Benign and Atypical Meningioma Metabolic Signatures by High-Resolution Magic-Angle Spinning Molecular Profiling Daniel Monleo ´ n,*,† Jose´ Manuel Morales,‡ Jose´ Gonzalez-Darder,§ Fernando Talamantes,§ § Oscar Corte´s, Rosario Gil-Benso,| Concepcio ´ n Lo ´ pez-Gine´s,| Miguel Cerda´-Nicola´s,|,# and Bernardo Celda⊥,∇ Fundacio´n de Investigacio´n del Hospital Clı´nico Universitario de Valencia, Valencia, Spain, Unidad Central de Investigacio´n en Medicina, Universitat de Vale´ncia, Valencia, Spain, Servicio de Neurocirugı´a, Hospital Clı´nico Universitario de Valencia, Valencia, Spain, Departamento de Patologı´a, Universitat de Valencia, Valencia, Spain, Departamento de Quı´mica Fı´sica, Universitat de Valencia, Valencia, Spain, Centro de Investigacio´n Biome´dica en Red de Enfermedades Respiratorias (CIBER-RES), and Centro de Investigacio´n Biome´dica en Red de Bioingenierı´a, Biomateriales y Nanomedicina (CIBER-BBN), Valencia, Spain Received February 11, 2008
Meningiomas are neoplasms that arise from the leptomeningeal covering of the brain and spinal cord, accounting for 15%-20% of CNS tumors. The WHO classifies meningiomas into three histological grades: benign, atypical, and anaplasic in accordance with the clinical prognosis. Atypical and anaplasic meningiomas tend to recur. Sometimes, meningiomas with histological diagnosis of benign meningioma show clinical characteristics of atypical meningioma. In this context, high-resolution magicangle spinning (HR-MAS) spectroscopy of intact tissue from brain tumor biopsies has shown great potential as a support diagnostic tool. In this work, we show differences between benign and atypical meningiomas in HR-MAS molecular profiles of meningioma biopsies. Metabolic differences between meningioma grades include changes in the levels of glutathione. Glutathione role in cancer is still unclear, as it may act both as protective and pathogenic factor. Glutamine and glutamate, which are related to glutathione metabolism and have been associated with tumor recurrence, are also increased in atypical meningiomas. Other metabolites associated with tumor malignancy that show statistically significant differences between benign and atypical meningiomas include phosphocholine and phosphoethanolamine. Overall, this work suggests that the additional information obtained by NMR metabolomics applied to biopsies of human meningiomas may be useful for assessing tumor grade and determining optimum treatment strategies. Keywords: atypical meningioma • molecular profiling • metabolomics • NMR • HR-MAS
Introduction Meningiomas are neoplasms arising from the leptomeningeal covering of the brain and spinal cord, accounting for 15%-20% of all central nervous system tumors. Although a majority of these tumors are histological benign, some meningiomas show signs of malignancy such as marked vascularity, loss of organoid structure, mitotic figures, nuclear pleomorphism, prominent nucleoli, focal necrosis, or infiltration to the adjacent brain.1 The World Health Organization (WHO) classifies men* To whom correspondence should be addressed. Dr. Daniel Monleo´n, Fundacio´n de Investigacio´n del Hospital Clı´nico Universitario de Valencia, Avda. Blasco Iba´n ˜ ez, 17, Valencia 46010, Spain. † Fundacio´n de Investigacio´n del Hospital Clı´nico Universitario de Valencia. ‡ Unidad Central de Investigacio´n en Medicina, Universitat de Vale´ncia. § Servicio de Neurocirugı´a, Hospital Clı´nico Universitario de Valencia. | Departamento de Patologı´a, Universitat de Valencia. # Centro de Investigacio´n Biome´dica en Red de Enfermedades Respiratorias (CIBER-RES). ⊥ Departamento de Quı´mica Fı´sica, Universitat de Valencia. ∇ Centro de Investigacio´n Biome´dica en Red de Bioingenierı´a, Biomateriales y Nanomedicina (CIBER-BBN).
