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Abstract: Melanocytic matricoma is a rare benign pilar tumor characterized by matrical differentiation and interspersed dendritic melanocytes. It may show cellular atypia and brisk mitotic activity. Histological characterization of some lesions may be difficult. In addition, because the reported cases are few and have limited follow-up, there is insufficient experience to define outcome-based criteria for malignancy. Some cases of melanocytic matricoma with more prominent atypia have been reported as malignant, but their clinical behavior is uncertain. We present a melanocytic matricoma with interspersed benign dendritic melanocytes, but moderate basaloid atypia, focally brisk mitotic activity, and atypical mitoses. Despite the apparently good delimitation of this tumor, higher magnification revealed a slightly irregular border. However, overt malignant features such as necrosis, frank asymmetry, deep infiltration, and ulceration were not present. This tumor showed a complex aberrant genomic profile with multiple whole chromosomes or chromosomal arms, losses, and duplications. The tumor mutational burden was high. A loss-of-function alteration in CDKN2A and a loss-of-function mutation in TP53 were also present. This unexpected molecular profile contrasts with the relatively bland histology of the tumor and is in line with the difficulties in microscopic differential diagnosis between melanocytic matricoma and an indolent malignant pilomatrical tumor. We suggest that molecular studies and longer follow-up periods may help to further understand and more precisely categorize borderline pilomatrical tumors with melanocytic hyperplasia., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Digital polymerase chain reaction (dPCR) is an emerging technology that enables accurate and sensitive quantification of nucleic acids. Most available dPCR systems have two channel optics, with ad hoc software limited to the analysis of single and duplex assays. Although multiplexing strategies were developed, variable assay designs, dPCR systems, and the analysis of low DNA input data restricted the ability for a universal automated clustering approach. To overcome these issues, we developed dPCR Cluster Predictor (dPCP), an R package and a Shiny app for automated analysis of up to 4-plex dPCR data. dPCP can analyse and visualize data generated by multiple dPCR systems carrying out accurate and fast clustering not influenced by the amount and integrity of input of nucleic acids. With the companion Shiny app, the functionalities of dPCP can be accessed through a web browser., (© The Author(s) 2023. Published by Oxford University Press.)

BRD4 is part of a multiprotein complex involved in loading the cohesin complex onto DNA, a fundamental process required for cohesin-mediated loop extrusion and formation of Topologically Associating Domains. Pathogenic variations in this complex have been associated with a growing number of syndromes, collectively known as cohesinopathies, the most classic being Cornelia de Lange syndrome. However, no cohort study has been conducted to delineate the clinical and molecular spectrum of BRD4-related disorder. We formed an international collaborative study, and collected 14 new patients, including two fetuses. We performed phenotype and genotype analysis, integrated prenatal findings from fetopathological examinations, phenotypes of pediatric patients and adults. We report the first cohort of patients with BRD4-related disorder and delineate the dysmorphic features at different ages. This work extends the phenotypic spectrum of cohesinopathies and characterize a new clinically relevant and recognizable pattern, distinguishable from the other cohesinopathies., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Patient-based cancer models are essential tools for studying tumor biology and for the assessment of drug responses in a translational context. We report the establishment a large cohort of unique organoids and patient-derived orthotopic xenografts (PDOX) of various glioma subtypes, including gliomas with mutations in IDH1, and paired longitudinal PDOX from primary and recurrent tumors of the same patient. We show that glioma PDOXs enable long-term propagation of patient tumors and represent clinically relevant patient avatars that retain histopathological, genetic, epigenetic, and transcriptomic features of parental tumors. We find no evidence of mouse-specific clonal evolution in glioma PDOXs. Our cohort captures individual molecular genotypes for precision medicine including mutations in IDH1, ATRX, TP53, MDM2/4, amplification of EGFR, PDGFRA, MET, CDK4/6, MDM2/4, and deletion of CDKN2A/B, PTCH, and PTEN. Matched longitudinal PDOX recapitulate the limited genetic evolution of gliomas observed in patients following treatment. At the histological level, we observe increased vascularization in the rat host as compared to mice. PDOX-derived standardized glioma organoids are amenable to high-throughput drug screens that can be validated in mice. We show clinically relevant responses to temozolomide (TMZ) and to targeted treatments, such as EGFR and CDK4/6 inhibitors in (epi)genetically defined subgroups, according to MGMT promoter and EGFR/CDK status, respectively. Dianhydrogalactitol (VAL-083), a promising bifunctional alkylating agent in the current clinical trial, displayed high therapeutic efficacy, and was able to overcome TMZ resistance in glioblastoma. Our work underscores the clinical relevance of glioma organoids and PDOX models for translational research and personalized treatment studies and represents a unique publicly available resource for precision oncology.

