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The primary olfactory nervous system is unique in that it continuously renews itself and regenerates after injury. These properties are attributed to the presence of olfactory glia, termed olfactory ensheathing cells (OECs). Evidence is now emerging that individual OEC populations exist with distinct anatomical localisations and physiological properties, but their differential roles have not been determined. Unlike other glia, OECs can migrate from the periphery into the central nervous system, and organised OEC migration can enhance axonal extension after injury. Despite this, the mechanisms regulating OEC migration are largely unknown. Here, we provide an overview of the roles of OECs in development and adulthood. We review the latest research describing the differences between individual OEC subpopulations and discuss potential regulatory mechanisms for OEC guidance and migration. Using advanced time lapse techniques, we have obtained novel insights into how OECs behave in a complex multicellular environment which we discuss here with particular focus on cell-cell interactions. Significantly, transplantation of OECs constitutes a promising novel therapy for nerve injuries, but results are highly variable and the method needs improvement. We here review the roles of transplanted OECs in neural repair of damaged neuronal tracts distinct from the primary olfactory nervous system.

Background The olfactory mucosa, the organ of smell in the nose, is a neural tissue that regenerates new sensory neurons throughout adult life. Based on this tissue, we previously demonstrated increased mitosis in olfactory biopsy cultures from schizophrenia patients compared with healthy control subjects. In addition, neural stem/progenitor cell cultures (neurosphere-derived cells) from nasal biopsies from individuals with schizophrenia show significantly altered gene and protein expression in key cell cycle control pathways. Methods The aim of this study was to investigate cell cycle dynamics in olfactory neurosphere-derived cells from nine male schizophrenia patients and nine male healthy control subjects. Cell cycles were arrested by serum deprivation after which cell population doubling time, proliferation fraction, and cell cycle period were calculated from cell counts over 96 hours. Cell cycle phase was investigated using flow cytometry. Cell lysates were analyzed for expression of cyclin proteins. Results Cell population proliferation rate was increased in schizophrenia through a larger pool of proliferating progenitors and a reduced cell cycle period. All phases of the cell cycle were phase-shifted by 2 hours in the schizophrenia-derived cells, which expressed higher levels of the cyclins D1, E, and A2. Conclusions Our observations indicate that schizophrenia is associated with subtle alterations in cell cycle dynamics, shortening of the cell cycle period, and increased expression of G1/S phase cyclins. We speculate that this underlying diathesis could alter the temporal and spatial cascade of brain development and contribute to an altered neurodevelopmental trajectory in schizophrenia.

Olfactory ensheathing cells (OECs) support the regeneration of olfactory sensory neurons throughout life, however, it remains unclear how OECs respond to a major injury. We have examined the proliferation and migration of OECs following unilateral bulbectomy in postnatal mice. S100ß-DsRed and OMP-ZsGreen transgenic mice were used to visualize OECs and olfactory neurons, respectively, and we used the thymidine analogue ethynyl deoxyuridine (EdU) to identify cells that were proliferating at the time of administration. Following unilateral bulbectomy, there was an initial phase of OEC proliferation throughout the olfactory pathway with a peak of proliferation occurring 2 to 7 days after the injury. A second phase of proliferation also occurred in which precursors localized within the olfactory mucosa divided to replenish the OEC population. We then tracked the positions of OECs that had proliferated and found that there was a progressive increase in OECs in the cavity for at least 12 to 16 days after injury which could not be accounted for solely by local proliferation of OECs within the cavity. These results suggest that OECs migrated from the peripheral olfactory nerve to populate the mass of cells that filled cavity left by bulbectomy. Our results demonstrate that following injury to the olfactory nervous system, the OEC population is replenished by migration of cells that arise from both local proliferation of OECs throughout the olfactory nerve pathway as well as from precursor cells in the olfactory mucosa.

Adenoid cystic carcinoma (AdCC) is a rare form of triple-negative and basal-like breast cancer that has an indolent clinical behaviour. Four breast AdCCs were recently shown to harbour the recurrent chromosomal translocation t(6;9)(q22-23;p23-24), which leads to the formation of the MYB-NFIB fusion gene. Our aims were (i) to determine the prevalence of the MYB-NFIB fusion gene in AdCCs of the breast; (ii) to characterize the gene copy number aberrations found in AdCCs; and (iii) to determine whether AdCCs are genomically distinct from histological grade-matched or triple-negative and basal-like invasive ductal carcinomas of no special type (IDC-NSTs). The presence of the MYB-NFIB fusion gene was investigated in 13 AdCCs of the breast by fluorescence in situ hybridization (FISH) and reverse transcriptase-PCR (RT-PCR), and MYB and BRCA1 RNA expression was determined by quantitative RT-PCR. Fourteen AdCCs, 14 histological grade-matched IDC-NSTs, and 14 IDC-NSTs of triple-negative and basal-like phenotype were microdissected and subjected to high-resolution microarray-based comparative genomic hybridization (aCGH). The MYB-NFIB fusion gene was detected in all but one AdCC. aCGH analysis demonstrated a relatively low number of copy number aberrations and a lack of recurrent amplifications in breast AdCCs. Contrary to grade-matched IDC-NSTs, AdCCs lacked 1q gains and 16q losses, and in contrast with basal-like IDC-NSTs, AdCCs displayed fewer gene copy number aberrations and expressed MYB and BRCA1 at significantly higher levels. Breast AdCCs constitute an entity distinct from grade-matched and triple-negative and basal-like IDC-NSTs, emphasizing the importance of histological subtyping of triple-negative and basal-like breast carcinomas.

Traumatic injury to the brain initiates an increase in astrocyte and microglial infiltration as part of an inflammatory response to injury. Increased astrogliosis around the injury impedes regeneration of axons through the injury, while activated microglia release inflammatory mediators. The persistent inflammatory response can lead to local progressive cell death. Modulating the astrocyte and microglial response to traumatic injury therefore has potential therapeutic benefit in brain repair. We examine the modulatory effect of a single bolus of vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) in combination on astrocytes and microglia to acute cerebral injury. A combination of VEGF and PDGF (20 pg) was injected into the striatum of adult male Sprague-Dawley rats. The effects of treatment were assessed by quantitative immunofluorescence microscopy analyzing astrocytes and microglia across the stab injury over time. Treatment delayed the onset of astrogliosis in the centre and edge of the stab injury up to day 5; however, increased astrogliosis at areas remote to the stab injury up to day 5 was observed. A persistent astrocytic response was observed in the centre and edge of the stab injury up to day 60. Treatment altered microglia cell morphology and numbers across the stab injury, with a decrease in ramified microglia, but an increase in activated and phagocytic microglia up to day 5 after stab injury. The increased microglial response from 10 until day 60 was comprised of the ramified morphology. Thus, VEGF and PDGF applied at the same time as a stab injury to the brain initially delayed the inflammatory response up to day 5 but evoked a persistent astrogliosis and microglial response up to 60 days.

Purpose: The oncogenic drivers of triple-negative (TN) and basal-like breast cancers are largely unknown. Substantial evidence now links aberrant signaling by the fibroblast growth factor receptors (FGFR) to the development of multiple cancer types. Here, we examined the role of FGFR signaling in TN breast cancer. Experimental Design: We examined the sensitivity of a panel of 31 breast cancer cell lines to the selective FGFR inhibitor PD173074 and investigated the potential mechanisms underlying sensitivity. Results: TN breast cancer cell lines were more sensitive to PD173074 than comparator cell lines (P = 0.011), with 47% (7/15) of TN cell lines showing significantly reduced growth. The majority of TN cell lines showed only modest sensitivity to FGFR inhibition in two-dimensional growth but were highly sensitive in anchorage-independent conditions. PD173074 inhibited downstream mitogen-activated protein kinase and PI3K–AKT signaling and induced cell-cycle arrest and apoptosis. Basal-like breast cancer cell lines were found to express FGF2 ligand (11/21 positive) and, similarly, 62% of basal-like breast cancers expressed FGF2, as assessed by immunohistochemistry compared with 5% of nonbasal breast cancers (P < 0.0001). RNA interference targeting of FGF2 in basal-like cell lines significantly reduced growth in vitro and reduced down stream signaling, suggesting an autocrine FGF2 signaling loop. Treatment with PD173074 significantly reduced the growth of CAL51 basal-like breast cancer cell line xenografts in vivo. Conclusions: Basal-like breast cancer cell lines, and breast cancers, express autocrine FGF2 and show sensitivity to FGFR inhibitors, identifying a potential novel therapeutic approach for these cancers. Clin Cancer Res; 17(16); 5275–86. ©2011 AACR.

