Dynamic urinary proteomic analysis in a Walker 256 intracerebral tumor model.

BACKGROUND: Patients with primary and metastatic brain cancer have an extremely poor prognosis, mostly due to the late diagnosis of disease. Urine, which lacks homeostatic mechanisms, is an ideal biomarker source that accumulates early and highly sensitive changes to provide information about the early stage of disease. METHODS: A rat model mimicking the local tumor growth process in the brain was established with intracerebral Walker 256 (W256) cell injection. Urine samples were collected on days 3, 5, and 8 after injection, and then analyzed by liquid chromatography coupled with tandem mass spectrometry. RESULTS: In the intracerebral W256 model, no obvious clinical manifestations or abnormal magnetic resonance imaging (MRI) signals were found on days 3 or 5; at these time points, 9 proteins were changed significantly in the urine of all eight tumor rats. On day 8, when tumors were detected by MRI, 25 differential proteins were identified, including 10 that have been reported to be closely related to brain metastasis or primary tumors. The differential urinary proteome was compared with those from the subcutaneous W256 model and the intracerebral C6 model. Few differential proteins overlapped, and specific differential protein patterns were observed among the three models. CONCLUSIONS: These findings demonstrate that early changes in the urine proteome can be detected in the intracerebral W256 model. The urinary proteome can reflect the difference when tumor cells with different growth characteristics are inoculated into the brain and when identical tumor cells are inoculated into different areas, specifically, the subcutis and the brain.

Using urinary bFGF and TIMP3 levels to predict the presence of juvenile pilocytic astrocytoma and establish a distinct biomarker signature.

OBJECTIVE The authors report the use of urinary biomarkers as a novel, noninvasive technique to detect juvenile pilocytic astrocytomas (JPAs), capable of distinguishing JPAs from other CNS diseases, including other brain tumors. Preliminary screening of an array of tumors implicated proteases (including matrix metalloproteinases [MMPs]) and their inhibitors (tissue inhibitors of metalloproteinase [TIMPs]) as well as growth factors (including basic fibroblast growth factor [bFGF]) as candidate biomarkers. These data led the authors to hypothesize that tissue inhibitor of metalloproteinase 3 (TIMP3) and bFGF would represent high-probability candidates as JPA-specific biomarkers. METHODS Urine was collected from 107 patients, which included children with JPA (n = 21), medulloblastoma (n = 17), glioblastoma (n = 9), arteriovenous malformations (n = 25), moyamoya (n = 14), and age- and sex-matched controls (n = 21). Biomarker levels were quantified with enzyme-linked immunosorbent assay, tumor tissue expression was confirmed with immunohistochemical analysis, and longitudinal biomarker expression was correlated with imaging. Results were subjected to univariate and multivariate statistical analyses. RESULTS Using optimal urinary cutoff values of bFGF > 1.0 pg/µg and TIMP3 > 3.5 pg/µg, multiplexing bFGF and TIMP3 predicts JPA presence with 98% accuracy. Multiplexing bFGF and MMP13 distinguishes JPA from other brain tumor subtypes with up to 98% accuracy. Urinary biomarker expression correlated with both tumor immunohistochemistry and in vitro tumor levels. Urinary bFGF and TIMP3 decrease following successful tumor treatment and correlate with changes in tumor size. CONCLUSIONS This study identifies 2 urinary biomarkers-bFGF and TIMP3-that successfully detect one of the most common pediatric brain tumors with high accuracy. These data highlight potential benefits of urinary biomarkers and support their utility as diagnostic tools in the treatment of children with JPA.

Secondary brain choriocarcinoma: a case report.

Choriocarcinoma metastasizes widely. One in every ten choriocarcinoma that leaves its primary site, metastasizes to the brain. This 27 years old patient presented with symptoms of space occupying lesion that was confirmed by CT-SCAN. There was no history of vaginal bleeding and amenorrhoea was concealed by unmarried patient. Chest X-ray was normal. Tumor was excised after craniotomy. Histology of tumor was that of secondary choriocarcinoma. Patient responded excellently to chemotherapy and was well one year after. We strongly recommend a high index of suspicion of choriocarcinoma in management of brain tumors. ß-HCG assay should be included in investigation of all patients with intracranial tumors irrespective of sex.

