Marie-Christine Guiot

Cystic fibrosis lung disease following infection with Pseudomonas aeruginosa in Cftr knockout mice using novel non-invasive direct pulmonary infection technique.

To better understand the mechanism of lung infection with Pseudomonas aeruginosa (P. aeruginosa), many techniques have been developed in order to establish lung infection in rodents. A model of chronic lung infection, using tracheotomy to inoculate the bacteria, has been extensively used in the cystic fibrosis (CF) mouse model of lung infection. The cystic fibrosis transmembrane channel (Cftr) knockout (KO) mice are smaller than normal mice and are more sensitive to housing and nutritional conditions, leading to small amounts of animals being available for experiments. Because of these characteristics, and because of the invasiveness of the infection procedure which we, and others, have been using to mimic the lung infection, we sought to find an alternative way to study the inflammatory response during lung P. aeruginosa infection. The technique we describe here consists of the injection of bacterial beads directly into the lungs through the mouth without the need of any tracheal incisions. This technique of direct pulmonary delivery enables much faster infection of the animals compared with the intratracheal technique previously used. The use of this less invasive technique allows the exclusion of the surgery-related inflammation. Our results show that, using the direct pulmonary delivery technique, the KO mice were more susceptible to P. aeruginosa lung infection compared with their wild-type (WT) controls, as shown by their increased weight loss, higher bacterial burden and more elevated polymorphonuclear (PMN) alveolar cell recruitment into the lungs. These differences are consistent with the pathological profiles observed in CF patients infected with P. aeruginosa. Overall, this method simplifies the infection procedure in terms of its duration and invasiveness, and improves the survival rate of the KO mice when compared with the previously used intratracheal procedure.

Sodium-channel defects in benign familial neonatal-infantile seizures

Ion-channel gene defects are associated with a range of paroxysmal disorders, including several monogenic epilepsy syndromes. Two autosomal dominant disorders present in the first year of life: benign familial neonatal seizures, which is associated with potassium-channel gene defects; and benign familial infantile seizures, for which no genes have been identified. Here, we describe a clinically intermediate variant, benign familial neonatal-infantile seizures, with mutations in the sodium-channel subunit gene SCN2A. This clinico-molecular correlation defines a new benign familial epilepsy syndrome beginning in early infancy, an age at which seizure disorders frequently have a sombre prognosis.

Comparison of polarization properties of human adult microglia and blood-derived macrophages.

Both microglia, the resident myeloid cells of the CNS parenchyma, and infiltrating blood‐derived macrophages participate in inflammatory responses in the CNS. Macrophages can be polarized into M1 and M2 phenotypes, which have been linked to functional properties including production of inflammation association molecules and phagocytic activity. We compare phenotypic and functional properties of microglia derived from the adult human CNS with macrophages derived from peripheral blood monocytes in response to M1 and M2 polarizing conditions. Under M1 conditions, microglia and macrophages upregulate expression of CCR7 and CD80. M2 treatment of microglia‐induced expression of CD209 but not additional markers CD23, CD163, and CD206 expressed by M2 macrophages. M1‐polarizing conditions induced production of IL‐12p40 by both microglia and macrophages; microglia produced higher levels of IL‐10 under M1 conditions than did macrophages. Under M2 conditions, microglia ± LPS produced comparable levels of IL‐10 under M1 conditions whereas IL‐10 was induced by LPS in M2 macrophages. Myelin phagocytosis was greater in microglia than macrophages under all conditions; for both cell types, activity was higher for M2 cells. Our findings delineate distinctive properties of microglia compared with exogenous myeloid cells in response to signals derived from an inflammatory environment in the CNS.

Intraoperative brain cancer detection with Raman spectroscopy in humans

A handheld Raman spectroscopy probe enabled detection of invasive brain cancer intraoperatively in patients with grade 2 to 4 gliomas. Probing for brain tumors Gliomas are invasive cancers, spreading quietly throughout the brain. They pose a formidable challenge to surgeons who try to remove all cancer cells during resection; leaving any cancer behind can lower the patient’s prospects for survival. Jermyn et al. adapted Raman spectroscopy for the operating room by developing an imaging technique that uses a commercially available, handheld contact fiber optic probe. The probe’s optic cables were connected to a near-infrared laser, for stimulating tissue molecules; in turn, these components were linked to a computer to visualize resulting spectra in real time. When held against human brain tissue, the probe measured the Raman scattering signal, which was separated from background signals and differentiated from “normal” tissues using certain algorithms. The authors tested the probe in 17 patients with grade 2 to 4 gliomas who were undergoing surgery and compared imaging results with 161 biopsy samples. Intraoperative Raman imaging allowed the authors to detect both invasive and dense cancer cells with an accuracy of 92%. By comparison, the surgeon, using standard surgical tools like the bright-field microscope and magnetic resonance imaging, identified cancer with 73% accuracy. Such label-free, portable, intraoperative imaging technologies will be important in improving the efficiency of tumor resections and, in turn, for extending survival times of glioma patients. Cancers are often impossible to visually distinguish from normal tissue. This is critical for brain cancer where residual invasive cancer cells frequently remain after surgery, leading to disease recurrence and a negative impact on overall survival. No preoperative or intraoperative technology exists to identify all cancer cells that have invaded normal brain. To address this problem, we developed a handheld contact Raman spectroscopy probe technique for live, local detection of cancer cells in the human brain. Using this probe intraoperatively, we were able to accurately differentiate normal brain from dense cancer and normal brain invaded by cancer cells, with a sensitivity of 93% and a specificity of 91%. This Raman-based probe enabled detection of the previously undetectable diffusely invasive brain cancer cells at cellular resolution in patients with grade 2 to 4 gliomas. This intraoperative technology may therefore be able to classify cell populations in real time, making it an ideal guide for surgical resection and decision-making.

