Identification of Novel Molecular and Clinical Biomarkers of Survival in Glioblastoma Multiforme Patients: A Study Based on The Cancer Genome Atlas Data.

Glioblastoma multiforme (GBM) is the most prevalent type of brain tumour; although advancements in treatment have been made, the median survival time for GBM patients has persisted at 15 months. This study is aimed at investigating the genetic alterations and clinical features of GBM patients to find predictors of survival. GBM patients' methylation and gene expression data along with clinical information from TCGA were retrieved. The most overrepresented pathways were identified independently for each omics dataset. From the genes found in at least 30% of these pathways, one gene that was identified in both sets was further examined using the Kaplan-Meier method for survival analysis. Additionally, three groups of patients who started radio and chemotherapy at different times were identified, and the influence of these variations in treatment modality on patient survival was evaluated. Four pathways that seemed to negatively impact survival and two with the opposite effect were identified. The methylation status of PRKCB was highlighted as a potential novel biomarker for patient survival. The study also found that treatment with chemotherapy prior to radiotherapy can have a significant impact on patient survival, which could lead to improvements in clinical management and therapeutic approaches for GBM patients.

Development of an in chemico high-throughput screening method for the identification of skin sensitization potential.

It is well established that chemical-peptide conjugation represents the molecular initiating event (MIE) in skin sensitization. This MIE has been successfully exploited in the development of in chemico peptide reactivity assays, with the Direct Peptide Reactivity Assay (DPRA) being validated as a screening tool for skin sensitization hazard as well as an OECD test guideline. This test relies on the use of a high-performance liquid chromatography/ultraviolet detection method to quantify chemical-peptide conjugation through measurement of the depletion of two synthetic peptides containing lysine or cysteine residues, which is labor-intensive and time-consuming. To improve assay throughput, sensitivity, and accuracy, we have developed a spectrophotometric assay for skin sensitization potential based on MIE measurement-the ProtReact assay. ProtReact is also a cheaper, faster, simpler, and more accessible alternative for the DPRA, giving comparable results. A set of 106 chemicals was tested with ProtReact and the peptide depletion values compared with those reported for the DPRA. The predictive capacity of both assays was evaluated with human reference data. ProtReact and DPRA assays show similar predictive capacities for hazard identification (75% and 74%, respectively), although ProtReact showed a higher specificity (86% versus 74%, respectively) and lower sensitivity (69% versus 73%). Overall, the results show that ProtReact assay described here represents an efficient, economic, and accurate assay for the prediction of skin sensitization potential of chemical haptens.

One Step Nucleic Acid Amplification (OSNA) Lysate Samples Are Suitable to Establish a Transcriptional Metastatic Signature in Patients with Early Stage Hormone Receptors-Positive Breast Cancer.

The One Step Nucleic Acid Amplification (OSNA) is being adopted worldwide for sentinel lymph nodes (SLNs) staging in breast cancer (BC). As major disadvantage, OSNA precludes prognostic information based on structural evaluation of SLNs. Our aim is to identify biomarkers related to tumor-microenvironment interplay exploring gene expression data from the OSNA remaining lysate. This study included 32 patients with early stage hormone receptors-positive BC. Remaining OSNA lysates were prepared for targeted RNA-sequencing analysis. Identification of differentially expressed genes (DEGs) was performed by DESeq2 in R and data analysis in STATA. The results show that, in metastatic SLNs, several genes were upregulated: KRT7, VTCN1, CD44, GATA3, ALOX15B, RORC, NECTIN2, LRG1, CD276, FOXM1 and IGF1R. Hierarchical clustering analysis revealed three different clusters. The identified DEGs codify proteins mainly involved in cancer aggressiveness and with impact in immune response. The overexpression of the immune suppressive genes VTCN1 and CD276 may explain that no direct evidence of activation of immune response in metastatic SLNs was found. We show that OSNA results may be improved incorporating microenvironment-related biomarkers that may be useful in the future for prognosis stratification and immunotherapy selection. As OSNA assay is being implemented for SLNs staging in other cancers, this approach could also have a wider utility.

