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BackgroundUp to 7% of patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) remain genetically undiagnosed after routine genetic testing. These patients are thought to carry deep intronic variants, structural variants or splicing alterations not detected through multiplex ligation-dependent probe amplification or exome sequencing.MethodsRNA was extracted from seven muscle biopsy samples of patients with genetically undiagnosed DMD/BMD after routine genetic diagnosis. RT-PCR of theDMDgene was performed to detect the presence of alternative transcripts. Droplet digital PCR and whole-genome sequencing were also performed in some patients.ResultsWe identified an alteration in the mRNA level in all the patients. We detected three pseudoexons inDMDcaused by deep intronic variants, two of them not previously reported. We also identified a chromosomal rearrangement between Xp21.2 and 8p22. Furthermore, we detected three exon skipping events with unclear pathogenicity.ConclusionThese findings indicate that mRNA analysis of theDMDgene is a valuable tool to reach a precise genetic diagnosis in patients with a clinical and anatomopathological suspicion of dystrophinopathy that remain genetically undiagnosed after routine genetic testing.

Late-onset Pompe disease (LOPD) is a rare genetic disorder produced by mutations in the GAA gene and is characterized by progressive muscle weakness. LOPD muscle biopsies show accumulation of glycogen along with the autophagic vacuoles associated with atrophic muscle fibers. The expression of molecules related to muscle fiber atrophy in muscle biopsies of LOPD patients was studied using immunofluorescence and real-time PCR. BCL2 and adenovirus E1B 19-kDa interacting protein 3 (BNIP3), a well-known atrogene, was identified as a potential mediator of muscle fiber atrophy in LOPD muscle biopsies. Vacuolated fibers in LOPD patient muscle biopsies were smaller than nonvacuolated fibers and expressed BNIP3. The current data suggested that BNIP3 expression is regulated by inhibition of the AKT-mammalian target of rapamycin pathway, leading to phosphorylation of Unc-51 like autophagy activating kinase 1 (ULK1) at Ser317 by AMP-activated protein kinase. Myoblasts and myotubes obtained from LOPD patients and age-matched controls were studied to confirm these results using different molecular techniques. Myotubes derived from LOPD patients were likewise smaller and expressed BNIP3. Conclusively, transfection of BNIP3 into control myotubes led to myotube atrophy. These findings suggest a cascade that starts with the inhibition of the AKT-mammalian target of rapamycin pathway and activation of BNIP3 expression, leading to progressive muscle fiber atrophy. These results open the door to potential new treatments targeting BNIP3 to reduce its deleterious effects on muscle fiber atrophy in Pompe disease.

List of primary GBM cell lines used in this study.

Analysis of differentiation genes after long term dacomitinib treatment.

Analysis of stemmness genes after long term dacomitinib treatment.

Effect of dacomitinib in the in vivo growth of GBMs.

Analysis of EGFR signaling after long term dacomitinib treatment.

Analysis of stemmness genes after 5 days of dacomitinib treatment.

Detailed description of methods used in this study.

Participation of EGFR mutations in the formation of glioma-derived pericytes.

List of glioma samples

Supplementary Materials and Methods

The extraordinary plasticity of glioma cells allows them to contribute to different cellular compartments in tumor vessels, reinforcing the vascular architecture. It was recently revealed that targeting glioma-derived pericytes, which represent a big percentage of the mural cell population in aggressive tumors, increases the permeability of the vessels and improves the efficiency of chemotherapy. However, the molecular determinants of this transdifferentiation process have not been elucidated. Here we show that mutations in EGFR stimulate the capacity of glioma cells to function as pericytes in a BMX- (bone marrow and X-linked) and SOX9-dependent manner. Subsequent activation of platelet-derived growth factor receptor beta in the vessel walls of EGFR-mutant gliomas stabilized the vasculature and facilitated the recruitment of immune cells. These changes in the tumor microenvironment conferred a growth advantage to the tumors but also rendered them sensitive to pericyte-targeting molecules such as ibrutinib or sunitinib. In the absence of EGFR mutations, high-grade gliomas were enriched in blood vessels, but showed a highly disrupted blood–brain barrier due to the decreased BMX/SOX9 activation and pericyte coverage, which led to poor oxygenation, necrosis, and hypoxia. Overall, these findings identify EGFR mutations as key regulators of the glioma-to-pericyte transdifferentiation, highlighting the intricate relationship between the tumor cells and their vascular and immune milieu. Our results lay the foundations for a vascular-dependent stratification of gliomas and suggest different therapeutic vulnerabilities determined by the genetic status of EGFR.Significance:This study identifies the EGFR-related mechanisms that govern the capacity of glioma cells to transdifferentiate into pericytes, regulating the vascular and immune phenotypes of the tumors.