[Protein malnutrition in patient in the early postoperative period after uncomplicated removal of the brain tumors].

Malnutrition leads to adverse effects and may worsen clinical outcome. Surgery as a stress factor activates pathological reactions changing metabolism structure. The aim of this study was to evaluate changes of protein metabolism in patients after elective neurosurgical operation. 24 patients were prepared for elective surgery and were enrolled in this study. Evaluation of each patient included: measurement of anthropometric indices--height, weight, arm circumference and the triceps skinfold thickness, the definition of protein loss by determining the loss of nitrogen in the urine, assessment of protein catabolism, determining the violations of nutritional status upon the base of laboratory parameters. During the course of the conducted investigation significant (p < 0.05) decrease in the indices of total protein, albumin, transferrin and the absolute numbers of lymphocytes in the postoperative period was revealed. All the patients developed severe protein catabolism. It became clear that uncomplicated elective surgical intervention, together with the adopted scheme of the nutritional therapy leads to severe protein catabolism in all patients.

A phase 1b trial of the combination of the antiangiogenic agent sunitinib and radiation therapy for patients with primary and metastatic central nervous system malignancies.

BACKGROUND: In this phase 1 trial, the authors evaluated sunitinib combined with radiation therapy (RT) for the treatment of primary or metastatic central nervous system (CNS) malignancies. METHODS: Eligible patients had CNS malignancies that required a (minimum) 2-week course of RT. Sunitinib (37.5 mg) was administered daily for the duration of RT with optional treatment extension of 1 month. Urine was collected at 3 time points for correlative biomarker studies. The primary endpoint was acute toxicity defined according to Common Toxicity Criteria version 3. RESULTS: Fifteen patients were enrolled (12 with CNS metastasis and 3 with primary tumors). RT doses ranged from 14 Gray (Gy) to 70 Gy (1.8-3.5 Gy per fraction). Acute toxicities included hematologic, nausea, hyperglycemia, fatigue, hypocalcemia, and diarrhea. Six patients (40%) developed grade ≤ 2 toxicities. Grade 3 toxicities occurred in 7 patients (47%) and included hematologic toxicity, fatigue, deep vein thrombosis, dysphasia, hyperglycemia, and hyponatremia. No grade 3 through 5 hypertensive events or intracerebral hemorrhages occurred. Two grade 5 adverse events attributed to disease progression occurred. The median follow-up was 34.2 months. Two patients (13%) achieved a partial response, 9 patients (60%) had stable disease, and 2 patients (13%) patients had progressive disease. The 6-month progression-free survival rate for patients who had brain metastasis was 58%. Grade 3 hematologic toxicity was correlated with greater changes in vascular endothelial growth factor levels changes between baseline and the completion of RT. CONCLUSIONS: Continuous 37.5-mg sunitinib combined with RT in patients who had CNS malignancies yielded acceptable toxicities and adverse events. The current results indicated that changes in urine vascular endothelial growth factor levels are associated with hematologic toxicity, and this association should be analyzed in a larger cohort. The feasibility, safety, and early response results warrant a phase 2 trial.

Detection of failure of bevacizumab treatment for malignant glioma based on urinary matrix metalloproteinase activity.

Although antiangiogenic treatment for malignant glioma using bevacizumab in combination with irinotecan chemotherapy has a promising effect on survival, the high incidence of increasing infiltrative tumors can be a problem in resistance to antiangiogenic therapy. In this study, we detected failure of bevacizumab treatment for malignant glioma through upregulation of metalloproteinase activity in the urine, as well as infiltrative tumors on MRI. In addition, MMP9 has been proved as a molecule that facilitates its infiltrative behavior in vivo in the brain animal model.

Can high-dose fotemustine reverse MGMT resistance in glioblastoma multiforme?