Accelerated hypofractionated intensity-modulated radiotherapy with concurrent and adjuvant temozolomide for patients with glioblastoma multiforme: a safety and efficacy analysis.

PURPOSE Despite multimodality treatments, the outcome of patients with glioblastoma multiforme remains poor. In an attempt to improve results, we have begun a program of accelerated hypofractionated intensity-modulated radiotherapy (hypo-IMRT) with concomitant and adjuvant temozolomide (TMZ). METHODS AND MATERIALS Between March 2004 and June 2006, 35 unselected patients with glioblastoma multiforme were treated with hypo-IMRT. During a 4-week period, using a concomitant boost technique, a dose of 60 Gy and 40 Gy were delivered in 20 fractions prescribed to the periphery of the gross tumor volume and planning target volume, respectively. TMZ was administered according to the regimen of Stupp et al. RESULTS The median follow-up was 12.6 months. Of the 35 patients, 29 (82.8%) completed the combined modality treatment, and 25 (71.4%) received a median of four cycles of adjuvant TMZ. The median overall survival was 14.4 months, and the median disease-free survival was 7.7 months. The median survival time differed significantly between patients who underwent biopsy and those who underwent partial or total resection (7.1 vs. 16.1 months, p = 0.035). The median survival was also significantly different between patients with methylated vs. unmethylated 0-6-methylguanine-DNA methyltransferase promoters (14.4 vs. 8.7 months, p = 0.049). The pattern of failure was predominantly central, within 2 cm of the initial gross tumor volume. Grade 3-4 toxicity was limited to 1 patient with nausea and emesis during adjuvant TMZ administration. CONCLUSION The results of our study have shown that hypo-IMRT with concomitant and adjuvant TMZ is well tolerated with a useful 2-week shortening of radiotherapy. Despite a high number of patients with poor prognostic features (74.3% recursive partitioning analysis class V or VI), the median survival was comparable to that after standard radiotherapy fractionation schedules plus TMZ.

Clinical Significance of Molecular Biomarkers in Glioblastoma

AIM To review the impact of molecular biomarkers on response to therapy and survival in patients with primary glioblastoma (GBM). MATERIALS & METHODS Tissue specimens were analyzed for p53 mutations, EGFR amplification, loss of PTEN and p16, and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. Demographic and clinical data were gathered from medical records. RESULTS Clinical and pathological data of 125 patients were collected and analysed. MGMT promoter methylation was associated with improved median overall survival (OS) (61 vs. 42 weeks, p = 0.01) and was an important prognosticator independent of age at diagnosis, extent of resection and post-operative ECOG performance status (HR 2.04, 95% CI 1.11-3.75). Among patients with MGMT promoter methylation, survival was significantly improved with chemoradiotherapy (CRT) over radiotherapy (RT) alone (71 vs. 14 weeks, p < 0.01). Furthermore, amongst those treated with temozolomide (TMZ) based CRT, the presence of EGFR amplification, maintenance of PTEN and wild-type p53 and p16 were each associated with trends towards improved survival. CONCLUSION MGMT promoter methylation is a strong, independent prognostic factor for OS in GBM. EGFR amplification, maintenance of PTEN, wild-type p53 and p16 all appear to be associated with improved survival in patients treated with CRT. However, the prognostic value of these biomarkers could not be ascertained and larger prospective studies are warranted.

Characterization of a Raman spectroscopy probe system for intraoperative brain tissue classification

A detailed characterization study is presented of a Raman spectroscopy system designed to maximize the volume of resected cancer tissue in glioma surgery based on in vivo molecular tissue characterization. It consists of a hand-held probe system measuring spectrally resolved inelastically scattered light interacting with tissue, designed and optimized for in vivo measurements. Factors such as linearity of the signal with integration time and laser power, and their impact on signal to noise ratio, are studied leading to optimal data acquisition parameters. The impact of ambient light sources in the operating room is assessed and recommendations made for optimal operating conditions. In vivo Raman spectra of normal brain, cancer and necrotic tissue were measured in 10 patients, demonstrating that real-time inelastic scattering measurements can distinguish necrosis from vital tissue (including tumor and normal brain tissue) with an accuracy of 87%, a sensitivity of 84% and a specificity of 89%.

