Survival After Newly-Diagnosed High-Grade Glioma Surgery: What Can We Learn From the French National Healthcare Database?

BACKGROUND: This study aimed to assess the overall survival (OS) of patients after high-grade glioma (HGG) resection and to search for associated prognostic factors. METHODS: A random sample of ad hoc cases was extracted from the French medico-administrative national database, Système National des Données de Santé (SNDS). We solely considered the patients who received chemoradiotherapy with temozolomide (TMZ/RT) after HGG surgery. Statistical survival methods were implemented. RESULTS: A total of 1,438 patients who had HGG resection at 58 different institutions between 2008 and 2019 were identified. Of these, 34.8% were female, and the median age at HGG resection was 63.2 years (interquartile range [IQR], 55.6-69.4 years). Median OS was 1.69 years (95% confidence interval [CI], 1.63-1.76), i.e., 20.4 months. Median age at death was 65.5 years (IQR, 58.5-71.8). OS at 1, 2, and 5 years was 78.5% (95% CI, 76.4-80.7), 40.3% (95% CI, 37.9-43), and 11.8% (95% CI, 10.2-13.6), respectively. In the adjusted Cox regression, female gender (HR=0.71; 95% CI, 0.63-0.79; p<0.001), age at HGG surgery (HR=1.02; 95% CI, 1.02-1.03; p<0.001), TMZ treatment over 6 months after HGG surgery (HR=0.36; 95% CI, 0.32-0.4; p<0.001), bevacizumab (HR=1.22; 95% CI, 1.09-1.37; p<0.001), and redo surgery (HR=0.79; 95% CI, 0.67-0.93; p=0.005) remained significantly associated with the outcome. CONCLUSION: The SNDS is a reliable source for studying the outcome of HGG patients. OS is better in younger patient, female gender, and those who complete concomitant chemoradiotherapy. Redo surgery for HGG recurrence was also associated with prolonged survival.

The landscape of cancer-associated transcript fusions in adult brain tumors: a longitudinal assessment in 140 patients with cerebral gliomas and brain metastases.

Background: Oncogenic fusions of neurotrophic receptor tyrosine kinase NTRK1, NTRK2, or NTRK3 genes have been found in different types of solid tumors. The treatment of patients with TRK fusion cancer with a first-generation TRK inhibitor (such as larotrectinib or entrectinib) is associated with high response rates (>75%), regardless of tumor histology and presence of metastases. Due to the efficacy of TRK inhibitor therapy of larotrectinib and entrectinib, it is clinically important to identify patients accurately and efficiently with TRK fusion cancer. In this retrospective study, we provide unique data on the incidence of oncogenic NTRK gene fusions in patients with brain metastases (BM) and gliomas. Methods: 140 samples fixed and paraffin-embedded tissue (FFPE) of adult patients (59 of gliomas [17 of WHO grade II, 20 of WHO grade III and 22 glioblastomas] and 81 of brain metastasis (BM) of different primary tumors) are analyzed. Identification of NTRK gene fusions is performed using next-generation sequencing (NGS) technology using Focus RNA assay kit (Thermo Fisher Scientific). Results: We identified an ETV6 (5)::NTRK3 (15) fusion event using targeted next-generation sequencing (NGS) in one of 59 glioma patient with oligodendroglioma-grade II, IDH-mutated and 1p19q co-deleted at incidence of 1.69%. Five additional patients harboring TMPRSS (2)::ERG (4) were identified in pancreatic carcinoma brain metastasis (BM), prostatic carcinoma BM, endometrium BM and oligodendroglioma (grade II), IDH-mutated and 1p19q co-deleted. A FGFR3 (17)::TACC3 (11) fusion was identified in one carcinoma breast BM. Aberrant splicing to produce EGFR exons 2-7 skipping mRNA, and MET exon 14 skipping mRNA were identified in glioblastoma and pancreas carcinoma BM, respectively. Conclusions: This study provides data on the incidence of NTRK gene fusions in brain tumors, which could strongly support the relevance of innovative clinical trials with specific targeted therapies (larotrectinib, entrectinib) in this population of patients. FGFR3 (17)::TACC3 (11) rearrangement was detected in breast carcinoma BM with the possibility of using some specific targeted therapies and TMPRSS (2)::ERG (4) rearrangements occur in a subset of patients with, prostatic carcinoma BM, endometrium BM, and oligodendroglioma (grade II), IDH-mutated and 1p19q co-deleted, where there are yet no approved ERG-directed therapies.

