RAS Mutation Clinical Risk Score to Predict Survival After Resection of Colorectal Liver Metastases.

OBJECTIVE: To determine the impact of RAS mutation status on the traditional clinical score (t-CS) to predict survival after resection of colorectal liver metastases (CLM). BACKGROUND: The t-CS relies on the following factors: primary tumor nodal status, disease-free interval, number and size of CLM, and carcinoembryonic antigen level. We hypothesized that the addition of RAS mutation status could create a modified clinical score (m-CS) that would outperform the t-CS. METHODS: Patients who underwent resection of CLM from 2005 through 2013 and had RAS mutation status and t-CS factors available were included. Multivariate analysis was used to identify prognostic factors to include in the m-CS. Log-rank survival analyses were used to compare the t-CS and the m-CS. The m-CS was validated in an international multicenter cohort of 608 patients. RESULTS: A total of 564 patients were eligible for analysis. RAS mutation was detected in 205 (36.3%) of patients. On multivariate analysis, RAS mutation was associated with poor overall survival, as were positive primary tumor lymph node status and diameter of the largest liver metastasis >50 mm. Each factor was assigned 1 point to produce a m-CS. The m-CS accurately stratified patients by overall and recurrence-free survival in both the initial patient series and validation cohort, whereas the t-CS did not. CONCLUSIONS: Modifying the t-CS by replacing disease-free interval, number of metastases, and CEA level with RAS mutation status produced an m-CS that outperformed the t-CS. The m-CS is therefore a simple validated tool that predicts survival after resection of CLM.

Endothelial trans-differentiation in glioblastoma recurring after radiotherapy.

We hypothesized that in glioblastoma recurring after radiotherapy, a condition whereby the brain endothelium undergoes radiation-induced senescence, tumor cells with endothelial phenotype may be relevant for tumor neovascularization. Matched glioblastoma samples obtained at primary surgery and at surgery for tumor recurrence after radiotherapy, all expressing epidermal growth factor receptor variant III (EGFRvIII), were assessed by a technique that combines fluorescent in situ hybridization (FISH) for the EGFR/CEP7 chromosomal probe with immunostaining for endothelial cells (CD31) and activated pericytes (α Smooth Muscle Actin). Five EGFRvIII-expressing paired primary/recurrent glioblastoma samples, in which the tumor cells showed EGFR/CEP7 amplification, were then assessed by CD31 and α Smooth Muscle Actin immunofluorescence. In glomeruloid bodies, the ratio between CD31+ cells with amplified EGFR/CEP7 signal and the total CD31+ cells was 0.23 ± 0.09 (mean ± sem) and 0.63 ± 0.07 in primary tumors and in recurrent ones, respectively (p < 0.002, Student-t test). In capillaries, the ratio of CD31+ cells with amplified EGFR/CEP7 over the total CD31+ cells lining the capillary lumen was 0.21 ± 0.06 (mean ± sem) and 0.42 ± 0.07 at primary surgery and at recurrence, respectively (p < 0.005, Student-t test). Expression of α Smooth Muscle Actin by cells with EGFR/CEP7 amplification was not observed. Then, in glioblastoma recurring after radiotherapy, where the brain endothelium suffers from radiation-induced cell senescence, tumor-derived endothelium plays a role in neo-vascularization.

Cytopathology of Follicular Cell Nodules.

The detection of thyroid nodules, consisting of different diseases, represents a common finding in population. Their evaluation and diagnosis are mostly achieved with fine-needle aspiration cytology (FNAC). Even though the majority of thyroid nodules are correctly diagnosed, a total of 25% to 30% of them are classified "indeterminate" comprising lesions with varying risk of malignancy and different types of management. Although the number of thyroid FNACs, including small lesions, is increasing due to the reliance upon sonographic and cytologic interpretations, there are issues concerning cytomorphologic interpretation and interobserver reproducibility. Different classification systems have tried to better define the criteria for inclusion in specific categories and to therefore reduce the rate of indeterminate diagnoses such as atypia of undetermined significance, follicular neoplasms, and suspicious for malignancy. However, the support of ancillary techniques (eg, immunocytochemistry and molecular analysis) are reshaping morphologic diagnoses made on materials obtained from FNAC.