Efficacy of different neoadjuvant treatment regimens in BRCA-mutated triple negative breast cancer: a systematic review and meta-analysis.

PURPOSE: Triple negative breast cancer (TNBC) is an aggressive breast cancer strongly associated with BRCA mutation. Standard neoadjuvant chemotherapy remains the standard of care for early stage TNBC, the optimal chemotherapy regimen is still a matter of discussion. Other agents, such as poly-ADP-ribosyl polymerase inhibitors (PARPi) and anti-vascular endothelial growth factor (VEGF) antibodies were evaluated in the neoadjuvant setting. This systematic review and meta-analysis intend to evaluate the impact of neoadjuvant treatments in pCR rates in TNBC gBRCA mutation, beyond traditional standard chemotherapy. METHODS: PubMed, Clinicaltrials.gov, Cochrane CENTRAL, Embase and key oncological meetings for trials were searched for studies reporting neoadjuvant chemo-immunotherapy in BRCA positive TNBC. RESULTS: Out of 1238 records reviewed, thirty-one trials were included, resulting in a total 619 BRCA-mutated TNBC patients. In BRCA mutated TNBC patients who received cisplatin in monotherapy the proportion of patients who achieved pCR was 0.53 (95%CI [0.30, 0.76]), and when treatment combined standard chemotherapy and platin derivatives the proportion of pCR increased to 0.62 (95% CI [0.48, 0.76]). The group of patients treated with platin derivatives, anthracyclines ± taxanes achieved the highest proportion of pCR, 0.66. Patients treated with PARPi alone show a pCR proportion of 0.55 (95% CI [0.30, 0.81]); and when standard chemotherapy and platin derivatives were combined with PARPi the proportion of pCR did not vary. CONCLUSIONS: Patients with BRCA mutated TNBC treated with cisplatin in monotherapy demonstrate inferior proportion in the pCR achievement when compared with standard chemotherapy plus platin derivates. The best pCR was achieved with platin derivates in association with anthracyclines ± taxanes. No difference in pCR was found between PARPi alone vs PARPi with standard chemotherapy.

Integrated Multi-Omics Signature Predicts Survival in Head and Neck Cancer.

Head and Neck Cancer (HNC) is characterized by phenotypic, biological, and clinical heterogeneity. Despite treatment modalities, approximately half of all patients will die of the disease. Several molecular biomarkers have been investigated, but until now, without clinical translation. Here, we identified an integrative nine-gene multi-omics signature correlated with HNC patients' survival independently of relapses or metastasis development. This prognosis multi-omic signature comprises genes mapped in the chromosomes 1q, 3p, 8q, 17q, 19p, and 19q and encompasses alterations at copy number, gene expression, and methylation. Copy number alterations in LMCD1-A1S and GRM7, the methylation status of CEACAM19, KRT17, and ST18, and the expression profile of RPL29, UBA7, FCGR2C, and RPSAP58 can predict the HNC patients' survival. The difference higher than two years observed in the survival of HNC patients that harbor this nine-gene multi-omics signature can represent a significant step forward to improve patients' management and guide new therapeutic targets development.

A Pilot Randomized Controlled Clinical Trial Comparing Piezo Versus Conventional Rotary Surgery for Removal of Impacted Mandibular Third Molars.