Background: Artificial genomic reference standards in a cytocentrifuge/cytospin format with well-annotated genomic data are useful for validating next-generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 × 10 5 ). This was done to better reflect the clinical challenge of working with insufficient cytological material., Methods: A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A-D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235_2249del15 p.E746_A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B-Raf proto-oncogene, serine/threonine kinase (BRAF) c.1798_1799GT>AA p.V600K mutations at various allele frequencies (AFs)., Results: EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second-look analysis highlighted the mutations (n = 10) that had been missed in the first-look analysis. BRAF c.1798_1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification., Conclusions: The data show that the detection of low-abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low-AF mutations., (© 2019 American Cancer Society.)

The identity and unique capacity of cancer stem cells (CSC) to drive tumor growth and resistance have been challenged in brain tumors. Here we report that cells expressing CSC-associated cell membrane markers in Glioblastoma (GBM) do not represent a clonal entity defined by distinct functional properties and transcriptomic profiles, but rather a plastic state that most cancer cells can adopt. We show that phenotypic heterogeneity arises from non-hierarchical, reversible state transitions, instructed by the microenvironment and is predictable by mathematical modeling. Although functional stem cell properties were similar in vitro, accelerated reconstitution of heterogeneity provides a growth advantage in vivo, suggesting that tumorigenic potential is linked to intrinsic plasticity rather than CSC multipotency. The capacity of any given cancer cell to reconstitute tumor heterogeneity cautions against therapies targeting CSC-associated membrane epitopes. Instead inherent cancer cell plasticity emerges as a novel relevant target for treatment.

Background: Molecular testing of cytological lung cancer specimens includes, beyond epidermal growth factor receptor (EGFR), emerging predictive/prognostic genomic biomarkers such as Kirsten rat sarcoma viral oncogene homolog (KRAS), neuroblastoma RAS viral [v-ras] oncogene homolog (NRAS), B-Raf proto-oncogene, serine/threonine kinase (BRAF), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA). Next-generation sequencing (NGS) and other multigene mutational assays are suitable for cytological specimens, including smears. However, the current literature reflects single-institution studies rather than multicenter experiences., Methods: Quantitative cytological molecular reference slides were produced with cell lines designed to harbor concurrent mutations in the EGFR, KRAS, NRAS, BRAF, and PIK3CA genes at various allelic ratios, including low allele frequencies (AFs; 1%). This interlaboratory ring trial study included 14 institutions across the world that performed multigene mutational assays, from tissue extraction to data analysis, on these reference slides, with each laboratory using its own mutation analysis platform and methodology., Results: All laboratories using NGS (n = 11) successfully detected the study's set of mutations with minimal variations in the means and standard errors of variant fractions at dilution points of 10% (P = .171) and 5% (P = .063) despite the use of different sequencing platforms (Illumina, Ion Torrent/Proton, and Roche). However, when mutations at a low AF of 1% were analyzed, the concordance of the NGS results was low, and this reflected the use of different thresholds for variant calling among the institutions. In contrast, laboratories using matrix-assisted laser desorption/ionization-time of flight (n = 2) showed lower concordance in terms of mutation detection and mutant AF quantification., Conclusions: Quantitative molecular reference slides are a useful tool for monitoring the performance of different multigene mutational assays, and this could lead to better standardization of molecular cytopathology procedures. Cancer Cytopathol 2017;125:615-26. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