PDGFRA-mutant syndrome.

Germline PDGFRA mutations cause multiple heterogeneous gastrointestinal mesenchymal tumors. In its familial form this disease, which was formerly termed intestinal neurofibromatosis/neurofibromatosis 3b (INF/NF3b), has been included among familial gastrointestinal stromal tumors (GISTs) because of its genotype, described when GIST was the only known PDGFRA-mutant gastrointestinal tumor. Shortly afterwards, however, inflammatory fibroid polyps also revealed PDGFRA mutations. Subsequently, gastrointestinal CD34+ 'fibrous tumors' of uncertain classification were described in a germline PDGFRA-mutant context. Our aim was to characterize the syndrome produced by germline PDGFRA mutations and establish diagnostic criteria and management strategies for this hitherto puzzling disease. We studied a kindred displaying multiple gastrointestinal mesenchymal tumors, comparing it with published families/individuals with possible analogous conditions. We identified a novel inherited PDGFRA mutation (P653L), constituting the third reported example of familial PDGFRA mutation. In adult mutants we detected inflammatory fibroid polyps, gastric GISTs and gastrointestinal fibrous tumors of uncertain nosology. We demonstrate that the syndrome formerly defined as INF/NF3b (exemplified by the family reported herein) is simplistically considered a form of familial GIST, because inflammatory fibroid polyps often prevail. Fibrous tumors appear variants of inflammatory fibroid polyps. 'INF/NF3b' and 'familial GIST' are misleading terms which we propose changing to 'PDGFRA-mutant syndrome'. In this condition, unlike KIT-dependent familial GIST syndromes, if present, GISTs are stomach-restricted and diffuse Cajal cell hyperplasia is not observed. This restriction of GISTs to the stomach in PDGFRA-mutant syndrome: (i) focuses oncological concern on gastric masses, as inflammatory fibroid polyps are benign; (ii) supports a selective role of gastric environment for PDGFRA mutations to elicit GISTs, justifying the known predilection for stomach of sporadic PDGFRA-mutant GISTs. An awareness that inflammatory fibroid polyps, relatively common among gastrointestinal mesenchymal tumors, may be the prevailing tumor in PDGFRA-mutant syndrome could eventually reveal an unsuspected prevalence of this condition.

Megakaryocytic emperipolesis and platelet function abnormalities in five patients with gray platelet syndrome.

The gray platelet syndrome (GPS) is a rare congenital platelet disorder characterized by mild to moderate bleeding diathesis, macrothrombocytopenia and lack of azurophilic α-granules in platelets. Some platelet and megakaryocyte (MK) abnormalities have been described, but confirmative studies of the defects in larger patient cohorts have not been undertaken. We studied platelet function and bone marrow (BM) features in five GPS patients with NBEAL2 autosomal recessive mutations from four unrelated families. In 3/3 patients, we observed a defect in platelet responses to protease-activated receptor (PAR)1-activating peptide as the most consistent finding, either isolated or combined to defective responses to other agonists. A reduction of PAR1 receptors with normal expression of major glycoproteins on the platelet surface was also found. Thrombin-induced fibrinogen binding to platelets was severely impaired in 2/2 patients. In 4/4 patients, the BM biopsy showed fibrosis (grade 2-3) and extensive emperipolesis, with many (36-65%) MKs containing 2-4 leukocytes engulfed within the cytoplasm. Reduced immunolabeling for platelet factor 4 together with normal immunolabeling for CD63 in MKs of two patients demonstrated that GPS MKs display an alpha granule-specific defect. Increased immunolabeling for P-selectin and decreased immunolabeling for PAR1, PAR4 and c-MPL were also observed in MKs of two patients. Marked emperipolesis, specific defect of MK alpha-granule content and defect of PAR1-mediated platelet responses are present in all GPS patients that we could study in detail. These results help to further characterize the disease.

