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Background Metaplastic breast cancer (MBC) is a rare form of breast cancer characterized by an aggressive clinical presentation, with a poor response to standard chemotherapy. MBCs are typically triple-negative breast cancers (TNBCs), frequently with alterations to genes of the PI3K-AKT-mTOR and RTK-MAPK signaling pathways. The objective of this study was to determine the response to PI3K and MAPK pathway inhibitors in patient-derived xenografts (PDXs) of MBCs with targetable alterations. Methods We compared survival between triple-negative MBCs and other histological subtypes, in a clinical cohort of 323 TNBC patients. PDX models were established from primary breast tumors classified as MBC. PI3K-AKT-mTOR and RTK-MAPK pathway alterations were detected by targeted next-generation sequencing (NGS) and analyses of copy number alterations. Activation of the PI3K-AKT-mTOR and RTK-MAPK signaling pathways was analyzed with reverse-phase protein arrays (RPPA). PDXs carrying an activating mutation of PIK3CA and genomic changes to the RTK-MAPK signaling pathways were treated with a combination consisting of a PI3K inhibitor and a MEK inhibitor. Results In our clinical cohort, the patients with MBC had a worse prognosis than those with other histological subtypes. We established nine metaplastic TNBC PDXs. Three had a pathogenic mutation of PIK3CA and additional alterations to genes associated with RTK-MAPK signaling. The MBC PDXs expressed typical EMT and stem cell genes and were of the mesenchymal or mesenchymal stem-like TNBC subtypes. On histological analysis, MBC PDXs presented squamous or chondroid differentiation. RPPA analysis showed activation of the PI3K-AKT-mTOR and RTK-MAPK signaling pathways. In vivo, the combination of PI3K and MAPK inhibitors displayed marked antitumor activity in PDXs carrying genomic alterations of PIK3CA , AKT1 , BRAF , and FGFR4 . Conclusion The treatment of metaplastic breast cancer PDXs by activation of the PI3K-AKT-mTOR and RTK-MAPK pathways at the genomic and protein levels with a combination of PI3K and MEK inhibitors resulted in tumor regression in mutated models and may therefore be of interest for therapeutic purposes.

The transition of ductal carcinoma in situ (DCIS) to invasive breast carcinoma requires tumor cells to cross the basement membrane (BM). However, mechanisms underlying BM transmigration are poorly understood. Here, we report that expression of membrane-type 1 (MT1)-matrix metalloproteinase (MMP), a key component of the BM invasion program, increases during breast cancer progression at the in situ to invasive breast carcinoma transition. In the intraductal xenograft model, MT1-MMP is required for BM transmigration of MCF10DCIS.com breast adenocarcinoma cells and is overexpressed in cell clusters overlying focal BM disruptions and at the invasive tumor front. Mirrored upregulation of p63 and MT1-MMP is observed at the edge of MCF10DCIS.com xenograft tumors and p63 is required for induction of MT1-MMP-dependent invasive program in response to microenvironmental signals. Immunohistochemistry and analysis of public database reveal that p63 and MT1-MMP are upregulated in human basal-like breast tumors suggesting that p63/MT1-MMP axis contributes to progression of basal-like breast cancers with elevated p63 and MT1-MMP levels.

Background: RSPO ligands, activators of the Wnt/ β -catenin pathway, are overexpressed in different cancers. The objective of this study was to investigate the role of RSPOs in breast cancer (BC). Methods: Expression of RSPO and markers of various cancer pathways were measured in breast tumours and cell lines by qRT–PCR. The effect of RSPO on the Wnt/ β -catenin pathway activity was determined by luciferase assay, western blotting, and qRT–PCR. The effect of RSPO2 inhibition on proliferation was determined by using RSPO2 siRNAs. The effect of IWR-1, an inhibitor of the Wnt/ β -catenin pathway, was examined on the growth of an RSPO2 -positive patient-derived xenograft (PDX) model of metaplastic triple-negative BC. Results: We detected RSPO2 and RSPO4 overexpression levels in BC, particularly in triple-negative BC (TNBC), metaplastic BC, and triple-negative cell lines. Various mechanisms could account for this overexpression: presence of fusion transcripts involving RSPO , and amplification or hypomethylation of RSPO genes. Patients with RSPO2 -overexpressing tumours have a poorer metastasis-free survival ( P =3.6 × 10 −4 ). RSPO2 and RSPO4 stimulate Wnt/ β -catenin pathway activity. Inhibition of RSPO expression in a TN cell line inhibits cell growth, and IWR-1 significantly inhibits the growth of an RSPO2- overexpressing PDX. Conclusions: RSPO overexpression could therefore be a new prognostic biomarker and therapeutic target for TNBC.