BACKGROUND: The extraction of impacted mandibular third molars is a frequent dental surgery, interfering with patients' quality of life. Ultrasonic surgery is an alternative to osteotomy with conventional rotary instruments. This study compares postoperative signals and symptoms after extracting impacted mandibular third molars using ultrasonic surgery or conventional rotary osteotomy. METHODS: A pilot randomized controlled clinical trial was conducted. Thirty patients were randomly divided into the test group (ultrasonic technique) and a control group (conventional rotatory technique). All surgeries were timed. Swelling parameters, trismus and paraesthesia were evaluated on the day of surgery and the third, fifth and seventh postoperative days. Intraoperative bleeding was evaluated during surgery. Postoperative pain was evaluated daily by the patient through a visual analogue scale and the number of ingested analgesics. RESULTS: Pain, swelling and trismus present beneficial results with the ultrasonic technique but without statistical significance. Intraoperative bleeding was significantly lower with ultrasonic surgery (t(28) = 3.258; p = 0.003). Operating time was significantly higher in extractions involving osteotomy and cutting crown and roots either with the conventional technique (p = 0.020) or ultrasonic technique (p = 0.039). Regardless of the surgical difficulty, no statistically significant results were detected between techniques regarding the procedure duration. CONCLUSIONS: The beneficial postoperative signs and symptoms make ultrasonic surgery a favourable therapeutic option, especially when the integrity of noble anatomical structures is the most important risk factor. Further studies with larger samples are needed to support the use of piezosurgery as a valid option for impacted mandibular third molar extraction.

Pharyngeal Airspace Alterations after Using the Mandibular Advancement Device in the Treatment of Obstructive Sleep Apnea Syndrome.

BACKGROUND: Mandibular Advancement Devices (MADs), inserted in non-surgical treatments for obstructive sleep apnea and hypopnea syndrome (OSAHS), are used intra-orally during the sleep period, with the aim of promoting mandibular protrusion. The aim of the study is to analyze the changes in the upper airway after the use of an MAD in the treatment of OSAHS. METHODS: 60 patients diagnosed with OSAHS, as established by the Sleep Medicine Service, underwent treatment with the Silensor SL device at the Stomatology Service of the University Hospital Center of Coimbra, from January 2018 to January 2019. All patients completed two polysomnographies and two lateral teleradiographies: one before starting treatment (T0) and one after 1 year of treatment (T1). In the lateral teleradiography performed after one year of treatment, the patient had the MAD placed intra-orally. The linear measurements of the airspace proposed by the Arnett/Gunson FAB Surgery cephalometric analysis were measured at four craniometric points: A, MCI, B, Pog. RESULTS: The results demonstrate an anteroposterior airway enlargement in two of the four points studied with the MAD placed intra-orally (B and Pog point). The greatest average increase is observed at point Pog (3 mm), followed by B (1 mm), and finally, point A (0.6 mm). CONCLUSIONS: This study proved that there is an improvement in anteroposterior measurements at various points in the upper airways after treatment with MAD.

The accuracy of magnetic resonance imaging in predicting the size of pure ductal carcinoma in situ: a systematic review and meta-analysis.

Ductal carcinoma in situ (DCIS) is a putative precursor of invasive breast cancer and MRI is considered the most sensitive imaging technique for its detection. This study aims to evaluate the accuracy of MRI measuring the pure DCIS size, against pathology, to better understand the role of MRI in the management of this intraductal neoplasm.Potentially eligible studies in MEDLINE, Embase and Google Scholar, up to January 2021 were considered, and a systematic review and meta-analysis according to the published protocol (Prospero-CRD42021232228) was performed. Outcomes of mean differences and accuracy rates were analysed using IBM® SPSS® v26 and random-effect models in platform R v3.3.Twenty-two cross-sectional studies were selected and 15 proceeded to meta-analysis. MRI accurately predicted 55% of the tumours' sizes and, according to Bland-Altman plots, concordance between MRI and pathology was greater for smaller tumours. In the meta-analysis, difference of the means between MRI and pathology was 3.85 mm (CI 95% [-0.92;8.60]) with considerable heterogeneity (I2 = 96.7%). Subgroup analysis showed similar results for sizes between different MRI fields, temporal resolution, slice thickness and acquisition times, but lower heterogeneity in studies using 3-T MRI (I2 = 57.2%). Results were concordant with low risk of bias studies (2.46, CI 95% [0.57-4.36]), without heterogeneity (I2 = 0%).Therefore, MRI is shown to be an accurate method in pure DCIS size assessment. Once the best MRI protocol is established, evaluation of the impact of pure DCIS size in predicting treatment outcomes will contribute to clarifying current issues related to intraductal breast carcinoma.