Systematic Review of Cerebrospinal Fluid Biomarker Discovery in Neuro-Oncology: A Roadmap to Standardization and Clinical Application.

Effective diagnosis, prognostication, and management of CNS malignancies traditionally involves invasive brain biopsies that pose significant risk to the patient. Sampling and molecular profiling of cerebrospinal fluid (CSF) is a safer, rapid, and noninvasive alternative that offers a snapshot of the intracranial milieu while overcoming the challenge of sampling error that plagues conventional brain biopsy. Although numerous biomarkers have been identified, translational challenges remain, and standardization of protocols is necessary. Here, we systematically reviewed 141 studies (Medline, SCOPUS, and Biosis databases; between January 2000 and September 29, 2022) that molecularly profiled CSF from adults with brain malignancies including glioma, brain metastasis, and primary and secondary CNS lymphomas. We provide an overview of promising CSF biomarkers, propose CSF reporting guidelines, and discuss the various considerations that go into biomarker discovery, including the influence of blood-brain barrier disruption, cell of origin, and site of CSF acquisition (eg, lumbar and ventricular). We also performed a meta-analysis of proteomic data sets, identifying biomarkers in CNS malignancies and establishing a resource for the research community.

Use of 5-ALA fluorescence-guided surgery versus white-light conventional microsurgery for the resection of newly diagnosed glioblastomas (RESECT study): a French multicenter randomized phase III study.

OBJECTIVE: Only one phase III prospective randomized study, published in 2006, has assessed the performance of 5-aminolevulinic acid (5-ALA) fluorescence-guided surgery (FGS) for glioblastoma resection. The aim of the RESECT study was to compare the onco-functional results associated with 5-ALA fluorescence and with white-light conventional microsurgery in patients with glioblastoma managed according to the current standards of care. METHODS: This was a phase III prospective randomized single-blinded study, involving 21 French neurosurgical centers, comparing 5-ALA FGS with white-light conventional microsurgery in patients with glioblastoma managed according to the current standards of care, including neuronavigation use and postoperative radiochemotherapy. Randomization was performed in a 1:1 ratio stratified by institution. 5-ALA (20 mg/kg) or placebo (ascorbic acid) was administered orally 3-5 hours before the incision. The primary endpoint was the rate of gross-total resection (GTR) blindly assessed by an independent committee. Patients without a confirmed pathological diagnosis of glioblastoma or with unavailable postoperative MRI studies were excluded from the per-protocol analysis. RESULTS: Between March 2013 and August 2016, a total of 171 patients were assigned to the 5-ALA fluorescence group (n = 88) or to the placebo group (n = 83). Twenty-four cases were excluded because the WHO histological criteria of grade 4 glioma were not met. The proportion of GTR was significantly higher in the 5-ALA fluorescence group (53/67, 79.1%) than in the placebo group (33/69, 47.8%; p = 0.0002). After adjustment for age, preoperative Karnofsky Performance Scale score, and tumor location, GTR was still associated with 5-ALA fluorescence (OR 4.13 [95% CI 1.94-8.79]). The mean 7-day postoperative Karnofsky Performance Scale score (≥ 80% in 49/71, 69.0% [5-ALA group]; 50/71, 70.4% [placebo group], p = 0.86) and the proportion of patients with a worsened neurological status 3 months postoperatively (9/68, 13.2% [5-ALA group]; 9/70, 12.9% [placebo group], p = 0.95) were similar between groups. Adverse events related to 5-ALA intake were rare and consisted of photosensitization in 4/87 (4.6%) patients and hepatic cytolysis in 1/87 (1.1%) patients. The 6-month PFS (70.2% [95% CI 57.7%-79.6%] and 68.4% [95% CI 55.7%-78.1%]; p = 0.39) and 24-month OS (30.1% [95% CI 18.9%-42.0%] and 37.7% [95% CI 25.8%-49.5%]; p = 0.89) did not significantly differ. In multivariate analysis, GTR was an independent predictor of PFS (hazard ratio 0.56 [95% CI 0.36-0.86], p = 0.008) and OS (hazard ratio 0.65 [95% CI 0.42-1.01], p = 0.05). The use of 5-ALA FGS generates a significant extra cost of 2732.36€ (95% CI 1658.40€-3794.11€). CONCLUSIONS: The authors found that 5-ALA FGS is an easy-to-use, cost-effective, and minimally time-consuming technique that safely optimizes the extent of resection in patients harboring glioblastoma amenable to a large resection.

