CNS tumors with PLAGL1-fusion: beyond ZFTA and YAP1 in the genetic spectrum of supratentorial ependymomas.

A novel methylation class, "neuroepithelial tumor, with PLAGL1 fusion" (NET-PLAGL1), has recently been described, based on epigenetic features, as a supratentorial pediatric brain tumor with recurrent histopathological features suggesting an ependymal differentiation. Because of the recent identification of this neoplastic entity, few histopathological, radiological and clinical data are available. Herein, we present a detailed series of nine cases of PLAGL1-fused supratentorial tumors, reclassified from a series of supratentorial ependymomas, non-ZFTA/non-YAP1 fusion-positive and subependymomas of the young. This study included extensive clinical, radiological, histopathological, ultrastructural, immunohistochemical, genetic and epigenetic (DNA methylation profiling) data for characterization. An important aim of this work was to evaluate the sensitivity and specificity of a novel fluorescent in situ hybridization (FISH) targeting the PLAGL1 gene. Using histopathology, immunohistochemistry and electron microscopy, we confirmed the ependymal differentiation of this new neoplastic entity. Indeed, the cases histopathologically presented as "mixed subependymomas-ependymomas" with well-circumscribed tumors exhibiting a diffuse immunoreactivity for GFAP, without expression of Olig2 or SOX10. Ultrastructurally, they also harbored features reminiscent of ependymal differentiation, such as cilia. Different gene partners were fused with PLAGL1: FOXO1, EWSR1 and for the first time MAML2. The PLAGL1 FISH presented a 100% sensitivity and specificity according to RNA sequencing and DNA methylation profiling results. This cohort of supratentorial PLAGL1-fused tumors highlights: 1/ the ependymal cell origin of this new neoplastic entity; 2/ benefit of looking for a PLAGL1 fusion in supratentorial cases of non-ZFTA/non-YAP1 ependymomas; and 3/ the usefulness of PLAGL1 FISH.

Brain exposure to SARS-CoV-2 virions perturbs synaptic homeostasis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with short- and long-term neurological complications. The variety of symptoms makes it difficult to unravel molecular mechanisms underlying neurological sequalae after coronavirus disease 2019 (COVID-19). Here we show that SARS-CoV-2 triggers the up-regulation of synaptic components and perturbs local electrical field potential. Using cerebral organoids, organotypic culture of human brain explants from individuals without COVID-19 and post-mortem brain samples from individuals with COVID-19, we find that neural cells are permissive to SARS-CoV-2 to a low extent. SARS-CoV-2 induces aberrant presynaptic morphology and increases expression of the synaptic components Bassoon, latrophilin-3 (LPHN3) and fibronectin leucine-rich transmembrane protein-3 (FLRT3). Furthermore, we find that LPHN3-agonist treatment with Stachel partially restored organoid electrical activity and reverted SARS-CoV-2-induced aberrant presynaptic morphology. Finally, we observe accumulation of relatively static virions at LPHN3-FLRT3 synapses, suggesting that local hindrance can contribute to synaptic perturbations. Together, our study provides molecular insights into SARS-CoV-2-brain interactions, which may contribute to COVID-19-related neurological disorders.

Micro-CT and high-field MRI for studying very early post-mortem human fetal anatomy at 8 weeks of gestation.

OBJECTIVE: This study involved very early post-mortem (PM) examination of human fetal anatomy at 8 weeks of gestation (WG) using whole-body multimodal micro-imaging: micro-CT and high-field MRI (HF-MRI). We discuss the potential place of this imaging in early first-trimester virtual autopsy. METHODS: We performed micro-CT after different contrast-bath protocols including diffusible iodine-based contrast-enhanced (dice) and HF-MRI with a 9.4 T machine with qualitative and quantitative evaluation and obtained histological sections. RESULTS: Nine fetuses were included: the crown-rump length was 10-24 mm and corresponded to 7 and 9 WG according to the Robinson formula. The Carnegie stages were 17-21. Dice micro-CT and HF-MRI presented high signal to noise ratio, >5, according to the Rose criterion, and for allowed anatomical phenotyping in these specimens. Imaging did not alter the histology, allowing immunostaining and pathological examination. CONCLUSION: PM non-destructive whole-body multimodal micro-imaging: dice micro-CT and HF-MRI allows for PM human fetal anatomy study as early as 8 WG. It paves the way to virtual autopsy in the very early first trimester. Obtaining a precision phenotype, even regarding miscarriage products, allows a reverse phenotyping to select variants of interest in genome-wide analysis, offering potential genetic counseling for bereaved parents.