Background: Ex vivo colospheres have been previously characterised as a colorectal cancer (CRC) well-rounded multicellular model, exclusively formed by carcinoma cells, and derived from fresh CRC tissue after mechanical dissociation. The ability to form colospheres was correlated with tumour aggressiveness. Their three-dimensional conformation prompted us to further investigate their potential interest as a preclinical cancer tool. Methods: Patient-derived CRC xenografts were used to produce numerous colospheres. Mechanism of formation was elucidated by confocal microscopy. Expression analysis of a panel of 64 selected cancer-related genes by real-time qRT–PCR and hierarchical clustering allowed comparison of colospheres with parent xenografts. In vitro and in vivo assays were performed for migration and chemosensitivity studies. Results: Colospheres, formed by tissue remodelling and compaction, remained viable several weeks in floating conditions, escaping anoikis through their strong cell–cell interactions. Colospheres matched the gene expression profile of the parent xenograft tissue. Colosphere-forming cells migrated in collagen I matrix and metastasised when subrenally implanted in nude mice. Besides, the colosphere responses to 5-fluorouracil and irinotecan, two standard drugs in CRC, reproduced those of the in vivo original xenografts. Conclusion: Colospheres closely mimic biological characteristics of in vivo CRC tumours. Consequently, they would be relevant ex vivo CRC models.

Background: Ataxia telangiectasia mutated (ATM) is a kinase that has a central role in the maintenance of genomic integrity by activating cell cycle checkpoints and promoting repair of DNA double-strand breaks (DSB). In breast cancer, a low level of ATM was correlated with poor outcome; however, the molecular mechanism of this downregulation is still unclear. Methods: We used qRT–PCR assay to quantify mRNA levels of ATM gene in 454 breast tumours from patients with known clinical/pathological status and outcome; reverse phase protein arrays (RPPA) were used to assess the levels of ATM and 14 proteins in 233 breast tumours. Results: ATM mRNA was associated with poor metastasis-free survival (MFS) ( P =0.00012) on univariate analysis. ATM mRNA and protein levels were positively correlated ( P =0.00040). A low level of ATM protein was correlated with poorer MFS ( P =0.000025). ATM expression at mRNA or protein levels are independent prognostic factors on multivariate analysis ( P =0.00046 and P =0.00037, respectively). The ATM protein level was positively correlated with the levels of six proteins of the DSB repair pathway: H2AX ( P <0.0000001), XRCC5 ( P <0.0000001), NBN ( P <0.0000001), Mre11 ( P =0.0000029), Rad50 ( P =0.0064), and TP53BP1 ( P =0.026), but not with proteins involved in other pathways that are altered in cancer. Low expression of ATM protein was significantly associated with high miR-203 expression ( P =0.011). Conclusion: We confirmed that ATM expression is an independent prognostic marker at both RNA and protein levels. We showed that alteration of ATM is involved in dysregulation of the DSB repair pathway. Finally, miR-203 may be responsible for downregulation of ATM in breast cancers.

Molecular methods are one of the most effective tools to monitor fungicide resistance. Long-read sequencing is an emerging technology in the field of plant pathology. We developed a PCR-based Oxford Nanopore Technologies amplicon sequencing method allowing the simultaneous detection and quantification of Plasmopara viticola variants conferring fungicide resistance to complex III inhibitors, zoxamide and oxathiapiprolin in the same vineyard population. Analysis of cyt b gene variants in natural P. viticola populations showed that almost all samples (23 out of 24 populations) collected in France contain variants G143A, S34L and/or E203-DE-V204 insertion. In the analysed populations, only cyt b reads with both substitutions S34L and G134A were detected at significant levels, suggesting the selection of resistant strains to both QoI fungicides and ametoctradin. French P. viticola population P36 with low sensitivity to oxathiapiprolin did not contain oxysterol binding protein sequences with both variants G770V and N837I, suggesting the presence of two different genotypes of P. viticola strain in this population. Zoxamide insensitivity associated with β-tubulin variants carrying the C239S substitution was detected in Italian vineyard populations but not in France.