Glioblastoma multiforme (GBM), the highest grade malignant glioma, is associated with a grim prognosis-median overall survival is in the range 12-15 months, despite optimum treatment. Surgery to the maximum possible extent, external beam radiotherapy, and systemic temozolomide chemotherapy are current standard treatments for newly diagnosed GBM, with intracerebral delivery of carmustine wafers (Gliadel). Unfortunately, the effectiveness of chemotherapy can be hampered by the DNA repair enzyme O6-methylguanine methyltransferase (MGMT), which confers resistance both to temozolomide and nitrosoureas, for example fotemustine and carmustine. MGMT activity can be measured by PCR and immunohistochemistry, with the former being the current validated technique. High-dose chemotherapy can deplete MGMT levels in GBM cells and has proved feasible in various trials on temozolomide, in both newly diagnosed and recurrent GBM. We here report the unique case of a GBM patient, with high MGMT expression by immunohistochemistry, who underwent an experimental, high-dose fotemustine schedule after surgery and radiotherapy. Although treatment caused two episodes of grade 3-4 thrombocytopenia, a complete response and survival of more than three years were achieved, with a 30% increase in dose intensity compared with the standard fotemustine schedule.

Urinary biomarkers predict brain tumor presence and response to therapy.

PURPOSE: A major difficulty in treating brain tumors is the lack of effective methods of identifying novel or recurrent disease. In this study, we have evaluated the efficacy of urinary matrix metalloproteinases (MMP) as diagnostic biomarkers for brain tumors. EXPERIMENTAL DESIGN: Urine, cerebrospinal fluid, and tissue specimens were collected from patients with brain tumors. Zymography, ELISA, and immunohistochemistry were used to characterize the presence of MMP-2, MMP-9, MMP-9/neutrophil gelatinase-associated lipocalin (NGAL), and vascular endothelial growth factor (VEGF). Results were compared between age- and sex-matched controls and subjected to univariate and multivariate statistical analyses. RESULTS: Evaluation of a specific panel of urinary biomarkers by ELISA showed significant elevations of MMP-2, MMP-9, MMP-9/NGAL, and VEGF (all P < 0.001) in samples from brain tumor patients compared with controls. Multiplexing MMP-2 and VEGF provided superior accuracy compared with any other combination or individual biomarker. Receiver-operating characteristics curves for MMP-2 and VEGF showed excellent discrimination. Immunohistochemistry identified these same proteins in the source tumor tissue. A subset of patients with longitudinal follow-up revealed subsequent clearing of biomarkers after tumor resection. CONCLUSION: We report, for the first time, the identification of a panel of urinary biomarkers that predicts the presence of brain tumors. These biomarkers correlate with presence of disease, decrease with treatment, and can be tracked from source tissue to urine. These data support the hypothesis that urinary MMPs and associated proteins are useful predictors of the presence of brain tumors and may provide a basis for a novel, noninvasive method to identify new brain tumors and monitor known tumors after treatment.

[Detection and clinical significance of urinary epidermal growth factor in brain tumor patients].

OBJECTIVE: To investigate the clinical significance of urinary epidermal growth factor (EGF) in patients with brain tumors. METHODS: The levels of EGF in urine samples collected from 20 patients (9 low grade astrocytomas, 6 anaplastic astrocytomas, and 5 meningiomas) and 5 healthy individuals were determined. EGF levels were measured by radioimmunoassay technique. A preoperative and one postoperative determination were performed. RESULTS: Preoperative urinary EGF levels of astrocytoma patients were statistically higher than those of meningioma patients and the controls (P < 0.01). Preoperative urinary EGF levels showed a positive correlation with the degree of malignance in the astrocytoma patients (P < 0.05). A significant decrease of the postoperative levels of EGF was observed in the astrocytoma patients who underwent gross total resection (P < 0.01). The pre/postoperative urinary EGF levels of the meningioma patients showed no significant fluctuations and showed no significant difference with those of healthy individuals (P > 0.05). CONCLUSION: The urinary EGF levels of astrocytoma patients correlate with the WHO grade of malignance and significantly decrease after gross total removal. Urinary EGF may be of practical value in diagnosing and evaluating the surgical efficacy of astrocytomas.

Role of GSTM1, GSTP1, and GSTT1 gene polymorphism in ifosfamide metabolism affecting neurotoxicity and nephrotoxicity in children.

