Potential of UCHL1 as biomarker for destruction of pancreatic beta cells.

There is a clinical need for plasma tests for real-time detection of beta cell destruction, as surrogate endpoint in islet transplantation and immunoprevention trials in type 1 diabetes. This study reports on the use of label-free LC-MS/MS proteomics for bottom-up selection of candidate biomarkers. Ubiquitin COOH-terminal hydrolase 1 (UCHL1) was identified as abundant protein in rat and human beta cells, showing promising beta cell-selectivity, and was selected for further validation in standardized toxicity models. In vitro, H2O2-induced necrosis of INS-1 cells and human islets resulted in intracellular UCHL1 depletion and its extracellular discharge. In vivo, streptozotocin progressively depleted UCHL1 from islet cores and in 50% of animals, an associated plasma UCHL1 surge was detected preceding the GAD65 peak. UCHL1 was cleared with a half-life of 20min. Whole-body dynamic planar imaging of (99m)-Technetium-labeled UCHL1 indicated a rapid UCHL1 uptake in the liver and spleen, followed by urinary excretion of mainly proteolytic UCHL1 fragments. We conclude that LC-MS/MS proteomics is a useful tool to prioritize biomarkers for beta cell injury with promising molar abundance. Despite its consistent UCHL1 discharge by damaged beta cells in vitro, its in vivo use might be restrained by its rapid elimination from plasma. BIOLOGICAL SIGNIFICANCE: Our bottom-up LC-MS/MS proteomics represents a pragmatic approach to identify protein-type biomarkers of pancreatic beta cell injury. UCHL1 successfully passed sequential validation steps of beta cell-selectivity, antigenicity and toxic discharge in vitro. Whole-body dynamic planar imaging of radiolabeled recombinant UCHL1 indicated rapid clearance through the liver, spleen and urinary excretion of proteolytic fragments, likely explaining non-consistent detection in vivo. Integration of kinetic biomarker clearance studies in the a priori selection criteria is recommended before engaging in resource-intensive custom development of sensitive immunoassays for clinical translation.

Phase II study of sunitinib malate in patients with recurrent high-grade glioma.

Receptor tyrosine kinase signaling causes profound neo-angiogenesis in high-grade gliomas (HGG). The KIT, PDGFR-α, and VEGFR2 genes are frequently amplified and expressed in HGG and are molecular targets for therapeutic inhibition by the small-molecule kinase inhibitor sunitinib malate. Twenty-one patients with progressive HGG after prior radiotherapy and chemotherapy received a daily dose of 37.5 mg sunitinib until progression or unacceptable toxicity. Magnetic resonance imaging (MRI) and dynamic susceptibility contrast (DSC)-enhanced perfusion measurements were performed before and during therapy. Cerebral blood volume (CBV) and cerebral blood flow (CBF) lesion-to-normal-white matter ratios were measured to evaluate the antiangiogenic effects of sunitinib. The most frequent grade ≥3 adverse events were skin toxicity, neutropenia, thrombocytopenia, and lymphocytopenia. None of the patients achieved an objective response, whereas a decrease in CBV and CBF within the lesion compared with the normal brain was documented in four out of 14 (29%) patients evaluable for DSC-enhanced perfusion measurements. All patients experienced progression of their disease before or after eight weeks of therapy. Median time-to-progression and overall survival were 1.6 (95%CI 0.8-2.5) and 3.8 (95% CI 2.2-5.3) months, respectively. No correlation could be established between VEGFR2, PDGFR-α, and KIT gene copy numbers or protein expression and the effects of sunitinib. Single-agent sunitinib at 37.5 mg/day had insufficient activity to warrant further investigation of this monotherapy regimen in recurrent HGG.

Primary leptomeningeal anaplastic oligodendroglioma with a 1p36-19q13 deletion: report of a unique case successfully treated with Temozolomide.

Primary leptomeningeal oligodendroglioma occurs very rarely and in only one patient a deletion of chromosome 1p has been reported. We describe a 60-year-old man with a prior history of an epileptic seizure three years earlier, who was referred because of depression and a rapid evolving cognitive impairment. Brain MRI showed a diffuse right parieto-occipital subarachnoid enhancing lesion without intra-axial extension. The diagnosis of an anaplastic oligodendroglioma (WHO grade 3) was made on pathological examination. Molecular analysis using the FISH technique revealed a combined deletion of chromosomes 1p36 and 19q13. A rapid progression of the lesion was shown on MRI with leptomeningeal spinal metastases. The patient was treated with Temozolomide (TMZ) 150 mg/m(2) for 5 days every 4 weeks and showed a marked clinical recovery. Serial MRI disclosed a near complete regression of the lesions with no residual enhancement left after 6 cycles of chemotherapy. At progression following 8 cycles of TMZ the patient underwent craniospinal radiotherapy with complete response of his disease. To our knowledge this is the first report of a patient with a primary leptomeningeal anaplastic oligodendroglioma with diffuse spinal seeding bearing a 1p36/19q13 deletion. Our patient achieved a durable clinical and radiological remission following TMZ treatment. Molecular analysis with determination of chromosome 1p/19q deletions should be performed in all cases of leptomeningeal gliomas to select those patients who might benefit from TMZ chemotherapy.

