Genetic relationships and introgression events between wild and cultivated grapevines (Vitis vinifera L.): focus on Italian Lambruscos.

Research efforts on genomic structure and ecology of wild populations of Vitis vinifera L. offer insights on grape domestication processes and on the assortment evolution of the cultivated forms. Attention is also paid to the origin of traditional, long-cultivated varieties, often producing renowned and valuable wines. The genetic relationships between 283 Vitis vinifera cultivated varieties (subsp. sativa) and 65 individuals from 9 populations of the sylvestris subspecies mainly from northern Italy were explored by means of molecular markers (27 nuclear and 4 chloroplastic microsatellites). Several episodes of contamination of the wild germplasm by the pollen of specific grape cultivars were detected, implying concern for maintaining the purity of the wild form. At the same time, events of introgression from the wild subspecies resulted playing a crucial role in the emergence of several cultivated varieties with a clear admixed genome ancestry sativa-sylvestris. These included Lambruscos originated from the flat areas crossed by the Po and Adige rivers in northern Italy, while other cultivars still called Lambrusco but typical of hilly areas did not show the same admixed genome. Historical and ecological evidences suggesting an adaptative recent post-domestication process in the origin of several Italian Lambruscos are discussed.

Melanoma in children and adolescents: analysis of susceptibility genes in 123 Italian patients.

BACKGROUND: A polygenic inheritance involving high, medium and low penetrance genes has been suggested for melanoma susceptibility in adults, but genetic information is scarce for paediatric patients. OBJECTIVE: We aim to analyse the major high and intermediate melanoma risk genes, CDKN2A, CDK4, POT1, MITF and MC1R, in a large multicentre cohort of Italian children and adolescents in order to explore the genetic context of paediatric melanoma and to reveal potential differences in heritability between children and adolescents. METHODS: One-hundred-twenty-three patients (<21 years) from nine Italian centres were analysed for the CDKN2A, CDK4, POT1, MITF, and MC1R melanoma predisposing genes. The rate of gene variants was compared between sporadic, familial and multiple melanoma patients and between children and adolescents, and their association with clinico-pathological characteristics was evaluated. RESULTS: Most patients carried MC1R variants (67%), while CDKN2A pathogenic variants were found in 9% of the cases, the MITF E318K in 2% of patients and none carried CDK4 or the POT1 S270N pathogenic variant. Sporadic melanoma patients significantly differed from familial and multiple cases for the young age at diagnosis, infrequent red hair colour, low number of nevi, low frequency of CDKN2A pathogenic variants and of the MC1R R160W variant. Melanoma in children (≤12 years) had more frequently spitzoid histotype, were located on the head/neck and upper limbs and had higher Breslow thickness. The MC1R V92M variant was more common in children than in adolescents. CDKN2A common polymorphisms and MC1R variants were associated with a high number of nevi. CONCLUSION: Our results confirm the scarce involvement of the major high-risk susceptibility genes in paediatric melanoma and suggest the implication of MC1R gene variants especially in the children population.

Acute and intermediate toxicity of 3-week radiotherapy with simultaneous integrated boost using TomoDirect: prospective series of 287 early breast cancer patients.

