Abstract P3-08-15: Proteomic profile of PAM50 intermediate risk early breast cancers

International audienceBackgroundBreast cancer is a heterogeneous disease with a wide range of outcomes that are not fully predicted by routine clinical and pathologic features. The risk of recurrence of hormone receptor positive early-stage breast cancer, the most frequent tumor subtype, increases continuously over time. Genomic-based signatures have been developed to categorize patients according to their risk of recurrence and guide therapeutic decisions in this tumor subtype. However, the main genomic-based assays used in clinical practice classify up to 40% of the patients in an intermediate risk group. Many interrogations remain about the optimal strategy in this group. The aim of this study was to refine the molecular characterization of the intermediate risk group and determine the proportion of shared features with the low or high-risk groups using a mass spectrometry based proteomic approach.MethodsTumors with available routine PAM50 assay (PROSIGNA) results were selected from a cohort of breast cancer patients treated at Oscar Lambret Cancer Center (France). Fifteen tumors evenly split between PAM50 low, intermediate and high-risk groups were analyzed to determine the proteomic profiles of both cancer cells and stroma using MALDI mass spectrometry imaging combined with microproteomics, a spatially resolved proteomic technology.ResultsPAM50-Intermediate risk tumors had a distinctive proteomic profile compared to low and high-risk tumors. Heterogeneous nuclear ribonucleoproteins, 4-aminobutyrate aminotransferase, and pleckstrin homology-like domain family A member 1 are discriminating proteins between intermediate risk and low risk tumors. Differences were observed in expression of integrin beta-1, DNA replication licensing factors, splicing factors and interleukin enhancer-binding factor 2 between intermediate and high-risk tumors. Proteomic profiles of stroma according to tumor risk groups also showed differential protein expressions mainly between intermediate and high-risk groups. Breast cancer markers such as nuclear mitotic apparatus protein 1(NUMA1), C-1-tetrahydrofolate synthase (MTHFD1), cystatin-C (CST3), and T-cell immune regulator 1 (TCIRG1) were identified in high-risk tumors. Specific protein profiles were identified in stroma versus tumor. Immunoglobulin kappa chain, IGHG1, IGHM, IGHM, MMP2, ORM1 & ORM2, podocan, asprorin, immunoglobulin superfamily containing leucine-rich repeat protein (ISLR) were detected in stroma. By contrast, squamous cell carcinoma antigen recognized by T-cells 3 (SART3), shootin-1, mitotic checkpoint protein BUB3, XRCC5 &XRCC6, membrane-associated progesterone receptor component 2 (PGRMC) and hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) were specifically detected in tumor. Further analyses on an expanded cohort of patients will be presented.ConclusionMALDI mass spectrometry proteomics reveal distinctive tumor and microenvironment profiles in PAM50 intermediate risk early breast cancers

Abstract P3-08-19: Proteomic tracking of breast cancer metastasis progression

International audienceBackgroundMetastases from breast cancers have not yet reached curability mainly because of the continuous evolution of cancer cells leading to treatment resistance and tumor progression. Despite the identification of the main genomic drivers of breast cancer resistance, and the development of specific targeted therapies, resistance has not been eradicated. A deeper understanding of tumor biology underlying treatment escape and tumor progression is necessary to find new targets. The aim of this study was to determine whether mass spectrometry based imaging and microproteomics are able to capture changes in protein expression and pathways occurring during metastasis progression.MethodsTen tumor samples from five progressing metastatic breast cancer patients treated at Oscar Lambret Cancer Center (France) were selected based on the availability of paired metastasis biopsy performed at two time points during the evolution of the disease. The proteomic profiles of paired tumor samples were analyzed using MALDI mass spectrometry imaging combined with microproteomics, a spatially resolved proteomic technology.ResultsComparison of paired samples showed gain and loss of proteins associated with tumor progression. However, few were shared between patients. The pathways and biological processes mainly represented during tumor progression, and which were found in at least two patients, were those involved in the negative regulation of leukocyte mediated cytotoxicity, the metabolism of carboxilic acid, peptides and nucleosides, in response to stress, oxidation-reduction process, endocytosis, catabolic processes, actin regulation, and extracellular matrix organization. During metastasis evolution, few shared proteins were identified in at least 3 patients such as SRPX2, CILP1, collagen alpha-2(V) chain, Ras-related C3 botulinum toxin substrate 1, filamin-C, PDZ and LIM domain protein 2 and chloride intracellular channel protein 4. The main proteins lost during progression and found in at least 2 patients were cell surface glycoprotein MUC18, collagen alpha-2(IV) chain, polyadenylate-binding protein 2, and latent-transforming growth factor beta-binding protein 4 (LTAGP4). Results from an expanded cohort will be presented at the meeting.ConclusionMALDI mass spectrometry proteomics identified private and shared changes in proteins and biological processes associated with breast cancer metastasis progression

Proteomic characterisation of leech microglia extracellular vesicles (EVs): comparison between differential ultracentrifugation and Optiprep™ density gradient isolation

International audienceIn Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons