Calliope Maris

Apparent Diffusion Coefficient and Cerebral Blood Volume in Brain Gliomas: Relation to Tumor Cell Density and Tumor Microvessel Density Based on Stereotactic Biopsies

BACKGROUND AND PURPOSE: MR imaging–based apparent diffusion coefficient (ADC) and regional cerebral blood volume (rCBV) measurements have been related respectively to both cell and microvessel density in brain tumors. However, because of the high degree of heterogeneity in gliomas, a direct correlation between these MR imaging–based measurements and histopathologic features is required. The purpose of this study was to correlate regionally ADC and rCBV values with both cell and microvessel density in gliomas, by using coregistered MR imaging and stereotactic biopsies. MATERIALS AND METHODS: Eighteen patients (9 men, 9 women; age range, 19–78 years) with gliomas underwent diffusion-weighted and dynamic susceptibility contrast-enhanced MR imaging before biopsy. Eighty-one biopsy samples were obtained and categorized as peritumoral, infiltrated tissue, or bulk tumor, with quantification of cell and microvessel density. ADC and rCBV values were measured at biopsy sites and were normalized to contralateral white matter on corresponding maps coregistered with a 3D MR imaging dataset. ADC and rCBV ratios were compared with quantitative histologic features by using the Spearman correlation test. RESULTS: The highest correlations were found within bulk tumor samples between rCBV and cell density (r=0.57, P < .001) and rCBV and microvessel density (r=0.46, P < .01). An inverse correlation was found between ADC and microvessel density within bulk tumor (r=−0.36, P < .05), whereas no significant correlation was found between ADC and cell density. CONCLUSION: rCBV regionally correlates with both cell and microvessel density within gliomas, whereas no regional correlation was found between ADC and cell density.

VEGFR1 and VEGFR2 Involvement in Extracellular Galectin-1- and Galectin-3-Induced Angiogenesis

Aim Accumulating evidence suggests that extracellular galectin-1 and galectin-3 promote angiogenesis. Increased expression of galectin-1 and/or galectin-3 has been reported to be associated with tumour progression. Thus, it is critical to identify their influence on angiogenesis. Methods We examined the individual and combined effects of galectin-1 and galectin-3 on endothelial cell (EC) growth and tube formation using two EC lines, EA.hy926 and HUVEC. The activation of vascular endothelial growth factor receptors (VEGFR1 and VEGFR2) was determined by ELISA and Western blots. We evaluated the VEGFR1 and VEGFR2 levels in endosomes by proximity ligation assay. Results We observed different responses to exogenous galectins depending on the EC line. An enhanced effect on EA.hy926 cell growth and tube formation was observed when both galectins were added together. Focusing on this enhanced effect, we observed that together galectins induced the phosphorylation of both VEGFR1 and VEGFR2, whereas galectin-1 and −3 alone induced VEGFR2 phosphorylation only. In the same way, the addition of a blocking VEGFR1 antibody completely abolished the increase in tube formation induced by the combined addition of both galectins. In contrast, the addition of a blocking VEGFR2 antibody only partially inhibited this effect. Finally, the addition of both galectins induced a decrease in the VEGFR1 and VEGFR2 endocytic pools, with a significantly enhanced effect on the VEGFR1 endocytic pool. These results suggest that the combined action of galectin-1 and galectin-3 has an enhanced effect on angiogenesis via VEGFR1 activation, which could be related to a decrease in receptor endocytosis.

Exploring the distinctive biological characteristics of pilocytic and low-grade diffuse astrocytomas using microarray gene expression profiles.

Abstract Although World Health Organization (WHO) grade I pilocytic astrocytomas and grade II diffuse astrocytomas have been classified for decades as different clinicopathologic entities, few, if any, data are available on the biologic features explaining these differences. Although more than 50 microarray-related studies have been carried out to characterize the molecular profiles of astrocytic tumors, we have identified only 11 that provide sound data on low-grade astrocytomas. We have incorporated these data into a comparative analysis for the purpose of identifying the most relevant molecular markers characterizing grade I pilocytic and grade II diffuse astrocytomas. Our analysis has identified various interesting genes that are differentially expressed in either grade I or grade II astrocytomas when compared with normal tissue and/or high-grade (WHO grade III and IV) astrocytomas. A large majority of these genes encode adhesion, extracellular matrix, and invasion-related proteins. Interestingly, a group of 6 genes (TIMP4, C1NH, CHAD, THBS4, IGFBP2, and TLE2) constitute an expression profile characteristic of grade I astrocytomas as compared with all other categories of tissue (normal brain, grade II, and high-grade astrocytomas). The end products (proteins) of these genes act as antimigratory compounds, a fact that could explain why pilocytic astrocytomas behave as compact (well-circumscribed) tumors as opposed to all the other astrocytic tumor types that diffusely invade the brain parenchyma. Having validated these molecular markers by means of real-time reverse transcriptase-polymerase chain reaction, an integrated model was proposed illustrating how and why pilocytic astrocytomas constitute a distinct biologic and pathologic entity when compared with diffuse astrocytomas.

