Automatic quantification of the microvascular density on whole slide images, applied to paediatric brain tumours

Angiogenesis is a key phenomenon for tumour progression, diagnosis and treatment in brain tumours and more generally in oncology. Presently, its precise, direct quantitative assessment can only be done on whole tissue sections immunostained to reveal vascular endothelial cells. But this is a tremendous task for the pathologist and a challenge for the computer since digitised whole tissue sections, whole slide images (WSI), contain typically around ten gigapixels. We define and implement an algorithm that determines automatically, on a WSI at objective magnification $40\times$, the regions of tissue, the regions without blur and the regions of large puddles of red blood cells, and constructs the mask of blur-free, significant tissue on the WSI. Then it calibrates automatically the optical density ratios of the immunostaining of the vessel walls and of the counterstaining, performs a colour deconvolution inside the regions of blur-free tissue, and finds the vessel walls inside these regions by selecting, on the image resulting from the colour deconvolution, zones which satisfy a double-threshold criterion. A mask of vessel wall regions on the WSI is produced. The density of microvessels is finally computed as the fraction of the area of significant tissue which is occupied by vessel walls. We apply this algorithm to a set of 186 WSI of paediatric brain tumours from World Health Organisation grades I to IV. The segmentations are of very good quality although the set of slides is very heterogeneous. The computation time is of the order of a fraction of an hour for each WSI on a modest computer. The computed microvascular density is found to be robust and strongly correlates with the tumour grade. This method requires no training and can easily be applied to other tumour types and other stainings

Comparison between fresh and fixed human biopsies using spectral and lifetime measurements: Fluorescence analysis using one and two photon excitations

International audienceThe purpose of this study is to make a comparison between the fluorescence emissions of fresh extracted human biopsies and fixed human biopsies, in order to evaluate the impact of fixation on autofluoresence signal. Our group is developing an endo-microscope to image brain tissues in-vivo, however to date, in order to validate our technology the easiest type of samples we can access are fixed samples. However, the fixation is still challenging. For that, we aim through this study to determine whether we should pursue to work on fixed samples or we should shift to work on fresh biopsies. Data were collected on spectroscopic, lifetime measurement and fluorescence imaging setups with visible and two-photon excitations wavelengths. Five fresh and five fixed samples are involved in the experiment. Endogenous fluorescence of fixed biopsies were calculated. Experimental results reveal that at 405 nm and 810 nm, the fresh samples have an intensity of fluorescence two times higher than that of fixed samples. However, for each fluorophore and each excitation wavelength, the lifetime for fresh samples is shorter than that for fixed samples. Still, further studies and investigations involving the comparison between different samples are required to strengthen our findings

Imaging of non tumorous and tumorous human brain tissue with full-field optical coherence tomography

A prospective study was performed on neurosurgical samples from 18 patients to evaluate the use of Full-Field Optical Coherence Tomography (FF-OCT) in brain tumor diagnosis. FF-OCT captures en face slices of tissue samples at 1\mum resolution in 3D with a typical 200\mum imaging depth. A 1cm2 specimen is scanned at a single depth and processed in about 5 minutes. This rapid imaging process is non-invasive and 30 requires neither contrast agent injection nor tissue preparation, which makes it particularly well suited to medical imaging applications. Temporal chronic epileptic parenchyma and brain tumors such as meningiomas, low- grade and high-grade gliomas, and choroid plexus papilloma were imaged. A subpopulation of neurons, myelin fibers and CNS vasculature were clearly identified. Cortex could be discriminated from white matter, but individual glial cells as astrocytes (normal or reactive) or oligodendrocytes were not observable. This study reports for the first time on the feasibility of using FF-OCT in a real-time manner as a label-free non-invasive imaging technique in an intra-operative neurosurgical clinical setting to assess tumorous glial and epileptic margins

A case report of pseudoprogression followed by complete remission after proton-beam irradiation for a low-grade glioma in a teenager: the value of dynamic contrast-enhanced MRI

A fourteen years-old boy was treated post-operatively with proton therapy for a recurrent low-grade oligodendroglioma located in the tectal region. Six months after the end of irradiation (RT), a new enhancing lesion appeared within the radiation fields. To differentiate disease progression from radiation-induced changes, dynamic susceptibility contrast-enhanced (DSCE) MRI was used with a T2* sequence to study perfusion and permeability characteristics simultaneously. Typically, the lesion showed hypoperfusion and hyperpermeability compared to the controlateral normal brain. Without additional treatment but a short course of steroids, the image disappeared over a six months period allowing us to conclude for a pseudo-progression. The patient is alive in complete remission more than 2 years post-RT

Portrait of Ependymoma Recurrence in Children: Biomarkers of Tumor Progression Identified by Dual-Color Microarray-Based Gene Expression Analysis