Newly Diagnosed High-Grade Glioma Surgery with Carmustine Wafers Implantation. A Long-Term Nationwide Retrospective Study.

BACKGROUND: Widespread use of carmustine wafers (CWs) to treat high-grade gliomas (HGG) has been limited by uncertainties about their efficacy. We sought to assess the outcome of patients after newly diagnosed HGG surgery with CW implantation and search for associated factors. METHODS: We processed the French medico-administrative national database between 2008 and 2019 to retrieve ad hoc cases. Survival methods were implemented. RESULTS: In total, 1608 patients who had CW implantation after HGG resection at 42 different institutions between 2008 and 2019 were identified; 36.7% were female and, median age at HGG resection with CW implantation was 61.5 years, interquartile range (IQR) [52.9-69.1]. A total of 1460 patients (90.8%) had died at data collection at a median age at death of 63.5 years, IQR [55.3-71.2]. Median overall survival (OS) was 1.42 years, 95% confidence interval [CI] 1.35-1.49, i.e., 16.8 months. Median age at death was 63.5 years, IQR [55.3-71.2]. OS at 1, 2, and, 5 years was 67.4%, 95% CI 65.1-69.7; 33.1%, 95% CI 30.9-35.5; and 10.7%, 95% CI 9.2-12.4, respectively. In the adjusted regression, sex (hazard ratio [HR] 0.82, 95% CI 0.74-0.92, P < 0.001), age at HGG surgery with CW implantation (HR 1.02, 95% CI 1.02-1.03, P < 0.001), adjuvant radiotherapy (HR 0.78, 95% CI 0.7-0.86, P < 0.001), chemotherapy by temozolomide (HR 0.7, 95% CI 0.63-0.79, P < 0.001), and redo surgery for HGG recurrence (HR 0.81, 95% CI 0.69-0.94, P = 0.005) remained significantly associated with the outcome. CONCLUSIONS: OS of patients with newly diagnosed HGG who underwent surgery with CW implantation is better in young patients, those of the female sex, and for those who complete concomitant chemoradiotherapy. Redo surgery for HGG recurrence also was associated with prolonged survival.

Comparison of Three Transcytotic Pathways for Distribution to Brain Metastases of Breast Cancer.