BackgroundBiallelic pathogenic variants inFXR1have recently been associated with two congenital myopathy phenotypes: a severe form associated with hypotonia, long bone fractures, respiratory insufficiency and infantile death, and a milder form characterised by proximal muscle weakness with survival into adulthood.ObjectiveWe report eight patients from four unrelated families with biallelic pathogenic variants in exon 15 ofFXR1.MethodsWhole exome sequencing was used to detect variants inFXR1.ResultsCommon clinical features were noted for all patients, which included proximal myopathy, normal serum creatine kinase levels and diffuse muscle atrophy with relative preservation of the quadriceps femoris muscle on muscle imaging. Additionally, some patients withFXR1-related myopathy had respiratory involvement and required bilevel positive airway pressure support. Muscle biopsy showed multi-minicores and type I fibre predominance with internalised nuclei.ConclusionFXR1-related congenital myopathy is an emerging entity that is clinically recognisable. Phenotypic variability associated with variants inFXR1can result from differences in variant location and type and is also observed between patients homozygous for the same variant, rendering specific genotype–phenotype correlations difficult. Our work broadens the phenotypic spectrum ofFXR1-related congenital myopathy.

Rhabdoid meningiomas (RM) shows heterogeneous histological findings, and a wide variety of chromosomal copy number alterations (CNA) are associated with an unpredictable course of the disease. In this study, we analyzed a series of 305 RM samples from patients previously reported in the literature and 33 samples from 23 patients studied in our laboratory. Monosomy 22-involving the minimal but most common recurrent region loss of the 22q11.23 chromosomal region was the most observed chromosomal alteration, followed by losses of chromosomes 14, 1, 6, and 19, polysomies of chromosomes 17, 1q, and 20, and gains of 13q14.2, 10p13, and 21q21.2 chromosomal regions. Based on their CNA profile, RM could be classified into two genetic subgroups with distinct clinicopathologic features characterized by the presence of (1) chromosomal losses only and (2) combined losses and gains of several chromosomes. The latter displays a higher frequency of WHO grade 3 tumors and poorer clinical outcomes. This research was funded by Consejería de Sanidad JCYL, Gerencia Regional de Salud, Spain grant numbers GRS 2132/A/20 and GRS 2315/A/21 and Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain grant number CIBERONC CB16/12/00400.

BackgroundGliomas with IDH1/2 mutations without 1p19q codeletion have been identified as the distinct diagnostic entity of IDH mutant astrocytoma (IDHmut astrocytoma). Homozygous deletion of Cyclin-dependent kinase 4 inhibitor A/B (CDKN2A/B) has recently been incorporated in the grading of these tumors. The question of whether histologic parameters still contribute to prognostic information on top of the molecular classification, remains unanswered. Here we evaluated consensus histologic parameters for providing additional prognostic value in IDHmut astrocytomas.MethodsAn international panel of seven neuropathologists scored 13 well-defined histologic features in virtual microscopy images of 192 IDHmut astrocytomas from EORTC trial 22033-26033 (low-grade gliomas) and 263 from EORTC 26053 (CATNON) (1p19q non-codeleted anaplastic glioma). For 192 gliomas the CDKN2A/B status was known. Consensus (agreement ≥ 4/7 panelists) histologic features were tested together with homozygous deletion (HD) of CDKN2A/B for independent prognostic power.ResultsAmong consensus histologic parameters, the mitotic count (cut-off of 2 mitoses per 10 high power fields standardized to a field diameter of 0.55 mm and an area of 0.24 mm2) significantly influences PFS (P = .0098) and marginally the OS (P = .07). Mitotic count also significantly affects the PFS of tumors with HD CDKN2A/B, but not the OS, possibly due to limited follow-up data.ConclusionThe mitotic index (cut-off 2 per 10 40× HPF) is of prognostic significance in IDHmut astrocytomas without HD CDKN2A/B. Therefore, the mitotic index may direct the therapeutic approach for patients with IDHmut astrocytomas with native CDKN2A/B status.

