Next-Generation Sequencing Comparative Analysis of DNA Mutations between Blood-Derived Extracellular Vesicles and Matched Cancer Tissue in Patients with Grade 4 Glioblastoma.

The biological heterogeneity of glioblastoma, IDH-wildtype (GBM, CNS WHO grade 4), the most aggressive type of brain cancer, is a critical hallmark, caused by changes in the genomic mutational asset and influencing clinical progression over time. The understanding and monitoring of the mutational profile is important not only to reveal novel therapeutic targets in this set of patients, but also to ameliorate the clinical stratification of subjects and the prognostic significance. As neurosurgery represents the primary technique to manage GBM, it is of utmost importance to optimize alternative and less invasive methods to monitor the dynamic mutation profile of these patients. Extracellular vesicles (EVs) are included in the liquid biopsy analysis and have emerged as the biological mirror of escaping and surviving mechanisms by many tumors, including glioblastoma. Very few studies have investigated the technical feasibility to detect and analyze the genomic profile by Next-Generation Sequencing (UMI system) in circulating EVs of patients with grade IV glioblastoma. Here, we attempted to characterize and to compare the corresponding matched tissue samples and potential variants with pathogenic significance of the DNA contained in peripheral-blood-derived EVs. The NGS analysis has revealed that patients with grade IV glioblastoma exhibited lesser DNA content in EVs than controls and that, both in EVs and matched cancer tissues, the NF1 gene was consistently mutated in all patients, with the c.2568C>G as the most common pathogenic variant expressed. This study supports the clinical utility of circulating EVs in glioblastoma as an eligible tool for personalized medicine.

Circulating Exosomal-DNA in Glioma Patients: A Quantitative Study and Histopathological Correlations-A Preliminary Study.

Glial neoplasms are a group of diseases with poor prognoses. Not all risk factors are known, and no screening tests are available. Only histology provides certain diagnosis. As already reported, DNA transported by exosomes can be an excellent source of information shared by cells locally or systemically. These vesicles seem to be one of the main mechanisms of tumor remote intercellular signaling used to induce immune deregulation, apoptosis, and both phenotypic and genotypic modifications. In this study, we evaluated the exosomal DNA (exoDNA) concentration in blood samples of patients affected by cerebral glioma and correlated it with histological and radiological characteristics of tumors. From 14 patients with diagnosed primary or recurrent glioma, we obtained MRI imaging data, histological data, and preoperative blood samples that were used to extract circulating exosomal DNA, which we then quantified. Our results demonstrate a relationship between the amount of circulating exosomal DNA and tumor volume, and mitotic activity. In particular, a high concentration of exoDNA was noted in low-grade gliomas. Our results suggest a possible role of exoDNAs in the diagnosis of brain glioma. They could be particularly useful in detecting early recurrent high-grade gliomas and asymptomatic low-grade gliomas.

Concomitant Mutations G12D and G13D on the Exon 2 of the KRAS Gene: Two Cases of Women with Colon Adenocarcinoma.

Colorectal cancer (CRC) is rapidly increasing representing the second most frequent cause of cancer-related deaths. From a clinical-molecular standpoint the therapeutically management of CRC focuses on main alterations found in the RAS family protein, where single mutations of KRAS are considered both the hallmark and the target of this tumor. Double and concomitant alterations of KRAS are still far to be interpreted as molecular characteristics which could potentially address different and more personalized treatments for patients. Here, we firstly describe the case of two patients at different stages (pT2N0M0 and pT4cN1cM1) but similarly showing a double concurrent mutations G12D and G13D in the exon 2 of the KRAS gene, normally mutually exclusive. We also evaluated genetic testing of dihydropyrimidine dehydrogenase (DPYD) and microsatellite instability (MSI) by real-time PCR and additional molecular mutations by next generation sequencing (NGS) which resulted coherently to the progression of the disease. Accordingly, we reinterpreted and discuss the clinical history of both cases treated as single mutations of KRAS but similarly progressing towards a metastatic asset. We concluded that double mutations of KRAS cannot be interpreted as univocal genomic alterations and that they could severely impact the clinical outcome in CRC, requiring a tighter monitoring of patients throughout the time.

An uncommon clinical presentation of primary pancreatic lymphoma: Bleeding. Case report and literature review.