Advances in drug treatments for brain metastases of breast cancer have improved progression-free survival but new, more efficacious strategies are needed. Most chemotherapeutic drugs infiltrate brain metastases by moving between brain capillary endothelial cells, paracellular distribution, resulting in heterogeneous distribution, lower than that of systemic metastases. Herein, we tested three well-known transcytotic pathways through brain capillary endothelial cells as potential avenues for drug access: transferrin receptor (TfR) peptide, low-density lipoprotein receptor 1 (LRP1) peptide, albumin. Each was far-red labeled, injected into two hematogenous models of brain metastases, circulated for two different times, and their uptake quantified in metastases and uninvolved (nonmetastatic) brain. Surprisingly, all three pathways demonstrated distinct distribution patterns in vivo. Two were suboptimal: TfR distributed to uninvolved brain but poorly in metastases, while LRP1 was poorly distributed. Albumin distributed to virtually all metastases in both model systems, significantly greater than in uninvolved brain (P < 0.0001). Further experiments revealed that albumin entered both macrometastases and micrometastases, the targets of treatment and prevention translational strategies. Albumin uptake into brain metastases was not correlated with the uptake of a paracellular probe (biocytin). We identified a novel mechanism of albumin endocytosis through the endothelia of brain metastases consistent with clathrin-independent endocytosis (CIE), involving the neonatal Fc receptor, galectin-3, and glycosphingolipids. Components of the CIE process were found on metastatic endothelial cells in human craniotomies. The data suggest a reconsideration of albumin as a translational mechanism for improved drug delivery to brain metastases and possibly other central nervous system (CNS) cancers.In conclusion, drug therapy for brain metastasis needs improvement. We surveyed three transcytotic pathways as potential delivery systems in brain-tropic models and found that albumin has optimal properties. Albumin used a novel endocytic mechanism.

Recurrent high grade glioma surgery with carmustine wafers implantation: a long-term nationwide retrospective study.

PURPOSE: Widespread use of carmustine wafers (CW) to treat high-grade gliomas (HGG) has been limited by uncertainties about its efficacy. To assess the outcome of patients after recurrent HGG surgery with CW implantation and, search for associated factors. METHODS: We processed the French medico-administrative national database between 2008 and 2019 to retrieve ad hoc cases. Survival methods were implemented. RESULTS: 559 patients who had CW implantation after recurrent HGG resection at 41 different institutions between 2008 and 2019 were identified. 35.6% were female and, median age at HGG resection with CW implantation was 58.1 years, IQR [50-65.4]. 520 patients (93%) had died at data collection with a median age at death of 59.7 years, IQR [51.6-67.1]. Median overall survival (OS) was 1.1 years, 95%CI[0.97-1.2], id est 13.2 months. Median age at death was 59.7 years, IQR [51.6-67.1]. OS at 1, 2 and 5 years was 52.1%, 95%CI[48.1-56.4], 24.6%, 95%CI[21.3-28.5] & 8%, 95%CI[5.9-10.7] respectively. In the adjusted regression, bevacizumab given before CW implantation, (HR = 1.98, 95%CI[1.49-2.63], p < 0.001), a longer delay between the first and the second HGG surgery (HR = 1, 95%CI[1-1], p < 0.001), RT given before and after CW implantation (HR = 0.59, 95%CI[0.39-0.87], p = 0.009) and TMZ given before and after CW implantation (HR = 0.81, 95%CI[0.66-0.98], p = 0.034) remained significantly associated with a longer survival. CONCLUSION: OS of patients with recurrent HGG that underwent surgery with CW implantation is better in case of prolonged delay between the two resections and, for the patients who had RT and TMZ before and after CW implantation.

Natural history of spinal cord metastasis from brain glioblastomas.

BACKGROUND AND OBJECTIVES: Spinal cord metastasis arising from an intracranial glioblastoma is a rare and late event during the natural course of the disease. These pathological entities remain poorly characterized. This study aimed to identify and investigate the timeline, clinical and imaging findings, and prognostic factors of spinal cord metastasis from a glioblastoma. METHODS: Consecutive histopathological cases of spinal cord metastasis from glioblastomas in adults entered in the French nationwide database between January 2004 and 2016 were screened. RESULTS: Overall, 14 adult patients with a brain glioblastoma (median age 55.2 years) and harboring a spinal cord metastasis were included. The median overall survival as 16.0 months (range, 9.8-22.2). The median spinal cord Metastasis Free Survival (time interval between the glioblastoma diagnosis and the spinal cord metastasis diagnosis) was 13.6 months (range, 0.0-27.9). The occurrence of a spinal cord metastasis diagnosis greatly impacted neurological status: 57.2% of patients were not ambulatory, which contributed to dramatically decreased Karnofsky Performance Status (KPS) scores (12/14, 85.7% with a KPS score ≤ 70). The median overall survival following spinal cord metastasis was 3.3 months (range, 1.3-5.3). Patients with a cerebral ventricle effraction during the initial brain surgery had a shorter spinal cord Metastasis Free Survival (6.6 vs 18.3 months, p = 0.023). Out of the 14 patients, eleven (78.6%) had a brain IDH-wildtype glioblastoma. CONCLUSIONS: Spinal cord metastasis from a brain IDH-wildtype glioblastoma has a poor prognosis. Spinal MRI can be proposed during the follow-up of glioblastoma patients especially those who have benefited from cerebral surgical resection with opening of the cerebral ventricles.

