Ultra-fast deep-learned CNS tumour classification during surgery.

Central nervous system tumours represent one of the most lethal cancer types, particularly among children1. Primary treatment includes neurosurgical resection of the tumour, in which a delicate balance must be struck between maximizing the extent of resection and minimizing risk of neurological damage and comorbidity2,3. However, surgeons have limited knowledge of the precise tumour type prior to surgery. Current standard practice relies on preoperative imaging and intraoperative histological analysis, but these are not always conclusive and occasionally wrong. Using rapid nanopore sequencing, a sparse methylation profile can be obtained during surgery4. Here we developed Sturgeon, a patient-agnostic transfer-learned neural network, to enable molecular subclassification of central nervous system tumours based on such sparse profiles. Sturgeon delivered an accurate diagnosis within 40 minutes after starting sequencing in 45 out of 50 retrospectively sequenced samples (abstaining from diagnosis of the other 5 samples). Furthermore, we demonstrated its applicability in real time during 25 surgeries, achieving a diagnostic turnaround time of less than 90 min. Of these, 18 (72%) diagnoses were correct and 7 did not reach the required confidence threshold. We conclude that machine-learned diagnosis based on low-cost intraoperative sequencing can assist neurosurgical decision-making, potentially preventing neurological comorbidity and avoiding additional surgeries.

Medulloblastoma in adults: evaluation of the Dutch society for neuro-oncology treatment protocol.

PURPOSE: Medulloblastoma is a rare tumor in adults. The objective of this nationwide, multicenter study was to evaluate the toxicity and efficacy of the Dutch treatment protocol for adult medulloblastoma patients. METHODS: Adult medulloblastoma patients diagnosed between 2010 and 2018 were identified in the Dutch rare tumors registry or nationwide pathology database. Patients with intention to treat according to the national treatment protocol were included. Risk stratification was performed based on residual disease, histological subtype and extent of disease. All patients received postoperative radiotherapy [craniospinal axis 36 Gy/fossa posterior boost 19.8 Gy (14.4 Gy in case of metastases)]. High-risk patients received additional neoadjuvant (carboplatin-etoposide), concomitant (vincristine) and adjuvant chemotherapy (carboplatin-vincristine-cyclophosphamide) as far as feasible by toxicity. Methylation profiling, and additional next-generation sequencing in case of SHH-activated medulloblastomas, were performed. RESULTS: Forty-seven medulloblastoma patients were identified, of whom 32 were treated according to the protocol. Clinical information and tumor material was available for 28 and 20 patients, respectively. The histological variants were mainly classic (43%) and desmoplastic medulloblastoma (36%). Sixteen patients (57%) were considered standard-risk and 60% were SHH-activated medulloblastomas. Considerable treatment reductions and delays in treatment occurred due to especially hematological and neurotoxicity. Only one high-risk patient could complete all chemotherapy courses. 5-years progression-free survival (PFS) and overall survival (OS) for standard-risk patients appeared worse than for high-risk patients (PFS 69% vs. 90%, OS 81% vs. 90% respectively), although this wasn't statistically significant. CONCLUSION: Combined chemo-radiotherapy is a toxic regimen for adult medulloblastoma patients that may result in improved survival.

Brain tumour diagnostics using a DNA methylation-based classifier as a diagnostic support tool.

AIMS: Methylation profiling (MP) is increasingly incorporated in the diagnostic process of central nervous system (CNS) tumours at our centres in The Netherlands and Scandinavia. We aimed to identify the benefits and challenges of MP as a support tool for CNS tumour diagnostics. METHODS: About 502 CNS tumour samples were analysed using (850 k) MP. Profiles were matched with the DKFZ/Heidelberg CNS Tumour Classifier. For each case, the final pathological diagnosis was compared to the diagnosis before MP. RESULTS: In 54.4% (273/502) of all analysed cases, the suggested methylation class (calibrated score ≥0.9) corresponded with the initial pathological diagnosis. The diagnosis of 24.5% of these cases (67/273) was more refined after incorporation of the MP result. In 9.8% of cases (49/502), the MP result led to a new diagnosis, resulting in an altered WHO grade in 71.4% of these cases (35/49). In 1% of cases (5/502), the suggested class based on MP was initially disregarded/interpreted as misleading, but in retrospect, the MP result predicted the right diagnosis for three of these cases. In six cases, the suggested class was interpreted as 'discrepant but noncontributory'. The remaining 33.7% of cases (169/502) had a calibrated score <0.9, including 7.8% (39/502) for which no class indication was given at all (calibrated score <0.3). CONCLUSIONS: MP is a powerful tool to confirm and fine-tune the pathological diagnosis of CNS tumours, and to avoid misdiagnoses. However, it is crucial to interpret the results in the context of clinical, radiological, histopathological and other molecular information.