AIMS: To report toxicity of a hypofractionated scheme of whole-breast (WB) intensity-modulated radiotherapy (IMRT) with simultaneous integrated boost (SIB) to the tumor bed (TB) using Tomotherapy® with Direct modality. METHODS: Patients with early breast cancer, undergoing radiotherapy (RT) in 15 daily fractions to WB (prescription dose 40.05 Gy) and SIB to the TB (48 Gy), between 2013 and 2017, was analyzed. Primary endpoint was acute and intermediate toxicity assessed at the end and within 6 months from RT, according to Radiation Therapy Oncology Group (RTOG) scale. Secondary endpoints included early chronic toxicity at 12-months follow-up, using the Late Effects Normal Tissue Task Subjective, Objective, Management, and Analytic (LENT-SOMA) scale, and cosmesis using Harvard criteria. RESULTS: The study population was of 287 patients. Acute and intermediate toxicity was collected among 183 patients with data available at the end of RT and within 6 months, 85 (46%) experienced G2 toxicity and 84 (46%) G1 toxicity, while 14 (8%) did not report toxicity at any time. A significant reduction of any grade toxicity was observed between the two time points, with the majority of patients reporting no clinically relevant toxicity at 6 months. At univariate analysis, age < 40 years, breast volume > 1000 cm3 and Dmax ≤ 115% of prescription dose were predictive factors of clinically relevant acute toxicity (G ≥ 2) at any time. At multivariable analysis, only age and breast volume were confirmed as predictive factors, with Relative Risks (95% Confidence Intervals): 2.02 (1.13-3.63) and 1.84 (1.26-2.67), respectively. At 12-month follow-up, 113 patients had complete information on any toxicity with 53% of toxicity G < 2, while cosmetic evaluation, available for 102 patients, reported a good-excellent result for 86% of patients. CONCLUSIONS: Hypofractionated WB IMRT with a SIB to the TB, delivered with TomoDirect modality, is safe and well-tolerated. Most patients reported no toxicity after 6 months and good-excellent cosmesis. Predictive factors of clinically relevant toxicity might be considered during treatment planning in order to further reduce side effects.

How many roads lead to stillbirth rate reduction? A 30-year analysis of risk factors in a Northern Italy University care center.

BACKGROUND: Stillbirths affect more than 2.5 million pregnancies worldwide every year and the progress in reducing stillbirth rates is slower than that required by World Health Organization. The aim of the present study was to investigate which factors were associated with stillbirths in a University Hospital in the North of Italy, over a time span of 30 years. The goal was to identify which factors are potentially modifiable to reduce stillbirth rate. METHODS: Retrospective case-control study (358 stillbirths, 716 livebirths) subdivided into two study periods (1987-2006 and 2007-2017). RESULTS: The prevalence of conception obtained by assisted reproductive technologies, pregnancy at advanced maternal age, and complications of pregnancy such as preeclampsia, fetal growth restriction (FGR), and other fetal diseases (abnormal fetal conditions including fetal anemia, fetal hydrops, TORCH infections) increased through the years of the study. Despite a rising prevalence, the last 10 years showed a significant reduction in stillbirths associated with preeclampsia and FGR. Similarly, the risk of stillbirth related to abnormal fetal conditions decreased in the second study period and a history of previous stillbirth becomes a nonsignificant risk factor. CONCLUSIONS: Altogether these results suggest that in pregnancies perceived as "high risk" (i.e. previous stillbirth, preeclampsia, FGR, abnormal fetal conditions) appropriate care and follow-up can indeed lower stillbirth rates. In conclusion, the road to stillbirth prevention passes inevitably through awareness and recognition of risk factors.

Association of quantitative MRI-based radiomic features with prognostic factors and recurrence rate in oropharyngeal squamous cell carcinoma.

Radiomics focuses on extracting a large number of quantitative imaging features and testing both their correlation with clinical characteristics and their prognostic and predictive values. We propose a radiomic approach using magnetic resonance imaging (MRI) to decode the tumor phenotype and local recurrence in oropharyngeal squamous cell carcinoma (OPSCC). The contrast-enhanced T1-weighted sequences from baseline MRI examinations of OPSCC patients treated between 2008 and 2016 were retrospectively selected. Radiomic features were extracted using the IBEX software, and hiegrarchical clustering was applied to reduce features redundancy. The association of each radiomic feature with tumor grading and stage, HPV status, loco-regional recurrence within 2 years, considered as main endpoints, was assessed by univariate analysis and then corrected for multiple testing. Statistical analysis was performed with SAS/STAT® software. Thirty-two eligible cases were identified. For each patient, 1286 radiomic features were extracted, subsequently grouped into 16 clusters. Higher grading (G3 vs. G1/G2) was associated with lower values of GOH/65Percentile and GOH/85Percentile features (p=0.04 and 0.01, respectively). Positive HPV status was associated with higher values of GOH/10Percentile (p=0.03) and lower values of GOH/90Percentile (p=0.03). Loco-regional recurrence within 2 years was associated with higher values of GLCM3/4-7Correlation (p=0.04) and lower values of GLCM3/2-1InformationMeasureCorr1 (p=0.04). Results lost the statistical significance after correction for multiple testing. T stage was significantly correlated with 9 features, 4 of which (GLCM25/180-4InformationMeasureCorr2, Shape/MeanBreadth, GLCM25/90-1InverseDiffMomentNorm, and GLCM3/6-1InformationMeasureCorr1) retained statistical significance after False Discovery Rate correction. MRI-based radiomics is a feasible and promising approach for the prediction of tumor phenotype and local recurrence in OPSCC. Some radiomic features seem to be correlated with tumor characteristics and oncologic outcome however, larger collaborative studies are warranted in order to increase the statistical power and to obtain robust and validated results.