2882 Journal of Proteome Research 2008, 7, 2882–2888 Published on Web 05/29/2008
ingiomas into three histological grades: grade I (benign), grade II (atypical), and grade III (anaplastic) in accordance with the clinical prognosis.2 Atypical and anaplastic meningiomas show a high index of recurrence 5 years after complete resection and are associated with lower survival rates compared to benign meningiomas.3 Sometimes, meningiomas with histological diagnosis of benign meningioma show clinical and genetical characteristics of atypical meningioma.4 The ability to distinguish benign from atypical tumors is important because of its impact on treatment decisions. Moreover, this distinction may also aid in determining the aggressiveness of surgical resection and necessity of combined radiation therapy. Additional criteria for better classification of meningiomas will improve these clinical decisions as well as patient follow-up strategy after surgery. High-resolution magic-angle spinning NMR spectroscopy (HR-MAS NMR) is a powerful technique for the investigation of metabolites within different intact tissues.5–10 The precise determination of biochemical and metabolic profiles in intact tissue promises to extend the possibilities of NMR as a medical 10.1021/pr800110a CCC: $40.75
2008 American Chemical Society
research articles
Benign and Atypical Meningiomas Molecular Profiling by HR-MAS 5,11–14
diagnostic tool. For nonsolid or highly viscous liquids, HR-MAS NMR spectroscopy allows the reduction of most of the line broadening associated to restricted molecular motion, chemical shift anisotropy, dipolar couplings and field inhomogeneity by high-rate spinning of the sample at the magic angle θ ) 54.7°.15–17 The potential of HR-MAS applications to the study of biological tissues has been widely demonstrated in the investigation of different cellular alterations.18,19 In addition, HR-MAS NMR spectroscopy of intact tissues (ex vivo) provides further advantages over traditional high resolution liquid NMR of tissue extracts (in vitro).11,20,21 High resolution NMR on extracts of excised tissues requires large amounts of sample.22 Likewise, the extraction process via protein precipitation methods disables the direct observation of small cellular proteins and membrane semimobile lipids. Moreover, extraction methods usually discriminate metabolites on the basis of solubility in a particular solvent. Although it has some minor limitations associated mainly to the spinning of the sample (spinning side-bands, spinning degradation effects and spinning temperature gradients among others), HR-MAS NMR is a nondestructive technique, which requires minimal sample preparation, and allows the observation of most of the tissue metabolites and dynamic interactions in an extremely reduced sample quantity. This technology can supplement histopathological examination and potentially improve brain tumor diagnostics.5,11,20 The similarities between ‘ex vivo’ and ‘in vivo’ spectra found in studies of different primary brain tumors allow better interpretation of in vivo MR spectra and increase the clinical potential of the method. HR-MAS spectra generate metabolic profiles that contain information on physiological and pathological status. This approach can be used to define the metabolomic phenotype of a tissue. The aim of our study was to obtain metabolic profiles of benign and atypical meningiomas by HR-MAS, to further characterize possible differences between these two types of tumors and to identify potential diagnosis biomarkers. Thirty human meningioma tissue samples underwent HR-MAS spectroscopy measurements and subsequent histopathological examination. Principal component analysis (PCA) performed over the HR-MAS spectra allowed to identify differences between meningioma types. These differences provided a set of relevant metabolites, which are potential atypical meningioma biomarkers.
Experimental Procedures Patients. Thirty human meningiomas biopsies were obtained from 30 patients at the Department of Neurosurgery of the Hospital Clinico Universitario de Valencia. This study was reviewed and approved by the local ethics committee. During surgery, most of the resected tissue was sent for routine histological analysis, and the remainder was immediately put in cryogenic vials and snap-frozen in liquid nitrogen. All snapfrozen samples were stored in a freezer at -80 °C until further analysis. Meningioma types analyzed include 14 meningothelial, 8 transitional, 5 fibroblastic, 1 secretory, 1 psammomatous, and 1 angiomatous, from 10 male and 20 female patients aged 29-82. Patient 9 showed some iatrogenic necrosis and was excluded from the Principal Component Analysis. However, resonance quantification was performed for most relevant metabolites. Tumoral grade was also assessed. Table 1 gives a summary of the clinical characterization of the patients and biopsy samples.
Table 1. Data for the meningioma patients and samples studied in this work. patient no.
histopathological diagnosis
grade
sex/age (years)
Ki-67 %
mitosis indexa
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Transitional Meningothelial Meningothelial Fibroblastic Transitional Transitional Psammomatous Meningothelial Meningothelial Secretory Transitional Meningothelial Meningothelial Meningothelial Transitional Fibroblastic Transitional Transitional Meningothelial Meningothelial Fibroblastic Meningothelial Meningothelial Meningothelial Angiomatous Transitional Fibroblastic Meningothelial Fibroblastic Meningothelial
I I I I I I I I I I I I I I I I I I I I I I I II II II II II II II
M/68 F/65 F/56 F/69 F/60 F/68 F/36 F/51 F/68 F/82 F/49 M/34 F/65 M/29 F/58 F/41 F/41 M/58 F/71 M/47 F/73 M/66 M/43 M/70 F/70 F/34 F/36 M/60 M/78 F/41