The 11q13-q14 locus is frequently amplified in human cancers, with a complex structure harbouring multiple potential oncogenic drivers. The EMSY gene has been proposed as a driver of the third core of the 11q13-q14 amplicon. This gene encodes a protein reported to be a BRCA2-binding partner, which when over-expressed would lead to impairment of BRCA2 functions and could constitute a mechanism for BRCA2 inactivation in non-hereditary breast and ovarian cancers. We hypothesized that if EMSY amplification abrogates BRCA2 functions, cells harbouring this aberration would be unable to elicit competent homologous recombination DNA repair and, therefore, may have increased sensitivity to genotoxic therapies and potent PARP inhibitors. Microarray-based comparative genomic hybridization of cell lines from distinct tumour sites, including breast, ovary, pancreas, oesophagus, lung and the oral cavity, led to the identification of 10 cell lines with EMSY amplification and 18 without. EMSY amplification was shown to correlate with EMSY mRNA levels, although not all cell lines harbouring EMSY amplification displayed EMSY mRNA or protein over-expression. RNA interference-mediated silencing of EMSY did not lead to a reduction in cell viability in tumour models harbouring EMSY amplification. Cell lines with and without EMSY amplification displayed a similar ability to elicit RAD51 foci in response to DNA damaging agents, and similar sensitivity to cisplatin and olaparib. Taken together, this suggests that EMSY is unlikely to be a driver of the 11q13-q14 amplicon and does not have a dominant role in modulating the response to agents targeting cells with defective homologous recombination.

Triple-negative breast cancers account for 12-17% of all invasive breast carcinomas and comprise a heterogeneous group of tumours, with varying histological features and clinical behaviours. Focal apocrine differentiation can be associated with a subset of these lesions. To establish whether morphological diversity is associated with divergent genetic aberrations the genomic profiles of microdissected, morphologically distinct components from an invasive ductal carcinoma of no special type with triple-negative phenotype and a region of apocrine differentiation were determined by high-resolution microarray-based comparative genomic hybridisation and validated by fluorescence in-situ hybridisation. Morphologically distinct components were found to harbour differing genetic aberrations, with the region of apocrine differentiation demonstrating genomic gains and losses on chromosome arms 9p and 9q, respectively, not present in non-apocrine areas. The results provide additional direct evidence of intra-tumour genetic heterogeneity in breast cancer and demonstrate that morphologically distinct regions can be associated with distinct genetic aberrations.

Injury to cervical dorsal roots mimics the deafferentation component of brachial plexus injury in humans, with intractable neuropathic pain in the deafferented limb being a common consequence. Such lesions are generally not amenable to surgical repair. The use of olfactory ensheathing cells (OECs) for dorsal root repair, via acute transplantation, has been successful in several studies. From a clinical point of view, delayed transplantation of OECs would provide a more realistic timeframe for repair. In this study we investigated the effect of delayed OEC transplantation on functional recovery of skilled forepaw movements and amelioration of neuropathic pain, using a C7 and C8 dorsal root injury rat model previously established in our lab. We found that OEC transplantation to the dorsal horn 1. week after root injury effectively attenuated neuropathic disturbances associated with dorsal root injury, including spontaneous pain behavior, tactile allodynia and thermal hyperalgesia. The sensory controls of complex, goal-oriented skilled reaching and ladder walking, however, were not improved by delayed OEC transplantation. We did not detect any significant influence of transplanted OECs on injury-induced central reorganisation and afferent sprouting. The anti-nociceptive effect mediated by OEC transplants may therefore be explained by alternative mechanisms such as modification of inflammation and astrogliosis. The significant effect of OEC transplants in mitigating neuropathic pain may be clinically useful in intractable pain syndromes arising from deafferentation. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair. © 2010.

Purpose: Ovarian clear cell carcinomas (OCCC) are a drug-resistant and aggressive type of epithelial ovarian cancer. We analyzed the molecular genetic profiles of OCCCs to determine whether distinct genomic subgroups of OCCCs exist. Experimental design: Fifty pure primary OCCCs were subjected to high-resolution microarray-based comparative genomic hybridization (aCGH). Unsupervised hierarchical clustering using Ward's linkage analysis was performed to identify genomic subgroups of OCCCs. Survival analysis was performed using Kaplan–Meier method and log-rank test. Cox-regression analysis was used to identify independent predictors of outcome. Differentially amplified regions between genomic subgroups of OCCCs were identified using a multi-Fisher's exact test. Results: Hierarchical cluster analysis revealed two distinct clusters of OCCCs with different clinical outcomes. Patients from cluster-1 had a significantly shorter median progression-free survival (PFS) than those from cluster-2 (11 vs. 65 months, P = 0.009), although estimates for ovarian cancer–specific survival (OCS) did not reach statistical significance (P = 0.065). In multivariate analysis, suboptimal debulking surgery and genomic cluster were independently prognostic for PFS. Recurrently amplified genomic regions with a significantly higher prevalence in cluster-1 than cluster-2 OCCCs were identified and validated. HER2 gene amplification and protein overexpression was observed in 14% of OCCCs, suggesting that this may constitute a potential therapeutic target for a subgroup of these tumors. Conclusions: OCCCs constitute a heterogeneous disease at the genomic level despite having similar histological features. The pattern of genomic aberrations in subgroups of OCCCs is of clinical significance. We have identified recurrently amplified regions that may harbor potential therapeutic targets for subgroups of OCCCs. Clin Cancer Res; 17(6); 1521–34. ©2011 AACR.

Background Breast cancers can be classified by hierarchical clustering using an "intrinsic" gene list into one of at least five molecular subtypes: basal-like, HER2, luminal A, luminal B, and normal breast-like. Five different intrinsic gene lists composed of varying numbers of genes have been used for molecular subtype identification and classification of breast cancers. The aim of this study was to determine the objectivity and interobserver reproducibility of the assignment of molecular subtype classes by hierarchical cluster analysis. Methods Three publicly available breast cancer datasets (n = 779) were subjected to two-way average-linkage hierarchical cluster analysis using five distinct intrinsic gene lists. We used free-marginal Kappa statistics to analyze interobserver agreement among five breast cancer researchers for the whole classification and for each molecular subtype separately according to each intrinsic gene list for each breast cancer dataset. Results None of the classification systems tested produced almost perfect agreement (Kappa ≥ 0.81) among observers. However, substantial interobserver agreement (70.8% to 76.1% of the samples and free-marginal Kappa scores from 0.635 to 0.701) was consistently observed in all datasets for four molecular subtypes (luminal, basal-like, HER2, and normal breast-like). When luminal cancers were subdivided (luminal A, B, and C), none of the classification systems produced substantial agreement (Kappa ≥ 0.61) in all the datasets analyzed. Analysis of each subtype separately revealed that only two (basal-like and HER2) could be reproducibly identified by independent observers (Kappa ≥ 0.81). Conclusions Assignment of molecular subtype classes of breast cancer based on the analysis of dendrograms obtained with hierarchical cluster analysis is subjective and shows modest interobserver reproducibility. For the development of a molecular taxonomy, objective definitions for each molecular subtype and standardized methods for their identification are required.

Research into the biology of the mammalian olfactory system would be greatly enhanced by transgenic reporter mice with cell-specific fluorescence. To this end we previously generated a mouse whose olfactory ensheathing cells (OECs) express DsRed driven by the S100ß promoter. We present here a transgenic reporter mouse whose olfactory sensory neurons express ZsGreen, driven by the olfactory marker protein (OMP) promoter. ZsGreen was very strongly expressed throughout the cytoplasm of olfactory sensory neurons labelling them in living cells and after fixation. Labelled sensory neurons were seen in all olfactory regions in the nose and fluorescent axons coursed through the lamina propria and into the main and accessory bulbs. We developed methods for culturing embryonic and postnatal olfactory sensory neurons using these mice to visualise living cells in vitro. ZsGreen was expressed along the length of axons providing exceptional detail of the growth cones. The ZsGreen fluorescence was very stable, without fading during frequent imaging. The combination of OMP-ZsGreen and S100ß-DsRed transgenic mice is ideal for developmental studies and neuron-glia assays and they can be bred with mutant mice to dissect the roles of various molecules in neurogenesis, differentiation, axon growth and targeting and other aspects of olfactory sensory neuron and glia biology.

Axons of primary olfactory neurons are intimately associated with olfactory ensheathing cells (OECs) from the olfactory epithelium until the final targeting of axons within the olfactory bulb. However, little is understood about the nature and role of interactions between OECs and axons during development of the olfactory nerve pathway. We have used high resolution time-lapse microscopy to examine the growth and interactions of olfactory axons and OECs in vitro. Transgenic mice expressing fluorescent reporters in primary olfactory axons (OMP-ZsGreen) and ensheathing cells (S100ß-DsRed) enabled us to selectively analyse these cell types in explants of olfactory epithelium. We reveal here that rather than providing only a permissive substrate for axon growth, OECs play an active role in modulating the growth of pioneer olfactory axons. We show that the interactions between OECs and axons were dependent on lamellipodial waves on the shaft of OEC processes. The motility of OECs was mediated by GDNF, which stimulated cell migration and increased the apparent motility of the axons, whereas loss of OECs via laser ablation of the cells inhibited olfactory axon outgrowth. These results demonstrate that the migration of OECs strongly regulates the motility of axons and that stimulation of OEC motility enhances axon extension and growth cone activity.