Diffuse glioneuronal tumour with oligodendroglioma-like features and nuclear clusters (DGONC) - a molecularly defined glioneuronal CNS tumour class displaying recurrent monosomy 14.

AIMS: DNA methylation-based central nervous system (CNS) tumour classification has identified numerous molecularly distinct tumour types, and clinically relevant subgroups among known CNS tumour entities that were previously thought to represent homogeneous diseases. Our study aimed at characterizing a novel, molecularly defined variant of glioneuronal CNS tumour. PATIENTS AND METHODS: DNA methylation profiling was performed using the Infinium MethylationEPIC or 450 k BeadChip arrays (Illumina) and analysed using the 'conumee' package in R computing environment. Additional gene panel sequencing was also performed. Tumour samples were collected at the German Cancer Research Centre (DKFZ) and provided by multinational collaborators. Histological sections were also collected and independently reviewed. RESULTS: Genome-wide DNA methylation data from >25 000 CNS tumours were screened for clusters separated from established DNA methylation classes, revealing a novel group comprising 31 tumours, mainly found in paediatric patients. This DNA methylation-defined variant of low-grade CNS tumours with glioneuronal differentiation displays recurrent monosomy 14, nuclear clusters within a morphology that is otherwise reminiscent of oligodendroglioma and other established entities with clear cell histology, and a lack of genetic alterations commonly observed in other (paediatric) glioneuronal entities. CONCLUSIONS: DNA methylation-based tumour classification is an objective method of assessing tumour origins, which may aid in diagnosis, especially for atypical cases. With increasing sample size, methylation analysis allows for the identification of rare, putative new tumour entities, which are currently not recognized by the WHO classification. Our study revealed the existence of a DNA methylation-defined class of low-grade glioneuronal tumours with recurrent monosomy 14, oligodendroglioma-like features and nuclear clusters.

Molecular pathology of tumors of the central nervous system.

Since the update of the 4th edition of the WHO Classification of Central Nervous System (CNS) Tumors published in 2016, particular molecular characteristics are part of the definition of a subset of these neoplasms. This combined 'histo-molecular' approach allows for a much more precise diagnosis of especially diffuse gliomas and embryonal CNS tumors. This review provides an update of the most important diagnostic and prognostic markers for state-of-the-art diagnosis of primary CNS tumors. Defining molecular markers for diffuse gliomas are IDH1/IDH2 mutations, 1p/19q codeletion and mutations in histone H3 genes. Medulloblastomas, the most frequent embryonal CNS tumors, are divided into four molecularly defined groups according to the WHO 2016 Classification: wingless/integrated (WNT) signaling pathway activated, sonic hedgehog (SHH) signaling pathway activated and tumor protein p53 gene (TP53)-mutant, SHH-activated and TP53-wildtype, and non-WNT/non-SHH-activated. Molecular characteristics are also important for the diagnosis of several other CNS tumors, such as RELA fusion-positive subtype of ependymoma, atypical teratoid rhabdoid tumor (AT/RT), embryonal tumor with multilayered rosettes, and solitary fibrous tumor/hemangiopericytoma. Immunohistochemistry is a helpful alternative for further molecular characterization of several of these tumors. Additionally, genome-wide methylation profiling is a very promising new tool in CNS tumor diagnostics. Much progress has thus been made by translating the most relevant molecular knowledge into a more precise clinical diagnosis of CNS tumors. Hopefully, this will enable more specific and more effective therapeutic approaches for the patients suffering from these tumors.

WHO 2016 Classification of gliomas.