Since the advent of monoclonal antibodies against epidermal growth factor receptor (EGFR) in colorectal cancer therapy, the determination of RAS mutational status is needed for therapeutic decision-making. Most prevalent in colorectal cancer are KRAS exon 2 mutations (40% prevalence); lower prevalence is observed for KRAS exon 3 and 4 mutations (6%) and NRAS exon 2, 3, and 4 mutations (5%). The Idylla™ KRAS Mutation Test on the molecular diagnostics Idylla™ platform is a simple (<2 minutes hands-on time), highly reliable, and rapid (approximately 2 hours turnaround time) in vitro diagnostic sample-to-result solution. This test enables qualitative detection of 21 mutations in codons 12, 13, 59, 61, 117, and 146 of the KRAS oncogene being clinically relevant according to the latest clinical guidelines. Here, the performance of the Idylla™ KRAS Mutation Assay, for Research Use Only, was assessed on archived formalin-fixed paraffin-embedded (FFPE) tissue sections by comparing its results with the results previously obtained by routine reference approaches for KRAS genotyping. In case of discordance, samples were assessed further by additional methods. Among the 374 colorectal cancer FFPE samples tested, the overall concordance between the Idylla™ KRAS Mutation Assay and the confirmed reference routine test results was found to be 98.9%. The Idylla™ KRAS Mutation Assay enabled detection of 5 additional KRAS-mutated samples not detected previously with reference methods. As conclusion the Idylla™ KRAS Mutation Test can be applied as routine tool in any clinical setting, without needing molecular infrastructure or expertise, to guide the personalized treatment of colorectal cancer patients., Competing Interests: Peter Sillekens is an employee of Biocartis. He is senior scientist at the Research & Development department. Biocartis has no financial contribution to this study, besides free of charge Idylla™ KRAS Mutations Assay cartridges.This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The histopathological and molecular heterogeneity of glioblastomas represents a major obstacle for effective therapies. Glioblastomas do not develop autonomously, but evolve in a unique environment that adapts to the growing tumour mass and contributes to the malignancy of these neoplasms. Here, we show that patient-derived glioblastoma xenografts generated in the mouse brain from organotypic spheroids reproducibly give rise to three different histological phenotypes: (i) a highly invasive phenotype with an apparent normal brain vasculature, (ii) a highly angiogenic phenotype displaying microvascular proliferation and necrosis and (iii) an intermediate phenotype combining features of invasion and vessel abnormalities. These phenotypic differences were visible during early phases of tumour development suggesting an early instructive role of tumour cells on the brain parenchyma. Conversely, we found that tumour-instructed stromal cells differentially influenced tumour cell proliferation and migration in vitro, indicating a reciprocal crosstalk between neoplastic and non-neoplastic cells. We did not detect any transdifferentiation of tumour cells into endothelial cells. Cell type-specific transcriptomic analysis of tumour and endothelial cells revealed a strong phenotype-specific molecular conversion between the two cell types, suggesting co-evolution of tumour and endothelial cells. Integrative bioinformatic analysis confirmed the reciprocal crosstalk between tumour and microenvironment and suggested a key role for TGFβ1 and extracellular matrix proteins as major interaction modules that shape glioblastoma progression. These data provide novel insight into tumour-host interactions and identify novel stroma-specific targets that may play a role in combinatorial treatment strategies against glioblastoma., Competing Interests: Authors have no conflict of interest to disclose

Anti-angiogenic therapy in glioblastoma (GBM) has unfortunately not led to the anticipated improvement in patient prognosis. We here describe how human GBM adapts to bevacizumab treatment at the metabolic level. By performing (13)C6-glucose metabolic flux analysis, we show for the first time that the tumors undergo metabolic re-programming toward anaerobic metabolism, thereby uncoupling glycolysis from oxidative phosphorylation. Following treatment, an increased influx of (13)C6-glucose was observed into the tumors, concomitant to increased lactate levels and a reduction of metabolites associated with the tricarboxylic acid cycle. This was confirmed by increased expression of glycolytic enzymes including pyruvate dehydrogenase kinase in the treated tumors. Interestingly, L-glutamine levels were also reduced. These results were further confirmed by the assessment of in vivo metabolic data obtained by magnetic resonance spectroscopy and positron emission tomography. Moreover, bevacizumab led to a depletion in glutathione levels indicating that the treatment caused oxidative stress in the tumors. Confirming the metabolic flux results, immunohistochemical analysis showed an up-regulation of lactate dehydrogenase in the bevacizumab-treated tumor core as well as in single tumor cells infiltrating the brain, which may explain the increased invasion observed after bevacizumab treatment. These observations were further validated in a panel of eight human GBM patients in which paired biopsy samples were obtained before and after bevacizumab treatment. Importantly, we show that the GBM adaptation to bevacizumab therapy is not mediated by clonal selection mechanisms, but represents an adaptive response to therapy.