The association between pesticide use and cutaneous melanoma: a systematic review and meta-analysis.

BACKGROUND: The incidence of cutaneous melanoma (CM), the deadliest form of skin cancer, has gradually increased in the last decades among populations of European origin. Epidemiological studies suggested that farmers and agricultural workers are at an increased risk of CM because they were exposed to pesticides. However, little is known about the relationship between pesticides and CM. OBJECTIVES: To investigate the association between exposure to pesticides and CM by systematically reviewing the literature. Secondary aim was to determine the categories of pesticides mainly involved in CM development. METHODS: A systematic review of the literature was performed up to September 2018 using MEDLINE, Embase and Web of Science. Studies assessing CM risk in licensed pesticide applicators were considered. Strict criteria were established to select independent studies and risk estimates; random effect models, taking into account heterogeneity, were applied. A pooled risk estimate for CM was calculated for the use of each type of pesticide and type of exposure. Between-study and estimate heterogeneity was assessed and publication bias investigated. RESULTS: A total of nine studies (two case-controls and seven cohorts) comprising 184 389 unique subjects were included. The summary relative risks for the categories 'herbicides - ever exposure', 'insecticides - ever exposure', 'any pesticide - ever exposure' and 'any pesticide - high exposure' resulted 1.85 [95% confidence interval (CI): 1.01, 3.36], 1.57 (95% CI: 0.58, 4.25), 1.31 (95% CI: 0.85, 2.04) and 2.17 (95% CI: 0.45, 10.36), respectively. Herbicides and insecticides had no between-study heterogeneity (I2  = 0%), while a significant heterogeneity (I2  > 50%) was detected for the high exposure to any pesticide. No indication for publication bias was found. CONCLUSIONS: Individuals exposed to herbicides are at an increased risk of CM. Future properly designed observational studies are required to confirm this finding.

Impact of chlorpyrifos on human villous trophoblasts and chorionic villi.

Placental barrier regulates maternal-fetal interchange protecting the baby from damage caused by substances found in the uterine environment or circulating in the vascular system. Organophosphate (OP) pesticides are a paramount group of environmental pollutants used in intensive agriculture for protection against diseases and pests. While many studies have reported an increased risk of pregnancy alterations in pregnant women exposed to OPs, few have analyzed the effects caused by these pesticides in the placenta. Herein, we evaluated the effects of chlorpyrifos (CPF), one of the most widely used OP insecticides, on human placenta using in vitro and ex vivo exposure models. Villous cytotrophoblast cells isolated from normal human term placentas maintained their cell viability, differentiated into syncytiotrophoblast-like structures, and increased the expression of ß-hCG, ABCG2, and P-gp in the presence of CPF at concentrations of 10 to 100µM. The same doses of CPF induced marked changes in chorionic villi samples. Indeed, CPF exposure increased stroma cell apoptosis, altered villi matrix composition, basement membrane thickness, and trophoblastic layer integrity. Histomorphological and ultrastructural alterations are compatible with those found in placentas where maternal-placenta injury is chronic and able to impair the placental barrier function and nutrient transport from mother to the fetus. Our study shows that placental ex vivo exposure to CPF produces tissue alterations and suggest that human placenta is a potential target of CPF toxicity. In addition, it highlights the importance of using different models to assess the effects of a toxic on human placenta.