The genetics of Richter syndrome reveals disease heterogeneity and predicts survival after transformation.

Richter syndrome (RS) represents the development of diffuse large B-cell lymphoma in the context of chronic lymphocytic leukemia. The scarcity of biologic information about RS has hampered the identification of molecular predictors of RS outcome. We addressed this issue by performing a comprehensive molecular characterization of 86 pathologically proven RS. TP53 disruption (47.1%) and c-MYC abnormalities (26.2%) were the most frequent alterations, whereas common genetic lesions of de novo diffuse large B-cell lymphoma were rare or absent. By multivariate analysis, lack of TP53 disruption (hazard ratio, 0.43; P = .003) translated into significant survival advantage with 57% reduction in risk of death. An algorithm based on TP53 disruption, response to RS treatment, and Eastern Cooperative Oncology Group performance status had 80.9% probability of correctly discriminating RS survival (c-index = .809). RS that were clonally unrelated to the paired chronic lymphocytic leukemia phase were clinically and biologically different from clonally related RS because of significantly longer survival (median, 62.5 months vs 14.2 months; P = .017) and lower prevalence of TP53 disruption (23.1% vs 60.0%; P = .018) and B-cell receptor stereotypy (7.6% vs 50.0%; P = .009). The molecular dissection of RS into biologically distinct categories highlights the genetic heterogeneity of this disorder and provides clinically relevant information for refining the prognostic stratification of patients.

Image-enhanced endoscopy with I-scan technology for the evaluation of duodenal villous patterns.

BACKGROUND: I-scan technology is the newly developed endoscopic tool that works in real time and utilizes a digital contrast method to enhance endoscopic image. AIMS: We performed a feasibility study aimed to determine the diagnostic accuracy of i-scan technology for the evaluation of duodenal villous patterns, having histology as the reference standard. METHODS: In this prospective, single center, open study, patients undergoing upper endoscopy for an histological evaluation of duodenal mucosa were enrolled. All patients underwent upper endoscopy using high resolution view in association with i-scan technology. During endoscopy, duodenal villous patterns were evaluated and classified as normal, partial villous atrophy, or marked villous atrophy. Results were then compared with histology. RESULTS: One hundred fifteen subjects were recruited in this study. The endoscopist was able to find marked villous atrophy of the duodenum in 12 subjects, partial villous atrophy in 25, and normal villi in the remaining 78 individuals. The i-scan system was demonstrated to have great accuracy (100 %) in the detection of marked villous atrophy patterns. I-scan technology showed quite lower accuracy in determining partial villous atrophy or normal villous patterns (respectively, 90 % for both items). CONCLUSIONS: Image-enhancing endoscopic technology allows a clear visualization of villous patterns in the duodenum. By switching from the standard to the i-scan view, it is possible to optimize the accuracy of endoscopy in recognizing villous alteration in subjects undergoing endoscopic evaluation.

Tailored therapy for recurrent glioblastoma: report of a personalized molecular approach.

BACKGROUND: Failure of clinical trials with targeted therapies in glioblastoma (GBM) is probably related to the enrollment of molecularly unselected patients. In this study we report the results of a precision medicine protocol in recurrent GBM. METHODS: We prospectively evaluated 34 patients with recurrent GBM. We determined the expression of vascular endothelial growth factor (VEGF), epidermal growth factor receptor variant III (EGFRvIII), and phosphatase and tensin homolog (PTEN). According to the molecular pattern we administered bevacizumab alone in patients with VEGF overexpression, absence of EGFRvIII, and normal PTEN (group A; N.=16); bevacizumab + erlotinib in patients with VEGF overexpression, expression of EGFRvIII, and normal PTEN (group B; N.=14); and bevacizumab + sirolimus in patients with VEGF overexpression and loss of PTEN, irrespective of the EGFRvIII status (group C; N.=4). We evaluated the response rate, the clinical benefit rate, the 6-month progression-free survival (PFS-6), the 12-month PFS (PFS-12) and the safety profile of the treatment. Moreover, we compared our results with the ones of EORTC 26101 trial. RESULTS: Response rate was 50% in the whole cohort with the highest rate in group C (75%). Clinical benefit rate was 71% with the highest rate in group C (75%). PFS-6 was 56% in the whole cohort with the highest rate in group B (64%). PFS-12 was 21% in the whole cohort with the highest rate in group B (29%). When comparing our results with those from the combination arm of the EORTC 26101 trial we found a significantly higher PFS-6 and PFS-12 in our cohort. CONCLUSIONS: The precision medicine protocol for recurrent GBM is feasible and leads to improved results if compared with studies lacking molecular selection.

