Phase 1 trial of olaparib and oral cyclophosphamide in BRCA breast cancer, recurrent BRCA ovarian cancer, non-BRCA triple-negative breast cancer, and non-BRCA ovarian cancer.

BACKGROUND: We conducted a Phase 1 study to evaluate safety and activity of olaparib tablets and oral cyclophosphamide. METHODS: Patients had metastatic breast cancer (BC) or recurrent high-grade serous ovarian cancer (HGSOC), performance status 0-2, and ≤3 lines of prior therapy. Patients were treated using a dose escalation strategy with cohort expansion once maximal tolerated dose (MTD) was determined. Dose level 1 (DL1): olaparib 300 mg bid, cyclophosphamide 50 mg on days 1, 3, and 5, weekly. DL2: olaparib 300 mg bid, cyclophosphamide 50 mg, days 1-5 weekly. RESULTS: Of 32 patients, 23 had HGSOC (germline BRCA mutation [gBRCAm] 70%) and 9 had BC (gBRCAm 67%). Four were treated at DL1 and 28 at DL2, the MTD. Haematological adverse events (AEs) were most common: grade 3/4 AEs: lymphopenia 75%, anaemia 31%, neutropenia 37%, thrombocytopenia 47%. Two permanently discontinued treatment due to haematological AEs. In BC, no objective response was reported. Unconfirmed objective response was 48% and 64% for all HGSOC and gBRCAm subset, respectively. CA125 responses were 70% (all HGSOC) and 92% (gBRCAm). CONCLUSIONS: In HGSOC and BC, olaparib 300 mg bid and cyclophosphamide 50 mg on days 1-5 weekly were tolerable and active, particularly in gBRCAm, and is worthy of further investigation.

Central nervous system expression of the exon 9 skipping form of the glutamate transporter GLAST.

We have raised antibodies that selectively recognize an exon 9 skipping form of GLAST. We demonstrate expression of this protein in brains of rats, cats, monkeys and humans. Immunolabelling was present in scattered populations of neurons, particularly in cerebral cortex and colliculi. Neurons were often present in small clusters and exhibited a range of morphologies, from apparently normal to highly degenerate. GLAST1b was also expressed by some glial cells. Cortical neurons expressing the exon 9 skipping form of GLAST also labelled with antibodies against the C- or N-terminal regions of GLAST. We suggest that alternate splicing of GLAST by subpopulations of neurons may indicate some dysfunction in these cells, and may be an indicator of inappropriate local excitation.

EAAT1 and D-serine expression are early features of human retinal development.

In the developing central nervous system (CNS), the activation of N-methyl-D-aspartate (NMDA) receptors is probably an important regulator of processes such as synaptogenesis and neurite growth. NMDA receptor activation is dependent upon the homeostasis of glutamate and the presence of co-agonists such as D-serine. We have investigated the expression of the glutamate transporter excitatory amino acid transporter-1 (EAAT1 or GLAST) as the key regulator of retinal extracellular glutamate levels, and the ontogeny of D-serine expression in the developing human retina. The expression of EAAT1 and D-serine was compared to the temporal and spatial distribution of the synaptic vesicle marker synaptophysin and the synaptic vesicle glutamate transporter vGLUT1. We also examined the co-expression of EAAT1 and cellular retinaldehyde-binding protein (CRALBP), and the co-expression of EAAT1 and D-serine. Human retinae aged 10-20 weeks' gestation (WG) were prepared for immunocytochemistry or for Western blotting. Expression of EAAT1 was evident at 10 WG in cell bodies, processes and end-feet of radial glia-like cells at all retinal eccentricities. D-serine immunolabelling was also evident in radial glia-like cells by 12 WG. In contrast, immunoreactivity for synaptophysin only started to appear in the central retina at 12 WG whilst immunoreactivity for vGLUT was slightly later. EAAT1 and d-serine were co-localised to the same cell population. In addition, EAAT1 and CRALBP were also co-localised to the same cell population of radial glia-like cells, suggesting that the EAAT1 and D-serine-positive cells may be Müller cells. This study shows that key potential modifiers of NMDA receptor activity are present before synaptic vesicle proteins are evident and may thus play a role in shaping synaptogenesis in the developing human retina.

Expression of the exon 3 skipping form of GLAST, GLAST1a, in brain and retina.