Clinical impact of minimal residual disease in children with different subtypes of acute lymphoblastic leukemia treated with Response-Adapted therapy.

To determine the clinical significance of minimal residual disease (MRD) in patients with prognostically relevant subtypes of childhood acute lymphoblastic leukemia (ALL), we analyzed data from 488 patients treated in St Jude Total Therapy Study XV with treatment intensity based mainly on MRD levels measured during remission induction. MRD levels on day 19 predicted treatment outcome for patients with hyperdiploid >50 ALL, National Cancer Institute (NCI) standard-risk B-ALL or T-cell ALL, while MRD levels on day 46 were prognostic for patients with NCI standard-risk or high-risk B-ALL. Patients with t(12;21)/(ETV6-RUNX1) or hyperdiploidy >50 ALL had the best prognosis; those with a negative MRD on day 19 had a particularly low risk of relapse: 1.9% and 3.8%, respectively. Patients with NCI high-risk B-ALL or T-cell ALL had an inferior outcome; even with undetectable MRD on day 46, cumulative risk of relapse was 12.7% and 15.5%, respectively. Among patients with NCI standard-risk B-ALL, the outcome was intermediate overall but was poor if MRD was ⩾1% on day 19 or MRD was detectable at any level on day 46. Our results indicate that the clinical impact of MRD on treatment outcome in childhood ALL varies considerably according to leukemia subtype and time of measurement.

Low oxygen tension induces Krüppel-Like Factor 6 expression in trophoblast cells.

The transcription factor Krüppel-Like Factor 6 (KLF6) has important roles in cell differentiation, angiogenesis, apoptosis, and proliferation. Furthermore, there is evidence that KLF6 is required for proper placental development. While oxygen is a critical mediator of trophoblast differentiation and function, the involvement of oxygen in the regulation of KLF6 expression remains unexplored. In the present study we examined the expression of KLF6 in placental tissue from uncomplicated and preeclamptic pregnancies, the latter often characterized by an inadequately perfused placenta. We also determined the effect of hypoxia and the involvement of Hypoxia-Inducible Factor 1α (HIF-1α) on the expression of KLF6 in cultured trophoblast cells and placental tissues. Results revealed that villous, interstitial and endovascular extravillous cytotrophoblasts from placentas from normal and preeclamptic pregnancies express KLF6. In addition, KLF6 immunoreactivity was higher in the placental bed of preeclamptic pregnancies than in those of uncomplicated pregnancies. We demonstrated that hypoxia induced an early and transient increase in KLF6 protein levels in HTR8/SVneo extravillous cytotrophoblast cells and in placental explants. Reoxygenation returned KLF6 protein to basal levels. Moreover, hypoxia-induced up-regulation of KLF6 expression was dependent on HIF-1α as revealed by siRNA knockdown in HTR8/SVneo cells. These results indicate that KLF6 may mediate some of the effects of hypoxia in placental development. The regulation of KLF6 protein levels by oxygen has significant implications for understanding its putative role in diseases affected by tissue hypoxia.

DNA damage and oxidative stress response to selenium yeast in the non-smoking individuals: a short-term supplementation trial with respect to GPX1 and SEPP1 polymorphism.

PURPOSE: Selenium, both essential and toxic element, is considered to protect against cancer, though human supplementation trials have generated many inconsistent data. Genetic background may partially explain a great variability of the studies related to selenium and human health. The aim of this study was to assess whether functional polymorphisms within two selenoprotein-encoding genes modify the response to selenium at the level of oxidative stress, DNA damage, and mRNA expression, especially in the individuals with a relatively low selenium status. METHODS: The trial involved 95 non-smoking individuals, stratified according to GPX1 rs1050450 and SEPP1 rs3877899 genotypes, and supplemented with selenium yeast (200 µg) for 6 weeks. Blood was collected at four time points, including 4 weeks of washout. RESULTS: After genotype stratification, the effect of GPX1 rs1050450 on lower GPx1 activity responsiveness was confirmed; however, in terms of DNA damage, we failed to indicate that individuals homozygous for variant allele may especially benefit from the increased selenium intake. Surprisingly, considering gene and time interaction, GPX1 polymorphism was observed to modify the level of DNA strand breaks during washout, showing a significant increase in GPX1 wild-type homozygotes. Regardless of the genotype, selenium supplementation was associated with a selectively suppressed selenoprotein mRNA expression and inconsistent changes in oxidative stress response, indicating for overlapped, antioxidant, and prooxidant effects. Intriguingly, DNA damage was not influenced by supplementation, but it was significantly increased during washout. CONCLUSIONS: These results point to an unclear relationship between selenium, genotype, and DNA damage.