"Hemispheric pilocytic astrocytoma" revisited: A comprehensive clinicopathological and molecular series emphasizing their overlap with other glioneuronal tumors.

Pilocytic astrocytomas (PA) typically exhibit distinct clinical, radiological, histopathological, and genetic features. DNA-methylation profiling distinguishes PA according to their location (infratentorial, midline, hemispheric, or spinal). In the hemispheric location, distinguishing PA from glioneuronal tumors remains a common diagnostic challenge for neuropathologists. Furthermore, the current version of the DKFZ classifier seems to have difficulty separating them from gangliogliomas. In this study, after central radiological review, we identified a histopathologically defined set of PA (histPA, n = 11) and a cohort of DNA-methylation defined PA (mcPA, n = 11). Nine out of the 11 histPA matched the methylation class of hemispheric PA, whereas 2 cases were classified at the end of the study as dysembryoplastic neuroepithelial tumors. Similarly, the mcPA cohort contained tumors mainly classified as PA (7/11), but 4 cases were classified as glioneuronal. The analysis of the 16 tumors with an integrated diagnosis of PA revealed that they affect mainly children with a wide spectrum of radiological, histopathological (i.e. a predominantly diffuse growth pattern), and genetic characteristics (large range of mitogen-activated protein kinase alterations). Based on these results, we consider hemispheric PA to be different from their counterparts in other locations and to overlap with other glioneuronal tumors, reinforcing the necessity of interpreting all data to obtain an accurate diagnosis.

Prognostic impact of the number and total tumor burden of secondary cerebral lesions in patients with resected brain metastases of non-small cell lung cancers.

OBJECTIVE: Systemic therapeutic advancements have improved the prognosis of cancer patients, leading to surgery more frequently being carried out for patients with multiple brain metastases (BM). The underlying evidence for the strategy is currently lacking. This study aimed to evaluate the prognostic significance of the number of BM and total tumor burden (TTB) on the overall survival (OS) of patients with resected BM of non-small cell lung cancer (NSCLC) in a modern series. METHODS: In this monocentric retrospective series, patients who underwent resection of BM of NSCLC between 2015 and 2021 were included. Demographic, clinical, and histological parameters were collected, and formal radiological volumetric analyses were performed. Prognostic biomarkers for cerebral progression-free survival (C-PFS) and OS were analyzed with univariate and multivariate Cox proportional hazards analysis. RESULTS: One hundred eighty-four patients were included in the study. Among these, 108 patients (58.7%) presented with a single brain metastasis, 36 patients (19.6%) with 2 BM, 22 patients (11.9%) with 3 BM, and 18 patients (9.8%) with more than 3 BM (maximum 15 BM). The mean ± SD (range) preoperative tumor burden was 23.1 ± 25.3 (1.1-145.3) cm3. The mean residual tumor burden after surgery was 0.3 ± 0.8 (0.0-6.3) cm3. By the time of the analysis, 128 patients (69.6%) had died. The median follow-up duration was 49.0 months (95% CI 39.6-63.6). The median OS was 19.2 months (95% CI 13.2-24.0), and the survival rates at 6 months, 1 year, and 2 years were 76% (95% CI 69%-82%), 61% (95% CI 53%-67%), and 43% (95% CI 35%-50%), respectively. The median C-PFS was 8.4 months (95% CI 7.2-12.0). In the Cox multivariate regression model, younger age (< 65 years), single brain metastasis, adjuvant brain radiation therapy, adjuvant use of targeted therapy, and TTB < 7 cm3 were all independent predictors of longer OS. CONCLUSIONS: In this era of modern systemic treatments for cancer, the number of BM and total cerebral tumor burden remain significant prognostic factors of OS. However, resection should be considered as an option even in those patients with multiple BM in order to enhance patient clinical status, enable further local and systemic treatment delivery, and improve their survival and quality of life.

Identification of Metabolomic Markers in Frozen or Formalin-Fixed and Paraffin-Embedded Samples of Diffuse Glioma from Adults.