GLAST is a glial glutamate transporter; mRNA for a splice variant, GLAST1a, which lacks exon 3, has previously been identified. To detect GLAST1a protein, we generated antibodies against a peptide sequence encompassing the splice site. We demonstrate by Western blotting and immunocytochemistry the expression of GLAST1a in brains and retinae. Robust immunolabelling was present in the cerebellar Bergmann glia, and weaker labelling was evident in the retinal Müller cells. GLAST1a is differentially targeted to some cellular compartments such as the end feet of the Müller cells. As GLAST1a protein may interfere with the transport of glutamate by normally spliced GLAST, differentially targeted expression of GLAST1a may represent a mechanism for selectively regulating GLAST function in the mammalian nervous system.

Evoked expression of the glutamate transporter GLT-1c in retinal ganglion cells in human glaucoma and in a rat model.

PURPOSE: Glaucoma is a common disease of the eye, a key characteristic consequence of which is the death of retinal ganglion cells. The cause of this loss is unknown, though glutamate-mediated toxicity has been implicated. Glutamate transporters are key regulators of glutamate; therefore, the purpose of the study was to determine whether unusual excitation is associated with unusual expression of one or more transporters. METHODS: The expression of a splice variant of the glutamate transporter GLT-1 (EAAT2) was examined in normal and glaucomatous retinas from humans and rats. RESULTS: In normal eyes of humans and rats, GLT-1c was expressed only in photoreceptors. In glaucoma, there was additional robust expression of GLT-1c in retinal ganglion cells, including occasional displaced ganglion cells. Conversely, cells such as displaced amacrine cells and amacrine cells were unlabeled. CONCLUSIONS: The induction of GLT-1c expression by retinal ganglion cells supports the notion that an anomaly or anomalies in glutamate homeostasis may be evident in glaucoma and that such anomalies selectively influence retinal ganglion cells. By analogy to in vitro experiments in which elevated glutamate levels induce expression of glutamate transporters, the authors hypothesize that expression of GLT-1c may represent an attempt by retinal ganglion cells to protect themselves against elevated levels of glutamate. Such anomalies in glutamate levels cannot be restricted to the ganglion cell layer, as this would not have affected displaced ganglion cells. GLT-1c may be a useful indicator of the extent of stress of the retinal ganglion cells and thus a tool for examining outcomes of potential therapeutic and experimental interventions.

Immunocytochemical analysis of D-serine distribution in the mammalian brain reveals novel anatomical compartmentalizations in glia and neurons.

D-Serine is a co-agonist at the NMDA receptor glycine-binding site. Early studies have emphasized a glial localization for D-serine. However the nature of the glial cells has not been fully resolved, because previous D-serine antibodies needed glutaraldehyde-fixation, precluding co-localization with fixation-sensitive antigens. We have raised a new D-serine antibody optimized for formaldehyde-fixation. Light and electron microscopic observations indicated that D-serine was concentrated into vesicle-like compartments in astrocytes and radial glial cells, rather than being distributed uniformly in the cytoplasm. In aged animals, patches of cortex and hippocampus were devoid of immunolabeling for D-serine, suggesting that impaired glial modulation of forebrain glutamatergic signaling might occur. Dual immunofluorescence labeling for glutamate and D-serine revealed D-serine in a subset of glutamatergic neurons, particularly in brainstem regions and in the olfactory bulbs. Microglia also contain D-serine. We suggest that some D-serine may be derived from the periphery. Collectively, our data suggest that the cellular compartmentation and distribution of D-serine may be more complex and extensive than previously thought and may have significant implications for our understanding of the role of D-serine in disease states including hypoxia and schizophrenia.

Cryptic expression of functional glutamate transporters in the developing rodent brain.

The co-ordinate functioning of neurons and glia is required for glutamate-mediated neurotransmission. In this study, we show by immunocytochemical detection of D-aspartate uptake, that functional glutamate transporters are present in the developing CNS of fetal and neonatal rats, including forebrain, midbrain and hindbrain, at least as early as embryonic day 12 (E12). Use of the transport inhibitor dihydrokainic acid revealed a significant role for GLT-1 in the uptake process.Immunolabelling for the glutamate transporters GLAST, GLT-1alpha and GLT-1v showed that each of these proteins are expressed early in development and appear to be restricted to glial-like cells throughout the development period examined (except in the retina, where neuronal elements were also labelled). Our capacity to detect very early expression of the variant forms of GLT-1 contrasts with other studies, a feature that we attribute to the use of antigen-recovery techniques that unmask protein epitopes that are otherwise undetectable. These studies illustrate the widespread presence of functional glutamate transporters in the developing CNS, in many cases before the onset of periods of synaptogenesis and indicate that regulation of extracellular glutamate by multiple excitatory amino acid transporters might be crucial in early CNS development.