Gliomas are the most frequent intrinsic tumours of the central nervous system and encompass two principle subgroups: diffuse gliomas and gliomas showing a more circumscribed growth pattern ('nondiffuse gliomas'). In the revised fourth edition of the WHO Classification of CNS tumours published in 2016, classification of especially diffuse gliomas has fundamentally changed: for the first time, a large subset of these tumours is now defined based on presence/absence of IDH mutation and 1p/19q codeletion. Following this approach, the diagnosis of (anaplastic) oligoastrocytoma can be expected to largely disappear. Furthermore, in the WHO 2016 Classification gliomatosis cerebri is not an entity anymore but is now considered as a growth pattern. The most important changes in the very diverse group of 'nondiffuse' gliomas and neuronal-glial tumours are the introduction of anaplastic pleomorphic xanthoastrocytoma, of diffuse leptomeningeal glioneuronal tumour and of RELA fusion-positive ependymoma as entities. In the last part of this review, after very briefly touching upon classification of neuronal, choroid plexus and pineal region tumours, some practical implications and challenges associated with the WHO 2016 Classification of gliomas are discussed.

Overcoming the blood-brain tumor barrier for effective glioblastoma treatment.

Gliomas are the most common primary brain tumors. Particularly in adult patients, the vast majority of gliomas belongs to the heterogeneous group of diffuse gliomas, i.e. glial tumors characterized by diffuse infiltrative growth in the preexistent brain tissue. Unfortunately, glioblastoma, the most aggressive (WHO grade IV) diffuse glioma is also by far the most frequent one. After standard treatment, the 2-year overall survival of glioblastoma patients is approximately only 25%. Advanced knowledge in the molecular pathology underlying malignant transformation has offered new handles and better treatments for several cancer types. Unfortunately, glioblastoma multiforme (GBM) patients have not yet profited as although numerous experimental drugs have been tested in clinical trials, all failed miserably. This grim prognosis for GBM is at least partly due to the lack of successful drug delivery across the blood-brain tumor barrier (BBTB). The human brain comprises over 100 billion capillaries with a total length of 400 miles, a total surface area of 20 m(2) and a median inter-capillary distance of about 50 µm, making it the best perfused organ in the body. The BBTB encompasses existing and newly formed blood vessels that contribute to the delivery of nutrients and oxygen to the tumor and facilitate glioma cell migration to other parts of the brain. The high metabolic demands of high-grade glioma create hypoxic areas that trigger increased expression of VEGF and angiogenesis, leading to the formation of abnormal vessels and a dysfunctional BBTB. Even though the BBTB is considered 'leaky' in the core part of glioblastomas, in large parts of glioblastomas and, even more so, in lower grade diffuse gliomas the BBTB more closely resembles the intact blood-brain barrier (BBB) and prevents efficient passage of cancer therapeutics, including small molecules and antibodies. Thus, many drugs can still be blocked from reaching the many infiltrative glioblastoma cells that demonstrate 'within-organ-metastasis' away from the core part to brain areas displaying a more organized and less leaky BBTB. Hence, drug delivery in glioblastoma deserves explicit attention as otherwise new experimental therapies will continue to fail. In the current review we highlight different aspects of the BBTB in glioma patients and preclinical models and discuss the advantages and drawbacks of drug delivery approaches for the treatment of glioma patients. We provide an overview on methods to overcome the BBTB, including osmotic blood-brain barrier disruption (BBBD), bradykinin receptor-mediated BBTB opening, inhibition of multidrug efflux transporters, receptor-mediated transport systems and physiological circumvention of the BBTB. While our knowledge about the molecular biology of glioma cells is rapidly expanding and is, to some extent, already assisting us in the design of tumor-tailored therapeutics, we are still struggling to develop modalities to expose the entire tumor to such therapeutics at pharmacologically meaningful quantities. Therefore, we must expand our knowledge about the fundamentals of the BBTB as a step toward the design of practical and safe devices and approaches for enhanced drug delivery into the diseased brain area.

Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer.