MC1R gene variants and non-melanoma skin cancer: a pooled-analysis from the M-SKIP project.

BACKGROUND: The melanocortin-1-receptor (MC1R) gene regulates human pigmentation and is highly polymorphic in populations of European origins. The aims of this study were to evaluate the association between MC1R variants and the risk of non-melanoma skin cancer (NMSC), and to investigate whether risk estimates differed by phenotypic characteristics. METHODS: Data on 3527 NMSC cases and 9391 controls were gathered through the M-SKIP Project, an international pooled-analysis on MC1R, skin cancer and phenotypic characteristics. We calculated summary odds ratios (SOR) with random-effect models, and performed stratified analyses. RESULTS: Subjects carrying at least one MC1R variant had an increased risk of NMSC overall, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC): SOR (95%CI) were 1.48 (1.24-1.76), 1.39 (1.15-1.69) and 1.61 (1.35-1.91), respectively. All of the investigated variants showed positive associations with NMSC, with consistent significant results obtained for V60L, D84E, V92M, R151C, R160W, R163Q and D294H: SOR (95%CI) ranged from 1.42 (1.19-1.70) for V60L to 2.66 (1.06-6.65) for D84E variant. In stratified analysis, there was no consistent pattern of association between MC1R and NMSC by skin type, but we consistently observed higher SORs for subjects without red hair. CONCLUSIONS: Our pooled-analysis highlighted a role of MC1R variants in NMSC development and suggested an effect modification by red hair colour phenotype.

Hyaluronan as drug carrier. The in vitro efficacy and selectivity of Hyaluronan-Doxorubicin complexes to affect the viability of overexpressing CD44 receptor cells.

We report Doxorubicin ionic complexes with hyaluronic acid (HiH-Dx) or its sodium salt (HiNa-Dx) as tumor-targeting delivery system. The complexes were prepared in situ by mixing aqueous solutions of Dx.HCl with HiH or HiNa. Clear colloidal dispersions with a high degree of counterionic condensation (cc) were obtained. Affinity constants (logKcc) of HiNa-Dx and HiH-Dx were 7.96 and 8.08, respectively. Delivery rates of Dx from the complexes were measured in a Franz-type bicompartimental device. In line with the high affinity constants, loaded Dx was slowly released from the complexes. To test the targeting potential of the complexes, carcinogenic A549 cells overexpressing the CD44 receptors were used. HTR8/SVneo cells without overexpression of CD44 were used as control. In A549 cells, cytotoxicity of both HiH-Dx and HiNa-Dx complexes was 3-fold higher than that of the reference solution. However, no differences were observed between the complexes and free Dx solution in HTR8/SVneo cells. Flow cytometry data suggested successful uptake of Dx in cells, with a greater internalization of Dx in A549 cells than in HTR8/SVneo cells when the complexes were used. Similarly, microscopy imagines revealed a higher concentration of Dx in A549 cells with the complexes. This work provides more detailed information in order to contribute to more solid bases to evaluate the potentiality of Hi as an antineoplastic drug carrier convenient for being used in specific therapeutic indications with minimal side effects.

A revised definition for cure of childhood acute lymphoblastic leukemia.