BACKGROUND: Primary pancreatic lymphoma (PPL) represents less than 0.5% of all pancreatic neoplasms. Clinical manifestations are non-specific and diagnosis is delayed in the majority of patients. CASE REPORT: 85-year-old woman reporting accidental fall at home 20-days earlier, was admitted with diagnosed of acute abdomen from suspected two-stage rupture of the spleen. The patient complained of pain in the upper abdomen. Blood-chemical tests did not show anemia and leukocytosis, but showed increased CA19.9, CA125, LDH and beta2- microglobulin. Contrast-enhanced CT showed left pleural, perisplenic, perihepatic, and Douglas blood effusion, a neoformation of the body-tail of the pancreas with peri-pancreatic blood layer, splenomegaly due to the presence of a hypodense area as from intraparenchymal hematoma, with an apparently undamaged splenic capsule. The patient underwent emergency exploratory laparotomy, that revealed the presence of modest free serohematic effusion from oozing of the pancreatic neoformation. The local spread of the disease prevented any attempt at surgical resection. Bleeding was checked with the addition of topical hemostats (Tabotamp®) and biopsy sampling of the pancreatic mass was performed. A final histological diagnosis of large cell NHL of centro-follicular origin, double expressor for the CMYC and BCL2 protein, was achieved. The age of the patient, the poor general conditions, the associated pathologies, the locally advanced spread of the disease and the histological aggressiveness, were contraindications to chemo-radiotherapy treatments. CONCLUSION: The initial misdiagnosis was due to the history of recent trauma, the uncommon clinical presentation, the underestimation of the serum increase in markers and the interpretation of the CT. KEY WORDS: Acute Abdomen, Hemoperitoneum, Primary Pancreatic Lymphoma.

Localization of lipopolysaccharide from Escherichia Coli into human atherosclerotic plaque.

Experimental studies showed that gut-derived lipopolysaccharide (LPS) is pro-atherogenic, however, its relationship with human atherosclerosis is still to be defined. We investigate if gut-derived LPS from Escherichia Coli localizes in human carotid plaque and its potential role as pro-inflammatory molecule in the atherosclerotic lesion. LPS from Escherichia Coli and Toll-like receptor 4 (TLR4) were studied in specimens from carotid and thyroid arteries of 10 patients undergoing endarterectomy and 15 controls matched for demographic and clinical characteristics. Blood LPS were significantly higher in patients compared to controls. Immunochemistry analysis revealed positivity for antibodies against LPS and TLR4 coincidentally with positivity for CD68 only in the atherosclerotic plaque of carotid arteries but not in thyroid arteries; the positivity for LPS and TLR4 was greater in the area with activated macrophages. LPS concentration similar to that detected in atherosclerotic plaque resulted in a dose-dependent TLR4-mediated Nox2 up-regulation by human monocytes. These data provide the first evidence that LPS from Escherichia Coli localizes in human plaque and may contribute to atherosclerotic damage via TLR4-mediated oxidative stress.

The expression of B23 and EGR1 proteins is functionally linked in tumor cells under stress conditions.

BACKGROUND: The nucleolus is a multi-domain enriched with proteins involved in ribosome biogenesis, cell cycle and apoptosis control, viral replication and differentiation of stem cells. Several authors have suggested a role for the nucleolus also in malignant transformation. We have recently demonstrated that under specific circumstances the transcriptional factor EGR1 is shuttled to the nucleolus where it functions as a negative regulator of RNA polymerase I. Since this activity is hampered in ARF -/- cells, and ARF transcription is regulated by EGR1 while the turnover of ARF protein is under the control of B23, we speculated that some sort of cooperation between EGR1 and B23 might also exist. RESULTS: In this work we identified a canonical EGR1 binding site on the B23 promoter through experiments of transactivation and in vitro DNA binding assay. We then found that the levels of B23 expression are directly correlated with those of EGR1, and that this correlation applies to several cellular types and to different stress conditions. Furthermore, we showed that EGR1 stability and accumulation within the nucleolus is in turn regulated by B23 through proteasome involvement, similarly to ARF turnover. CONCLUSION: Our results highlight EGR1 as a regulator of B23 expression actively playing within the newly discovered nucleolar B23-ARF-EGR1 network.