BACKGROUND: O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. PATIENTS AND METHODS: We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA (cfDNA) from plasma samples using an ultra-sensitive two-step digital PCR technique (methyl-BEAMing). Results were compared with two established techniques, methylation-specific PCR (MSP) and Bs-pyrosequencing. RESULTS: Thresholds for MGMT methylated status for each technique were established in a training set of 98 glioblastoma (GBM) patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 GBM patients treated with temozolomide in which methyl-BEAMing displayed a better specificity than the other techniques. Cutoff values of MGMT methylation specific for metastatic colorectal cancer (mCRC) tissue samples were established in a cohort of 60 patients treated with dacarbazine. In mCRC, both quantitative assays methyl-BEAMing and Bs-pyrosequencing outperformed MSP, providing better prediction of treatment response and improvement in progression-free survival (PFS) (P < 0.001). Ability of methyl-BEAMing to identify responding patients was validated in a cohort of 23 mCRC patients treated with temozolomide and preselected for MGMT methylated status according to MSP. In mCRC patients treated with dacarbazine, exploratory analysis of cfDNA by methyl-BEAMing showed that MGMT methylation was associated with better response and improved median PFS (P = 0.008). CONCLUSIONS: Methyl-BEAMing showed high reproducibility, specificity and sensitivity and was applicable to formalin-fixed paraffin-embedded tissues and cfDNA. This study supports the quantitative assessment of MGMT methylation for clinical purposes since it could refine prediction of response to alkylating agents.

T2-FLAIR Mismatch: An Imaging Biomarker for Children's MYB/MYBL1-Altered Diffuse Astrocytoma or Angiocentric Glioma.

BACKGROUND AND PURPOSE: T2-FLAIR mismatch is a highly specific imaging biomarker of IDH-mutant diffuse astrocytoma in adults. It has however also been described in MYB/MYBL1-altered low grade tumors. Our aim was to assess the diagnostic power of the T2-FLAIR mismatch in IDH-mutant astrocytoma and MYB/MYBL1-altered low-grade tumors in children and correlate this mismatch with histology. MATERIALS AND METHODS: We evaluated MR imaging examinations of all pediatric patients, performed at the Princess Máxima Center for Pediatric Oncology and the University Medical Center Utrecht between January 2012 and January 2023, with the histomolecular diagnosis of IDH-mutant astrocytoma, diffuse astrocytoma MYB/MYBL1-altered, or angiocentric glioma, and the presence of T2-FLAIR mismatch was assessed. Histologically, the presence of microcysts in the tumor (a phenomenon suggested to be correlated with T2-FLAIR mismatch in IDH-mutant astrocytomas in adults) was evaluated. RESULTS: Nineteen pediatric patients were diagnosed with either IDH-mutant astrocytoma (n = 8) or MYB/MYBL1-altered tumor (n = 11: diffuse astrocytoma, MYB- or MYBL1-altered n = 8; or angiocentric glioma n = 3). T2-FLAIR mismatch was present in 11 patients, 3 (38%) in the IDH-mutant group and 8 (73%) in the MYB/MYBL1 group. No correlation was found between T2-FLAIR mismatch and the presence of microcysts or an enlarged intercellular space in either IDH-mutant astrocytoma (P = .38 and P = .56, respectively) or MYB/MYBL1-altered tumors (P = .36 and P = .90, respectively). CONCLUSIONS: In our pediatric population, T2-FLAIR mismatch was more often found in MYB/MYBL1-altered tumors than in IDH-mutant astrocytomas. In contrast to what has been reported for IDH-mutant astrocytomas in adults, no correlation was found with microcystic changes in the tumor tissue. This finding challenges the hypothesis that such microcystic changes and/or enlarged intercellular spaces in the tissue of these tumors are an important part of explaining the occurrence of the T2-FLAIR mismatch.

Discordance in ERα, PR and HER2 receptor status across different distant breast cancer metastases within the same patient.

BACKGROUND: We studied discordance in estrogen receptor alpha (ERα), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status between multiple distant metastases from the same breast cancer patient. MATERIAL AND METHODS: Multiple distant metastases from 55 female patients were stained for ERα, PR and HER2 by immunohistochemistry and in situ hybridization for confirmation of the HER2 status. RESULTS: Different metastatic sites within the same patient showed discordance in ERα receptor status in 7.3% or 10.9% of patients (using a 10% or 1% threshold for positivity, respectively). For PR, 29.1% or 30.9% of patients showed discordance. Taking ERα and PR together, 36.4% of cases (both thresholds) showed discrepancy between metastases. In 10.9% (10% threshold) or 14.5% of patients (1% threshold), such discordance could have clinical consequences with regard to hormonal treatment. For HER2, there was 3.6% discordance on the immunohistochemical level but 0% on the gene level. CONCLUSION: In a significant proportion of metastatic breast cancer patients, discordance in ERα and PR receptor status between different metastatic sites was observed. This implies that multiple metastases may need to be biopsied to optimally reassess receptors.