Natural Course and Prognosis of Primary Spinal Glioblastoma: A Nationwide Study.

BACKGROUND AND OBJECTIVES: Primary spinal glioblastoma (PsGBM) is extremely rare. The dramatic neurologic deterioration and unresectability of PsGBM makes it a particularly disabling malignant neoplasm. Because it is a rare and heterogeneous disease, the assessment of prognostic factors remains limited. METHODS: PsGBMs were identified from the French Brain Tumor Database and the Club de Neuro-Oncologie of the Société Française de Neurochirurgie retrospectively. Inclusion criteria were age 18 years or older at diagnosis, spinal location, histopathologic diagnosis of newly glioblastoma according to the 2016 World Health Organization classification, and surgical management between 2004 and 2016. Diagnosis was confirmed by a centralized neuropathologic review. The primary outcome was overall survival (OS). Therapeutic interventions and neurologic outcomes were also collected. RESULTS: Thirty-three patients with a histopathologically confirmed PsGBM (median age 50.9 years) were included (27 centers). The median OS was 13.1 months (range 2.5-23.7), and the median progression-free survival was 5.9 months (range 1.6-10.2). In multivariable analyses using Cox model, Eastern Cooperative Oncology Group (ECOG) performance status at 0-1 was the only independent predictor of longer OS (hazard ratio [HR] 0.13, 95% CI 0.02-0.801; p = 0.02), whereas a Karnofsky performance status (KPS) score <60 (HR 2.89, 95% CI 1.05-7.92; p = 0.03) and a cervical anatomical location (HR 4.14, 95% CI 1.32-12.98; p = 0.01) were independent predictors of shorter OS. The ambulatory status (Frankel D-E) (HR 0.38, 95% CI 0.07-1.985; p = 0.250) was not an independent prognostic factor, while the concomitant standard radiochemotherapy with temozolomide (Stupp protocol) (HR 0.35, 95% CI 0.118-1.05; p = 0.06) was at the limit of significance. DISCUSSION: Preoperative ECOG performance status, KPS score, and the location are independent predictors of OS of PsGBMs in adults. Further analyses are required to capture the survival benefit of concomitant standard radiochemotherapy with temozolomide.

Surgery for glioblastomas in the elderly: an Association des Neuro-oncologues d'Expression Française (ANOCEF) trial.