Cold Atmospheric Plasma Apoptotic and Oxidative Effects on MCF7 and HCC1806 Human Breast Cancer Cells.

Breast cancer (BC) is a malignant neoplasia with the highest incidence and mortality rates in women worldwide. Currently, therapies include surgery, radiotherapy, and chemotherapy, including targeted therapies in some cases. However, treatments are often associated with serious adverse effects. Looking for new options in BC treatment, we evaluated the therapeutic potential of cold atmospheric plasma (CAP) in two cell lines (MCF7 and HCC1806) with distinct histological features. Apoptosis seemed to be the most prevalent type of death, as corroborated by several biochemical features, including phosphatidylserine exposure, the disruption of mitochondrial membrane potential, an increase in BAX/BCL2 ratio and procaspase 3 loss. Moreover, the accumulation of cells in the G2/M phase of the cell cycle points to the loss of replication ability and decreased survival. Despite reported toxic concentrations of peroxides in culture media exposed to plasma, intracellular peroxide concentration was overall decreased accompanying a reduction in GSH levels shortly after plasma exposure in both cell lines. In HCC1806, elevated nitric oxide (NO) concentration accompanied by reduced superoxide levels suggests that these cells are capable of converting plasma-derived nitrites into NO that competes with superoxide dismutase (SOD) for superoxide to form peroxinitrite. The concomitant inhibition of the antioxidative activity of cells during CAP treatment, particularly the inhibition of cytochrome c oxidase with sodium azide, synergistically increased plasma toxicity. Thus, this in vitro research enlightens the therapeutic potential of CAP in the treatment of breast cancer, elucidating its possible mechanisms of action.

Implantoplasty Improves Clinical Parameters over a 2-Year Follow-Up: A Case Series.

Background and Objectives: Peri-implantitis treatment is still undefined. Regenerative treatment is expensive and technically demanding due to the need to handle biomaterials, membranes and different methodologies of decontamination. Resective treatment and implantoplasty might be a viable solution. This case series presents a 24 month retrospective observational study of 10 peri-implantitis patients treated with implantoplasty. Materials and Methods: In the present case series, 10 peri-implantitis patients (20 implants) were treated with a resective approach and implantoplasty. Previous to implantoplasty, all patients underwent non-surgical treatment. This surgery consisted in a full-thickness flap and implant surface exposure. The exposed non-osseointegrated implant body was submitted to implantoplasty. The flap was apically repositioned and sutured. Patients were accompanied for 24 months. Results: The mean initial probing depth (PD) (PD = 5.37 ± 0.86 mm), bleeding on probing (BoP = 0.12 ± 0.06%) and suppuration (Sup = 0.01 ± 0.01%) decreased significantly at the 12 month evaluation (PD = 2.90 ± 0.39 mm; BoP = 0.01 ± 0.01% and Sup = 0.00 ± 0.00%). Between the 12 and 24 month evaluations, there were no significant clinical changes (PD = 2.85 ± 0.45 mm; BoP = 0.01 ± 0.01% and Sup = 0.00 ± 0.00%). Mucosal recession (MR) had a significant increase between the baseline and the first 12 months (0.69 ± 0.99 mm vs. 1.96 ± 1.33 mm), but there were no significant changes between the 12th and 24th month (1.94 ± 1.48 mm). The success rate was 100% without implant fracture or loss. Conclusions: Resective surgery and implantoplasty might be a valid option in some specific peri-implantitis cases. Properly designed clinical trials are needed to confirm this possibility.

Development of a genomic predictive model for cholangiocarcinoma using copy number alteration data.