Aberrant β-catenin expression as determined by assessment of its subcellular localization constitutes a surrogate marker of Wnt signalling pathway activation and has been reported in a subset of breast cancers. The association of β-catenin/Wnt pathway activation with clinical outcome and the mechanisms leading to its activation in breast cancers still remain a matter of controversy. The aims of this study were to address the distribution of β-catenin expression in invasive breast cancers, the correlations between β-catenin expression and clinicopathological features and survival of breast cancer patients, and to determine whether aberrant β-catenin expression is driven by CTNNB1 (β-catenin encoding gene) activating mutations. Immunohistochemistry was performed on a tissue microarray containing 245 invasive breast carcinomas from uniformly treated patients, using two anti-β-catenin monoclonal antibodies. Selected samples were subjected to CTNNB1 exon 3 mutation analysis by direct gene sequencing. A good correlation between the two β-catenin antibodies was observed (Spearman's r >0.62, P

Paediatric high grade glioma and diffuse midline glioma (including DIPG) are comprised of multiple biological and clinical subgroups, the majority of which urgently require novel therapies. Patient-derived in vitro primary cell cultures represent potentially useful tools for mechanistic and preclinical investigation based upon their retention of key features of tumour subgroups under experimental conditions amenable to high-throughput approaches. We established 21 novel primary cultures derived from patients in London, Dublin and Rome, and together with cultures shared from Barcelona, Brisbane and Stanford we assembled a panel of 42 models under 2D (laminin matrix) and/or 3D (neurospheres) conditions, fully credentialed by phenotypic and molecular comparison to the original tumour sample (methylation BeadArray, panel/exome sequencing, RNAseq). Screening against a panel of ~400 approved chemotherapeutics and small molecules, we identified specific dependencies associated with tumour subgroups and/or specific molecular markers. This allowed for functional annotation of distinct variants in human tumours, for example cells with sensitizing (HSJD-GBM-001, PDGFRA_A385ins; HSJD-DIPG-008, PDGFRA_D846N) or resistance (HSJD-GBM-002, PDGFRA_D842Y) mutations to a range of PDGFRA inhibitors. We found individual models showing profound sensitivity to distinct kinase inhibitors based upon cell-specific mechanisms of activation, such as QCTB-R006 to multiple FGFR-targeted drugs, and HSJD-DIPG-012 to those directed against EGFR. Subclasses with functionally relevant pathway-based dependencies included sensitivity of DIPGs with PPM1Dmutation (HSJD-DIPG-008, HSJD-DIPG-014) to PARP and MDM2 inhibitors, and MAPK-dysregulated PXA-like cultures (ICR-CXJ-008, ICR-CXJ015) differentially responsive to inhibitors of upstream signalling viaPKC and CK2. Of note, all cultures were insensitive to temozolomide. In total, 85% cells were found to have at least one drug screening hit in short term assays linked to the underlying biology of the patient’s tumour, providing a rational approach for individualised clinical translation.

Olfactory ensheathing cells (OECs) migrate with olfactory axons that extend from the nasal epithelium into the olfactory bulb. Unlike other glia, OECs are thought to migrate ahead of growing axons instead of following defined axonal paths. However it remains unknown how the presence of axons and OECs influences the growth and migration of each other during regeneration. We have developed a regeneration model in neonatal mice to examine whether (i) the presence of OECs ahead of olfactory axons affects axonal growth and (ii) the presence of olfactory axons alters the distribution of OECs. We performed unilateral bulbectomy to ablate olfactory axons followed by methimazole administration to further delay neuronal growth. In this model OECs filled the cavity left by the bulbectomy before new axons extended into the cavity. We found that delaying axon growth increased the rate at which OECs filled the cavity. The axons subsequently grew over a significantly larger region and formed more distinct fascicles and glomeruli in comparison with growth in animals that had undergone only bulbectomy. In vitro, we confirmed (i) that olfactory axon growth was more rapid when OECs were more widely distributed than the axons and (ii) that OECs migrated faster in the absence of axons. These results demonstrate that the distribution of OECs can be increased by repressing by growth of olfactory axons and that olfactory axon growth is significantly enhanced if a permissive OEC environment is present prior to axon growth.

Rotenone, a mitochondrial complex 1 inhibitor, causes oxidative damage via production of reactive oxygen species. We examined the pathophysiology of neuronal and glial cells of the nigrostriatal pathway following unilateral infusion of varying doses of rotenone into the substantia nigra or medial forebrain bundle of adult male Sprague-Dawley rats, sacrificed 14 and 60 days after infusion. Immunofluorescence techniques were used to qualitatively and quantitatively assay dopaminergic neurons, their projections, glial cells, synapses, and oxidative stress. Rotenone infusion into the substantia nigra at all concentrations caused extensive damage and tissue necrosis, therefore of limited relevance for producing a Parkinson disease model. Infusion of 0.5μg of rotenone targeting the medial forebrain bundle induced oxidative stress in dopaminergic neurons causing ongoing cell stress as defined by an elevation of stress granule and oxidative stress markers. This treatment resulted in the loss of tyrosine hydroxylase immunoreactive cells in the substantia nigra (p≤0.01) and loss of tyrosine hydroxylase immunoreactive nerve fibres and synaptic specialisations in the striatum (p≤0.01). The infusion of 0.5μg of rotenone also caused an increase in astrocytes and microglial cells in the substantia nigra in comparison to control (p≤0.01). We examined the time-dependent reduction of tyrosine hydroxylase-positive nerve fibres and cell bodies in the striatum and substantia nigra respectively, with a progressive reduction evident 60days after infusion (p≤0.01, p≤0.05). Dopaminergic axons exposed to low-dose rotenone undergo oxidative stress, with a resultant ongoing loss of dopaminergic neurons, providing an animal model relevant to Parkinson disease.

SummaryBreast cancers in BRCA1 mutation carriers frequently have a distinctive basal-like phenotype. It has been suggested that this results from an origin in basal breast epithelial stem cells. Here, we demonstrate that deleting Brca1 in mouse mammary epithelial luminal progenitors produces tumors that phenocopy human BRCA1 breast cancers. They also resemble the majority of sporadic basal-like breast tumors. However, directing Brca1 deficiency to basal cells generates tumors that express molecular markers of basal breast cancers but do not histologically resemble either human BRCA1 or the majority of sporadic basal-like breast tumors. These findings support a derivation of the majority of human BRCA1-associated and sporadic basal-like tumors from luminal progenitors rather than from basal stem cells. They also demonstrate that when target cells for transformation have the potential for phenotypic plasticity, tumor phenotypes may not directly reflect histogenesis. This has important implications for cancer prevention strategies.

Liquid biopsy components from blood, such as cell free DNA (cfDNA) and circulating tumor cells (CTCs), are prognostic for overall survival in advanced prostate cancer patients and allow the study of clonal evolution. cfDNA is easily obtained and has been widely used for molecular characterization and reflects pooled genomic profiles in a patient, but has limitations regarding gene copy number calls. CTC single-cell genomic studies generate precise gene copy number calls and elucidate intrapatient intercellular genomic heterogeneity. The main limitation of CTC analyses has been the low CTC count found in many cancer patients. We elected to study whether liquid biopsy by apheresis in advanced prostate cancer patients increases the yield of CTC to study tumor genomics, intrapatient heterogeneity, and ex vivo organotypic 3D models. Advanced metastatic prostate cancer patients being considered for clinical trials were invited to consent to apheresis. Apheresis CTC counts using CellSearchTM (Menarini) were acquired from 16 patients. The contents of the CellSearch cartridges were sorted into pure single cells by fluorescence-activated cell sorting and subsequently assessed by array comparative genomic hybridization (aCGH, Agilent Technology) for copy number aberrations (CNA). Exome and aCGH from tissue biopsies were compared to the single cell aCGH results. We generated patient-derived organoid (PDOs) cultures from apheresis products by preenrichment using density gradient (Lymphoprep) and subsequent CTC enrichment by EpCAM positive selection (EasySep StemCell Technologies). PDOs were characterized by immunofluorescence (IF) as DAPI+/CK+/EpCAM+ and CD45- cells and subsequently by aCGH for CNA. All sixteen patients (median age of 70 years; range 60-77 years) tolerated apheresis without any adverse effects. CTC counts from peripheral blood (PB) prior to apheresis ranged from 13 to 711 (median = 96), and did not significantly change post apheresis. The estimated CTC yield per apheresis ranged from 660-35473 per apheresis product (median = 3351). This constitutes an increase of 102-fold when compared to median CTC capture from 7.5mL of PB. A total of 170 single CTCs from 15 apheresis patients were genomically profiled and the copy number aberration profiles confirmed prostate cancer with multiple genomic hallmarks including CNAs such as AR amplification, chromosome 8q gain (MYC locus), and PTEN, RB1, BRCA2, TP53, CHD1 loss. CNA profiles of PDOs showed similar genomic aberrations to same patient CTCs and also reflected intrapatient heterogeneity detected by single CTC analysis. In conclusion, apheresis from advanced prostate cancer patients is a well-tolerated procedure and in our study increased the CTC yield by 102-fold when compared to PB. CTC and PDOs from apheresis products shared similar CNA profile compared with tissues biopsies and furthermore gave us an insight of the tumor heterogeneity and clonal evolution. Citation Format: Maryou B.K. Lambros, Veronica Gil, Mateus Crespo, Mariane S. Fontes, Alan Mackay, Gemma Folwer, Gemma Folwer, Berni Ebbs, Rui Neves, Penny Flohr, Susana Miranda, Semini Sumanasuriya, Daniel N. Rodrigues, Rita Pereira, Geroge Seed, Wei Yuan, Joanne Hunt, Deirdre Moloney, Dionne Ayanda, Niven Mehra, Jane Goodall, Claudia Bertan, Suzanne Carreira, Nikolas H. Stoecklein, Leon W.M.M. Terstappen, Gunther Boysen, Joahnn S. De Bono. Liquid biopsy by apheresis: Molecular characterization of circulating tumor cells and their organoid culture reflects intrapatient heterogeneity and clonal evolution [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A051.