It is well known that in vitro subculture represents a selection pressure on cell lines, and over time this may result in a genetic drift in the cancer cells. In addition, long-term cultures harbor the risk of cross-contamination with other cell lines. The consequences may have major impact on experimental results obtained in various laboratories, where the cell lines no longer reflect the original tumors that they are supposed to represent. Much neglected in the scientific community is a close monitoring of cell cultures by regular phenotypic and genetic characterization. In this report, we present a thorough characterization of the commonly used glioblastoma (GBM) model U-251, which in numerous publications has been wrongly identified as U-373, due to an earlier cross-contamination. In this work, the original U-251 and three subclones of U-251, commonly referred to as U-251 or U-373, were analyzed with regard to their DNA profile, morphology, phenotypic expression, and growth pattern. By array comparative genomic hybridization (aCGH), we show that only the original low-passaged U-251 cells, established in the 1960s, maintain a DNA copy number resembling a typical GBM profile, whereas all long-term subclones lost the typical GBM profile. Also the long-term passaged subclones displayed variations in phenotypic marker expression and showed an increased growth rate in vitro and a more aggressive growth in vivo. Taken together, the variations in genotype and phenotype as well as differences in growth characteristics may explain different results reported in various laboratories related to the U-251 cell line., (© 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Malignant melanoma is the most lethal form of skin cancer, with a high propensity to metastasize to the brain. More than 60% of melanomas have the BRAFV600E mutation, which activates the mitogen-activated protein kinase (MAPK) pathway [1]. In addition, increased PI3K (phosphoinositide 3-kinase) pathway activity has been demonstrated, through the loss of activity of the tumor suppressor gene, PTEN [2]. Here, we treated two melanoma brain metastasis cell lines, H1&#95;DL2, harboring a BRAFV600E mutation and PTEN loss, and H3, harboring WT (wild-type) BRAF and PTEN loss, with the MAPK (BRAF) inhibitor vemurafenib and the PI3K pathway associated mTOR inhibitor temsirolimus. Combined use of the drugs inhibited tumor cell growth and proliferation in vitro in H1&#95;DL2 cells, compared to single drug treatment. Treatment was less effective in the H3 cells. Furthermore, a strong inhibitory effect on the viability of H1&#95;DL2 cells, when grown as 3D multicellular spheroids, was seen. The treatment inhibited the expression of pERK1/2 and reduced the expression of pAKT and p-mTOR in H1&#95;DL2 cells, confirming that the MAPK and PI3K pathways were inhibited after drug treatment. Microarray experiments followed by principal component analysis (PCA) mapping showed distinct gene clustering after treatment, and cell cycle checkpoint regulators were affected. Global gene analysis indicated that functions related to cell survival and invasion were influenced by combined treatment. In conclusion, we demonstrate for the first time that combined therapy with vemurafenib and temsirolimus is effective on melanoma brain metastasis cells in vitro. The presented results highlight the potential of combined treatment to overcome treatment resistance that may develop after vemurafenib treatment of melanomas.

Glioblastoma (GBM) is known to be a heterogeneous disease; however, the genetic composition of the cells within a given tumour is only poorly explored. In the advent of personalised medicine the understanding of intra-tumoural heterogeneity at the cellular and the genetic level is mandatory to improve treatment and clinical outcome. By combining ploidy-based flow sorting with array-comparative genomic hybridization we show that primary GBMs present as either mono- or polygenomic tumours (64 versus 36%, respectively). Monogenomic tumours were limited to a pseudodiploid tumour clone admixed with normal stromal cells, whereas polygenomic tumours contained multiple tumour clones, yet always including a pseudodiploid population. Interestingly, pseudodiploid and aneuploid fractions carried the same aberrations as defined by identical chromosomal breakpoints, suggesting that evolution towards aneuploidy is a late event in GBM development. Interestingly, while clonal heterogeneity could be recapitulated in spheroid-based xenografts, we find that genetically distinct clones displayed different tumourigenic potential. Moreover, we show that putative cancer stem cell markers including CD133, CD15, A2B5 and CD44 were present on genetically distinct tumour cell populations. These data reveal the clonal heterogeneity of GBMs at the level of DNA content, tumourigenic potential and stem cell marker expression, which is likely to impact glioma progression and treatment response. The combined knowledge of intra-tumour heterogeneity at the genetic, cellular and functional level is crucial to assess treatment responses and to design personalized treatment strategies for primary GBM.

Two of the signature genetic events that occur in human gliomas, EGFR amplification and IDH mutation, are poorly represented in experimental models in vitro. EGFR amplification, for example, occurs in 40 to 50% of GBM, and yet, EGFR amplification is rarely preserved in cell cultures derived from human tumors. To analyze the fate of EGFR amplified and IDH mutated cells in culture, we followed the development over time of cultures derived from human xenografts in nude rats enriched for tumor cells with EGFR amplification and of cultures derived from patient samples with IDH mutations, in serum monolayer and spheroid suspension culture, under serum and serum free conditions. We observed under serum monolayer conditions, that nestin positive or nestin and SMA double positive rat stromal cells outgrew EGFR amplified tumor cells, while serum spheroid cultures preserved tumor cells with EGFR amplification. Serum free suspension culture exhibited a more variable cell composition in that the resultant cell populations were either predominantly nestin/SOX2 co-expressing rat stromal cells or human tumor cells, or a mixture of both. The selection for nestin/SMA positive stromal cells under serum monolayer conditions was also consistently observed in human oligodendrogliomas and oligoastrocytomas with IDH mutations. Our results highlight for the first time that serum monolayer conditions can select for stromal cells instead of tumor cells in certain brain tumor subtypes. This result has an important impact on the establishment of new tumor cell cultures from brain tumors and raises the question of the proper conditions for the growth of the tumor cell populations of interest.