Implementation of a multi-institutional diffuse intrinsic pontine glioma autopsy protocol and characterization of a primary cell culture.

AIMS: Diffuse intrinsic pontine glioma (DIPG) is a fatal paediatric malignancy. Tumour resection is not possible without serious morbidity and biopsies are rarely performed. The resulting lack of primary DIPG material has made preclinical research practically impossible and has hindered the development of new therapies for this disease. The aim of the current study was to address the lack of primary DIPG material and preclinical models by developing a multi-institutional autopsy protocol. METHODS: An autopsy protocol was implemented in the Netherlands to obtain tumour material within a brief post mortem interval. A team of neuropathologists and researchers was available at any time to perform the autopsy and process the material harvested. Whole brain autopsy was performed and primary DIPG material and healthy tissue were collected from all affected brain areas. Finally, the study included systematic evaluation by parents. RESULTS: Five autopsies were performed. The mean time interval between death and time of autopsy was 3 h (range 2-4). All tumours were graded as glioblastoma. None of the parents regretted their choice to participate, and they all derived comfort in donating tissue of their child in the hope to help future DIPG patients. In addition, we developed and characterized one of the first DIPG cell cultures from post mortem material. CONCLUSION: Here we show that obtaining post mortem DIPG tumour tissue for research purposes is feasible with short delay, and that the autopsy procedure is satisfying for participating parents and can be suitable for the development of preclinical DIPG models.

Quantitative short echo time 1H MRSI of the peripheral edematous region of human brain tumors in the differentiation between glioblastoma, metastasis, and meningioma.

PURPOSE: To assess metabolite levels in peritumoral edematous (PO) and surrounding apparently normal (SAN) brain regions of glioblastoma, metastasis, and meningioma in humans with (1)H-MRSI to find biomarkers that can discriminate between tumors and characterize infiltrative tumor growth. MATERIALS AND METHODS: Magnetic resonance (MR) spectra (semi-LASER MRSI, 30 msec echo time, 3T) were selected from regions of interest (ROIs) under MRI guidance, and after quality control of MR spectra. Statistical testing between patient groups was performed for mean metabolite ratios of an entire ROI and for the highest value within that ROI. RESULTS: The highest ratios of the level of choline compounds and the sum of myo-inositol and glycine over N-acetylaspartate and creatine compounds were significantly increased in PO regions of glioblastoma versus that of metastasis and meningioma. In the SAN region of glioblastoma some of these ratios were increased. Differences were less prominent for metabolite levels averaged over entire ROIs. CONCLUSION: Specific metabolite ratios in PO and SAN regions can be used to discriminate glioblastoma from metastasis and meningioma. An analysis of these ratios averaged over entire ROIs and those with most abnormal values indicates that infiltrative tumor growth in glioblastoma is inhomogeneous and extends into the SAN region.

Contribution of CSF cytology in the diagnostic work-up of breast cancer patients with neurological symptoms: a retrospective analysis over two decades.

The aim of this study was to evaluate the contribution of cytological analysis of cerebrospinal fluid (CSF) in the diagnostic work-up of breast cancer patients who present with neurological symptoms suspected for central nervous system (CNS) metastases. In the period 1989-2009, a total of 81 patients with breast cancer underwent CSF cytological examination. Relevant tumour characteristics, clinical presentation and radiological findings were scored. The CSF cytological diagnosis was classified according to the 1996 NCI-sponsored conference approach as malignant, suspicious for malignancy, atypical, benign or inadequate. During the course of 20 years, 145 CSF cytological examinations were performed. Relatively common neurological symptoms resulting in cytological CSF examination were headache (n = 25), nausea and vomiting (n = 19), sensory disturbances (n = 16), and cranial nerve dysfunction (n = 16). Of these, headache and nausea/vomiting were most often associated with malignant cells in the CSF (CSF(+)) (in 48 and 53% of the cases, respectively). The 4 patients with both headache and confusion/altered mental status all had CSF(+). In 10 patients, CSF(+) was found despite the absence of radiological evidence for metastasis in/around the CNS. In our series, repeated CSF analysis appeared to have limited additional value, and CSF(+) was strongly correlated with shorter survival. A substantial number of patients with neurological symptoms but without radiological abnormalities can have CSF(+). In our series, the additional value of repeated cytological examination of CSF was limited. Our study underscores the value of CSF cytology as a tool for the unequivocal diagnosis of metastatic spread of breast cancer to the CNS, and confirms that CSF(+) is a strong predictor of poor survival.