Mucinous carcinomas are a rare entity accounting for up to 2% of all breast cancers, which have been shown to display a gene expression profile distinct from that of invasive ductal carcinomas of no special type (IDC-NSTs). Here, we have defined the genomic aberrations that are characteristic of this special type of breast cancer and have investigated whether mucinous carcinomas might constitute a genomic entity distinct from IDC-NSTs. Thirty-five pure and 11 mixed mucinous breast carcinomas were assessed by immunohistochemistry using antibodies against oestrogen receptor (ER), progesterone receptor, HER2, Ki67, cyclin D1, cortactin, Bcl-2, p53, E-cadherin, basal markers, neuroendocrine markers, and WT1. Fifteen pure mucinous carcinomas and 30 grade- and ER-matched IDC-NSTs were microdissected and subjected to high-resolution microarray-based comparative genomic hybridization (aCGH). In addition, the distinct components of seven mixed mucinous carcinomas were microdissected separately and subjected to aCGH. Pure mucinous carcinomas consistently expressed ER (100%), lacked HER2 expression (97.1%), and showed a relatively low level of genetic instability. Unsupervised hierarchical cluster analysis revealed that pure mucinous carcinomas were homogeneous and preferentially clustered together, separately from IDC-NSTs. They less frequently harboured gains of 1q and 16p and losses of 16q and 22q than grade- and ER-matched IDC-NSTs, and no pure mucinous carcinoma displayed concurrent 1q gain and 16q loss, a hallmark genetic feature of low-grade IDC-NSTs. Finally, both components of all but one mixed mucinous carcinoma displayed similar patterns of genetic aberrations and preferentially clustered together with pure mucinous carcinomas on unsupervised clustering analysis. Our results demonstrate that mucinous carcinomas are more homogeneous between themselves at the genetic level than IDC-NSTs. Both components of mixed mucinous tumours are remarkably similar at the molecular level to pure mucinous cancers, suggesting that mixed mucinous carcinomas may be best classified as variants of mucinous cancers rather than of IDC-NSTs. Copyright (C) 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Breast adenosquamous carcinomas are rare tumours characterized by well-developed gland formation intimately admixed with solid nests of squamous cells immersed in a highly cellular spindle cell stroma. A low-grade variant has been described that is associated with a better prognosis. Here we studied five cases of adenosquamous carcinomas to determine their genetic profiles and to investigate whether the spindle cell component of these cancers could at least in part stem from the glandular/epithelial components. Five adenosquamous carcinomas of the breast were subjected to (1) immunohistochemical analysis, (2) microdissection and genetic analysis with a high-resolution microarray comparative genomic hybridization platform, and (3) chromogenic in situ hybridization. All cases displayed a triple-negative immunophenotype, consistently expressed 'basal' keratins and showed variable levels of epidermal growth factor receptor expression. Microarray comparative genomic hybridization analysis of two of the cases revealed multiple low-level gains and losses affecting several chromosomal arms. Case 1 displayed gains of the whole of chromosome 7, and case 2 harboured a focal, high-level amplification of 7p12, encompassing the epidermal growth factor receptor gene, which was associated with strong and intense membranous epidermal growth factor receptor expression. Chromogenic in situ hybridization revealed that the genetic features found in the epithelial cells were also present in a minority of the spindle cells of the stromal component, in particular in those near the epithelial clusters, indicating that some of the spindle cells are clonal and derived from the epithelial component of the tumour. In conclusion, breast adenosquamous carcinomas are triple-negative cancers that express 'basal' keratins. These tumours harbour complex genetic profiles. Some of the spindle cells in adenosquamous carcinomas are derived from the epithelial component, suggesting that adenosquamous carcinomas may also be part of the group of metaplastic breast carcinomas with spindle cell metaplastic elements.

The growth and guidance of primary olfactory axons are partly attributed to the presence of olfactory ensheathing cells (OECs). However, little is understood about the differences between the subpopulations of OECs and what regulates their interactions. We used OEC-axon assays and determined that axons respond differently to peripheral and central OECs. We then further purified OECs from anatomically distinct regions of the olfactory bulb. Cell behaviour assays revealed that OECs from the olfactory bulb were a functionally heterogeneous population with distinct differences which is consistent with their proposed roles in vivo. We found that the heterogeneity was regulated by motile lamellipodial waves along the shaft of the OECs and that inhibition of lamellipodial wave activity via Mek1 abolished the ability of the cells to distinguish between each other. These results demonstrate that OECs from the olfactory bulb are a heterogeneous population that use lamellipodial waves to regulate cell-cell recognition.

This study examines the hypothesis that multipotent olfactory mucosal stem cells could provide a basis for the development of autologous cell transplant therapy for the treatment of heart attack. In humans, these cells are easily obtained by simple biopsy. Neural stem cells from the olfactory mucosa are multipotent, with the capacity to differentiate into developmental fates other than neurons and glia, with evidence of cardiomyocyte differentiationin vitroand after transplantation into the chick embryo. Olfactory stem cells were grown from rat olfactory mucosa. These cells are propagated as neurosphere cultures, similar to other neural stem cells. Olfactory neurospheres were grownin vitro, dissociated into single cell suspensions, and transplanted into the infarcted hearts of congeneic rats. Transplanted cells were genetically engineered to express green fluorescent protein (GFP) in order to allow them to be identified after transplantation. Functional assessment was attempted using echocardiography in three groups of rats: control, unoperated; infarct only; infarcted and transplanted. Transplantation of neurosphere-derived cells from adult rat olfactory mucosa appeared to restore heart rate with other trends towards improvement in other measures of ventricular function indicated. Importantly, donor-derived cells engrafted in the transplanted cardiac ventricle and expressed cardiac contractile proteins.

Background: Germline mutation screening of BRCA1 and BRCA2 genes is performed in suspected familial breast cancer cases, but a causative mutation is found in only 30% of patients. The development of additional methods to identify good candidates for BRCA1 and BRCA2 analysis would therefore increase the efficacy of diagnostic mutation screening. With this in mind, we developed a study to determine molecular signatures of BRCA1—or BRCA2—mutated breast cancers. Materials and Methods: Array-cgh (comparative genomic hybridization) and transcriptomic analysis were performed on a series of 103 familial breast cancers. The series included 7 breast cancers with a BRCA1 mutation and 5 breast cancers with a BRCA2 mutation. The remaining 91 cases were obtained from 73 families selected on the basis of at least 3 affected first-degree relatives or at least 2 affected first-degree relatives with breast cancer at an average age of 45 years. Array-cgh analyses were performed on a 4407 BAC-array (CIT-V8) manufactured by IntegraGen. Transcriptomic analyses were performed using an Affymetrix Human Genome U133 Plus 2.0 chip. Results: Using supervised clustering analyses we identified two transcriptomic signatures: one for BRCA1-mutated breast cancers consisting of 600 probe sets and another for BRCA2-mutated breast cancers also consisting of 600 probes sets. We also defined cgh-array signatures, based on the presence of specific genomic rearrangements, one for BRCA1-mutated breast cancers and one for BRCA2-mutated breast cancers. Conclusions: This study identified molecular signatures of breast cancers with BRCA1 or BRCA2 germline mutations. Genes present in these signatures could be exploited to find new markers for such breast cancers. We also identified specific genomic rearrangements in these breast cancers, which could be screened for in a diagnostic setting using fluorescence in situ hybridization, thus improving patient selection for BRCA1 and BRCA2 molecular genetic analysis.