AIMS: Cholangiocarcinoma (CC) is a rare tumour arising from the biliary tract epithelium. The aim of this study was to perform a genomic characterisation of CC tumours and to implement a model to differentiate extrahepatic (ECC) and intrahepatic (ICC) cholangiocarcinoma. METHODS: DNA extracted from tumour samples of 23 patients with CC, namely 10 patients with ECC and 13 patients with ICC, was analysed by array comparative genomic hybridisation. A support vector machine algorithm for classification was applied to the genomic data to distinguish between ICC and ECC. A survival analysis comparing both groups of patients was also performed. RESULTS: With these whole genome results, we observed several common alterations between tumour samples of the same CC anatomical type, namely gain of Xp and loss of 3p, 11q11, 14q, 16q, Yp and Yq in ICC tumours, and gain of 16p25.3 and loss of 3q26.1, 6p25.3-22.3, 12p13.31, 17p, 18q and Yp in ECC tumours. Gain of 2q37.3 was observed in the samples of both tumour subtypes, ICC and ECC. The developed genomic model comprised four chromosomal regions that seem to enable the distinction between ICC and ECC, with an accuracy of 71.43% (95% CI 43% to 100%). Survival analysis revealed that in our cohort, patients with ECC survived on average 8 months less than patients with ICC. CONCLUSIONS: This genomic characterisation and the introduction of genomic models to clinical practice could be important for patient management and for the development of targeted therapies. The power of this genomic model should be evaluated in other CC populations.

The challenge of ovarian tissue culture: 2D versus 3D culture.

BACKGROUND: Cryopreservation of ovarian tissue is a powerful technique for preserving female fertility, as it can restore fertility and endocrine function. To increase the longevity of the transplant and decrease the risk of reimplantation of neoplastic cells, several studies have been carried out with culture of ovarian tissue. The aim of this study was to compare a conventional (2D) culture with an alginate matrix three-dimensional (3D) model for ovarian tissue culture. RESULTS: The ovarian tissue culture within the alginate matrix (3D) was similar to 2D culture, regarding follicular density and cell apoptosis in follicles and stroma. The proliferation rate remained stable in both models for follicles, but for stromal cell proliferation it decreased only in 3D culture (p = 0.001). At 24 h of culture, cytotoxicity was lower in the 3D model (p = 0.006). As culture time increased, cytotoxicity seemed similar. Degradation of the tissue was suggested by the histological score analysis of tissue morphology after 72 h of culture. Tissue injury was greater (p = 0.01) in 3D culture due to higher interstitial oedema (p = 0.017) and tissue necrosis (p = 0.035). CONCLUSION: According to our results, 3D culture of ovarian tissue has no advantage over 2Dculture; it is more time consuming and difficult to perform and has worse reproducibility.

One-Abutment One-Time Effect on Peri-Implant Marginal Bone: A Prospective, Controlled, Randomized, Double-Blind Study.

OBJECTIVE: To evaluate the peri-implant hard tissue change at 6 and 12 months after implant placement between definitive abutment placed at the same time of implant surgery, never removing it, and healing abutment disconnected and reconnected three times until the placement of the final rehabilitation. MATERIAL AND METHODS: Each partial edentulous patient could receive between 1 and 4 platform-switched implants in the posterior regions. If the implants had primary stability-implant stability quotient (ISQ) equal to or greater than 50, they were randomized to the test group with the abutment inserted at the same time of implant placement (DA) or to the control group, receiving a healing abutment (PA). At 6 and 12 months after surgery, data related with vertical bone level changes (primary outcome) and other clinical parameters (implant mobility, bleeding on probing, probing depth, plaque index) were assessed. RESULTS: 53 implants were included in the trial and completed 12 months follow-up (overall survival rate: 100%). All implants achieved primary stability, with an average ISQ value of 80.9 on the day of surgery. From surgery to 6 months, the mean bone loss was 0.14 ± 0.18 mm for the DA group and 0.23 ± 0.29 mm for the PA group, without statistical significance difference. Between 6 and 12 months, the mean bone loss was 0.14 ± 0.21 mm for the DA group and 0.21 ± 0.27 mm for the PA group, also without statistical significance between the two groups. There were no statistically significant differences (p = 0.330) in total bone loss after 12 months between the control and the study groups. CONCLUSIONS: The one abutment one time protocol has at least an equivalent effect on the peri-implant bone level changes when compared with the use of healing abutments that are disconnected and reconnected at least three times.