Direct correlation between double K-RAS mutation and mucinous carcinoma. A case report.

Mutations at the K-RAS locus in colon cancer cells are frequently associated with lack of responsiveness to therapy with EGFR inhibitors, as a consequence of the activation of Ras-dependent intracellular signals. Here we report a colon cancer case carrying a novel combination of K-RAS mutations involving codon 13 (Gly to Asp) and codon 19 (Leu to Phe), on separate alleles. The double mutation was restricted to tumor cells bearing a mucinous-like phenotype.

BRAF and NRAS mutations are heterogeneous and not mutually exclusive in nodular melanoma.

Inhibitors of RAF inhibit the MAPK pathway that plays an important role in the development and progression of those melanoma carrying the V600E BRAF mutation, but there's a subset of such patients who do not respond to the therapy. Various mechanisms of drug resistance have been proposed which include the clonal heterogeneity of the tumor. We have studied a population of nodular melanoma to investigate the intratumor and intertumor heterogeneity by Laser Capture Microdissection (LCM) analysis. Our results showed that BRAF and NRAS mutations were detected in 47% and 33% of nodular melanoma, respectively, and that there is a discrepancy in mutational pattern of tumoral sample because in the 36% of patients a different mutation, in at least 1 area of the tumor, was found by LCM analysis, giving evidence of the presence of different clonal cells populations. Moreover, we found that mutations in BRAF and NRAS are not mutually exclusive because they were simultaneously present in the same tumor specimens and we observed that when the 2 different mutations were present one is a high-frequency mutation and the other is a low-frequency mutation. This was more evident in lymphonodal metastasis that resulted from wild type to mutational analysis, but showed different mutations following LCM analysis. Therefore, we believed that, when primary tumoral sample results negative to mutational analysis, if it is possible, metastases should be investigated to verify the presence of mutations. Generally, it should be searched for other mutations, in addition to BRAF V600E, so as to better understand the mechanism of drug resistance.

M2muscarinic receptors inhibit cell proliferation and migration in urothelial bladder cancer cells.

The role of muscarinic receptors in several diseases including cancer has recently emerged. To evaluate the hypothesis that muscarinic acetylcholine receptors may play a role in bladder cancer as well as in other tumor types, we investigated their expression in bladder tumor specimens. All examined samples expressed the M1, M2 and M3 receptor subtypes. We also found that the level of M2 transcripts, but not those of M1 or M3, significantly increased with the tumor histologic grade. In view of these results, we proceeded to investigate whether the M2 agonist Arecaidine had any effect on in vitro cell growth and migration of T24 cells, a bladder tumor cell line expressing the muscarinic receptors, including the M2 subtype. We observed that Arecaidine significantly reduced T24 and 5637 cell proliferation and migration in a concentration dependent manner. The silencing of M2 receptor by siRNA in T24 and 5637 cell lines showed the inability of Arecaidine (100 µM) to inhibit cell proliferation after 48 hours, whereas the use of M1 and M3 antagonists in T24 appeared not to counteract the Arecaidine effect, suggesting that the inhibition of cell proliferation was directly dependent on M2 receptor activation. These data suggest that M2 muscarinic receptors may play a relevant role in bladder cancer and represent a new attractive therapeutic target.

Axitinib affects cell viability and migration of a primary foetal lung adenocarcinoma culture.

Fetal lung adenocarcinoma (FLAC) is a rare variant of lung adenocarcinoma. Studies regarding FLAC have been based only on histopathological observations, thus representative in vitro models of FLAC cultures are unavailable. We have established and characterized a human primary FLAC cell culture, exploring its biology, chemosensitivity, and migration. FLAC cells and specimen showed significant upregulation of VEGF165 and HIF-1α mRNA levels. This observation was confirmed by in vitro chemosensitivity and migration assay, showing that only Axitinib was comparable to Cisplatin treatment. We provide a suitable in vitro model to further investigate the nature of this rare type of cancer.

Overactive bladder in diabetes mellitus patients: a questionnaire-based observational investigation.