The aim of this study was to evaluate the impact of GSTM1, GSTT1, and GSTP1 gene polymorphism on urinary excretion of unchanged ifosfamide, 2-dechloroethylifosfamide (2DCIF), and 3-dechloroethylifosfamide (3DCIF) with regard to the incidence of ifosfamide-related nephrotoxicity and neurotoxicity in children. The study comprised 76 children (38 girls, 38 boys) ages 9.84 to 210 months who were being treated for various malignant diseases with ifosfamide. The children were enrolled after identification of genotype coding for three classes of glutathione S-transferases (GSTM1, GSTT1, and GSTP1) at the initial stage of diagnosis. (P) nuclear magnetic resonance spectroscopy was used to analyze the urinary excretion of unchanged ifosfamide, 2DCIF, and 3DCIF metabolites on consecutive days after the end of the 3-hour infusion of ifosfamide. In children with polymorphic locus of the GSTP1 gene compared with children with homozygous wild alleles, increased urinary excretion of 3DCIF (P=0.029) and decreased creatinine clearance was found (Mann-Whitney P=0.03; median 81.1 mL/min/1.73 m vs. 105.0 mL/min/1.73 m, respectively). The authors' multidimensional analysis model revealed that besides the total ifosfamide dose and co-administration of other toxic drugs, polymorphic locus of GSTP1 gene may be one of the factors determining a higher toxicity of the cytostatic agent. The model was construed at P=0.029. Moreover, no correlation was found between the GSTM1 or GSTT1 genotype and ifosfamide toxicity and the urinary excretion of its metabolites. The results of this analysis indicate that individual reactions to ifosfamide can depend on inherited genetic polymorphisms, especially associated with the GSTP1 gene coding detoxifying enzyme.

Determination of boron-containing compounds in urine and blood plasma from boron neutron capture therapy patients. The importance of using coupled techniques.

The necessity of using coupled techniques to analyze samples from boron neutron capture therapy (BNCT) patients prior to element-specific detection has been demonstrated. BNCT patients were infused with p-boronophenylalanine (BPA)-fructose complex before the therapy started. Urine and blood plasma samples were collected at different times after the start of the BPA administration and were run on a porous graphitic carbon column coupled on-line to an inductively coupled plasma-atomic emission spectrometer (ICP-AES) and an ICP time-of-flight mass spectrometer (TOF-MS). In addition to BPA, a possible metabolite to BPA and some minor boron-containing compounds, eluting close to the front, were also found in the urine and plasma samples. Because only the total concentration of boron has been measured so far in earlier studies, the suspected metabolite could not be detected, and this is the first report indicating its presence in urine and plasma of BNCT patients. The abundance of 10B in urine was about the same for BPA and its possible metabolite (98-99%). The ratio between the possible metabolite and BPA was found to differ in the urine from different patients. Most of the patients had a metabolite concentration of approximately 10 mol % of the BPA content in their urine 5-11 h after the start of the BPA administration. This ratio increased to between 30 and 80% when 24 h had passed. The ratio of metabolite to BPA was found to be lower in the plasma than in the urine samples at comparable time after the start of BPA infusion. Preliminary results from micro-LC-electrospray ionization (ESI)-MS/MS measurements on four urine samples indicate that the metabolite has a higher mass than BPA.

A method to estimate urinary electrolyte excretion in patients at risk for developing cerebral salt wasting.

OBJECT: Two major criteria are necessary to diagnose cerebral salt wasting (CSW): a cerebral lesion and a large urinary excretion of Na+ and Cl- at a time when the extracellular fluid (ECF) volume is contracted. Nevertheless, it is difficult for the physician to confirm from bedside observation that a patient has a contracted ECF volume. Hyponatremia, although frequently present, should not be a criterion for a diagnosis of salt wasting. A contracted ECF volume is unlikely if there are positive balances of Na+ and Cl-. The goal of this study was to assess the accuracy of calculating balances for Na+ plus K+ and of Cl- over 1 to 10 days in an intensive care unit (ICU) setting. METHODS: A prospective comparison of measured and estimated quantities of Na+ plus K+ and of Cl- excreted over 1 to 10 days in 10 children and 12 adults who had recently received a traumatic brain injury or undergone recent neurosurgery. Plasma concentrations of electrolytes were recorded at the beginning and end of the study period. The total volumes infused and excreted and the concentrations of Na+, K+, and Cl- in the infusate were obtained from each patient's ICU chart. The electrolytes in the patients' urine were measured and calculated. Correlations between measured and calculated values for excretions of Cl- and of Na+ plus K+ were excellent. CONCLUSIONS: Mass balances for Na+ plus K+ and for Cl- can be accurately estimated. These data provide information to support or refute a clinical diagnosis of CSW. The danger of relying on balances for these electrolytes measured within a single day to diagnose CSW is illustrated.