Plasma activated media and direct exposition can selectively ablate retinoblastoma cells.

A new therapy based on atmospheric plasma, the fourth state of matter, has raised the medical community's attention by circumventing many undesirable effects of old anticancer treatments. This work aimed to evaluate the effect, selectivity, and mechanisms of action of cold atmospheric plasma (CAP) in human retinoblastoma cells. An electronic device was designed to generate CAP in the open air, 2 mm above seeded cell cultures. Three approaches were performed: direct use of CAP, plasma-activated media (PAM), and conditioned media (CM). Timely-resolved output voltage measurement, emission spectroscopy, and quantification of reactive species (RS) of PAM were performed. To evaluate cytotoxicity and selectivity, similarly treated Y79, fibroblasts HFF1, and retinal RPE-D407 cells were assessed. After 60 s of direct CAP treatment, the metabolic activity of retinoblastoma cells decreased more than 50%, mainly due to apoptosis, while HFF1 and RPE-D407 remained viable. Similar results were obtained with indirect treatment (PAM and CM). Cell survival was reduced, and cells accumulated in S and G2/M phases; however, no DNA strand breaks were detected. Regarding RS, plasma increased extracellular and intracellular concentrations of peroxides and nitric oxide, despite glutathione activation and lack of success in reverting cytotoxicity with some RS inhibitors. RS increase comes in two timely distant waves, the first one originating from the plasma itself with secondary solubilization and passive diffusion, the second wave deriving from the mitochondrion. The addition of low doses of carboplatin to CAP-treated cells resulted in a significant increase in cytotoxicity compared with either regimen alone. Additionally, maximal antiangiogenic effects were obtained with 60 s of plasma exposure. Direct and indirect treatment with CAP might be a selective therapy with the potential to target tumour cells and supporting the microenvironment.

A seven-gene signature to predict the prognosis of oral squamous cell carcinoma.

The prognosis of oral squamous cell carcinoma (OSCC) patients remains poor without implemented biomarkers in the clinical routine practice to help in the patient's management. With this study we aimed to identify specific prognostic biomarkers for OSCC using a whole genome technology as well as to verify the clinical utility of a head and neck cancer-specific multiplex ligation-dependent probe amplification (MLPA) panel. A genomic characterization of tumor samples from 62 OSCC patients was performed using array comparative genomic hybridization (aCGH) and a more straightforward and cost-effective molecular technology, MLPA. The identification of a genomic signature and prognosis biomarkers was carried out by applying several statistical methods. With aCGH we observed that the chromosomes most commonly altered were 3p, 3q, 5q, 6p, 7q, 8p, 8q, 11q, 15q, 17q, and 18q. The MLPA results showed that the chromosomes with a higher frequency of alterations were 3p, 3q, 8p, 8q, and 11q. We identified a genomic signature with seven genes OCLN (3p21.31), CLDN16 (3q29), SCRIB (3q29), IKBKB (3q22.3), PAK2 (8q22.3), PIK3CB (3q28), and YWHAZ (8q24.3) that together allow to differentiate the patients that developed metastases or relapses after primary tumor treatment, with an overall accuracy of 79%. Amplification of PIK3CB as a predictor of metastases or relapses development was validated using TCGA data. This amplified gene showed a reduction in more than 5 years in the median survival of the patients. The identified biomarkers might have a significant impact in the patients' management and could leverage the OSCC precision medicine.

Collagenated Porcine Heterologous Bone Grafts: Histomorphometric Evaluation of Bone Formation Using Different Physical Forms in a Rabbit Cancellous Bone Model.