PURPOSE: Bladder dysfunction, secondary to diabetes, is mainly characterized by poor bladder emptying and overflow incontinence. However, there is evidence in literature that storage symptoms, as those suggestive for overactive bladder (OAB), may also affect people with diabetes. The aim of this study was to evaluate the prevalence of overactive bladder, the complaint of urinary urgency with/without urge incontinence, usually with frequency and nocturia, in people with diabetes compared to healthy subjects (control group). METHODS: Symptoms were assessed through the overactive bladder questionnaire (OAB-q), an investigative tool, specifically developed for OAB diagnosis. RESULTS: OAB-q scores resulted higher in diabetic people than those of the control group. Age and disease duration resulted in measurements that showed a statistical correlation with the OAB-q scores. CONCLUSIONS: OAB symptoms are more prevalent in diabetic people than in non-diabetic people. This prompts further research to determine whether the onset of OAB symptoms can be considered as an indicator of diabetic neuropathy.

Altered calcium regulation in isolated cardiomyocytes from Egr-1 knock-out mice.

Early growth response-1 one gene (Egr-1), one of the immediate early response genes, plays an important role in the adaptive response of the myocardium to hypertrophic stimuli. We aimed to investigate the effects of Egr-1 deletion on cardiac function. Egr-1 knock-out (Egr-1(-/-)) homozygous mice were employed to evaluate the electrophysiological and molecular properties of left ventricular cardiomyocytes (VCM) by using patch-clamp technique, intracellular calcium measurements, real-time PCR, and Western blot. Action potential was prolonged and diastolic potential was positive-shifted in VCMs isolated from Egr-1(-/-) mice, in comparison with those from their wild-type (WT) littermates. The calcium content of the sarcoplasmic reticulum was reduced and the decay time for steady-state calcium transient slowed down. Serca2, Ryr, L-type Ca(2+)-channel, and PLB mRNA expression were reduced in Egr-1(-/-) mice compared with the controls. Moreover, Serca2 protein was reduced, while the amount of Ncx1 protein was increased in Egr-1(-/-) hearts compared with those of the WT littermates. Furthermore, genes involved in heart development (GATA-4, TGF-ß) and in Egr-1 regulation (Nab1, Nab2) were down regulated in Egr-1(-/-) mice. These results suggest that Egr-1 plays a pivotal role in regulating excitation-contraction coupling in cardiac myocytes.

A novel closed-chest porcine model of chronic ischemic heart failure suitable for experimental research in cardiovascular disease.

Cardiac pathologies are among the leading causes of mortality and morbidity in industrialized countries, with myocardial infarction (MI) representing one of the major conditions leading to heart failure (HF). Hitherto, the development of consistent, stable, and reproducible models of closed-chest MI in large animals, meeting the clinical realism of a patient with HF subsequent to chronic ischemic necrosis, has not been successful. We hereby report the design and ensuing application of a novel porcine experimental model of closed-chest chronic ischemia suitable for biomedical research, mimicking post-MI HF. We also emphasize the key procedural steps involved in replicating this unprecedented model, from femoral artery and vein catheterization to MI induction by permanent occlusion of the left anterior descending coronary artery through superselective deployment of platinum-nylon coils, as well as endomyocardial biopsy sampling for histologic analysis and cell harvesting. Our model could indeed represent a valuable contribution and tool for translational research, providing precious insights to understand and overcome the many hurdles concerning, and currently quenching, the preclinical steps mandatory for the clinical translation of new cardiovascular technologies for personalized HF treatments.

Prospective randomized study of radiofrequency versus ultrasound scalpels on functional outcomes of laparoscopic radical prostatectomy.

BACKGROUND AND PURPOSE: Surgical treatment of patients with prostate cancer currently involves laparoscopic radical prostatectomy (LRP) or robot-assisted LRP. Continence and nerve-sparing procedures in these techniques are supported by dissection and hemostatic surgical devices powered by different types of energy. The aim of this study was to assess recovery of continence and erectile function after laparoscopic extraperitoneal radical prostatectomy comparing two surgical devices for dissection and hemostasis-radiofrequency (RF) and ultrasound (US) scalpels. PATIENTS AND METHODS: A total of 132 men with localized prostate cancer were prospectively enrolled and scheduled for extraperitoneal LRP. Patients were randomly assigned to the RF group (LigaSure; n=66) or the US group (UltraCision; n=66). Outcomes were measured by the self-administered questionnaires (International Consultation on Incontinence Questionnaire-Urinary Incontinence [ICIQ-UI] and International Index of Erectile Function 5 [IIEF 5]) 15 days before surgery, 90 and 180 days after prostatectomy to assess recovery of urinary continence and erectile function. RESULTS: No significant difference was found between the two groups regarding operative time, intra- and perioperative complications, or time of hospital stay. At 180 days after surgery, patients in the RF-treated group showed better recovery in terms of continence and erectile function compared with patients in the US group (ICIQ-UI: p=0.0016; IIEF 5: p=0.0352). CONCLUSIONS: The use of the RF scalpel provided better functional outcomes compared with the US scalpel in patients undergoing extraperitoneal LRP. This might be attributed to the low contiguous damage of those tissues, which are not directly involved in dissection and hemostasis, achieved using the RF device.