BACKGROUND: Children with ependymoma may experience a relapse in up to 50% of cases depending on the extent of resection. Key biological events associated with recurrence are unknown. METHODOLOGY/PRINCIPAL FINDINGS: To discover the biology behind the recurrence of ependymomas, we performed CGHarray and a dual-color gene expression microarray analysis of 17 tumors at diagnosis co-hybridized with the corresponding 27 first or subsequent relapses from the same patient. As treatment and location had only limited influence on specific gene expression changes at relapse, we established a common signature for relapse. Eighty-seven genes showed an absolute fold change ≥2 in at least 50% of relapses and were defined as the gene expression signature of ependymoma recurrence. The most frequently upregulated genes are involved in the kinetochore (ASPM, KIF11) or in neural development (CD133, Wnt and Notch pathways). Metallothionein (MT) genes were downregulated in up to 80% of the recurrences. Quantitative PCR for ASPM, KIF11 and MT3 plus immunohistochemistry for ASPM and MT3 confirmed the microarray results. Immunohistochemistry on an independent series of 24 tumor pairs at diagnosis and at relapse confirmed the decrease of MT3 expression at recurrence in 17/24 tumor pairs (p = 0.002). Conversely, ASPM expression was more frequently positive at relapse (87.5% vs 37.5%, p = 0.03). Loss or deletion of the MT genes cluster was never observed at relapse. Promoter sequencing after bisulfite treatment of DNA from primary tumors and recurrences as well as treatment of short-term ependymoma cells cultures with a demethylating agent showed that methylation was not involved in MT3 downregulation. However, in vitro treatment with a histone deacetylase inhibitor or zinc restored MT3 expression. CONCLUSIONS/SIGNIFICANCE: The most frequent molecular events associated with ependymoma recurrence were over-expression of kinetochore proteins and down-regulation of metallothioneins. Metallothionein-3 expression is epigenetically controlled and can be restored in vitro by histone deacetylase inhibitors

Astrocytes reverted to a neural progenitor-like state with transforming growth factor alpha are sensitized to cancerous transformation.

International audienceGliomas, the most frequent primitive central nervous system tumors, have been suggested to originate from astrocytes or from neural progenitors/stem cells. However, the precise identity of the cells at the origin of gliomas remains a matter of debate because no pre-neoplastic state has been yet identified. Transforming growth factor (TGF)-alpha, an epidermal growth factor family member, is frequently overexpressed in the early stages of glioma progression. We previously demonstrated that prolonged exposure of astrocytes to TGF-alpha is sufficient to trigger their reversion to a neural progenitor-like state. To determine whether TGF-alpha dedifferentiating effects are associated with cancerous transforming effects, we grafted intracerebrally dedifferentiated astrocytes. We show that these cells had the same cytogenomic profile as astrocytes, survived in vivo, and did not give birth to tumors. When astrocytes dedifferentiated with TGF-alpha were submitted to oncogenic stress using gamma irradiation, they acquired cancerous properties: they were immortalized, showed cytogenomic abnormalities, and formed high-grade glioma-like tumors after brain grafting. In contrast, irradiation did not modify the lifespan of astrocytes cultivated in serum-free medium. Addition of TGF-alpha after irradiation did not promote their transformation but decreased their lifespan. These results demonstrate that reversion of mature astrocytes to an embryonic state without genomic manipulation is sufficient to sensitize them to oncogenic stress

Molecular, pathological, radiological, and immune profiling of non-brainstem pediatric high-grade glioma from the HERBY phase II randomized trial

The HERBY trial was a phase II open-label, randomized, multicenter trial evaluating bevacizumab (BEV) in addition to temozolomide/radiotherapy in patients with newly diagnosed non-brainstem high-grade glioma (HGG) between the ages of 3 and 18 years. We carried out comprehensive molecular analysis integrated with pathology, radiology, and immune profiling. In post-hoc subgroup analysis, hypermutator tumors (mismatch repair deficiency and somatic POLE/POLD1 mutations) and those biologically resembling pleomorphic xanthoastrocytoma ([PXA]-like, driven by BRAF_V600E or NF1 mutation) had significantly more CD8+ tumor-infiltrating lymphocytes, and longer survival with the addition of BEV. Histone H3 subgroups (hemispheric G34R/V and midline K27M) had a worse outcome and were immune cold. Future clinical trials will need to take into account the diversity represented by the term ‘‘HGG’’ in the pediatric population

Constitutional mismatch repair deficiency–associated brain tumors: report from the European C4CMMRD consortium

Abstract Background Malignant brain tumors (BT) are among the cancers most frequently associated with constitutional mismatch repair deficiency (CMMRD), a rare childhood cancer predisposition syndrome resulting from biallelic germline mutations in mismatch repair genes. This study analyzed data from the European "Care for CMMRD" (C4CMMRD) database to describe their clinical characteristics, treatments, and outcome with the aim of improving its diagnosis/treatment. Methods Retrospective analysis of data on patients with CMMRD and malignant BT from the C4CMMRD database up to July 2017. Results Among the 87 registered patients, 49 developed 56 malignant BTs: 50 high-grade gliomas (HGG) (with giant multinucleated cells in 16/21 histologically reviewed tumors) and 6 embryonal tumors. The median age at first BT was 9.2 years [1.1–40.6], with nine patients older than 18. Twenty-seven patients developed multiple malignancies (including16 before the BT). Most patients received standard treatment, and eight patients immunotherapy for relapsed HGG. The 3- and 5-year overall survival (OS) rates were 30% (95% CI: 19–45) and 22% (95% CI: 12–37) after the first BT, with worse prognosis for HGG (3-year OS = 20.5%). Six patients were alive (median follow-up 2.5 years) and 43 dead (38 deaths, 88%, were BT-related). Other CMMRD-specific features were café-au-lait macules (40/41), multiple BTs (5/15), developmental brain anomalies (11/15), and consanguinity (20/38 families). Conclusions Several characteristics could help suspecting CMMRD in pediatric malignant BTs: giant cells on histology, previous malignancies, parental consanguinity, café-au-lait macules, multiple BTs, and developmental brain anomalies. The prognosis of CMMRD-associated BT treated with standard therapies is poor requiring new therapeutic up-front approaches