The aim of this study was to identify metabolomic signatures associated with the gliomagenesis pathway (IDH-mutant or IDH-wt) and tumor grade of diffuse gliomas (DGs) according to the 2021 WHO classification on frozen samples and to evaluate the diagnostic performances of these signatures in tumor samples that are formalin-fixed and paraffin-embedded (FFPE). An untargeted metabolomic study was performed using liquid chromatography/mass spectrometry on a cohort of 213 DG samples. Logistic regression with LASSO penalization was used on the frozen samples to build classification models in order to identify IDH-mutant vs. IDH-wildtype DG and high-grade vs low-grade DG samples. 2-Hydroxyglutarate (2HG) was a metabolite of interest to predict IDH mutational status and aminoadipic acid (AAA) and guanidinoacetic acid (GAA) were significantly associated with grade. The diagnostic performances of the models were 82.6% AUC, 70.6% sensitivity and 80.4% specificity for 2HG to predict IDH status and 84.7% AUC, 78.1% sensitivity and 73.4% specificity for AAA and GAA to predict grade from FFPE samples. Thus, this study showed that AAA and GAA are two novel metabolites of interest in DG and that metabolomic data can be useful in the classification of DG, both in frozen and FFPE samples.

Two novel tumours with NTRK2 fusion in the methylation class of extraventricular neurocytomas, including one intraventricular.

We report here about two novel tumours classified as extraventricular neurocytomas (EVN) using DNA-methylation profiling, associated with NTRK2 fusions instead of the usual FGFR1 alterations so far attributed to this tumoural entity. We present the second detailed case of an intraventricular presentation in the MC EVN. Our findings broaden the spectrum of MC EVN and have implications in terms of diagnosis, therapy and terminology.

Chronological interplay, clinical features, and treatments among patients with cancer and primary Sjögren's syndrome.

OBJECTIVE: While the incidence and type of blood malignancies are well documented amid primary Sjögren's syndrome patients (pSS), data focusing on solid neoplasms are more conflicting. We aimed to describe clinical, pathological, and immunological characteristics of pSS patients with cancers, along with the chronological interplay between the two conditions. METHODS: Outcomes concerning both pSS and cancer were retrospectively collected from Montpellier University Hospital (tertiary center) between 2019 and 2020. pSS characteristics were compared to a control group of pSS patients without cancer. RESULTS: A total of 165 patients with pSS were included: 55 patients with cancer (52 female, mean age 58.4 ± 10.4 years at pSS diagnosis; mean follow-up 10.5 ± 10.1 years, 12 patients had multiple cancers) and 110 controls without cancer. Characteristics of pSS patients with cancers were different from controls mostly for lymphoma prognosis factors. Among the 70 cancers, we recorded 55 solid neoplasms (whom 27 breast cancers and 8 lung cancers, and 82% of adenocarcinomas), with no evidence of disease at the end of follow-up in 85% of them. Among the 15 recorded blood malignancies, ten were lymphomas with an excellent prognosis. Regarding chronological interplay between cancer and pSS, most cancers (43%) were diagnosed close (± 5 years) to pSS diagnosis. Breast cancers were diagnosed before or close to pSS diagnosis (mean delay - 1.8 ± 13.0 years), at an early stage, with only two relapses (no cancer-related death), while lung cancers were diagnosed late after. CONCLUSIONS: The tight chronological interplay between breast cancer and pSS and the intriguing pathological and immunological pattern of pSS in these patients suggest a hypothesis of immune control of cancer.

Bevacizumab in recurrent WHO grades II-III glioma.

Purpose: The management of recurrent WHO grades II-III (rGII-III) glioma is not well established. This study describes the clinical outcomes in patients who received bevacizumab as rescue treatment. Methods: In this retrospective study, the main inclusion criteria were as follows: adult patients with histologicaly proved rGII-III glioma according 2016 WHO classification treated with bevacizumab from 2011 to 2019, T1 contrast enhancement on MRI. Efficacy was assessed using the high-grade glioma 2017 Response Assessment in Neuro-Oncology criteria. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Results: Eighty-one patients were included (M/F ratio: 1.7, median age at diagnosis: 38 years) among whom 46 (56.8%) had an initial diagnosis of grade II glioma. Previous treatments included at least one surgical intervention, radiotherapy (98.8%), and ≥ 2 chemotherapy lines (64.2%). After bevacizumab initiation, partial response, stable disease, and progressive disease were observed in 27.2%, 22.2%, and 50.6% of patients. The median PFS and OS were 4.9 months (95% confidence interval [CI] 3.7-6.1) and 7.6 months (95% CI 5.5-9.9). Bevacizumab severe toxicity occurred in 12.3%. Twenty-four (29.6%) patients discontinued bevacizumab without radiological progression. Oligodendroglioma and age ≥ 38 years at diagnosis were more frequent in this subgroup (odds ratio = 0.24, 95% CI 0.07-0.84, p = 0.023 and 0.36, 95% CI 0.13-0.99, p = 0.042). Ten of these 24 patients were alive at 12 months and two patients at 8 years after bevacizumab initiation, without any subsequent treatment. Conclusion: Bevacizumab can be an option for heavily pretreated patients with rGII-III glioma with contrast enhancement. In our study, bevacizumab displayed prolonged activity in a subgroup of patients.