A simplified approach for the molecular classification of glioblastomas.

Glioblastoma (GBM) is the most common malignant primary brain tumors in adults and exhibit striking aggressiveness. Although GBM constitute a single histological entity, they exhibit considerable variability in biological behavior, resulting in significant differences in terms of prognosis and response to treatment. In an attempt to better understand the biology of GBM, many groups have performed high-scale profiling studies based on gene or protein expression. These studies have revealed the existence of several GBM subtypes. Although there remains to be a clear consensus, two to four major subtypes have been identified. Interestingly, these different subtypes are associated with both differential prognoses and responses to therapy. In the present study, we investigated an alternative immunohistochemistry (IHC)-based approach to achieve a molecular classification for GBM. For this purpose, a cohort of 100 surgical GBM samples was retrospectively evaluated by immunohistochemical analysis of EGFR, PDGFRA and p53. The quantitative analysis of these immunostainings allowed us to identify the following two GBM subtypes: the “Classical-like” (CL) subtype, characterized by EGFR-positive and p53- and PDGFRA-negative staining and the “Proneural-like” (PNL) subtype, characterized by p53- and/or PDGFRA-positive staining. This classification represents an independent prognostic factor in terms of overall survival compared to age, extent of resection and adjuvant treatment, with a significantly longer survival associated with the PNL subtype. Moreover, these two GBM subtypes exhibited different responses to chemotherapy. The addition of temozolomide to conventional radiotherapy significantly improved the survival of patients belonging to the CL subtype, but it did not affect the survival of patients belonging to the PNL subtype. We have thus shown that it is possible to differentiate between different clinically relevant subtypes of GBM by using IHC-based profiling, a method that is advantageous in its ease of daily implementation and in large-scale clinical application.

The Differential Expression of Galectin-1 and Galectin-3 in Normal Lymphoid Tissue and Non-Hodgkin's and Hodgkin's Lymphomas

The WHO classification of lymphomas was established on the basis of clinical, morphological, immunohistochemical and genetic criteria. However, each entity displays its own spectrum of clinical aggressiveness. Treatment success varies widely and is not predictable. Since galectins are involved in oncogenesis and the physiology of immune cells, we investigated whether galectin-1 and galectin-3 immunohistochemical expression could differ in 25 normal lymphoid tissues, 42 non-Hodgkins and 14 Hodgkins lymphomas. Immunohistochemical galectin expression was submitted to semi-quantitative and quantitative (computer-assisted microscopy) evaluations. This study is completed by an analysis (by means of quantitative RT-PCR) of galectin-3 mRNA expression in 3 normal lymph nodes, 3 follicular lymphomas (FLs) and 3 diffuse large B-cell lymphomas (DLBCLs). The data show that in normal lymphoid tissue, lymphocytes do not express galectin-1 and rarely express galectin-3. In contrast, galectin-3 was expressed in 8 of the 16 DLBCL cases and in 1 of the 8 FL cases. Furthermore, galectin-3 mRNA was expressed 3-times more in the DLBCLs than in the FLs. While the blood vessel walls of the lymphomas expressed galectin-1, the vessel walls of normal lymphoid tissues did not. This expression of galectin-1 in blood vessel walls was correlated with vascular density. The present study thus shows that DLBCL can be distinguished from normal lymphoid tissue and other lymphomas on the basis of galectin-3 expression.

Comparison of clinical and post-mortem findings in intensive care unit patients

The autopsy has long been regarded as an important tool for clinical confrontation, education and quality assurance. The aims of this study were to examine the correlation between the clinical diagnosis and autopsy findings in adult patients who died in an intensive care unit (ICU) and to identify the types of errors in diagnosis to improve quality of care. Autopsies from 289 patients who died in the ICU during a 2-year period were studied. Post-mortem examination revealed unexpected findings in 61 patients (21%) including malignancy, pulmonary embolism, aspergillosis, myocardial or mesenteric infarction and unsuspected bacterial, viral or fungal infection. These unexpected findings were classified as Goldman class I errors in 17 (6%), class II in 38 (13%) and class III in six (2%) cases. Although the incidence of unexpected findings with clinical significance was low, post-mortem examination remains a valuable source of pertinent information that may improve the management of ICU patients.

