New Antibody-Free Mass Spectrometry-Based Quantification Reveals That C9ORF72 Long Protein Isoform Is Reduced in the Frontal Cortex of Hexanucleotide-Repeat Expansion Carriers

Frontotemporal dementia (FTD) is a fatal neurodegenerative disease characterized by behavioral and language disorders. The main genetic cause of FTD is an intronic hexanucleotide repeat expansion (G4C2)n in the C9ORF72 gene. A loss of function of the C9ORF72 protein associated with the allele-specific reduction of C9ORF72 expression is postulated to contribute to the disease pathogenesis. To better understand the contribution of the loss of function to the disease mechanism, we need to determine precisely the level of reduction in C9ORF72 long and short isoforms in brain tissue from patients with C9ORF72 mutations. In this study, we developed a sensitive and robust mass spectrometry (MS) method for quantifying C9ORF72 isoform levels in human brain tissue without requiring antibody or affinity reagent. An optimized workflow based on surfactant-aided protein extraction and pellet digestion was established for optimal recovery of the two isoforms in brain samples. Signature peptides, common or specific to the isoforms, were targeted in brain extracts by multiplex MS through the parallel reaction monitoring mode on a Quadrupole–Orbitrap high resolution mass spectrometer. The assay was successfully validated and subsequently applied to frontal cortex brain samples from a cohort of FTD patients with C9ORF72 mutations and neurologically normal controls without mutations. We showed that the C9ORF72 short isoform in the frontal cortices is below detection threshold in all tested individuals and the C9ORF72 long isoform is significantly decreased in C9ORF72 mutation carriers

A surprising tumor after cardiac surgery

International audienc

Métastase d'un carcinome mammaire dans un carcinome rénal. [Breast carcinoma metastasis into a renal cell carcinoma].

International audienceWe report the case of a patient carrying a right breast carcinoma whose imaging exams showed lung and bone metastasic release, and incidentally synchronous right renal tumor. Histologic examination of the renal tumor found a mammary carcinoma metastasis into a clear renal cell carcinoma. This is the second case report of breast cancer with metastasis in a resected renal clear cell carcinoma

Antifibrotics effects of nintedanib on lung fibroblasts derived from patients with progressive fibrosing intertitial lung diseases

International audienc

Immune genes are associated with human glioblastoma pathology and patient survival

International audienceBackground: Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults. Several recent transcriptomic studies in GBM have identified different signatures involving immune genes associated with GBM pathology, overall survival (OS) or response to treatment. Methods: In order to clarify the immune signatures found in GBM, we performed a co-expression network analysis that grouped 791 immune-associated genes (IA genes) in large clusters using a combined dataset of 161 GBM specimens from published databases. We next studied IA genes associated with patient survival using 3 different statistical methods. We then developed a 6-IA gene risk predictor which stratified patients into two groups with statistically significantly different survivals. We validated this risk predictor on two other Affymetrix data series, on a local Agilent data series, and using RT-Q-PCR on a local series of GBM patients treated by standard chemo-radiation therapy. Results: The co-expression network analysis of the immune genes disclosed 6 powerful modules identifying innate immune system and natural killer cells, myeloid cells and cytokine signatures. Two of these modules were significantly enriched in genes associated with OS. We also found 108 IA genes linked to the immune system significantly associated with OS in GBM patients. The 6-IA gene risk predictor successfully distinguished two groups of GBM patients with significantly different survival (OS low risk: 22.3 months versus high risk: 7.3 months; p < 0.001). Patients with significantly different OS could even be identified among those with known good prognosis (methylated MGMT promoter-bearing tumor) using Agilent (OS 25 versus 8.1 months; p < 0.01) and RT-PCR (OS 21.8 versus 13.9 months; p < 0.05) technologies. Interestingly, the 6-IA gene risk could also distinguish proneural GBM subtypes. Conclusions: This study demonstrates the immune signatures found in previous GBM genomic analyses and suggests the involvement of immune cells in GBM biology. The robust 6-IA gene risk predictor should be helpful in establishing prognosis in GBM patients, in particular in those with a proneural GBM subtype, and even in the well-known good prognosis group of patients with methylated MGMT promoter-bearing tumors

La protéinose alvéolaire pulmonaire

National audienc

Proteomic analysis underlines the usefulness of both primary adherent and stem-like cell lines for studying proteins involved in human glioblastoma