Mitochondria Transfer from Mesenchymal Stem Cells Confers Chemoresistance to Glioblastoma Stem Cells through Metabolic Rewiring.

Glioblastomas (GBM) are heterogeneous tumors with high metabolic plasticity. Their poor prognosis is linked to the presence of glioblastoma stem cells (GSC), which support resistance to therapy, notably to temozolomide (TMZ). Mesenchymal stem cells (MSC) recruitment to GBM contributes to GSC chemoresistance, by mechanisms still poorly understood. Here, we provide evidence that MSCs transfer mitochondria to GSCs through tunneling nanotubes, which enhances GSCs resistance to TMZ. More precisely, our metabolomics analyses reveal that MSC mitochondria induce GSCs metabolic reprograming, with a nutrient shift from glucose to glutamine, a rewiring of the tricarboxylic acid cycle from glutaminolysis to reductive carboxylation and increase in orotate turnover as well as in pyrimidine and purine synthesis. Metabolomics analysis of GBM patient tissues at relapse after TMZ treatment documents increased concentrations of AMP, CMP, GMP, and UMP nucleotides and thus corroborate our in vitro analyses. Finally, we provide a mechanism whereby mitochondrial transfer from MSCs to GSCs contributes to GBM resistance to TMZ therapy, by demonstrating that inhibition of orotate production by Brequinar (BRQ) restores TMZ sensitivity in GSCs with acquired mitochondria. Altogether, these results identify a mechanism for GBM resistance to TMZ and reveal a metabolic dependency of chemoresistant GBM following the acquisition of exogenous mitochondria, which opens therapeutic perspectives based on synthetic lethality between TMZ and BRQ. Significance: Mitochondria acquired from MSCs enhance the chemoresistance of GBMs. The discovery that they also generate metabolic vulnerability in GSCs paves the way for novel therapeutic approaches.

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.

Clinico-pathological and epigenetic heterogeneity of diffuse gliomas with FGFR3::TACC3 fusion.

BACKGROUND: Gliomas with FGFR3::TACC3 fusion mainly occur in adults, display pathological features of glioblastomas (GB) and are usually classified as glioblastoma, IDH-wildtype. However, cases demonstrating pathological features of low-grade glioma (LGG) lead to difficulties in classification and clinical management. We report a series of 8 GB and 14 LGG with FGFR3:TACC3 fusion in order to better characterize them. METHODS: Centralized pathological examination, search for TERT promoter mutation and DNA-methylation profiling were performed in all cases. Search for prognostic factors was done by the Kaplan-Meir method. RESULTS: TERT promoter mutation was recorded in all GB and 6/14 LGG. Among the 7 cases with a methylation score > 0.9 in the classifier (v12.5), 2 were classified as glioblastoma, 4 as ganglioglioma (GG) and 1 as dysembryoplastic neuroepithelial tumor (DNET). t-SNE analysis showed that the 22 cases clustered into three groups: one included 12 cases close to glioblastoma, IDH-wildtype methylation class (MC), 5 cases each clustered with GG or DNET MC but none with PLNTY MC. Unsupervised clustering analysis revealed four groups, two of them being clearly distinct: 5 cases shared age (< 40), pathological features of LGG, lack of TERT promoter mutation, FGFR3(Exon 17)::TACC3(Exon 10) fusion type and LGG MC. In contrast, 4 cases shared age (> 40), pathological features of glioblastoma, and were TERT-mutated. Relevant factors associated with a better prognosis were age < 40 and lack of TERT promoter mutation. CONCLUSION: Among gliomas with FGFR3::TACC3 fusion, age, TERT promoter mutation, pathological features, DNA-methylation profiling and fusion subtype are of interest to determine patients' risk.

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.

GAB1 as a Marker of Recurrence in Anterior Skull Base Meningioma.