The pathogenesis of late normal tissue fibrosis after high-dose ionizing radiation involves multiple cell types and signalling pathways but is not well understood. To identify the molecular changes occurring after radiotherapy, paired normal tissue samples were collected from the non-irradiated breast and from the treated breast of women who had undergone curative radiotherapy for early breast cancer months or years previously. As radiation may induce distinct transcriptional changes in the different components of the breast, laser capture microdissection and gene expression microarray profiling were performed separately for epithelial and stromal components and selected genes were validated using immunohistochemistry. In the epithelial compartment, a reduction of KIT (c-Kit; CD117) and a reciprocal increase in ESR1 (oestrogen receptor-alpha, ERalpha) mRNA and protein levels were seen in irradiated compared to non-irradiated samples. In the stromal compartment, extracellular matrix genes including FN1 (fibronectin 1) and CTGF (connective tissue growth factor; CCN2) were increased. Further investigation revealed that c-Kit and ERalpha were expressed in distinct subpopulations of luminal epithelial cells. Interlobular c-Kit-positive mast cells were also increased in irradiated cases not showing features of post-radiation atrophy. Pathway analysis revealed 'cancer, reproductive system disease and tumour morphology' as the most significantly enriched network in the epithelial compartment, whereas in the stromal component, a significant enrichment for 'connective tissue disorders, dermatological diseases and conditions, genetic disorder' and 'cancer, tumour morphology, infection mechanism' networks was observed. These data identify previously unreported changes in the epithelial compartment and show altered expression of genes implicated in late normal tissue injury in the stromal compartment of normal breast tissue. The findings are relevant to both fibrosis and atrophy occurring after radiotherapy for early breast cancer.

Expression, mutation and copy number analysis of platelet-derived growth factor receptor A (PDGFRA) and its ligand PDGFA in gliomas, BACKGROUND: Malignant gliomas are the most prevalent type of primary brain tumours but the therapeutic armamentarium for these tumours is limited. Platelet-derived growth factor (PDGF) signalling has been shown to be a key regulator of glioma development. Clinical trials evaluating the efficacy of anti-PDGFRA therapies on gliomas are ongoing. In this study, we intended to analyse the expression of PDGFA and its receptor PDGFRA, as well as the underlying genetic (mutations and amplification) mechanisms driving their expression in a large series of human gliomas. METHODS: PDGFA and PDGFRA expression was evaluated by immunohistochemistry in a series of 160 gliomas of distinct World Health Organization (WHO) malignancy grade. PDGFRA-activating gene mutations (exons 12, 18 and 23) were assessed in a subset of 86 cases by PCR-single-strand conformational polymorphism (PCR-SSCP), followed by direct sequencing. PDGFRA gene amplification analysis was performed in 57 cases by quantitative real-time PCR (QPCR) and further validated in a subset of cases by chromogenic in situ hybridisation (CISH) and microarray-based comparative genomic hybridisation (aCGH). RESULTS: PDGFA and PDGFRA expression was found in 81.2% (130 out of 160) and 29.6% (48 out of 160) of gliomas, respectively. Its expression was significantly correlated with histological type of the tumours; however, no significant association between the expression of the ligand and its receptor was observed. The absence of PDGFA expression was significantly associated with the age of patients and with poor prognosis. Although PDGFRA gene-activating mutations were not found, PDGFRA gene amplification was observed in 21.1% (12 out of 57) of gliomas. No association was found between the presence of PDGFRA gene amplification and expression, excepting for grade II diffuse astrocytomas. CONCLUSION: The concurrent expression of PDGFA and PDGFRA in different subtypes of gliomas, reinforce the recognised significance of this signalling pathway in gliomas. PDGFRA gene amplification rather than gene mutation may be the underlying genetic mechanism driving PDGFRA overexpression in a portion of gliomas. Taken together, our results could provide in the future a molecular basis for PDGFRA-targeted therapies in gliomas., OM is a recipient of a PhD fellowship (SFRH/BD/36463/2007) from Fundação para a Ciência e a Tecnologia (FCT), Portugal. JSRF,MBKL, AM, NT, KF and AA are funded by Breakthrough BreastCancer. This study was supported in part by Pfizer/Sociedade de Ciências dicas de Lisboa with the prize: Research in oncology diseases, Professor Francisco Gentil’ and Breakthrough Breast Cancer., info:eu-repo/semantics/publishedVersion

Background In humans, lower back pain is one of the most common causes of morbidity. Many studies implicate degeneration of intervertebral discs as the cause. In the normal intervertebral disc, the nucleus pulposus exerts a hydrostatic pressure against the constraining annulus fibrosus, which allows the disc to maintain flexibility between adjacent vertebrae, while absorbing necessary compressive forces. The nucleus pulposus performs this role because of its hydrophilic gel-like structure. The extracellular matrix of the nucleus pulposus is up to 80% hydrated, as a result of large amounts of the aggregating proteoglycan, chondroitin sulfate proteoglycan (CSPG). This proteoglycan is enmeshed in a randomly orientated network of fine collagen Type II (CT2) fibers. Study design and purpose A useful adult tissue-derived stem cell is that from the olfactory mucosa, the organ of smell. These cells, accessible in humans from nasal biopsies, are multipotent and are able to make many cell types from all germ layers. They are easily grown in vitro and can be expanded to large numbers and stored frozen. These qualities indicate the potential for autologous transplantation for disc repair. In this article, using a rat model, we explore the hypothesis that olfactory stem cells can differentiate into a nucleus pulposus chondrocyte phenotype in vitro, as well as in vivo after transplantation into the injured intervertebral disc. Patient sample Female rats (14 weeks) were anesthetized with xylazine/ketamine. The abdominal wall was shaved and injected with local anesthetic (lidocaine) before incision. The ventral part of the lumbar spine, including two intervertebral discs, was exposed. Disc degeneration was then induced in the two exposed discs by needle aspiration of the nucleus pulposus. The prominent spina iliaca posterior superior was used as an anatomical landmark for identification of the first disc. Two weeks later, one injured intervertebral disc was exposed in a second, similar, surgery and 20,000 olfactory neurosphere-derived cells were transplanted with a 25-G needle. Outcome measures In vitro induction of nucleus pulposus chondrocyte phenotype is measured by the percentage of cells expressing CT2 and CSPG. In vivo, a successful outcome is evidence of engraftment of donor-derived cells and their expression of CT2 and CSPG. Methods In this article, we tested two hypotheses: the first that progenitor cells within olfactory neurospheres could be induced to express markers distinctive of the nucleus pulposus when placed in vitro in a coculture experiment. The second hypothesis tested the same induction in genetically labeled transplanted cells within damaged vertebral discs in vivo. The two markers measured are those held by current literature to engender the necessary cushioning characteristics of nucleus pulposus, CT2 and CSPG. Results Our experiments demonstrated virtually 100% induction of these two markers in vitro. Also, this induction was achieved in donor-derived cells after delivery to the nucleus pulposus region of animals whose discs had previously been lesioned 2 weeks before transplant. Conclusions These results provide a rationale for moving toward more extensive larger animal studies for assessment of regeneration before human trials where relief of symptoms can be more easily assessed.

Background. Burkholderiapseudomallei,thecausativeagentofmelioidosis,isgenerallyconsideredtobeacquired via inhalation of dust or water droplets from the environment. In this study, we show that infection of the nasal mucosa is potentially an important portal of entry in melioidosis. Methods. After intranasal inoculation of mice, infection was monitored by bioluminescence imaging and by immunohistological analysis of coronal sections. The bacterial loads in organ and tissue specimens were also monitored. Results. Bioluminescence imaging showed colonization and replication in the nasal cavity, including the nasalassociated lymphoid tissue (NALT). Analysis of coronal sections and immunofluorescence microscopy further demonstrated the presence of infection in the respiratory epithelium and the olfactory epithelium (including associated nerve bundles), as well as in the NALT. Of significance, the olfactory epithelium and the brain were rapidly infected before bacteria were detected in blood, and a capsule-deficient mutant infected the brain without significantly infecting blood. Conclusions. These data suggest that the olfactory nerve is the route of entry into the brain and that this route of entry may be paralleled in cases of human neurologic melioidosis. This study focuses attention on the upper respiratory tract as a portal of entry, specifically focusing on NALT as a route for the development of systemic infection via the bloodstream and on the olfactory epithelium as a direct route to the brain. Burkholderia pseudomallei, a member of the -proteobacteria, is an environmental organism and the causative agent of melioidosis. Melioidosis in humans is a potentially fatal disease with many presentations varying from subacute to chronic and acute disease. Death may occur rapidly after septicshock[1].ThemainfociofendemicityareSoutheast Asia and northern Australia, where the mortality rates are

Olfactory dysfunction has been reported in clinical and preclinical phases of Alzheimer's disease. Subjective memory complaints have been proposed as a potential early indicator for increased risk of Alzheimer's disease, but have also been associated with depression, personality characteristics, and health problems. In this study, we aimed to determine which of these putative markers can predict memory complaints in community-dwelling elderly individuals, focusing on olfactory symptoms. A cohort of 144 elderly volunteers (42 males and 102 females), aged 50 to 86, was recruited from an ongoing longitudinal study. Participants were assessed for olfactory capacities (threshold, discrimination, and identification), subjective memory complaints, depression, and cognitive functions. Subjective memory complaints were significantly associated with olfactory discrimination and identification but not with threshold. Olfactory functions and depressive symptoms were both significantly associated with subjective memory complaints. In addition, memory complainers were significantly worse than non-complainers with respect to olfactory discrimination, identification, and overall olfactory functioning. The findings suggest that olfactory capacity may be a potentially significant biomarker for identifying community-dwelling elderly with memory complaints who are at increased risk for age-related cognitive decline and Alzheimer's disease.