Localized or isolated neurofibromas are peripheral nerve sheath tumors. They are rare in the orbit and occur without a systemic neurofibromatosis. There are few cases of bilateral tumors reported but none affecting both supraorbital and infraorbital nerves. We report a 45-year-old female who presented an extraconal mass in the right orbit as an incidental finding in a head computer tomography, without ocular symptoms. Magnetic resonance image showed a well-defined oval mass in the right supraorbital and infraorbital nerves, of similar characteristics, as well as smaller masses in the left supraorbital and infraorbital nerves. A progressive increase in size of the left supraorbital and infraorbital tumor motivated their surgical excision. The histological result was compatible with a neurofibroma. These uncommon orbital tumors are slow growing and affect the sensory nerves of the trigeminal nerve. Neurofibromas usually present progressive symptoms due to the orbital mass, proptosis, or visual changes although not in this case. Surgical removal is the only definitive treatment.

Brain metastasis is an unmet clinical need, affecting between 10-30% of cancer patients with 200000 to 400000 newly diagnosis per annum in the US. Recently, several clinical trials have reported benefits using immunotherapy to treat brain metastasis. However, variability of the responses is broad and high benefit is found mainly in asymptomatic brain metastasis, while the benefit is dramatically reduced in the clinically relevant stage. Thus, it is currently unknown how to effectively target symptomatic brain metastases with immunotherapy. We previously reported a clinically relevant protumoral program driven by STAT3 activation in a subpopulation of reactive astrocytes in these advanced stages of the disease. Our current study further exploited the heterogeneity within the metastasis-associated microenvironment as a resource to identify novel therapeutic vulnerabilities to improve the benefits of immunotherapies based on immune checkpoint blocking antibodies (ICB) in symptomatic brain metastasis. Our results demonstrate that reactive astrocytes are strong immunomodulatory cells in brain tumors. We have identified the molecular profile of disease-associated glial cells and defined its connection to modulatory activities on specific lymphocyte populations in experimental brain metastasis as well as human-derived samples. scRNASeq and high content multiplex immunofluorescence allowed us to report a novel local immunomodulatory axis dependent on TIMP1 (astrocytes)/CD63 (CD8 T cells), which is present in brain metastasis patients with high immunoscore and would imply an additional immunosuppressive signal for potential ICB responders in brain metastasis. Genetic and pharmacologic approaches targeting this STAT3-dependent local immunomodulatory axis have allowed us to define the rationale to combine immune checkpoint blockade with a STAT3 inhibitor, which we previously used in patients. We proved that such combined immunotherapy boost the systemic activation of T cells while also preventing the local blockade. Additionally, our comprehensive strategy includes the possibility to stratify patients that are best qualified to benefit from this therapy by measuring TIMP1 in liquid biopsies from CSF. Even more, our data using Patient Derived Organotypic Cultures (PDOC) from fresh brain metastasis neurosurgeries confirms that our therapeutic strategy might benefit brain metastases generated from any primary source. In conclusion, we describe an immunosuppressive mechanism in the brain microenvironment that could explain the lack of response to ICB in patients with advanced brain metastasis. Our finding provides the rationale to implement complementary approaches targeting local immunosuppression to increase the benefit of immunotherapy in symptomatic brain metastasis. Citation Format: Neibla Priego, Pedro García-Gómez, Ana de Pablos-Aragoneses, María Perea-García, Laura Álvaro-Espinosa, Carolina Hernández-Oliver, Elena Martínez-Saez, Ángel Pérez-Núñez, Aurelio Hernández-Laín, Rebeca Sanz-Pamplona, Marc Schmitz, Stephen J. Crocker, Diego Serrano, Asís Palazón, RENACER Red Nacional de Metástasis Cerebral, Manuel Valiente. TIMP1 mediates astrocyte-dependent local immunosuppression in brain metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5105.