Background Friedreich ataxia (FRDA) is the most frequent form of inherited ataxias. Vestibular and auditory assessments are not commonly part of the check up for these patients despite hearing and balance complaints. Screening of vestibular and auditory function was performed in a large group of young patients with genetically confirmed FRDA. Methods Our study included 43 patients (7–24 years of age). A complete vestibular assessment was performed including the canals function evaluation at 3 head velocities (bithermal caloric test, earth vertical axis rotation (EVAR) and head impulse test (HIT)) and otolith function evaluation (cervical vestibular evoked myogenic potentials). Information regarding the hearing evaluation of the patients were also retrieved including impedance tympanometry, distortion product otoacoustic emissions (DPOAEs), air and bone conduction audiometry and auditory brainstem response (ABR). Results Vestibular responses were impaired for canal responses (only at high and middle head velocities) and vestibulospinal otolithic responses. Abnormal neural conduction in the central auditory pathways was frequently observed. Oculomotor abnormalities were frequent, mostly hypermetric saccades and gaze instability. Inhibition of the vestibulo-ocular reflex by fixation was normal. Conclusions We show that Friedreich ataxia, even at onset, frequently associate saccadic intrusions, abnormal ABRs and decreased vestibulo-ocular and vestibulospinal responses progressing over time. These sensory impairments combined with ataxia further impair patient’s autonomy. These vestibular, auditory and visual impairments could be used as markers of the severity and progression of the disease. Adding vestibular and auditory testing to Friedreich patient’s evaluation may help physicians improve patient’s management.

Background: The Hedgehog (Hh) signalling pathway functions as an organiser in embryonic development. Recent studies have shown constitutive activation of this pathway in various malignancies, but its role in bladder cancer remains poorly studied. Methods: Expression levels of 31 genes and 9 microRNAs (miRNAs) involved in the Hh pathway were determined by quantitative real-time RT–PCR in 71 bladder tumour samples (21 muscle-invasive (MIBC) and 50 non-muscle-invasive (NMIBC) bladder cancers), as well as in 6 bladder cancer cell lines. Results: The SHH ligand gene and Gli-inducible target genes ( FOXM1, IGF2, OSF2, H19, and SPP1) were overexpressed in tumour samples as compared with normal bladder tissue. SHH overexpression was found in 96% of NMIBC and 52% of MIBC samples, as well as in two bladder cancer cell lines. Altered expression of miRNAs supported their oncogene or tumour-suppressor gene status. In univariate analysis, high expression levels of PTCH2 , miRNA-92A, miRNA-19A, and miRNA-20A were associated with poorer overall survival in MIBC ( P =0.02, P =0.012, P =0.047, and P =0.036, respectively). Conclusion: We observed constitutive activation of the Hh pathway in most NMIBC and about 50% of MIBC. We also found that some protein-coding genes and miRNAs involved in the Hh pathway may have prognostic value at the individual level.

Vergence abnormalities could lead to inappropriate vestibulo-ocular reflex (VOR), causing vertigo and imbalance (Brandt 1999). Indeed, a recent study by Anoh-Tanon et al. (2000) reported the existence of a population of children with symptoms of vertigo in the absence of vestibular dysfunction but with abnormal vergence findings in orthoptic tests. The purpose of this study was to examine in such children the accuracy, duration and mean velocity of vergence and saccades; additionally, for a few subjects, the effect of orthoptic vergence training on these parameters was also investigated. LEDs were used to stimulate saccades, pure vergence along the median plane and combined saccade-vergence movements. Movements from both eyes were recorded with a photoelectric device (Bouis). The results show that children with vertigo perform saccades as normal subjects of comparable age. In contrast, vergence, particularly convergence, shows abnormalities: poor accuracy, long duration and low speed. During combined movements, the well known reciprocal interaction between the saccade and the vergence is present only for saccades combined with divergence; for saccades combined with convergence such interaction is abnormal: the saccade is slowed down by the convergence but the convergence is not accelerated by the saccade. Orthoptic training improves significantly the accuracy of all eye movements; such improvement was significant for all types of eye movements except for divergence (pure and combined). Furthermore, convergence remains abnormal and the lack of acceleration by the saccade persists. These specific convergence deficits could be of both subcortical and cortical origin. Orthoptic training improves the accuracy presumably via visual attentional mechanisms, but cannot completely override deficits related to subcortical deficiencies.

Human pharmaceuticals, such as nonsteroidal anti-inflammatory drugs (NSAIDs), are an emerging threat to marine organisms. NSAIDs act through inhibition of cyclooxygenase (COX) conversion of arachidonic acid into prostaglandins. One experiment was carried out whereby marine mussels were exposed for 72 h to 1 and 100 μg/L diclofenac (DCF). A specific and sensitive method using liquid chromatography high-resolution tandem mass spectrometry was developed to quantify DCF in mussel tissues. The developed method could also clearly identify and quantify COX products, i.e., prostaglandin levels, and be used to assess their modulation following DCF exposure. Prostaglandin-D 2 (PGD 2 ) was always found below the detection limit (20 μg/kg dry weight (dw)). Basal prostaglandin-E 2 (PGE 2 ) concentrations ranged from below the detection limit to 202 μg/kg dw. Exposure of 100 μg/L resulted in a significant reduction in PGE 2 levels, whereas a downward trend was observed at 1 μg/L exposure. No difference was observed for prostaglandin-F 2 α (PGF 2α ) levels between controls and exposed organisms.