Background: Deregulated growth factor signaling is a major driving force in the initiation and progression of glioblastoma. The tumor suppressor and stem cell marker Lrig1 is a negative regulator of the epidermal growth factor receptor (EGFR) family. Here, we addressed the therapeutic potential of the soluble form of Lrig1 (sLrig1) in glioblastoma treatment and the mechanism of sLrig1-induced growth inhibition., Methods: With use of encapsulated cells, recombinant sLrig1 was locally delivered in orthotopic glioblastoma xenografts generated from freshly isolated patient tumors. Tumor growth and mouse survival were evaluated. The efficacy of sLrig1 and the affected downstream signaling was studied in vitro and in vivo in glioma cells displaying variable expression of wild-type and/or a constitutively active EGFR mutant (EGFRvIII)., Results: Continuous interstitial delivery of sLrig1 in genetically diverse patient-derived glioma xenografts led to strong tumor growth inhibition. Glioma cell proliferation in vitro and tumor growth in vivo were potently inhibited by sLrig1, irrespective of EGFR expression levels. Of importance, tumor growth was also suppressed in EGFRvIII-driven glioma. sLrig1 induced cell cycle arrest without changing total receptor level or phosphorylation. Affected downstream effectors included MAP kinase but not AKT signaling. Of importance, local delivery of sLrig1 into established tumors led to a 32% survival advantage in treated mice., Conclusions: To our knowledge, this is the first report demonstrating that sLrig1 is a potent inhibitor of glioblastoma growth in clinically relevant experimental glioma models and that this effect is largely independent of EGFR status. The potent anti-tumor effect of sLrig1, in combination with cell encapsulation technology for in situ delivery, holds promise for future treatment of glioblastoma.

Background: Point mutations in genes encoding NADP+-dependent isocitrate dehydrogenases (especially IDH1) are common in lower grade diffuse gliomas and secondary glioblastomas and occur early during tumor development. The contribution of these mutations to gliomagenesis is not completely understood and research is hampered by the lack of relevant tumor models. We previously described the development of the patient-derived high-grade oligodendroglioma xenograft model E478 that carries the commonly occurring IDH1-R132H mutation. We here report on the analyses of E478 xenografts at the genetic, histologic and metabolic level., Results: LC-MS and in situ mass spectrometric imaging by LESA-nano ESI-FTICR revealed high levels of the proposed oncometabolite D-2-hydroxyglutarate (D-2HG), the product of enzymatic conversion of α-ketoglutarate (α-KG) by IDH1-R132H, in the tumor but not in surrounding brain parenchyma. α-KG levels and total NADP+-dependent IDH activity were similar in IDH1-mutant and -wildtype xenografts, demonstrating that IDH1-mutated cancer cells maintain α-KG levels. Interestingly, IDH1-mutant tumor cells in vivo present with high densities of mitochondria and increased levels of mitochondrial activity as compared to IDH1-wildtype xenografts. It is not yet clear whether this altered mitochondrial activity is a driver or a consequence of tumorigenesis., Conclusions: The oligodendroglioma model presented here is a valuable model for further functional elucidation of the effects of IDH1 mutations on tumor metabolism and may aid in the rational development of novel therapeutic strategies for the large subgroup of gliomas carrying IDH1 mutations.

Angiogenesis is regarded as a hallmark of cancer progression and it has been postulated that solid tumor growth depends on angiogenesis. At present, however, it is clear that tumor cell invasion can occur without angiogenesis, a phenomenon that is particularly evident by the infiltrative growth of malignant brain tumors, such as glioblastomas (GBMs). In these tumors, amplification or overexpression of wild-type (wt) or truncated and constitutively activated epidermal growth factor receptor (EGFR) are regarded as important events in GBM development, where the complex downstream signaling events have been implicated in tumor cell invasion, angiogenesis and proliferation. Here, we show that amplification and in particular activation of wild-type EGFR represents an underlying mechanism for non-angiogenic, invasive tumor growth. Using a clinically relevant human GBM xenograft model, we show that tumor cells with EGFR gene amplification and activation diffusely infiltrate normal brain tissue independent of angiogenesis and that transient inhibition of EGFR activity by cetuximab inhibits the invasive tumor growth. Moreover, stable, long-term expression of a dominant-negative EGFR leads to a mesenchymal to epithelial-like transition and induction of angiogenic tumor growth. Analysis of human GBM biopsies confirmed that EGFR activation correlated with invasive/non-angiogenic tumor growth. In conclusion, our results indicate that activation of wild-type EGFR promotes invasion and glioblastoma development independent of angiogenesis, whereas loss of its activity results in angiogenic tumor growth.