With improved contemporary therapy, we reassess long-term outcome in patients completing treatment for childhood acute lymphoblastic leukemia (ALL) to determine when cure can be declared with a high degree of confidence. In six successive clinical trials between 1984 and 2007, 1291 (84.5%) patients completed all therapies in continuous complete remission. The post-therapy cumulative risk of relapse or development of a second neoplasm and the event-free survival rate and overall survival were analyzed according to the presenting features and the three treatment periods defined by relative outcome. Over the three treatment periods, there has been progressive increase in the rate of event-free survival (65.2% vs 74.8% vs 85.1% (P<0.001)) and overall survival (76.5% vs 81.1% vs 91.7% (P<0.001)) at 10 years. The most important predictor of outcome after completion of therapy was the type of treatment. In the most recent treatment period, which omitted the use of prophylactic cranial irradiation, the post-treatment cumulative risk of relapse was 6.4%, death in remission 1.5% and development of a second neoplasm 2.3% at 10 years, with all relapses except one occurring within 4 years of therapy. None of the 106 patients with the t(9;22)/BCR-ABL1, t(1;19)/TCF3-PBX1 or t(4;11)/MLL-AFF1 had relapsed after 2 years from completion of therapy. These findings demonstrate that with contemporary effective therapy that excludes cranial irradiation, approximately 6% of children with ALL may relapse after completion of treatment, and those who remain in remission at 4 years post treatment may be considered cured (that is, less than 1% chance of relapse).

An international study of intrachromosomal amplification of chromosome 21 (iAMP21): cytogenetic characterization and outcome.

Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct cytogenetic subgroup of childhood B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). To date, fluorescence in situ hybridisation (FISH), with probes specific for the RUNX1 gene, provides the only reliable detection method (five or more RUNX1 signals per cell). Patients with iAMP21 are older (median age 9 years) with a low white cell count. Previously, we demonstrated a high relapse risk when these patients were treated as standard risk. Recent studies have shown improved outcome on intensive therapy. In view of these treatment implications, accurate identification is essential. Here we have studied the cytogenetics and outcome of 530 iAMP21 patients that highlighted the association of specific secondary chromosomal and genetic changes with iAMP21 to assist in diagnosis, including the gain of chromosome X, loss or deletion of chromosome 7, ETV6 and RB1 deletions. These iAMP21 patients when treated as high risk showed the same improved outcome as those in trial-based studies regardless of the backbone chemotherapy regimen given. This study reinforces the importance of intensified treatment to reduce the risk of relapse in iAMP21 patients. This now well-defined patient subgroup should be recognised by World Health Organisation (WHO) as a distinct entity of BCP-ALL.

The organophosphate chlorpyrifos disturbs redox balance and triggers antioxidant defense mechanisms in JEG-3 cells.

INTRODUCTION: Reactive oxygen species (ROS) are produced by a number of physiological and pathological processes which influence the function of a diverse array of cellular events. An imbalance between ROS generation and elimination was reported for different environmental xenobiotics exposure. Here, we analyzed the effect of chlorpyrifos (CPF) on the JEG-3 cell antioxidant defense in conditions where cell viability and morphology were not altered. METHODS: Acetylcholinesterase (AChE) activity, reduced glutathione (GSH) content and catalase (CAT) antioxidant enzyme activity were measured by biochemical studies. ROS production was detected using the fluorogenic probe 2',7'-dichlorodihydrofluorescein diacetate. The transcript level of superoxide dismutase enzyme 1 (SOD1), glutathione reductase (GR), heme oxygenase-1 (HO-1), and nuclear factor E2-related factor 2 (Nrf2) as well as Nrf2 protein amount were analyzed by quantitative real time PCR and Western blot, respectively. RESULTS: The results showed that CPF inhibited AChE activity, induced ROS production, upregulated CAT activity, and decreased GSH concentration. In response to CPF exposure, GR and HO-1 mRNA levels were increased with no changes in SOD1 mRNA. Furthermore, CPF significantly augmented Nrf2 at both mRNA and protein levels trigging the antioxidant status by increasing nuclear Nrf2 translocation. DISCUSSION AND CONCLUSION: Taken together, these data indicate that JEG-3 cells are able to attenuate the oxidative stress induced by CPF through the adaptive activation of the Nrf2/ARE pathway.