Molecular history of Richter syndrome: origin from a cell already present at the time of chronic lymphocytic leukemia diagnosis.

Richter syndrome (RS) represents the transformation of chronic lymphocytic leukemia to aggressive lymphoma. We explored intraclonal diversification (ID) of immunoglobulin genes in order to (i) follow the evolutionary history of the RS clone (ii) compare the role of ID in clonally related RS vs. clonally unrelated cases. Most (10/11, 90.9%) clonally related RS stem from the predominant clone observed at CLL diagnosis. One single RS had a transformation pattern compatible with sequential evolution from a secondary CLL subclone. Once RS transformation had occurred, all secondary CLL subclones disappeared and were substituted by the dominant RS clone with its own descendants. These observations suggest that genetic lesions associated with RS transformation are acquired by a cell belonging to the original CLL clone, rather than being progressively accumulated by later CLL subclones. Accordingly, most (9/11, 81.1%) clonally related RS harbored a genetic lesion disrupting TP53 that was already present, though at subclonal levels, in 5/11 (45.5%) samples of the paired CLL phase. A fraction of clonally related RS switched off ID (4/11, 36.4%) or reduced the levels of ID (5/11, 45.4%) at transformation. Conversely, all clonally unrelated RS harbored ID and were characterized by a significantly higher mutation frequency compared to clonally related RS (median: 1.18 × 10(-3) vs. 0.13 × 10(-3); p =0.002). These data indicate that (i) clonally related RS stems from a cell that is already present within the initial CLL clone and (ii) clonally unrelated and clonally related RS are biologically distinct disorders also in terms of antigen affinity maturation.

Intralesional interferon-α for conjunctival mucosa-associated lymphoid tissue lymphoma: long-term results.

OBJECTIVE: To report the long-term therapeutic results for patients with conjunctival mucosa-associated lymphoid tissue (MALT) lymphoma who were treated with intralesional injections of interferon-α (IFN-α). DESIGN: Prospective, nonrandomized, interventional case series. PARTICIPANTS: Twenty eyes of 16 patients with histologically proven conjunctival MALT lymphoma in the absence of systemic disease. METHODS: Patients were given 1,500,000 international units (IU) of IFN-α (Roferon-A; Roche s.p.a., Milano, Italy) subconjunctivally inside the lesion 3 times weekly for 4 weeks. If there was even a minimal response, a further cycle of 1,000,000 IU 3 times weekly for 4 weeks was administered. MAIN OUTCOME MEASURES: Patients were followed up clinically using slit-lamp examination to determine evidence of tumor disappearance or recurrence. In 10 eyes, an incisional biopsy was performed 6 months after therapy to verify the histologic absence of the lesion. RESULTS: A complete response was obtained in 15 eyes (75%) at the end of first cycle treatment, and in 5 eyes (25%) after further cycles. Seventeen eyes (85%) showed no local recurrence after a median follow-up of 65 months (range, 15-136 months). Three eyes (15%) demonstrated recurrence at variable points after treatment. One patient with stage IIA lymphoma exhibited systemic lymphoma progression. CONCLUSIONS: Local immunotherapy with IFN-α seems to be an effective and lasting treatment method and provides an alternative to radiotherapy for conjunctival MALT lymphomas. Very few transient side effects were detected.