The identification and significance of cancer stem-like cells in malignant gliomas remains controversial. It has been proposed that cancer stem-like cells display increased drug resistance, through the expression of ATP-binding cassette transporters that detoxify cells by effluxing exogenous compounds. Here, we investigated the 'side population' phenotype based on efflux properties of ATP-binding cassette transporters in freshly isolated human glioblastoma samples and intracranial xenografts derived thereof. Using fluorescence in situ hybridization analysis on sorted cells obtained from glioblastoma biopsies, as well as human tumour xenografts developed in immunodeficient enhanced green fluorescence protein-expressing mice that allow an unequivocal tumour-stroma discrimination, we show that side population cells in human glioblastoma are non-neoplastic and exclusively stroma-derived. Tumour cells were consistently devoid of efflux properties regardless of their genetic background, tumour ploidy or stem cell associated marker expression. Using multi-parameter flow cytometry we identified the stromal side population in human glioblastoma to be brain-derived endothelial cells with a minor contribution of astrocytes. In contrast with their foetal counterpart, neural stem/progenitor cells in the adult brain did not display the side population phenotype. Of note, we show that CD133-positive cells often associated with cancer stem-like cells in glioblastoma biopsies, do not represent a homogenous cell population and include CD31-positive endothelial cells. Interestingly, treatment of brain tumours with the anti-angiogenic agent bevacizumab reduced total vessel density, but did not affect the efflux properties of endothelial cells. In conclusion our findings contribute to an unbiased identification of cancer stem-like cells and stromal cells in brain neoplasms, and provide novel insight into the complex issue of drug delivery to the brain. Since efflux properties of endothelial cells are likely to compromise drug availability, transiently targeting ATP-binding cassette transporters may be a valuable therapeutic strategy to improve treatment effects in brain tumours.

Oligodendroglioma poses a biological conundrum for malignant adult human gliomas: it is a tumor type that is universally incurable for patients, and yet, only a few of the human tumors have been established as cell populations in vitro or as intracranial xenografts in vivo. Their survival, thus, may emerge only within a specific environmental context. To determine the fate of human oligodendroglioma in an experimental model, we studied the development of an anaplastic tumor after intracranial implantation into enhanced green fluorescent protein (eGFP) positive NOD/SCID mice. Remarkably after nearly nine months, the tumor not only engrafted, but it also retained classic histological and genetic features of human oligodendroglioma, in particular cells with a clear cytoplasm, showing an infiltrative growth pattern, and harboring mutations of IDH1 (R132H) and of the tumor suppressor genes, FUBP1 and CIC. The xenografts were highly invasive, exhibiting a distinct migration and growth pattern around neurons, especially in the hippocampus, and following white matter tracts of the corpus callosum with tumor cells accumulating around established vasculature. Although tumors exhibited a high growth fraction in vivo, neither cells from the original patient tumor nor the xenograft exhibited significant growth in vitro over a six-month period. This glioma xenograft is the first to display a pure oligodendroglioma histology and expression of R132H. The unexpected property, that the cells fail to grow in vitro even after passage through the mouse, allows us to uniquely investigate the relationship of this oligodendroglioma with the in vivo microenvironment.

Unlabelled: The hypothesis of this study was that distinct experimental glioblastoma phenotypes resembling human disease can be noninvasively distinguished at various disease stages by imaging in vivo., Methods: Cultured spheroids from 2 human glioblastomas were implanted into the brains of nude rats. Glioblastoma growth dynamics were followed by PET using (18)F-FDG, (11)C-methyl-l-methionine ((11)C-MET), and 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) and by MRI at 3-6 wk after implantation. For image validation, parameters were coregistered with immunohistochemical analysis., Results: Two tumor phenotypes (angiogenic and infiltrative) were obtained. The angiogenic phenotype showed high uptake of (11)C-MET and (18)F-FLT and relatively low uptake of (18)F-FDG. (11)C-MET was an early indicator of vessel remodeling and tumor proliferation. (18)F-FLT uptake correlated to positive Ki67 staining at 6 wk. T1- and T2-weighted MR images displayed clear tumor delineation with strong gadolinium enhancement at 6 wk. The infiltrative phenotype did not accumulate (11)C-MET and (18)F-FLT and impaired the (18)F-FDG uptake. In contrast, the Ki67 index showed a high proliferation rate. The extent of the infiltrative tumors could be observed by MRI but with low contrast., Conclusion: For angiogenic glioblastomas, noninvasive assessment of tumor activity corresponds well to immunohistochemical markers, and (11)C-MET was more sensitive than (18)F-FLT at detecting early tumor development. In contrast, infiltrative glioblastoma growth in the absence of blood-brain barrier breakdown is difficult to noninvasively follow by existing imaging techniques, and a negative (18)F-FLT PET result does not exclude the presence of proliferating glioma tissue. The angiogenic model may serve as an advanced system to study imaging-guided antiangiogenic and antiproliferative therapies.