Approximately 8% of breast cancers show increased copy numbers of chromosome 17 centromere (CEP17) by fluorescence in situ hybridization (FISH) (ie average CEP17 >3.0 per nucleus). Currently, this pattern is believed to represent polysomy of chromosome 17. HER2-amplified cancers have been shown to harbour complex patterns of genetic aberrations of chromosome 17, in particular involving its long arm. We hypothesized that aberrant copy numbers of CEP17 in FISH assays may not necessarily represent true chromosome 17 polysomy. Eighteen randomly selected CEP17 polysomic cases and a control group of ten CEP17 disomic cases, as defined by dual-colour FISH, were studied by microarray-based comparative genomic hybridization (aCGH), which was performed on microdissected samples using a 32K tiling-path bacterial artificial chromosome microarray platform. Additional FISH probes were employed for SMS (17p11.2) and RARA (17q21.2) genes, as references for chromosome 17 copy number. Microarray-based comparative genomic hybridization revealed that 11 out of the 18 polysomic cases harboured gains of 17q with involvement of the centromere, one displayed 17q gain sparing the centromeric region, and only one could be defined as polysomic. The remaining five cases displayed amplification of the centromeric region. Among these, one case, showing score 2+ by immunohistochemistry and 8.5 HER2 mean copy number, was classified as not amplified by HER2/CEP17 ratio and as amplified by HER2/SMS ratio. Our results suggest that true chromosome 17 polysomy is likely to be a rare event in breast cancer and that CEP17 copy number greater than 3.0 in FISH analysis is frequently related to gain or amplification of the centromeric region. Larger studies investigating the genetic profiles of CEP17 polysomic cases are warranted.

Purpose: To identify therapeutic targets in ovarian clear cell carcinomas, a chemoresistant and aggressive type of ovarian cancer. Experimental Design: Twelve ovarian clear cell carcinoma cell lines were subjected to tiling path microarray comparative genomic hybridization and genome-wide expression profiling analysis. Regions of high-level amplification were defined and genes whose expression levels were determined by copy number and correlated with gene amplification were identified. The effects of inhibition of PPM1D were assessed using short hairpin RNA constructs and a small-molecule inhibitor (CCT007093). The prevalence of PPM1D amplification and mRNA expression was determined using chromogenic in situ hybridization and quantitative real-time reverse transcription-PCR in a cohort of pure ovarian clear cell carcinomas and on an independent series of unselected epithelial ovarian cancers. Results: Array-based comparative genomic hybridization analysis revealed regions of high-level amplification on 1q32, 1q42, 2q11, 3q24-q26, 5p15, 7p21-p22, 11q13.2-q13.4, 11q22, 17q21-q22, 17q23.2, 19q12-q13, and 20q13.2. Thirty-four genes mapping to these regions displayed expression levels that correlated with copy number gains/amplification. PPM1D had significantly higher levels of mRNA expression in ovarian clear cell carcinoma cell lines harboring gains/amplifications of 17q23.2. PPM1D inhibition revealed that PPM1D expression and phosphatase activity are selectively required for the survival of ovarian clear cell carcinoma cell lines with 17q23.2 amplification. PPM1D amplification was significantly associated with ovarian clear cell carcinoma histology (P = 0.0003) and found in 10% of primary ovarian clear cell carcinomas. PPM1D expression levels were significantly correlated with PPM1D gene amplification in primary ovarian clear cell carcinomas. Conclusion: Our data provide strong circumstantial evidence that PPM1D is a potential therapeutic target for a subgroup of ovarian clear cell carcinomas.

Based on clues from epidemiology, it has been proposed that low prenatal vitamin D may be a risk factor for schizophrenia. In order to explore this hypothesis, our group has undertaken an integrated research program linking analytic epidemiology and rodent experiments. There is consistent evidence from rodents that offspring exposed to low developmental vitamin D deficiency have altered brain structure and function as adults. This chapter provides a concise summary of the evidence linking vitamin D to brain development and function. In addition, the epidemiological evidence linking hypovitaminosis D and various neuropsychiatric disorders is outlined.

Purpose: Perilobar nephrogenic rests (PLNRs) are abnormally persistent foci of embryonal immature blastema that have been associated with dysregulation at the 11p15 locus by genetic/epigenetic means and are thought to be precursor lesions of Wilms tumor. The precise genomic events are, however, largely unknown. Experimental Design: We used array comparative genomic hybridization to analyze a series of 50 PLNRs and 25 corresponding Wilms tumors characterized for 11p15 genetic/epigenetic alterations and insulin-like growth factor-II expression. Results: The genomic profiles of PLNRs could be subdivided into three categories: those with no copy number changes (22 of 50, 44%); those with single, whole chromosome alterations (8 of 50, 16%); and those with multiple gains/losses (20 of 50, 40%). The most frequent aberrations included 1p- (7 of 50, 14%) +18 (6 of 50, 12%), +13 (5 of 50, 10%), and +12 (3 of 50, 6%). For the majority (19 of 25, 76%) of cases, the rest harbored a subset of the copy number changes in the associated Wilms tumor. We identified a temporal order of genomic changes, which occur during the insulin-like growth factor-II/PLNR pathway of Wilms tumorigenesis, with large-scale chromosomal alterations such as 1p-, +12, +13, and +18 regarded as “early” events. In some of the cases (24%), the PLNRs harbored large-scale copy number changes not observed in the concurrent Wilms tumor, including +10p, +14q, and +18. Conclusions: These data suggest that although the evidence for PLNRs as precursors is compelling, not all lesions must necessarily undergo malignant transformation.

Objective: To determine the incidence of olfactory dysfunction after head trauma using clinical and radiologic findings, quantitative assessment, and electro-physiologic methods. Participants: A total of 190 patients with head trauma of different severity (n = 32 with mild traumatic brain injury (TBI), n = 94 with signs of moderate TBI, and n = 64 with severe TBI) 6 to 32 months prior to the study. Design: Patients were selected retrospectively, surveyed by telephone (n = 190), and screened for olfactory function with Brief Smell Identification Test (n = 82). Those with olfactory dysfunction were assessed as outpatients using the Sniffin' Sticks (n = 19) and olfactory-evoked potential recording (n = 16). Results: Twenty-one participants (11%) reported a decreased sense of smell after trauma. The incidence of olfactory dysfunction after head injury was 12.8%. The results of the odor-evoked potentials were heterogeneous. A significant correlation was found between olfactory dysfunction and the appearance of skull base fractures and intracranial hemorrhage or hematoma. Conclusion: The site of trauma may be more relevant to prognosis than a simple probability (of olfactory loss) based on incidence. Odor-evoked potentials indicate that functional anosmia can occur even when there is some evidence of intact olfactory nerve function.

HER2 and TOP2A are targets for the therapeutic agents trastuzumab and anthracyclines and are frequently amplified in breast cancers. The aims of this study were to provide a detailed molecular genetic analysis of the 17q12-q21 amplicon in breast cancers harbouring HER2/TOP2A co-amplification and to investigate additional recurrent co-amplifications in HER2/TOP2A-co-amplified cancers. In total, 15 breast cancers with HER2 amplification, 10 of which also harboured TOP2A amplification, as defined by chromogenic in situ hybridisation, and 6 breast cancer cell lines known to be amplified for HER2 were subjected to high-resolution microarray-based comparative genomic hybridisation analysis. This revealed that the genomes of 12 cases were characterised by at least one localised region of clustered, relatively narrow peaks of amplification, with each cluster confined to a single chromosome arm (ie 'firestorm' pattern) and 3 cases displayed many narrow segments of duplication and deletion affecting the vast majority of chromosomes (ie 'sawtooth' pattern). The smallest region of amplification (SRA) on 17q12 in the whole series extended from 34.73 to 35.48 Mb, and encompassed HER2 but not TOP2A. In HER2/TOP2A-co-amplified samples, the SRA extended from 34.73 to 36.54 Mb, spanning a region of approximately 1.8 Mb. Apart from HER2 and TOP2A, this region encompassed four additional genes whose expression levels as defined by quantitative real-time PCR are significantly higher in HER2/TOP2A-co-amplified vs HER2-amplified breast cancers: CASC3, CDC6, RARA and SMARCE1. Of the cell lines studied, SKBR3 and UACC812 showed HER2/TOP2A co-amplification. In conclusion, this is the first detailed genome-wide characterisation of HER2/TOP2A-amplified breast cancers; cell lines were identified that can be used to model these cancers in vitro. The 17q12 amplicon is complex and harbours multiple genes that may be associated with breast cancer development and progression, and potentially exploitable as therapeutic targets.

