additional data allowed us to conclude that genetic characteristics may be associated with both gender and age of the patients: there are polymorphisms predictive of risk related to one of the two parameters (MTHFR 1298A>C in young women); polymorphisms localized in repair genes are mainly associated with a greater risk and a lower survival in the elderly population. Translational research is a fundamental step for the application of experimental research to clinical practice. One of the most important aspects in this area is the customization of the therapy that should be considerate important to get treatment optimization especially in the field of oncology.