OBJECTIVE: The role of surgery in the treatment of malignant gliomas in the elderly is not settled. The authors conducted a randomized trial that compared tumor resection with biopsy only-both followed by standard therapy-in such patients. METHODS: Patients ≥ 70 years of age with a Karnofsky Performance Scale (KPS) score ≥ 50 and presenting with a radiological suspicion of operable glioblastoma (GBM) were randomly assigned between tumor resection and biopsy groups. Subsequently, they underwent standard radiotherapy during the first years of the trial (2008-2017), with the addition of adjunct therapy with temozolomide when this regimen became standard (2017-2019). The primary endpoint was survival, and secondary endpoints were progression-free survival (PFS), cognitive status (Mini-Mental State Examination), autonomy (KPS), quality of life (European Organisation for Research and Treatment of Cancer [EORTC] QLQ-C30 and QLQ-BN20), and perioperative morbidity and mortality. RESULTS: Between 2008 and 2019, 107 patients from 9 centers were enrolled in the study; 101 were evaluable for analysis because a GBM was histologically confirmed (50 in the surgery arm and 51 in the biopsy arm). There was no statistically significant difference in median survival between the surgery (9.37 months) and the biopsy (8.96 months, p = 0.36) arms (adjusted HR 0.79, 95% CI 0.52-1.21, p = 0.28). However, the surgery group had an increased PFS (5.06 vs 4.02 months; p = 0.034) (adjusted HR 0.50, 95% CI 0.32-0.78, p = 0.002). Less deterioration of quality of life and KPS score evolution than in the biopsy group was observed. Surgery was not associated with increased mortality or morbidity. CONCLUSIONS: This study suggests that debulking surgery is safe, and-compared to biopsy-is associated with a less severe deterioration of quality of life and autonomy, as well as a significant although modest improvement of PFS in elderly patients suffering from newly diagnosed malignant glioma. Although resection does not provide a significant survival benefit in the elderly, the authors believe that the risk/benefit analysis favors an attempt at optimal tumor resection in this population, provided there is careful preoperative geriatric evaluation. Clinical trial registration no.: NCT02892708 (ClinicalTrials.gov).

Characteristics and management of hydrocephalus in adult patients with cerebellar glioblastoma: lessons from a French nationwide series of 118 cases.

The characteristics of hydrocephalus associated with cerebellar glioblastoma (cGB) remain poorly known. The objectives were to describe the occurence of hydrocephalus in a French nationwide series of adult patients with cGB, to identify the characteristics associated with hydrocephalus and to analyze the outcomes associated with the different surgical strategies, in order to propose practical guidelines. Consecutive cases of adult cGB patients prospectively recorded into the French Brain Tumor Database between 2003 and 2017 were screened. Diagnosis was confirmed by a centralized neuropathological review. Among 118 patients with cGB (mean age 55.9 years), 49 patients (41.5%) presented with pre-operative hydrocephalus. Thirteen patients (11.0%) developed acute (n=7) or delayed (n=6) hydrocephalus postoperatively. Compared to patients without hydrocephalus at admission, patients with hydrocephalus were younger (52.0 years vs 58.6 years, p=0.03) and underwent more frequently tumor resection (93.9% vs 73.9%, p=0.006). A total of 40 cerebrospinal-fluid diversion procedures were performed, including 18 endoscopic third ventriculostomies, 12 ventriculoperitoneal shunts and 10 external ventricular drains. The different cerebrospinal-fluid diversion options had comparable functional results and complication rates. Among the 89 patients surgically managed for cGB without prior cerebrospinal-fluid diversion, 7 (7.9%) were long-term shunt-dependant. Hydrocephalus is frequent in patients with cGB and has to be carefully managed in order not to interfere with adjuvant oncological treatments. In case of symptomatic hydrocephalus, a cerebrospinal-fluid diversion is mandatory, especially if surgical resection is not feasible. In case of asymptomatic hydrocephalus, a cerebrospinal-fluid diversion has to be discussed only if surgical resection is not feasible.

Machine learning random forest for predicting oncosomatic variant NGS analysis.