Collagenated porcine-derived bone graft materials exhibit osteoconductive properties and the development of different formulations intends to enhance bone regeneration. This study aims to evaluate bone healing in a rabbit cancellous bone defect in response to grafting with different physicochemical forms of heterologous porcine bone. Twenty-six adult male New Zealand White rabbits received two critical size femoral bone defects per animal (n = 52), each randomly assigned to one of the five tested materials (Apatos, Gen-Os, mp3, Putty, and Gel 40). Animals were sacrificed at 15- and 30-days post-surgery. Qualitative and quantitative (new bone, particle and connective tissue percentages) histological analyses were performed. Histomorphometry showed statistically significant differences in all evaluated parameters between mp3 and both Putty and Gel 40 groups, regardless of the timepoint (p < 0.05). Moreover, statistical differences were observed between Apatos and both Putty (p = 0.014) and Gel 40 (p = 0.007) groups, at 30 days, in regard to particle percentage. Within each group, regarding new bone formation, mp3 showed significant differences (p = 0.028) between 15 (40.93 ± 3.49%) and 30 (52.49 ± 11.04%) days. Additionally, intragroup analysis concerning the percentage of particles revealed a significant reduction in particle occupied area from 15 to 30 days in mp3 and Gen-Os groups (p = 0.009). All mp3, Gen-Os and Apatos exhibited promising results in terms of new bone formation, thus presenting suitable alternatives to be used in bone regeneration.

Kinetics of radium-223 and its effects on survival, proliferation and DNA damage in lymph-node and bone metastatic prostate cancer cell lines.

BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) is associated with a very unfavorable prognosis. At this advanced stage of the disease, there are several therapeutic strategies approved in recent times, being one of them Radium-223 dichloride (Radium-223). However, its mechanisms of action and the process that conducts to cell death are not fully understood. Given this, our main goal is to characterize the radiobiological effects induced by Radium-223 and to evaluate its kinetics on metastatic Prostate Cancer (mPCa) cells. MATERIALS AND METHODS: In vitro studies were conducted using two mPCa cell lines, the LNCaP and PC3, the first being derived from lymph node metastasis and the second from bone metastasis. Kinetic studies were conducted to access the capacity of these cell lines to uptake, retain and internalize the Radium-223. For the assessment of radiobiological effects, cells were first exposed to different doses of Radium-223 and the clonogenic assay was done to evaluate cell survival and to determine lethal doses (LD50). Then, the effects were also evaluated in terms of proliferation, oxidative stress, morphological changes and cell damage. RESULTS: Radium-223 is uptaken by mPCa cells and reaches the nucleus, where it is retained over time. Irradiation decreases cell survival and proliferation, with LNCaP cells (LD50 = 1.73mGy) being more radiosensitive than PC3 cells (LD50 = 4.20mGy). Irradiated cells showed morphological changes usually associated with apoptosis and a dose-dependent increase in DNA damage. Moreover, activation of cell cycle checkpoints occurs through ATM/CHK2 pathway, which is involved in cell cycle arrest and cell death. CONCLUSIONS: The cytotoxic and anti-proliferative effects on both cell lines showed that Radium-223 can decrease the aggressiveness of tumor cells by decreasing the cell survival and proliferation and, also, by increasing the DNA damage. The similar results observed in both cell lines indicated that Radium-223 may have the potential to be used as a therapeutic option also for mCRPC patients with lymph node metastasis. The activation of DNA Damage Response pathways allows the possibility to understand the importance of these checkpoints as targets for new combined therapies.

Cold Atmospheric Plasma, a Novel Approach against Bladder Cancer, with Higher Sensitivity for the High-Grade Cell Line.