BACKGROUND: About one-third of anterior skull base meningiomas show Hedgehog pathway activation. We have recently identified GAB1 as a surrogate marker for Hedgehog pathway-activated meningiomas. OBJECTIVE: To determine the reproducibility and prognostic value of GAB1 marker in anterior skull base meningiomas. METHODS: A retrospective bicentric cohort of anterior skull base meningiomas, operated from 2005 to 2015, was constituted. GAB1 immunohistochemistry was performed in 2 centers, and the GAB1 score was assessed. Clinical and pathological data were reviewed to determine the prognostic value of the GAB1 score, along with classical factors of recurrence. RESULTS: One hundred forty-eight patients were included (median follow-up of 72 ± 46 months). 78% of patients had gross total resection. Eighty-four percentage of patients harbored grade 1 meningiomas. GAB1 immunohistochemistry was positive (ie, GAB1 staining score was >250) in 53 cases (35%). GAB1-positive cases were mainly at olfactory groove, of meningothelial grade 1 subtype, and showed greater recurrence (36% vs 14%, P = .002), greater requirement for multiple surgeries (17% vs 4.2%, P = .014), and more likely evolution toward diffuse skull base infiltration (15% vs 3%, P = .0017). By multivariable Cox regression analysis, incomplete surgical resection (hazard ratios [HR] = 8.3, 95% IC [3.7-18.2], P < .001), male sex (HR = 5.4, 95% IC [2.2-13.5], P < .001), GAB1 positivity (HR = 3.2, 95% CI [1.5-6.9], P = .004), and Ki67 index >4 (HR = 2.2, 95% IC [1.2-4.6], P = .035) were independent prognostic factors for recurrence. CONCLUSION: GAB1 marker is an independent prognostic factor for anterior skull base meningioma and could be useful for both prognostic evaluation and identification of Hedgehog-activated meningiomas.

Addressing the quality challenge of a human biospecimen biobank through the creation of a quality management system.

BACKGROUND: The objective of the COMET (COllection of MEtabolic Tissues) biobank project is to create a high-quality collection of insulin-sensitive tissues (liver, muscle, adipose tissues, and epiploic artery) and blood sample derivatives (plasma, serum, DNA and RNA), collected from 270 grade 2-3 obese patients undergoing bariatric surgery. Relevant data on patient such as clinical/biological characteristics and sample handling are also collected. For this, our aim was to establish a Quality Management System (QMS) to meet the reliability and quality requirements necessary for its scientific exploitation. MATERIALS AND METHODS: The COMET QMS includes: (1) Quality Assurance to standardize all stages of the biobanking process, (2) Quality Controls on samples from the first patients included in order to validate the sample management process and ensure reproducible quality; and 3) "in process" Quality Controls to ensure the reliability of the storage procedures and the stability of the samples over time. RESULTS: For serum and plasma, several corrective actions, such as temperature handling and centrifugation conditions, were made to the protocol and led to improvement of the volume and quality of samples. Regarding DNA, all samples evaluated achieved a satisfactory level of purity and integrity and most of them yielded the required DNA quantity. All frozen tissue samples had RNAs of good purity. RNA quality was confirmed by RIN, achieving values in most cases over 7 and efficient amplification of housekeeping genes by RT-qPCR, with no significant differences among samples from the same tissue type. In the "in process" Quality Controls, DNA, RNA, and histological integrity of tissues showed no differences among samples after different preservation times. CONCLUSION: Quality Control results have made it possible to validate the entire biobank process and confirm the utility of implementing QMS to guarantee the quality of a biospecimen collection.

A comprehensive profiling of the immune microenvironment of breast cancer brain metastases.