The oncometabolite 2-hydroxyglutarate activates the mTOR signalling pathway.

The identification of cancer-associated mutations in the tricarboxylic acid (TCA) cycle enzymes isocitrate dehydrogenases 1 and 2 (IDH1/2) highlights the prevailing notion that aberrant metabolic function can contribute to carcinogenesis. IDH1/2 normally catalyse the oxidative decarboxylation of isocitrate into α-ketoglutarate (αKG). In gliomas and acute myeloid leukaemias, IDH1/2 mutations confer gain-of-function leading to production of the oncometabolite R-2-hydroxyglutarate (2HG) from αKG. Here we show that generation of 2HG by mutated IDH1/2 leads to the activation of mTOR by inhibiting KDM4A, an αKG-dependent enzyme of the Jumonji family of lysine demethylases. Furthermore, KDM4A associates with the DEP domain-containing mTOR-interacting protein (DEPTOR), a negative regulator of mTORC1/2. Depletion of KDM4A decreases DEPTOR protein stability. Our results provide an additional molecular mechanism for the oncogenic activity of mutant IDH1/2 by revealing an unprecedented link between TCA cycle defects and positive modulation of mTOR function downstream of the canonical PI3K/AKT/TSC1-2 pathway.

Low frequency of promoter methylation of O6-methylguanine DNA methyltransferase and hMLH1 in ulcerative colitis-associated tumors: comparison with sporadic colonic tumors.

To cast light on the contribution of methylation to genesis of ulcerative colitis (UC)-associated tumors, promoter methylation and expression of O6-methylguanine DNA methyltransferase (MGMT), hMLH1, p16INK4, and E-cadherin were examined in 14 low-grade dysplasias (LGDs), 15 high-grade dysplasias (HGDs), and 14 adenocarcinomas associated with UC and, for comparison, in 30 sporadic adenomas with LGD, 30 adenomas with HGD, and 60 adenocarcinomas, using methylation-specific polymerase chain reaction and immunohistochemical analysis. The frequency of MGMT and hMLH1 methylation in UC-associated tumors was low, with a significant difference between HGD and sporadic adenomas with HGD of the left hemicolon. The methylation frequency of p16INK4 in UC-associated tumors was also relatively low compared with sporadic colonic tumors. For E-cadherin, methylation was limited in both types of tumor. Decrease of expression of MGMT, hMLH1, and p16INK4 was significantly correlated with methylation. Thus, compared with the sporadic type, contribution of methylation to UC-associated tumorigenesis seems to be low.

Low-grade oligodendroglioma: an indolent but incurable disease? Clinical article.

OBJECT The authors reviewed their institutional experience with pure low-grade oligodendroglioma (LGO), correlating outcomes with several variables of possible prognostic values. METHODS Sixty-nine patients with WHO-classified LGOs were treated between 1992 and 2006 at the McGill University Health Center. Clinical, pathological, and radiological records were carefully reviewed. Demographic characteristics; the nature and duration of presenting symptoms; baseline neurological function; extent of resection; Karnofsky Performance Scale score; preoperative radiological findings including tumor size, location, and absence/presence of enhancement; and pathological data including chromosome arms 1p/19q codeletion and O-methylguanine-DNA methyltransferase promoter gene methylation status were all compiled. The timing and dose of radio- and/or chemotherapy, date of tumor progression, pathological finding at disease progression, treatment at time of disease progression, and status at the last follow-up were also recorded. RESULTS The median follow-up period was 6.1 years (range 1.3-16.3 years). The majority (78%) of patients presented with seizures; contrast enhancement was initially seen in 16 patients (25%). All patients had undergone an initial surgical procedure: gross-total resection in 27%, partial resection in 59%, and biopsy only in the remaining 13%. Fifteen patients received adjuvant radiotherapy. Data on O-methylguanine-DNA methyltransferase promoter gene methylation status was available in 47 patients (68%) and in all but 1 patient for 1p/19q status. Survival at 5, 10, and 15 years was 83, 63, and 29%, respectively. Multivariate analysis showed that seizures at presentation and the absence of contrast enhancement were the only independent favorable prognostic factors for survival. The 5-, 10-, and 15-year progression-free survival rates were 46, 7.7, and 0%, respectively. CONCLUSIONS This retrospective review confirms the indolent but progressively fatal nature of LGOs. Contrast enhancement was the most evident single prognostic factor. New treatment strategies are clearly needed in the management of this disease.