The Impact of KRAS Mutational Status on Long-Term Survival following Liver Resection for Hilar Cholangiocarcinoma.

KRAS mutation is reportedly associated with poor prognosis in patients with different cancer types. However, mutational data on hilar cholangiocarcinoma are few and controversial. The aim of this study was to evaluate the rate of KRAS mutations in a single-center homogeneous population resected for hilar cholangiocarcinoma and the subsequent impact on prognosis. KRAS mutation status was evaluated in 54 patients undergoing major hepatectomy combined with resection of the main biliary confluence and regional lymphadenectomy for hilar cholangiocarcinoma between 2001 and 2019. Among these 54 patients, 12 (22.2%) had a KRAS mutation. KRAS mutation was not related with pathologic characteristics of the tumor. Five-year overall survival (OS) in patients with KRAS mutation was significantly lower than that observed in patients with KRAS wild type (0 vs. 49.2%, respectively; p = 0.003). In the multivariable analysis; independent predictors of poor OS were KRAS mutation (HR = 5.384; p = 0.003) and lymph node metastases (HR = 2.805; p = 0.023). The results of our study suggested that KRAS mutation in hilar cholangiocarcinoma was not rarely observed. KRAS mutation was an independent strong predictor of poor OS. KRAS mutation analysis should be included in the routine pathologic evaluation of resected hilar cholangiocarcinoma in order to better stratify prognosis.

The bladder epicheck test and cytology in the follow-up of patients with non-muscle-invasive high grade bladder carcinoma.

BACKGROUND: The management of non-muscle invasive bladder carcinoma (NMIBC) after transurethral resection of a bladder tumor consists of adjuvant intravesical therapy and strict and long surveillance with urine cytology and cystoscopy. The Bladder EpiCheck test (Nucleix Ltd) (BE) is a newly developed urinary markers based on DNA methylation changes in a panel of 15 genomic biomarkers, with a promising performance in term of non-invasive NMIBC detection. METHODS: In this study we prospectively enrolled 151 consecutive patients with high grade NMIBC, treated with intravesical BCG and mitomycin C therapy and evaluated during the follow-up by voided urine cytology and white-light cystoscopy, according to the European Association of Urology Guidelines. The Bladder EpiCheck test was performed at the same time of urine cytology in voided specimen. In all cases with positive cytology the diagnosis was confirmed by histology and a diagnosis was made according to the 2017 tumor, node, metastasis (TNM) classification and graded using both the 1973 and the 2004 World Health Organization (WHO) classifications. RESULTS: At three months of follow-up, we reported similar overall specificity rates for BE and urine cytology (85,1% vs 86,3%). In the group of patients with carcinoma in situ (CIS), we found the same specificity for BE and urine cytology (81,4%), while in the groups of patients with papillary high grade NMIBC, the specificity of BE was higher compared to cytology (96,3% vs 90,4%). The sensitivity of BE was always higher compared to cytology during all the follow-up both for papillary NMIBC and CIS. CONCLUSION: In the early follow-up of NMIBC the EpiCheck test might replace urinary cytology.

Molecular Analysis in a Glioblastoma Cohort-Results of a Prospective Analysis.

The prognostic role of epidermal growth factor receptor variant III (EGFRvIII), a constitutively activated oncogenic receptor, in glioblastoma is controversial. We performed a prospective study enrolling 355 patients operated on for de novo glioblastoma at a large academic center. The molecular profile, including EGFRvIII status, MGMT promoter methylation, and VEGF expression, was assessed. Standard parameters (age, clinical status and extent of surgical resection) were confirmed to hold prognostic value. MGMT promoter methylation portended a slightly improved survival. In the whole series, confirming previous results, EGFRvIII was not associated with worsened prognosis. Interestingly, female sex was associated with a better outcome. Such findings are of interest for the design of future trials.