Introduction: Glioblastoma remains a highly aggressive primary brain cancer with very poor prognosis. The detection of mutations in the metabolic enzyme isocitrate dehydrogenase in gliomas, has broadened our view of tumourigenic mechanisms. Together with renewed awareness of tumour-specific energy metabolism, research is pointed towards novel ways for targeting brain cancer., Areas Covered: This paper reviews recent knowledge on the possible tumourigenic mechanism of mutant isocitrate dehydrogenase, and provides a detailed overview of cancer-specific metabolic enzymes associated with glycolysis and intracellular pH regulation. It also discusses available drugs that may serve as a basis for novel drug development to target metabolic transformation in gliomas., Expert Opinion: Despite the fact that energy metabolism is a very basic cellular process, tumour specific alterations in key metabolic processes represent promising targets for glioma treatment. Novel therapies against gliomas, including those that target metabolic transformation, need to consider the genetic background of the individual tumours, to allow the correlation of treatment response with the underlying biological status, both in preclinical and clinical studies.

Bevacizumab, an antibody against vascular endothelial growth factor (VEGF), is a promising, yet controversial, drug in human glioblastoma treatment (GBM). Its effects on tumor burden, recurrence, and vascular physiology are unclear. We therefore determined the tumor response to bevacizumab at the phenotypic, physiological, and molecular level in a clinically relevant intracranial GBM xenograft model derived from patient tumor spheroids. Using anatomical and physiological magnetic resonance imaging (MRI), we show that bevacizumab causes a strong decrease in contrast enhancement while having only a marginal effect on tumor growth. Interestingly, dynamic contrast-enhanced MRI revealed a significant reduction of the vascular supply, as evidenced by a decrease in intratumoral blood flow and volume and, at the morphological level, by a strong reduction of large- and medium-sized blood vessels. Electron microscopy revealed fewer mitochondria in the treated tumor cells. Importantly, this was accompanied by a 68% increase in infiltrating tumor cells in the brain parenchyma. At the molecular level we observed an increase in lactate and alanine metabolites, together with an induction of hypoxia-inducible factor 1α and an activation of the phosphatidyl-inositol-3-kinase pathway. These data strongly suggest that vascular remodeling induced by anti-VEGF treatment leads to a more hypoxic tumor microenvironment. This favors a metabolic change in the tumor cells toward glycolysis, which leads to enhanced tumor cell invasion into the normal brain. The present work underlines the need to combine anti-angiogenic treatment in GBMs with drugs targeting specific signaling or metabolic pathways linked to the glycolytic phenotype.

We previously defined quantitative trait loci (QTLs) that control susceptibility to 7,12-dimethylbenz(alpha)anthracene-induced mammary carcinoma in SPRD-Cu3 (susceptible) and WKY (resistant) rats. Two of these QTLs, assigned to chromosomes (Chr) 10 and 18, control tumor growth rate and invasiveness. In this study we characterized a congenic strain in which a large segment of WKY Chr 10 was introduced in the SPRD-Cu3 genetic background and demonstrated that this chromosome segment controls this tumor trait. The WKY allele at this QTL (Mcsta1) reduces the growth rate of the fastest growing tumors by 26%. We also previously showed that two SPRD-Cu3-WKY congenic strains containing a WKY chromosome segment derived either from Chr 5 or from Chr 18 exhibit a reduction in tumor multiplicity (QTLs Msctm1 and Mcstm2, respectively) (with no reduction in tumor growth rate in the Chr 18 congenic). In this study we generated a double congenic strain, which contains the two WKY differential segments from Chr 5 and 18, to determine how these two segments interact with one another. Interestingly, two types of epistatic interactions were found: no additive effect was seen with respect to tumor multiplicity, while a reduction in tumor growth rate was observed. It thus appears that WKY alleles located on Chr 5 and Chr 8 interact epistatically in a contrasting manner to modulate tumor multiplicity (in a nonadditive manner) and growth rate (in a synergic manner). Tumor growth rate is thus influenced by two QTLs, on Chr 10 (Mcsta1) and on Chr 18 (Mcsta2), the action of the latter being dependent on the presence of the Chr5 QTL (Mcstm1). The expression level of positional and functional candidate genes was also analyzed. On Chr 5, Pla2g2a is subject to a syntenic control while expression of the Tp53 (Chr 10) and Pmai1/Noxa (Chr 18) genes appears to be controlled by several mammary cancer resistance QTLs.