Transurethral resection of prostate and the role of pharmacological treatment with dutasteride in decreasing surgical blood loss.

PURPOSE: Transurethral resection of prostate (TURP) still represents the gold standard in the surgical treatment of symptomatic benign prostatic hyperplasia (BPH). The most frequent complication is represented by intra- and perioperative bleeding. Preoperative use of 5-alpha-reductase inhibitors (finasteride or dutasteride) to reduce surgical bleeding is still a topic of debate in literature. Previous studies provided favorable data on blood loss reduction by preoperative administration of finasteride or dutasteride. The aim of this study was to evaluate whether pretreatment with dutasteride for six weeks before surgery can reduce surgical blood loss. METHODS: A total of 142 patients with BPH-who were to undergo TURP-were enrolled and randomized into two groups. The dutasteride group comprising of 71 patients, was treated with dutasteride (0.5 mg/day) for 6 weeks before surgery and the control group, comprising of other 71 patients, did not receive dutasteride. Blood loss was evaluated in terms of a reduction in the serum hemoglobin level (ΔHb and ΔHCT), and was estimated by measuring the Hb and hematocrit levels before and 24 hours after surgery. RESULTS: None of the patients treated with dutasteride reported any side effects. A significantly lower mean blood loss was observed in the dutasteride group compared to the control group (ΔHb=-1.29 ± 0.81 v -1.83 ± 1.25, respectively, p<0.0027; ΔHCT=-5.67 ± 2.58 v -6.50 ± 2.40, respectively, p<0.0491). CONCLUSIONS: Our results showed that pretreatment with dutasteride for 6 weeks before TURP reduces the surgical bleeding considerably. This treatment schedule can be used routinely to decrease TURP surgical bleeding.

Evaluation of the oncogenic risk of diffuse gastric polyposis. A case report.

Benign polyps of the stomach undergo malignant transformation at a rate correlating to the histological type and size of the proliferative lesion. We report a case of a 50-year-old Caucasian woman, affected by a diffuse gastric polyposis of both hyperplastic and adenomatous type. At endoscopy polyps were more than 1,000, scattered over the entire gastric cavity. The patient underwent total gastrectomy. The perilesional gastric mucosa was characterized by the presence of either atrophic or metaplastic areas and by a mild dysplasia. A single tubulo-villous adenomatous polyp was also present in the ascending tract of the colon. The absence of both high-grade dysplastic lesions and outbreaks of neoplastic transformation well correlated with the histochemical and molecular features, confirming the highly proliferative pattern of the polyps in the lack of signs of malignant progression.

Evaluation of the oncogenic risk of diffuse gastric polyposis. A case report / Evaluación del riesgo oncogénico de la poliposis gástrica difusa. Un caso clínico

Benign polyps of the stomach undergo malignant transformation at a rate correlating to the histological type and size of the proliferative lesion. We report a case of a 50-year-old Caucasian woman, affected by a diffuse gastric polyposis of both hyperplastic and adenomatous type. At endoscopy polyps were more than 1,000, scattered over the entire gastric cavity. The patient underwent total gastrectomy. The perilesional gastric mucosa was characterized by the presence of either atrophic or metaplastic areas and by a mild dysplasia. A single tubulo- villous adenomatous polyp was also present in the ascending tract of the colon. The absence of both high-grade dysplastic lesions and outbreaks of neoplastic transformation well correlated with the histochemical and molecular features, confirming the highly proliferative pattern of the polyps in the lack of signs of malignant progression(AU)