BACKGROUND: Despite potential clinical implications, the complexity of breast cancer (BC) brain metastases (BM) immune microenvironment is poorly understood. Through multiplex immunofluorescence, we here describe the main features of BCBM immune microenvironment (density and spatial distribution) and evaluate its prognostic impact. METHODS: Sixty BCBM from patients undergoing neurosurgery at three institutions (2003-2018) were comprehensively assessed using two multiplex immunofluorescence panels (CD4, CD8, Granzyme B, FoxP3, CD68, pan-cytokeratin, DAPI; CD3, PD-1, PD-L1, LAG-3, TIM-3, CD163, pan-cytokeratin, DAPI). The prognostic impact of immune subpopulations and cell-to-cell spatial interactions was evaluated. RESULTS: Subtype-related differences in BCBM immune microenvironment and its prognostic impact were observed. While in HR-/HER2- BM and HER2+ BM, higher densities of intra-tumoral CD8+ lymphocytes were associated with significantly longer OS (HR 0.16 and 0.20, respectively), in HR+/HER2- BCBMs a higher CD4+FoxP3+/CD8+ cell ratio in the stroma was associated with worse OS (HR 5.4). Moreover, a higher density of intra-tumoral CD163+ M2-polarized microglia/macrophages in BCBMs was significantly associated with worse OS in HR-/HER2- and HR+/HER2- BCBMs (HR 6.56 and 4.68, respectively), but not in HER2+ BCBMs. In HER2+ BCBMs, multiplex immunofluorescence highlighted a negative prognostic role of PD-1/PD-L1 interaction: patients with a higher percentage of PD-L1+ cells spatially interacting with (within a 20 µm radius) PD-1+ cells presented a significantly worse OS (HR 4.60). CONCLUSIONS: Our results highlight subtype-related differences in BCBM immune microenvironment and identify two potential therapeutic targets, M2 microglia/macrophage polarization in HER2- and PD-1/PD-L1 interaction in HER2+ BCBMs, which warrant future exploration in clinical trials.

Primal-dual for classification with rejection (PD-CR): a novel method for classification and feature selection-an application in metabolomics studies.

BACKGROUND: Supervised classification methods have been used for many years for feature selection in metabolomics and other omics studies. We developed a novel primal-dual based classification method (PD-CR) that can perform classification with rejection and feature selection on high dimensional datasets. PD-CR projects data onto a low dimension space and performs classification by minimizing an appropriate quadratic cost. It simultaneously optimizes the selected features and the prediction accuracy with a new tailored, constrained primal-dual method. The primal-dual framework is general enough to encompass various robust losses and to allow for convergence analysis. Here, we compare PD-CR to three commonly used methods: partial least squares discriminant analysis (PLS-DA), random forests and support vector machines (SVM). We analyzed two metabolomics datasets: one urinary metabolomics dataset concerning lung cancer patients and healthy controls; and a metabolomics dataset obtained from frozen glial tumor samples with mutated isocitrate dehydrogenase (IDH) or wild-type IDH. RESULTS: PD-CR was more accurate than PLS-DA, Random Forests and SVM for classification using the 2 metabolomics datasets. It also selected biologically relevant metabolites. PD-CR has the advantage of providing a confidence score for each prediction, which can be used to perform classification with rejection. This substantially reduces the False Discovery Rate. CONCLUSION: PD-CR is an accurate method for classification of metabolomics datasets which can outperform PLS-DA, Random Forests and SVM while selecting biologically relevant features. Furthermore the confidence score provided with PD-CR can be used to perform classification with rejection and reduce the false discovery rate.

SLUG and Truncated TAL1 Reduce Glioblastoma Stem Cell Growth Downstream of Notch1 and Define Distinct Vascular Subpopulations in Glioblastoma Multiforme.

Glioblastomas (GBM) are high-grade brain tumors, containing cells with distinct phenotypes and tumorigenic potentials, notably aggressive and treatment-resistant multipotent glioblastoma stem cells (GSC). The molecular mechanisms controlling GSC plasticity and growth have only partly been elucidated. Contact with endothelial cells and the Notch1 pathway control GSC proliferation and fate. We used three GSC cultures and glioma resections to examine the expression, regulation, and role of two transcription factors, SLUG (SNAI2) and TAL1 (SCL), involved in epithelial to mesenchymal transition (EMT), hematopoiesis, vascular identity, and treatment resistance in various cancers. In vitro, SLUG and a truncated isoform of TAL1 (TAL1-PP22) were strongly upregulated upon Notch1 activation in GSC, together with LMO2, a known cofactor of TAL1, which formed a complex with truncated TAL1. SLUG was also upregulated by TGF-ß1 treatment and by co-culture with endothelial cells. In patient samples, the full-length isoform TAL1-PP42 was expressed in all glioma grades. In contrast, SLUG and truncated TAL1 were preferentially overexpressed in GBMs. SLUG and TAL1 are expressed in the tumor microenvironment by perivascular and endothelial cells, respectively, and to a minor extent, by a fraction of epidermal growth factor receptor (EGFR) -amplified GBM cells. Mechanistically, both SLUG and truncated TAL1 reduced GSC growth after their respective overexpression. Collectively, this study provides new evidence for the role of SLUG and TAL1 in regulating GSC plasticity and growth.