International audiencePrimary cell lines derived as neurospheres, enriched in cancer stem cells, are currently the focus of interest in glioblastoma to test new drugs, because of their tumor initiating abilities and resistance to conventional therapies. However, not all glioblastoma samples are propagatable under neurosphere culture and not all neurosphere cell lines are tumorigenic. These cells therefore cannot recapitulate the heterogeneity of glioblastoma samples. We have conducted a proteomic analysis of primary glioblastoma cell lines derived either as adherent cells in the presence of serum (n=11) or as neurospheres (n=12). A total of 963 proteins were identified by nano-LC/Q-TOF MS: 342 proteins were found only in neurosphere lines and were mostly implicated in various metabolic and cellular processes, while 112 proteins were found only in adherent cells and mostly linked to cell adhesion. A protein signature of 10 proteins, 9 of them involved in a cell adhesion pathway, characterized adherent lines. Neurospheres were characterized by 73 proteins mostly linked to DNA metabolic processes associated to cell cycle and protein metabolism. In the Repository of Molecular Brain Neoplasia Data, expression of genes coding for several proteins related to adherent cells or neurospheres were of prognostic relevance for glioblastoma. BIOLOGICAL SIGNIFICANCE: Primary cell lines enriched in cancer stem cells (CSC) have become popular models for testing new drugs for glioblastoma. In this proteomic study on an important number of cell lines obtained either as adherent cells in the presence of serum (a classic way to derive cell lines) or as neurospheres (enriched in CSC), we show that each type of cell line displays different GBM-specific features, highlighting that these two culture types are complementary tools for drug screening

Incidence of brain metastases in HER2+ gastric or gastroesophageal junction adenocarcinoma.

International audienceGastric or gastroesophageal junction (GEJ) adenocarcinoma represents a major health issue, being the 3rd cause of death by cancer in the world. In industrialized countries, the incidence of GEJ cancers continues to rise, while gastric cancers have declined over the past few decades.The human epidermal growth factor 2 (HER2) gene is a well-described proto-oncogene. HER2 gene amplification and protein overexpression have been mostly studied in breast cancer, where HER2 overexpression was reported in 15-20% of cases.In breast cancer, HER2 overexpression is associated with a higher risk of brain metastases and significantly worse survival [1]. The prognostic value of HER2 overexpression in gastric and GEJ adenocarcinomas remains controversial. A recently published systematic review was inconclusive in patients after curative surgery [2]. The standard of care in advanced gastric carcinoma patients has changed recently, with the results of the ToGa phase III trial adding trastuzumab to chemotherapy in patients with HER2 overexpression [3]. The study indicated that this subgroup of patients benefit from trastuzumab with a three months improvement of overall survival.The aim of this study was to seek if the correlation between HER2 overexpression and brain metastases exists in gastric or GEJ adenocarcinomas, where brain metastases are uncommon

Arsenic trioxide inhibits the functions of lung fibroblasts derived from patients with idiopathic pulmonary fibrosis

International audienceIdiopathic pulmonary fibrosis (IPF) is a chronic and fatal interstitial lung disease. Currently, no treatment can block or reverse the development of lung fibrosis in patients suffering from IPF. Recent studies indicate that arsenic trioxide (ATO), a safe, effective anti-cancer pro-oxidant drug, prevents the differentiation of normal human lung fibroblasts (NHLFs) in vitro and reduces experimental pulmonary fibrosis in vivo. In this context, we investigated the anti-fibrotic effects of ATO on the main fibrosis functions of human lung fibroblasts (HLFs) isolated from patients with IPF. IPF and non-IPF (control) HLFs were incubated with 0.01-1 μM ATO and stimulated with pro-fibrotic factors (PDGF-BB or TGF-β1). We measured their rates of proliferation, migration and differentiation and the cell stress response triggered by ATO. ATO did not affect cell viability but strongly inhibited the proliferation and migration of PDGF-BB-stimulated IPF and control HLFs. ATO also prevented myofibroblastic differentiation, as assessed by the expression of α-smooth muscle actin (α-SMA) and collagen-1, and the phosphorylation of SMAD2/3 in TGF-β1-stimulated HLFs. These antifibrotic effects were associated with increased expression of the transcription factor NRF2 and its target genes NQO1 and HMOX1. Genetic silencing of NRF2 inhibited the ATO-induced cell stress response but did not prevent the ATO-dependent inhibition of α-SMA expression in TGF-β1-stimulated HLFs. The results demonstrate that ATO, at concentrations similar to exposure in blood plasma of ATO-treated cancer patients, counteracted pro-fibrotic activities of HLFs from IPF patients. We propose to consider ATO for clinical exploration to define the therapeutic potential in patients with IPF