The COP and WKY rat strains are resistant to mammary cancer. It has shown previously that upon chemical carcinogen treatment, COP females exhibit mammary preneoplastic lesions which disappear within a few weeks. We show here that in similar conditions, WKY females do not exhibit any visible preneoplastic lesions. WKY females are characterized by precocious mammary tissue differentiation, including active expression of the beta-casein gene in young virgin females. This trait might be critical in resistance to mammary carcinogenesis of WKY rats. To test this hypothesis, we took advantage of 2 congenic strains that contain a limited chromosome segment of WKY origin, derived either from chromosome 5 or from chromosome 18, introgressed in the susceptible genetic background (SPRD-Cu3). Each of these congenic strains has been shown to be partially resistant to chemically induced mammary carcinogenesis (reduction in tumour multiplicity with respect to the susceptible SPRD-Cu3 rats). We show here that these 2 congenic strains also exhibit precocious mammary differentiation, though to a lower extent than the WKY females. The conclusion of this study is thus 2-fold: (i) eradication of preneoplastic lesions is not a general phenomenon in mammary cancer resistance; (ii) the same segment of rat chromosomes 5 or 18 that controls mammary cancer resistance also contains a quantitative trait locus imposing precocious mammary differentiation. These 2 traits are thus associated, supporting the hypothesis that there might be a cause-effect relationship between precocious mammary differentiation and cancer resistance., ((c) 2007 Wiley-Liss, Inc.)

We previously mapped several quantitative trait loci (QTLs) controlling DMBA-induced mammary tumor development in female rats derived from a SPRD-Cu3 (susceptible strain) x WKY (resistant strain) cross. Two of these QTLs were assigned to chromosomes 5 and 18. In the present study, we generated and characterized congenic strains in which a segment of WKY chromosomes 5 or 18 was introduced in the SPRD-Cu3 genetic background, thereby physically demonstrating that each of these two chromosomes controls mammary tumor multiplicity. The chromosome 5 QTL (Mcstm1) accounts for 7 tumors per animal (versus a total of 11 tumors per SPRD-Cu3 rat). The chromosome 18 QTL (Mcstm2) accounts for 3 tumors per animal and is the first chemically-induced mammary cancer susceptibility locus assigned to this chromosome. In addition, the Mcstm1 region was shown to also controls tumor latency. These loci thus play a major role in chemically-induced mammary tumor development. QTLs controlling chemically-induced or estrogen-induced mammary tumor development have independently been identified on chromosomes 5 and 18, using susceptible strains others than SPRD-Cu3. Therefore the haplotype structure of the relevant chromosome regions was analyzed in the different strains. Some chromosome regions were found to be highly mosaic (haplotype blocks < 1 Mb), while one region showed an apparently conserved haplotype block of 7.5 Mb. This analysis points to limited regions that could harbor the causative genes and also indicates that at least Mcstm2 is a novel QTL., ((c) 2007 Wiley-Liss, Inc.)

The rat is considered an excellent model for studying human breast cancer. Therefore, understanding the genetic basis of susceptibility to mammary cancer in this species is of great interest. Previous studies based on crosses involving the susceptible strain WF (crossed with the resistant strains COP or WKY) and focusing on tumor multiplicity as the susceptibility phenotype led to the identification of several loci that control chemically induced mammary cancer. The present study was aimed to determine whether other loci can be identified by analyzing crosses derived from another susceptible strain on the one hand, and by including phenotypes other than tumor multiplicity on the other hand. A backcross was generated between the susceptible SPRD-Cu3 strain and the resistant WKY strain. Female progeny were genotyped with microsatellite markers covering all rat autosomes, treated with a single dose of DMBA, and phenotyped with respect to tumor latency, tumor multiplicity, and tumor aggressiveness. Seven loci controlling mammary tumor development were detected. Different loci control tumor multiplicity, latency, and aggressiveness. While some of these loci colocalize with loci identified in crosses involving the susceptible strain WF, new loci have been uncovered, indicating that the use of distinct susceptible and resistant strain pairs will help in establishing a comprehensive inventory of mammary cancer susceptibility loci.

The rat strain COP is resistant to spontaneous and carcinogen-induced mammary cancer, whereas the strain WF is susceptible. Using genetic linkage analysis of (WF x COP) F1 x WF backcrosses, LC Hsu, LA Shepel and co-workers showed that a region at the centromeric end of Chromosome (Chr) 2 (2q1) segregates with the sensitivity to mammary cancer development. The responsible locus was named Mcs1 (for mammary cancer susceptibility 1). We have developed the chromosome map of the 2q1 region by localizing 18 genes, 4 ESTs, and several anonymous markers, using radiation hybrids and fluorescence in situ hybridization. The region containing Mcs1 was delineated to 2q12-q14. Five of the genes (Mef2c, Map1b, Ccnh, Rasa, Rasgrf2) were assigned to this region and were shown to be expressed in the rat mammary glands, while three possible functional candidate genes, Pi3kr1, Rad17, and Naip, were excluded from the critical region. Since cyclin H, encoded by Ccnh, plays an important role in the control of the cell cycle and since the proteins encoded by Rasa and Rasgrf2 control the activity of the RAS oncoprotein, the corresponding genes appeared as both functional and positional Mcs1 candidates. RT-PCR experiments on RNA extracted from mammary glands of the two rat strains (COP, WF) was done. No amino acid sequence difference was found between the two strains. These results argue against the hypothesis that any of these three genes is Mcs1.