Clinical and NGS predictors of response to regorafenib in recurrent glioblastoma.

Predictive factors for response to regorafenib in recurrent glioblastoma, IDH-wildtype, are scarcely recognized. The objective of this study was to identify molecular predictive factors for response to regorafenib using a clinically available platform. We analyzed a prospective cohort of 30 patients harboring recurrent glioblastoma, IDH-wildtype, and treated with regorafenib. Next-generation sequencing (NGS) analysis was performed on DNA extracted from paraffin-embedded tissues using a clinically available platform. Moreover, MGMT methylation and EGFRvIII expression analyses were performed. Six-month progression-free survival (PFS) was 30% and median overall survival (OS) was 7.5 months, in line with literature data. NGS analysis revealed a mutation in the EGFR pathway in 18% of cases and a mutation in the mitogen-activated protein-kinase (MAPK) pathway in 18% of cases. In the remaining cases, no mutations were detected. Patients carrying MAPK pathway mutation had a poor response to regorafenib treatment, with a significantly shorter PFS and a nonsignificantly shorter OS compared to EGFR-mutated patients (for PFS, 2.5 vs 4.5 months, p = 0.0061; for OS, 7 vs 9 months, p = 0.1076). Multivariate analysis confirmed that MAPK pathway mutations independently predicted a shorter PFS after regorafenib treatment (p = 0.0188). The negative prognostic role of MAPK pathway alteration was reinforced when we combined EGFR-mutated with EGFRvIII-positive cases. Recurrent glioblastoma tumors with an alteration in MAPK pathway could belong to the mesenchymal subtype and respond poorly to regorafenib treatment, while EGFR-altered cases have a better response to regorafenib. We thus provide a molecular selection criterion easy to implement in the clinical practice.

FMRP modulates the Wnt signalling pathway in glioblastoma.

Converging evidence indicates that the Fragile X Messenger Ribonucleoprotein (FMRP), which absent or mutated in Fragile X Syndrome (FXS), plays a role in many types of cancers. However, while FMRP roles in brain development and function have been extensively studied, its involvement in the biology of brain tumors remains largely unexplored. Here we show, in human glioblastoma (GBM) biopsies, that increased expression of FMRP directly correlates with a worse patient outcome. In contrast, reductions in FMRP correlate with a diminished tumor growth and proliferation of human GBM stem-like cells (GSCs) in vitro in a cell culture model and in vivo in mouse brain GSC xenografts. Consistently, increased FMRP levels promote GSC proliferation. To characterize the mechanism(s) by which FMRP regulates GSC proliferation, we performed GSC transcriptome analyses in GSCs expressing high levels of FMRP, and in these GSCs after knockdown of FMRP. We show that the WNT signalling is the most significantly enriched among the published FMRP target genes and genes involved in ASD. Consistently, we find that reductions in FMRP downregulate both the canonical WNT/ß-Catenin and the non-canonical WNT-ERK1/2 signalling pathways, reducing the stability of several key transcription factors (i.e. ß-Catenin, CREB and ETS1) previously implicated in the modulation of malignant features of glioma cells. Our findings support a key role for FMRP in GBM cancer progression, acting via regulation of WNT signalling.

Evaluation of intraorbital injection of rituximab for treatment of primary ocular adnexal lymphoma: a pilot study.

An interventional pilot study to assess the tolerability and activity of the intralesional injection of rituximab, a chimeric mAb that targets the CD20 antigen, in patients with orbital B-cell lymphoma. Five patients received four intralesional injections (one injection a week) of rituximab together with ropivicaine 2%. Side-effects and tumor response were assessed after each injection and during the follow-up (20 months). Two patients obtained complete remission of the intraorbital lesion. Two patients showed incomplete response after induction therapy and received planned escalating rituximab doses, obtaining regression of subjective symptoms. One patient did not achieve tumor regression after the first injection and underwent systemic treatment. This small exploratory study suggests that intralesional rituximab is a well-tolerated treatment for patients with primary ocular adnexal lymphoma. These preliminary findings suggest that intralesional rituximab is a well-tolerated strategy in anterior intraorbital lesion localization of lymphoma.