Aromatase inhibitors play a key role in the clinical management of hormone receptor–positive breast cancer and have potential utility as chemopreventive agents. Further understanding of the molecular effects of estrogen and its deprivation in normal breast tissue may allow the development of biomarkers of risk of breast cancer and help to predict the value of chemoprevention with aromatase inhibitors. Core biopsies of normal breast tissue were taken before and after letrozole treatment from postmenopausal women in the LITMaS pilot prevention study. RNA was extracted from these samples and used for cDNA microarray analysis. Gene expression changes induced by letrozole treatment were much less extensive than observed in estrogen receptor–positive malignant tissue; however, overall, they correlated to a highly significant degree (ρ = 0.511; P < 10−20). As well as some classically estrogen-associated genes, many genes associated with extracellular matrix remodeling were affected by estrogen deprivation in the normal breast in vivo. These data indicate for the first time that gene expression of normal breast tissue remains dependent on endogenous estrogens after the menopause. The modest degree of gene change suggests that intermediate markers of chemoprevention may be difficult to identify. (Cancer Epidemiol Biomarkers Prev 2008;17(4):855–63)

Developmental vitamin D (DVD) deficiency has been proposed as an environmental risk factor for a number of brain disorders. The absence of this vitamin during foetal development in the rat is known to alter behaviour in the adult, and many of these alterations are informative with respect to the clinical features of schizophrenia. Here we investigated whether DVD deficiency had a similar effect on 129/SvJ and C57BL/6J mice. Female mice were fed a diet deficient in vitamin D for 6 weeks prior to conception until birth, after which dams and their offspring were fed a normal diet (i.e. containing vitamin D). Control mice were fed a normal diet throughout the experiment. The adult offspring underwent a comprehensive behavioural test battery at 10 weeks of age. We found that DVD-deficient mice of both strains exhibited significantly higher levels of exploration, as measured by the frequency of head dipping on the hole board test. In addition, DVD-deficient 129/SvJ mice, but not C57BL/6J mice, displayed spontaneous hyperlocomotion. There was no effect of maternal diet on parameters assessed by the SHIRPA primary screen, or on tests of sensorimotor gating, social behaviour, anxiety or depression. Some of these findings resemble the rat phenotype (hyperlocomotion) but there are also novel effects of DVD deficiency on mouse behaviour (increased exploration). This study confirms that the developmental absence of this vitamin affects brain function in another species (mouse), and lends further weight to the hypothesis that DVD deficiency in humans may contribute to adverse neuropsychiatric outcomes.

Published reports of neurotrophin expression in the olfactory system are incomplete because of missing data and conflicting results. Previous studies used a variety of fixation procedures and antibodies on different species and different ages. The aim of the present study was to examine expression of neurotrophins and their receptors using optimized methodologies: five methods of fixation, multiple antibodies, a variety of immunochemical protocols, and RT-PCR. We show here that (i) transcripts for all neurotrophins and their receptors are found in the adult olfactory epithelium; (ii) all neurotrophins are expressed in the supporting cells and the neuronal layers of the undisturbed adult olfactory epithelium while NT4 is found additionally in the horizontal basal cells; (iii) neurotrophin immunoreactivity required a fixative that included parabenzoquinone (not used in previous studies of olfactory tissue); (iv) TrkB and TrkC are restricted to the globose basal cell and neuron layers while TrkA is found in the horizontal basal cells and in the supporting cells where it co-localizes with the low affinity receptor for NGF (p75NTR). These findings confirm that neurotrophins are produced within the olfactory epithelium, suggesting autocrine and paracrine regulation of olfactory neurogenesis.

Deletion of the TP53 gene on chromosome 17p13.1 is the prognostic factor associated with the shortest survival in CLL. We used array-based comparative genomic hybridisation (arrayCGH) to identify additional DNA copy number changes in peripheral blood samples from 74 LRF CLL4 trial patients, 37 withor=5% and 37 without TP53-deleted cells. ArrayCGH reliably detected deletions on 17p, including the TP53 locus, in cases withor=50%TP53-deleted cells detected by fluorescence in situ hybridisation, plus seven additional cases with deleted regions on 17p excluding TP53. Losses on chromosomal regions 18p and/or 20p were found exclusively in cases withor=5%TP53-deleted cells (p0.001), 38% having one or both losses. The incidence of additional cytogenetic abnormalities, reflecting an increased chromosomal instability, was higher inor=5%TP53-deleted cases (p=0.02). In particular, amplification of 2p and deletion of 6q were both more frequent. Cases with20%TP53-deleted cells had the worst prognosis in the LRF CLL4 trial.

Stem cell research is now a very broad field encompassing cells derived from all stages of life from the embryonic stem cells of the early blastocyst through to the adult stem cells of many tissues of the body. Adult stem cells from a variety of tissues are proving to be pluripotent and can differentiate into cell types different from the tissues from which they derive. Pre-clinical animal models indicate that adult stem cells do not cause tumours, not even, teratomas when transplanted. These properties, combined with the possibility of autologous transplantation, indicate significant advantages over embryonic stem cells in many proposed clinical applications.

During development, neuronal processes extend, branch and navigate to ultimately synapse with target tissue. We have shown a regulatory role for ARNO and ARF6 in dendritic branching and axonal elongation and branching during neuritogenesis, particularly with respect to cytoskeletal dynamics. Here, we have examined the role of ARF6 and the ARF GEF ARNO in endosomal dynamics during neurite elongation in hippocampal neurons. Axonal and dendritic endosomes were labeled by expression of the endosomal marker, endotubin. Expression of endotubin-green fluorescent protein resulted in targeting to tubular-vesicular structures throughout the somatodendritic and axonal domains. These endosomal structures did not colocalize with conventional early or late endosomal markers or with the synaptic vesicle marker, SV2. However, they did label with internalized lectin, indicating that they are endosomal structures. Expression of catalytically inactive ARNO (ARNO-E156K) or inactive ARF6 (ARF6-T27N) caused a redistribution of endotubin to the cell surface of the axons and dendrites. In contrast, expression of these constructs had no effect upon the distribution of SV2-positive structures. Furthermore, expression of inactive ARF1 (ARF1-T31N) did not change endotubin distribution. These results suggest that endotubin labels a distinct endosomal structure in neurons and that ARNO and ARF6 mediate neurite extension through the regulation of this compartment.

An increased risk for multiple sclerosis and schizophrenia is observed at increasing latitude and in patients born in winter or spring. To explore a possible link between maternal vitamin D deficiency and these brain disorders, we examined the impact of prenatal hypovitaminosis D on protein expression in the adult rat brain. Vitamin D-deficient female rats were mated with vitamin D normal males. Pregnant females were kept vitamin D-deficient until birth whereupon they were returned to a control diet. At week 10, protein expression in the progeny's prefrontal cortex and hippocampus was compared with control animals using silver staining 2-D gels associated with MS and newly devised data mining software. Developmental vitamin D (DVD) deficiency caused a dysregulation of 36 brain proteins involved in several biological pathways including oxidative phosphorylation, redox balance, cytoskeleton maintenance, calcium homeostasis, chaperoning, PTMs, synaptic plasticity and neurotransmission. A computational analysis of these data revealed that (i) nearly half of the molecules dysregulated in our animal model have also been shown to be misexpressed in either schizophrenia and/or multiple sclerosis and (ii) an impaired synaptic network may be a consequence of mitochondrial dysfunction.

It has become increasingly evident that morphologically similar gliomas may have distinct clinical phenotypes arising from diverse genetic signatures. To date, glial tumours from the Tunisian population have not been investigated. To address this, we correlated the clinico-pathology with molecular data of 110 gliomas by a combination of HM450K array, MLPA and TMA-IHC. PTEN loss and EGFR amplification were distributed in different glioma histological groups. However, 1p19q co-deletion and KIAA1549:BRAF fusion were, respectively, restricted to Oligodendroglioma and Pilocytic Astrocytoma. CDKN2A loss and EGFR overexpression were more common within high-grade gliomas. Furthermore, survival statistical correlations led us to identify Glioblastoma (GB) prognosis subtypes. In fact, significant lower overall survival (OS) was detected within GB that overexpressed EGFR and Cox2. In addition, IDH1R132H mutation seemed to provide a markedly survival advantage. Interestingly, the association of IDHR132H mutation and EGFR normal status, as well as the association of differentiation markers, defined GB subtypes with good prognosis. By contrast, poor survival GB subtypes were defined by the combination of PTEN loss with PDGFRa expression and/or EGFR amplification. Additionally, GB presenting p53-negative staining associated with CDKN2A loss or p21 positivity represented a subtype with short survival. Thus, distinct molecular subtypes with individualised prognosis were identified. Interestingly, we found a unique histone mutation in a poor survival young adult GB case. This tumour exceptionally associated the H3F3A G34R mutation and MYCN amplification as well as 1p36 loss and 10q loss. Furthermore, by exhibiting a remarkable methylation profile, it emphasised the oncogenic power of G34R mutation connecting gliomagenesis and chromatin regulation.