Antitumor therapies based on Cold Atmospheric Plasma (CAP) are an emerging medical field. In this work, we evaluated CAP effects on bladder cancer. Two bladder cancer cell lines were used, HT-1376 (stage III) and TCCSUP (stage IV). Cell proliferation assays were performed evaluating metabolic activity (MTT assay) and protein content (SRB assay). Cell viability, cell cycle, and mitochondrial membrane potential (Δψm) were assessed using flow cytometry. Reactive oxygen and nitrogen species (RONS) and reduced glutathione (GSH) were evaluated by fluorescence. The assays were carried out with different CAP exposure times. For both cell lines, we obtained a significant reduction in metabolic activity and protein content. There was a decrease in cell viability, as well as a cell cycle arrest in S phase. The Δψm was significantly reduced. There was an increase in superoxide and nitric oxide and a decrease in peroxide contents, while GSH content did not change. These results were dependent on the exposure time, with small differences for both cell lines, but overall, they were more pronounced in the TCCSUP cell line. CAP showed to have a promising antitumor effect on bladder cancer, with higher sensitivity for the high-grade cell line.

Multiple Basal Cell Carcinomas of the Scalp After Radiotherapy: Genomic Study in a Case With Latency Period Over 80 Years.

ABSTRACT: Basal cell carcinoma (BCC) has been linked mostly to ultraviolet radiation exposure, but ionizing radiation has also been implicated in the genesis of a subset of BCCs occurring after radiotherapy. We present a 93-year-old woman with 4 BCCs of the scalp after radiotherapy for tinea capitis, diagnosed after a latency period of over 80 years. The largest lesion was located on the right temporal region and corresponded to a BCC of mixed type, with nodular, infiltrative, and micronodular components. We performed genomic study with array comparative genomic hybridization in samples from each BCC, which revealed more imbalances in the largest lesion than in the remaining ones, correlating with its higher histological complexity. Furthermore, this was the only lesion presenting loss at 2p22.3, where is mapped the BIRC6 gene associated with regulation of apoptosis, and loss at 16q24.3, where is mapped FANCA gene, responsible for DNA repair and maintenance of chromosome stability. Despite these differences, there were aberrations shared by all tumor samples, suggesting a common genetic signature. Our report describes, to the best of our knowledge, the longest latency period between exposure to radiotherapy and the diagnosis of BCC. The genomic study showed imbalances common to all tumor samples but also differences that could explain their heterogeneity in terms of histological subtype and biological potential. In addition, these differences could also be a consequence of different times in the evolution of the lesions at the moment of presentation, thus having a diverse combination of accumulated genomic imbalances.

Basal cell carcinomas of the scalp after radiotherapy for tinea capitis in childhood: A genetic and epigenetic study with comparison with basal cell carcinomas evolving in chronically sun-exposed areas.

BACKGROUND: Basal cell carcinoma (BCC) has been mostly associated with sun exposure, but ionizing radiation is also a known risk factor. It is not clear if the pathogenesis of BCC, namely at a genomic and epigenetic level, differs according to the underlying triggering factors. OBJECTIVE: The present study aims to compare genetic and epigenetic changes in BCCs related to ionizing radiation and chronic sun exposure. METHODS: Tumor samples from BCCs of the scalp in patients submitted to radiotherapy to treat tinea capitis in childhood and BCCs from sun-exposed areas were analysed through array comparative genomic hybridization (array-CGH) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) to detect copy number alterations and methylation status of specific genes. RESULTS: Genomic characterization of tumor samples revealed several copy number gains and losses in all chromosomes, with the most frequent gains observed at 2p, 6p, 12p, 14q, 15q, 18q, Xp and Yp, and the most frequent losses observed at 3q, 14q, 16p, 17q, 22q, Xp, Yp and Yq. We developed a statistical model, encompassing gains in 3p and 16p and losses in 14q and 20p, with potential to discriminate BCC samples with sporadic aetiology from BCC samples that evolve after radiotherapy in childhood for the treatment of tinea capitis, which presented statistical significance (P = 0.003). Few methylated genes were detected through MS-MLPA, most frequently RARB and CD44. CONCLUSIONS: Our study represents a step forward in the understanding of the genetic mechanisms underlying the pathogenesis of BCC and suggests potential differences according to the underlying ris k factors.