Advances in understanding the pathogenesis of familial thrombocythaemia.

Familial thrombocytosis can be divided into two broad categories. The first includes inherited syndromes that affect only the megakaryocytic lineage with Mendelian inheritance, high penetrance and polyclonal haematopoiesis. The second category includes inherited predisposition to true Philadelphia-negative myeloproliferative neoplasms (MPN) and is characterized by low penetrance, clonal haematopoiesis and presence of somatic mutations such as JAK2 V617F. It must be underlined that these two categories represent two well separate entities, with different patterns of proliferation and different transmission modalities. This review will focus on the molecular pathogenesis of hereditary thrombocytosis, underlining those clinical pictures that are specifically associated with mutations in the genes of thrombopoietin or in its receptor. Moreover, we propose an approach for the diagnosis and therapy of these syndromes.

Upper urothelial tract high-grade carcinoma: comparison of urine cytology and DNA methylation analysis in urinary samples.

Numerous studies showed that bladder urothelial carcinoma and upper urothelial tract carcinoma (UTUC) display clinical and genomic similarities. In order to analyze that the same panel of biomarkers used in the diagnosis of bladder urothelial carcinoma could be suitable for early detection of UTUC, we performed a retrospective study in which we analyzed Bladder EpiCheck scores in the urinary samples obtained by selective ureteral catheterization in a high-grade UTUC cohort, correlating the results with urinary cytology and diagnostic urethral biopsies. The present study represents a retrospective analysis of 82 patients with clinically localized high-grade UTUC (60 renal pelvis UTUC, 22 ureter UTUC) who had undergone a radical nephroureterectomy (RNU) at our Urology department from June 2018 to November 2020. Before any surgical procedure, one sample of urine, obtained by selective ureteral catheterization, was collected for each patient for cytological examination, and the remaining material was stored for the Bladder EpiCheck test. Our results showed that the sensitivity of the methylation test for high-grade UTUC was about 97.4%, significantly higher than the sensitivity of urinary cytology either considering the HGUC cytological diagnosis or including in the positive cases the SHGUC cytological diagnosis (97.4% versus 59% or 70.5%). The methylation analysis of urinary samples may represent a valid tool in the diagnostic process of patients with suspected UTUC. In cases with a difficult clinical decision after upper urinary tract biopsy and cytology, the methylation test could assist in the clinical management of UTUC patients.

Dilation of Brain Veins and Perivascular Infiltration by Glioblastoma Cells in an In Vivo Assay of Early Tumor Angiogenesis.

The cranial window (CW) technique provides a simple and low-cost method to assess tumor angiogenesis in the brain. The CW combined with histology using selective markers for tumor and endothelial cells can allow a sensitive monitoring of novel antiangiogenesis therapies in preclinical models. The CW was established in cyclosporine immunosuppressed rats that were stereotactically grafted with fluorescent U87MG glioblastoma cells. One to 3 weeks after grafting, brain vasculature was visualized in vivo and assessed by immunofluorescence microscopy using antibodies against endothelial and smooth-muscle cells and blood brain barrier. At 1-2 weeks after grafting, the CW reliably detected the hypertrophy of venous-venous anastomoses and cortical veins. These structures increased highly significantly their pregrafting diameter. Arterialized veins and hemorrhages were seen by three weeks after grafting. Immunofluorescence microscopy showed significant branching and dilation of microvessels, particularly those surrounded by tumor cells. Mechanistically, these changes lead to loss of vascular resistance, increased venous outflow, and opening of venous-venous anastomoses on the cortical surface. Data from the present study, namely, the hypertrophy of cortical venous-venous anastomoses, microvessel branching, and dilation of the microvessels surrounded by tumor cells, indicate the power of this in vivo model for the sensitive monitoring of early tumor angiogenesis.