Stratified phase II trial of cetuximab in patients with recurrent high-grade glioma.

BACKGROUND: To evaluate the antitumor activity and toxicity of single-agent cetuximab in patients with recurrent high-grade glioma (HGG) after failure of surgery, radiation therapy, and chemotherapy. PATIENTS AND METHODS: In this two-arm, open-label, phase II study patients were stratified according to their epidermal growth factor receptor (EGFR) gene amplification status. Cetuximab was administered intravenously at a dose of 400 mg/m(2) on week 1 followed by weekly dose of 250 mg/m(2). The primary end point for this study was the response rate in both study arms separately. RESULTS: Fifty-five eligible patients (28 with and 27 without EGFR amplification) tolerated cetuximab well. Three patients (5.5%) had a partial response and 16 patients (29.6%) had stable disease. The median time to progression was 1.9 months [95% confidence interval (CI) 1.6-2.2 months]. Whereas the progression-free survival (PFS) was <6 months in the majority (n = 50/55) of patients, five patients (9.2%) had a PFS on cetuximab of >9 months. Median overall survival was 5.0 months (95% CI 4.2-5.9 months). No significant correlation was found between response, survival and EGFR amplification. CONCLUSIONS: Cetuximab was well tolerated but had limited activity in this patient population with progressive HGG. A minority of patients may derive a more durable benefit but were not prospectively identified by EGFR gene copy number.

MGMT promoter hypermethylation correlates with a survival benefit from temozolomide in patients with recurrent anaplastic astrocytoma but not glioblastoma.

AIMS: To investigate the correlation between O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter methylation and benefit from temozolomide in patients with recurrent high-grade glioma. PATIENTS AND METHODS: A real-time, quantitative, methylation-specific PCR assay was performed on archival tissue blocks from patients treated with temozolomide at the first recurrence. RESULTS: A subgroup of 38 patients who were chemotherapy-naive at recurrence was analysed (22 glioblastoma, 12 anaplastic astrocytoma [AA] and 4 anaplastic oligoastrocytoma [AOA]); none had 1p/19q loss. Among 10 (26%) patients with a hypermethylated MGMT promoter, none experienced disease progression within the first two treatment cycles compared with 12 of 28 (43%) patients with an unmethylated promoter (p=0.016). By Cox multivariate analysis, tumour grade and MGMT promoter methylation correlated with time to progression (p<0.05); MGMT promoter methylation correlated with superior overall survival in AA/AOA but not in glioblastoma. CONCLUSIONS: MGMT promoter methylation predicted a survival benefit in patients with 1p/19q intact AA/AOA treated with temozolomide at recurrence.

Neuropathological and molecular aspects of low-grade and high-grade gliomas.

Gliomas are the most frequent primary brain tumors. They are derived from glial cells of astrocytic, oligodendroglial and ependymal origin. According to the WHO classification of brain tumors gliomas are divided in low-grade (grades I and II) and high-grade (grades III and IV) tumors. Low-grade tumors are well-differentiated, slow-growing lesions. Grade I tumors are well-circumscribed and often surgically curable, whereas grade II tumors are diffuse, infiltrating lesions with a marked potential over time for progression towards a high-grade malignant tumor. The optimal management of low-grade gliomas is still debated. Important prognostic factors such as histology, grade and location of the tumor, age and functional status of the patient, must be taken into consideration to select the most appropriate treatment. Major advances in the molecular genetic assessment of brain tumors and of gliomas in particular have lead to the identification of several molecular markers playing a crucial role in the development of gliomas and in their malignant transformation. Some of those markers were found very useful to assist in the histological diagnosis and to predict survival and response to therapy. A combined deletion of chromosomes arms 1p and 19q can be found in more than 50% of Grade II and III oligodendrogliomas and has been associated with chemosensitivity and a better prognosis. Once limited to the field of research, molecular biology has now entered the daily neuropathological practice and will undoubtedly play an increasing role in future classification and treatment of brain tumors.