Non-invasively measured brain activity and radiological progression in diffuse glioma.

Non-invasively measured brain activity is related to progression-free survival in glioma patients, suggesting its potential as a marker of glioma progression. We therefore assessed the relationship between brain activity and increasing tumor volumes on routine clinical magnetic resonance imaging (MRI) in glioma patients. Postoperative magnetoencephalography (MEG) was recorded in 45 diffuse glioma patients. Brain activity was estimated using three measures (absolute broadband power, offset and slope) calculated at three spatial levels: global average, averaged across the peritumoral areas, and averaged across the homologues of these peritumoral areas in the contralateral hemisphere. Tumors were segmented on MRI. Changes in tumor volume between the two scans surrounding the MEG were calculated and correlated with brain activity. Brain activity was compared between patient groups classified into having increasing or stable tumor volume. Results show that brain activity was significantly increased in the tumor hemisphere in general, and in peritumoral regions specifically. However, none of the measures and spatial levels of brain activity correlated with changes in tumor volume, nor did they differ between patients with increasing versus stable tumor volumes. Longitudinal studies in more homogeneous subgroups of glioma patients are necessary to further explore the clinical potential of non-invasively measured brain activity.

Micronodular transformation as a novel mechanism of VEGF-A-induced metastasis.

How and why tumors metastasize is still a matter of debate. The assumption is that mutations render tumor cells with a metastatic phenotype, enabling entrance in and transport through lymph or blood vessels. Distant outgrowth is thought to occur only in a suitable microenvironment (the seed and soil hypothesis). However, the anatomical location of most metastases in cancer patients suggests entrapment of tumor cells in the first microcapillary bed that is encountered. We here investigated how vascular endothelial growth factor-A (VEGF-A) attributes to the metastatic process. We describe here that VEGF-A enhances spontaneous metastasis by inducing intravasation of heterogeneous tumor cell clusters, surrounded by vessel wall elements, via an invasion-independent mechanism. These tumor clusters generate metastatic tissue embolisms in pulmonary arteries. Treatment of tumor-bearing mice with the antiangiogenic compound ZD6474 prevented the development of this metastatic phenotype. This work shows that tumors with high constitutive VEGF-A expression metastasize via the formation of tumor emboli and provides an alternative rationale for anti-VEGF-A therapy, namely to inhibit metastasis formation.

Characterisation of tumour vasculature in mouse brain by USPIO contrast-enhanced MRI.

To enhance the success rate of antiangiogenic therapies in the clinic, it is crucial to identify parameters for tumour angiogenesis that can predict response to these therapies. In brain tumours, one such parameter is vascular leakage, which is a response to tumour-derived vascular endothelial growth factor-A and can be measured by Gadolinium-DTPA (Gd-DTPA)-enhanced magnetic resonance imaging (MRI). However, as vascular permeability and angiogenesis are not strictly coupled, tumour blood volume may be another potentially important parameter. In this study, contrast-enhanced MR imaging was performed in three orthotopic mouse models for human brain tumours (angiogenic melanoma metastases and E34 and U87 human glioma xenografts) using both Gd-DTPA to detect vascular leakage and ultrasmall iron oxide particles (USPIO) to measure blood volume. Pixel-by-pixel maps of the enhancement in the transverse relaxation rates (Delta R(2) and Delta R(2)(*)) after injection of USPIO provided an index proportional to the blood volume of the microvasculature and macrovasculature, respectively, for each tumour. The melanoma metastases were characterised by a blood volume and vessel leakage higher than both glioma xenografts. The U87 glioblastoma xenografts displayed higher permeability and blood volume in the rim than in the core. The E34 glioma xenografts were characterised by a relatively high blood volume, accompanied by only a moderate blood-brain barrier disruption. Delineation of the tumour was best assessed on post-USPIO gradient-echo images. These findings suggest that contrast-enhanced MR imaging using USPIOs and, in particular, Delta R(2) and Delta R(2)(*) quantitation, provides important additional information about tumour vasculature.