Endothelial hyperplasia and endothelial galectin-3 expression are prognostic factors in primary central nervous system lymphomas

Recently, considerable attention has been focused on the identification of clinically relevant prognostic markers for primary central nervous system lymphomas (PCNSL). The present study investigated whether three morphological features, i.e. necrosis, reactive perivascular T‐cell infiltrate and endothelial hyperplasia, and galectin‐1 and galectin‐3 immunohistochemical expression have prognostic roles in a series of 58 PCNSL samples from 44 immunocompetent and 14 immunocompromised patients. The presence of endothelial hyperplasia (identified in 21% of the assessable cases) was identified as a bad prognostic factor for immunocompetent PCNSL patients, whereas the other morphological features were not associated with any prognostic value. Lymphomatous cells of eight PCNSL cases expressed galectin‐3 without any prognostic value, and lymphomatous cells did not express galectin‐1. In contrast, endothelial expression of galectin‐3 was identified (by means of uni‐ and multi‐variate analyses) as a bad prognostic factor for immunocompetent PCNSL patients. In addition, a combination of endothelial hyperplasia and/or endothelial galectin‐3 expression was shown to be an independent prognostic factor for immunocompetent PCNSL patients treated with methotrexate‐based chemotherapy. In summary, this study suggests that endothelial‐related markers can identify risk groups of PCNSL patients and indicates that galectin‐3 could be involved in PCNSL angiogenesis.

In vitro and in vivo evaluation of a dry powder endotracheal insufflator device for use in dose-dependent preclinical studies in mice

The aim of this study was to evaluate the ability of the Penn-Century Dry Powder Insufflator for mice (DP-4M) to reproducibly, uniformly, and deeply deliver dry powders for inhalation in the mouse lung. Itraconazole-based dry powder formulations produced by spray-drying were different in terms of composition (different ratios of drug and mannitol, with or without phospholipids), but relatively similar in terms of particle size and mass median aerodynamic diameter. The ability of the dry powder insufflator to disaggregate each formulation was the same, indicated by the absence of a statistically significant difference between the particle size distribution parameters, as measured by laser scattering. The emitted fraction varied in vivo compared to the in vitro condition. Fluorescent particle distribution in the lungs was uniform and reached the alveolar spaces, as visualized by fluorescent microscopy. In terms of drug recovery in lung tissue, a minimum administered powder mass (in this case ∼1 mg) was necessary to recover at least 30% of the emitted dose in the lung and to obtain reproducible pulmonary concentrations. To reduce the dose administered in the lung, it was preferable to dilute the active ingredient within the carrier instead of reducing the dry powder mass inserted in the sampling chamber. Dry powder insufflators are devices usable in dose-dependent preclinical trials but have critical parameters to efficiently deliver reproducible doses depending on the type of formulation.

Clustering methods applied in the detection of Ki67 hot-spots in whole tumor slide images: An efficient way to characterize heterogeneous tissue-based biomarkers

Whole‐slide scanners allow the digitization of an entire histological slide at very high resolution. This new acquisition technique opens a wide range of possibilities for addressing challenging image analysis problems, including the identification of tissue‐based biomarkers. In this study, we use whole‐slide scanner technology for imaging the proliferating activity patterns in tumor slides based on Ki67 immunohistochemistry. Faced with large images, pathologists require tools that can help them identify tumor regions that exhibit high proliferating activity, called “hot‐spots” (HSs). Pathologists need tools that can quantitatively characterize these HS patterns. To respond to this clinical need, the present study investigates various clustering methods with the aim of identifying Ki67 HSs in whole tumor slide images. This task requires a method capable of identifying an unknown number of clusters, which may be highly variable in terms of shape, size, and density. We developed a hybrid clustering method, referred to as Seedlink. Compared to manual HS selections by three pathologists, we show that Seedlink provides an efficient way of detecting Ki67 HSs and improves the agreement among pathologists when identifying HSs.

TIMP-4 and CD63: new prognostic biomarkers in human astrocytomas.

Based on the molecular profiling of astrocytomas, we previously identified a series of genes involved in astrocytoma invasion. Of these, tissue inhibitor of metalloproteinase-4 (TIMP-4) was found to be overexpressed in pilocytic astrocytomas relative to diffuse astrocytomas of any histological grade. Although some data suggest that TIMP-4 may be an anti-tumoral actor in astrocytomas, recent findings challenge this concept. The present study aims to investigate the diagnostic and prognostic values of TIMP-4 and its putative partner CD63 in human astrocytomas. Tissue microarray and image analysis were first carried out to quantitatively analyze the immunohistochemical expression of these proteins in 471 gliomas including 354 astrocytomas. Pathological semi-quantitative scores of both markers’ expression were then established and correlated to astrocytoma diagnosis and patient prognosis. TIMP-4 and CD63 expressions were both overexpressed in astrocytomas compared with oligodendrogliomas (P<0.001) and in pilocytic astrocytomas compared with grade II diffuse astrocytomas (P<0.001). In glioblastomas, high TIMP-4/CD63 co-expression scores were identified as independent prognostic factors associated with progression and shorter survival. In conclusion, this work provides the first evidence of a TIMP-4/CD63 association in astrocytoma tumor cells. It identifies TIMP-4 and CD63 as markers of the astrocytic phenotype in patients with gliomas. In addition, this work highlights the contribution of high TIMP-4/CD63 co-expression to the adverse outcomes of patients with glioblastomas.