Since 2017, we have used IonTorrent NGS platform in our hospital to diagnose and treat cancer. Analyzing variants at each run requires considerable time, and we are still struggling with some variants that appear correct on the metrics at first, but are found to be negative upon further investigation. Can any machine learning algorithm (ML) help us classify NGS variants? This has led us to investigate which ML can fit our NGS data and to develop a tool that can be routinely implemented to help biologists. Currently, one of the greatest challenges in medicine is processing a significant quantity of data. This is particularly true in molecular biology with the advantage of next-generation sequencing (NGS) for profiling and identifying molecular tumors and their treatment. In addition to bioinformatics pipelines, artificial intelligence (AI) can be valuable in helping to analyze mutation variants. Generating sequencing data from patient DNA samples has become easy to perform in clinical trials. However, analyzing the massive quantities of genomic or transcriptomic data and extracting the key biomarkers associated with a clinical response to a specific therapy requires a formidable combination of scientific expertise, biomolecular skills and a panel of bioinformatic and biostatistic tools, in which artificial intelligence is now successful in developing future routine diagnostics. However, cancer genome complexity and technical artifacts make identifying real variants challenging. We present a machine learning method for classifying pathogenic single nucleotide variants (SNVs), single nucleotide polymorphisms (SNPs), multiple nucleotide variants (MNVs), insertions, and deletions detected by NGS from different types of tumor specimens, such as: colorectal, melanoma, lung and glioma cancer. We compared our NGS data to different machine learning algorithms using the k-fold cross-validation method and to neural networks (deep learning) to measure the performance of the different ML algorithms and determine which one is a valid model for confirming NGS variant calls in cancer diagnosis. We trained our machine learning with 70% of our data samples, extracted from our local database (our data structure had 7 parameters: chromosome, position, exon, variant allele frequency, minor allele frequency, coverage and protein description) and validated it with the 30% remaining data. The model offering the best accuracy was chosen and implemented in the NGS analysis routine. Artificial intelligence was developed with the R script language version 3.6.0. We trained our model on 70% of 102,011 variants. Our best error rate (0.22%) was found with random forest machine learning (ntree = 500 and mtry = 4), with an AUC of 0.99. Neural networks achieved some good scores. The final trained model with the neural network achieved an accuracy of 98% and an ROC-AUC of 0.99 with validation data. We tested our RF model to interpret more than 2000 variants from our NGS database: 20 variants were misclassified (error rate < 1%). The errors were nomenclature problems and false positives. After adding false positives to our training database and implementing our RF model routinely, our error rate was always < 0.5%. The RF model shows excellent results for oncosomatic NGS interpretation and can easily be implemented in other molecular biology laboratories. AI is becoming increasingly important in molecular biomedical analysis and can be very helpful in processing medical data. Neural networks show a good capacity in variant classification, and in the future, they may be useful in predicting more complex variants.

Role of the Tyrosine Phosphatase SHP-2 in Mediating Adrenomedullin Proangiogenic Activity in Solid Tumors.

VE-cadherin is an essential adhesion molecule in endothelial adherens junctions, and the integrity of these complexes is thought to be regulated by VE-cadherin tyrosine phosphorylation. We have previously shown that adrenomedullin (AM) blockade correlates with elevated levels of phosphorylated VE-cadherin (pVE-cadherinY731) in endothelial cells, associated with impaired barrier function and a persistent increase in vascular endothelial cell permeability. However, the mechanism underlying this effect is unknown. In this article, we demonstrate that the AM-mediated dephosphorylation of pVE-cadherinY731 takes place through activation of the tyrosine phosphatase SHP-2, as judged by the rise of its active fraction phosphorylated at tyrosine 542 (pSHP-2Y542) in HUVECs and glioblastoma-derived-endothelial cells. Both pre-incubation of HUVECs with SHP-2 inhibitors NSC-87877 and SHP099 and SHP-2 silencing hindered AM-induced dephosphorylation of pVE-cadherinY731 in a dose dependent-manner, showing the role of SHP-2 in the regulation of endothelial cell contacts. Furthermore, SHP-2 inhibition impaired AM-induced HUVECs differentiation into cord-like structures in vitro and impeded AM-induced neovascularization in in vivo Matrigel plugs bioassays. Subcutaneously transplanted U87-glioma tumor xenograft mice treated with AM-receptors-blocking antibodies showed a decrease in pSHP-2Y542 associated with VE-cadherin in nascent tumor vasculature when compared to control IgG-treated xenografts. Our findings show that AM acts on VE-cadherin dynamics through pSHP-2Y542 to finally modulate cell-cell junctions in the angiogenesis process, thereby promoting a stable and functional tumor vasculature.