Introduction Aromatase inhibitors (AIs) are a vital component of estrogen receptor positive (ER+) breast cancer treatment. De novo and acquired resistance, however, is common. The aims of this study were to relate patterns of copy number aberrations to molecular and proliferative response to AIs, to study differences in the patterns of copy number aberrations between breast cancer samples pre- and post-AI neoadjuvant therapy, and to identify putative biomarkers for resistance to neoadjuvant AI therapy using an integrative analysis approach. Methods Samples from 84 patients derived from two neoadjuvant AI therapy trials were subjected to copy number profiling by microarray-based comparative genomic hybridisation (aCGH, n = 84), gene expression profiling (n = 47), matched pre- and post-AI aCGH (n = 19 pairs) and Ki67-based AI-response analysis (n = 39). Results Integrative analysis of these datasets identified a set of nine genes that, when amplified, were associated with a poor response to AIs, and were significantly overexpressed when amplified, including CHKA, LRP5 and SAPS3. Functional validation in vitro, using cell lines with and without amplification of these genes (SUM44, MDA-MB134-VI, T47D and MCF7) and a model of acquired AI-resistance (MCF7-LTED) identified CHKA as a gene that when amplified modulates estrogen receptor (ER)-driven proliferation, ER/estrogen response element (ERE) transactivation, expression of ER-regulated genes and phosphorylation of V-AKT murine thymoma viral oncogene homolog 1 (AKT1). Conclusions These data provide a rationale for investigation of the role of CHKA in further models of de novo and acquired resistance to AIs, and provide proof of concept that integrative genomic analyses can identify biologically relevant modulators of AI response. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0532-0) contains supplementary material, which is available to authorized users.

This work was supported by the Chief Scientist Office, Scotland (ETM37; to G.D. Stewart, A.C.P. Riddick, M. Aitchison, and D.J. Harrison), Cancer Research UK (Experimental Cancer Medicine Centre; to T. Powles, London and D.J. Harrison, Edinburgh), Medical Research Council (to A. Laird and D.J. Harrison), Royal College of Surgeons of Edinburgh (to A. Laird), Melville Trust (to A. Laird), Medical Research Council (MC_UU_12018/25; to I.M. Overton), Royal Society of Edinburgh Scottish Government Fellowship cofunded by Marie Curie Actions (to I.M. Overton), Renal Cancer Research Fund (to G.D. Stewart), Kidney Cancer Scotland (to G.D. Stewart) and an educational grant from Pfizer (to T. Powles). Purpose: The aim of this study was to investigate the effect of VEGF targeted therapy (sunitinib) on molecular intratumoral heterogeneity (ITH) in metastatic clear cell renal cancer (mccRCC). Experimental design: Multiple tumor samples (n=187 samples) were taken from the primary renal tumors of mccRCC patients who were sunitinib treated (n=23, SuMR clinical trial) or untreated (n=23, SCOTRRCC study). ITH of pathological grade, DNA (aCGH), mRNA (Illumina Beadarray) and candidate proteins (reverse phase protein array) were evaluated using unsupervised and supervised analyses (driver mutations, hypoxia and stromal related genes). ITH was analysed using intratumoral protein variance distributions and distribution of individual patient aCGH and gene expression clustering. Results: Tumor grade heterogeneity was greater in treated compared to untreated tumors (P=0.002). In unsupervised analysis, sunitinib therapy was not associated with increased ITH in DNA or mRNA. However, there was an increase in ITH for the driver mutation gene signature (DNA and mRNA) as well as increasing variability of protein expression with treatment (p

We analysed the molecular genetic profiles of breast cancer samples before and after neoadjuvant chemotherapy with combination doxorubicin and cyclophosphamide (AC). DNA was obtained from microdissected frozen breast core biopsies from 44 patients before chemotherapy. Additional samples were obtained before the second course of chemotherapy (D21) and after the completion of the treatment (surgical specimens) in 17 and 21 patients, respectively. Microarray-based comparative genome hybridisation was performed using a platform containing approximately 5800 bacterial artificial chromosome clones (genome-wide resolution: 0.9 Mb). Analysis of the 44 pretreatment biopsies revealed that losses of 4p, 4q, 5q, 12q13.11-12q13.12, 17p11.2 and 17q11.2; and gains of 1p, 2p, 7q, 9p, 11q, 19p and 19q were significantly associated with oestrogen receptor negativity. 16q21-q22.1 losses were associated with lobular and 8q24 gains with ductal types. Losses of 5q33.3-q4 and 18p11.31 and gains of 6p25.1-p25.2 and Xp11.4 were associated with HER2 amplification. No correlations between DNA copy number changes and clinical response to AC were found. Microarray-based comparative genome hybridisation analysis of matched pretreatment and D21 biopsies failed to identify statistically significant differences, whereas a comparison between matched pretreatment and surgical samples revealed a statistically significant acquired copy number gain on 11p15.2-11p15.5. The modest chemotherapy-driven genomic changes, despite profound loss of cell numbers, suggest that there is little therapeutic selection of resistant non-modal cell lineages.

"Sniffin' Sticks" is a test of nasal chemosensory function that is based on pen-like odor dispensing devices, introduced some 10 years ago by Kobal and co-workers. It consists of tests for odor threshold, discrimination, and identification. Previous work established its test-retest reliability and validity. Results of the test are presented as "TDI score", the sum of results obtained for threshold, discrimination, and identification measures. While normative data have been established they are based on a relatively small number of subjects, especially with regard to subjects older than 55 years where data from only 30 healthy subjects have been used. The present study aimed to remedy this situation. Now data are available from 3,282 subjects as compared to data from 738 subjects published previously. Disregarding sex-related differences, the TDI score at the tenth percentile was 24.9 in subjects younger than 15 years, 30.3 for ages from 16 to 35 years, 27.3 for ages from 36 to 55 years, and 19.6 for subjects older than 55 years. Because the tenth percentile has been defined to separate hyposmia from normosmia, these data can be used as a guide to estimate individual olfactory ability in relation to subject's age. Absolute hyposmia was defined as the tenth percentile score of 16-35 year old subjects. Other than previous reports the present norms are also sex-differentiated with women outperforming men in the three olfactory tests. Further, the present data suggest specific changes of individual olfactory functions in relation to age, with odor thresholds declining most dramatically compared to odor discrimination and odor identification.

Background: Cells with oncocytic change (OC) are a common finding in salivary glands (SGs) and in SG tumours. When found within pleomorphic adenomas (PAs), cells with OC may be perceived as evidence of malignancy, and lead to a misdiagnosis of carcinoma ex pleomorphic adenoma (CaExPa). Aim: To describe a case of PA with atypical OC, resembling a CaExPa. A genomewide molecular analysis was carried out to compare the molecular genetic features of the two components and to determine whether the oncocytic cells originated from PA cells, entrapped normal cells, or whether these cells constitute an independent tumour. Materials and methods: Representative blocks were immunohistochemically analysed with antibodies raised against cytokeratin (Ck) 5/6, Ck8/18, Ck14, vimentin, p63, α-smooth muscle actin (ASMA), S100 protein, anti-mitochondria antibody, β-catenin, HER2, Ki67, p53 and epidermal growth factor receptor. Typical areas of PA and OC were microdissected and subjected to microarray-based comparative genomic hybridisation (aCGH). Chromogenic in situ hybridisation (CISH) was performed with in-house generated probes to validate the aCGH findings. Results: PA cells showed the typical immunohistochemical profile, including positivity for Ck5/6, Ck8/18, Ck14, vimentin, ASMA, S100 protein, p63, epidermal growth factor receptor and β-catenin, whereas oncocytic cells showed a luminal phenotype, expression of anti-mitochondria antibody and reduced β-catenin staining. Both components showed low proliferation rates and lacked p53 reactivity. aCGH revealed a similar amplification in both components, mapping to 12q13.3–q21.1, which was further validated by CISH. No HER2 gene amplification or overexpression was observed. The foci of oncocytic metaplasia showed an additional low-level gain of 6p25.2–p21.31. Conclusion: The present data demonstrate that the bizarre atypical cells of the present case show evidence of clonality but no features of malignancy. In addition, owing to the presence of a similar genome amplification pattern in both components, it is proposed that at least in some cases, OC may originate from PA cells.

We previously demonstrated that olfactory cultures from individuals with schizophrenia had increased cell proliferation compared to cultures from healthy controls. The aims of this study were to (a) replicate this observation in a new group of individuals with schizophrenia, (b) examine the specificity of these findings by including individuals with bipolar I disorder and (c) explore gene expression differences that may underlie cell cycle differences in these diseases. Compared to controls (n = 10), there was significantly more mitosis in schizophrenia patient cultures (n = 8) and significantly more cell death in the bipolar I disorder patient cultures (n = 8). Microarray data showed alterations to the cell cycle and phosphatidylinositol signalling pathways in schizophrenia and bipolar I disorder, respectively. Whilst caution is required in the interpretation of the array results, the study provides evidence indicating that cell proliferation and cell death in olfactory neuroepithelial cultures is differentially altered in schizophrenia and bipolar disorder.