Carboxypeptidase G2 rescue in delayed methotrexate elimination in renal failure.

We report here the case of a 68-year-old woman who presented severe renal failure following the first cycle of high dose methotrexate (HDMTX) for the treatment of a cerebral malignant lymphoma. Before HDMTX administration, serum creatinine value was normal and three days after HDMTX, it reached 457 micromol/L. Leucovorin rescue, hemodialysis and cholestyramine did not increase MTX clearance. Because of the persistence of renal failure, and the high risk of important hematological side-effects associated with high MTX plasma levels, the patient received carboxypeptidase G2 (CPDG2). This allowed MTX plasma levels to decrease by 80% in 15 minutes. No side effects were observed and renal function normalized rapidly. In some patients, when high-dose leucovorin associated with hemodialysis and cholestyramine are unable to restore normal MTX clearance, CPDG2 should be considered because it may represent a safe and efficient alternative for the management of MTX intoxication.

Two approaches for determining the urinary excretion patterns of nucleosides--HPLC and CE.

Cancer patients excrete in their urine increased amounts of modified purines and pyrimidines. These modified mucleosides are primary constituents of RNA and are synthesized at the macromolecular level. When RNA is catabolized, most of these modified nucleosides cannot be reutilized; consequently, they are excreted. Due to their increased urinary excretion in conjunction with altered RNA turnover in carcinogenesis, they have been proposed as tumour markers. We developed both reversed-phase high performance liquid chromatographic and capillary electrophoretic approaches to determine normal and modified nucleosides in urine. Data obtained with CE are in good accordance with those from HPLC. The reference excretion levels of urinary nucleosides from healthy volunteers were established, and elevated ones of modified nucleosides from 34 patients with different kinds of cancer were observed. The developed methods are suitable for the analysis of large series of samples for clinical studies.

PCR detection of JC virus DNA in the brain tissue of a 9-year-old child with pleomorphic xanthoastrocytoma.

Pleomorphic xanthoastrocytoma (PXA) is a rare cerebral tumor of young adults with a slow growth and a good prognosis. Due to its peculiar histopathological findings, the tumor resemble to the lytic phase of progressive multifocal leukoencephalopathy (PML), a JC Virus (JCV) induced disease. For these reasons, the presence of JCV genoma and viral particles were searched for by means of nested polymerase chain reaction (nPCR) and electron microscopy (EM) in a 9-year-old child with PXA. Although EM did not reveal any viral particles, nPCR did reveal genomic sequences of the LT, R, and VP1 regions of JCV. Sequence analysis showed that the R region was mutated with respect to the archetypal form thus yielding the Mad 4 variant of JCV previously reported as being oncogenic in animals. We suggest that JCV may have played a role in the development of this tumor.

The diuretic effect of borocaptate sodium in rats and in patients with brain tumors.

Kidney function changes after single-dose administration of borocaptate sodium were studied in rats and in patients with brain tumors. Changes of glomerular filtration rate (GFR) measured as 14C-inulin clearance and urine flow rate (UFR) after a slow intravenous injection of BSH (25 and 50 mg/kg b.w., respectively) were investigated in rats under pentobarbital anesthesia. The effect of BSH has been compared with that of its disulfide (BSSB) which is spontaneously generated by oxidation of BSH during storage. It was found that BSH decreases GFR in relation to dose and, in the same way, causes a temporary increase of UFR. On the other hand, BSSB (50 mg/kg) induced a large reversible decrease of GFR as well as a decrease of urine excretion. Measurements of GFR (inulin clearance), renal plasma flow (PAH clearance) and urine excretion were taken in a group of patients with brain tumors in which boron disposition after an infusion of BSH (25 mg/kg b.w. over 1 h) had been studied. An increase in urine production was the dominant effect (up to 200% of the initial value), with the alterations of GFR and RPF being of minor significance except in one patient with a GFR reduction up to almost 50% the original value. Kidney function changes after BSH or BSSB administration are supposedly related to the high retention of BSH in kidney.