The brain is endowed with a unique cellular composition and organization, embedded within a vascular network and isolated from the circulating blood by a specialized frontier, the so-called blood-brain barrier (BBB), which is necessary for its proper function. Recent reports have shown that increments in the permeability of the blood vessels facilitates the entry of toxic components and immune cells to the brain parenchyma and alters the phenotype of the supporting astrocytes. All of these might contribute to the progression of different pathologies such as brain cancers or neurodegenerative diseases. Although it is well known that BBB breakdown occurs due to pericyte malfunctioning or to the lack of stability of the blood vessels, its participation in the diverse neural diseases needs further elucidation. This review summarizes what it is known about BBB structure and function and how its instability might trigger or promote neuronal degeneration and glioma progression, with a special focus on the role of pericytes as key modulators of the vasculature. Moreover, we will discuss some recent reports that highlights the participation of the BBB alterations in glioma growth. This pan-disease analysis might shed some light into these otherwise untreatable diseases and help to design better therapeutic approaches. Work was supported by Ministerio de Economía y Competitividad and FEDER funds: PI13/01258 to AHL, by “Asociación Española contra el Cancer (AECC) grant: INVES192GARG to RG and by Ministerio de Ciencia, Innovación y Universidades and FEDER funds: RTI2018-093596 to PSG. Sí

Cocaine consumption is associated with a variety of clinical manifestations. Though cocaine intranasal inhalation always determines nasal mucosal damages, extensive septum perforations, and midline destructions-known as cocaine-induced midline destructive lesions (CIMDL)-affect only a limited fraction of patients. CIMDL is viewed as a cocaine-associated autoimmune phenomenon in which the presence of atypical anti-neutrophil cytoplasmic antibody (ANCA) promotes and/or defines the disease phenotype. A 51-year-old man presented with an intracranial tumor-like lesion by its space-occupying effect. CT also revealed the destruction of the nasal septum and skull base. A diagnosis of CIMDL was made in light of the patient's history as well as findings of the physical and endoscopic examinations, imaging studies, and laboratory testing. There was no evidence of other pathologies. Histopathological results from cerebral biopsy led us to consider the intracranial pathology as an extension of the CIMDL. CIMDL is the result of a necrotizing inflammatory tissue response triggered by cocaine abuse in a subset of predisposed patients. The reported case is the first CIMDL consistent with brain extension mimicking a tumor-like lesion. While the presence of atypical ANCA seems to promote and/or define the disease phenotype, the specific role of these and other circulating autoantibodies needs further investigation.

BackgroundMitochondrial progressive external ophthalmoplegia (PEO) encompasses a broad spectrum of clinical and genetic disorders. We describe the phenotypic subtypes of PEO and its correlation with molecular defects and propose a diagnostic algorithm.MethodsRetrospective analysis of the clinical, pathological and genetic features of 89 cases.ResultsThree main phenotypes were found: ‘pure PEO’ (42%), consisting of isolated palpebral ptosis with ophthalmoparesis; Kearns-Sayre syndrome (10%); and ‘PEO plus’, which associates extraocular symptoms, distinguishing the following subtypes: : myopathic (33%), bulbar (12%) and others (3%). Muscle biopsy was the most accurate test, showing mitochondrial changes in 95%. Genetic diagnosis was achieved in 96% of the patients. Single large-scale mitochondrial DNA (mtDNA) deletion was the most frequent finding (63%), followed by multiple mtDNA deletions (26%) due to mutations in TWNK (n=8), POLG (n=7), TK2 (n=6) or RRM2B (n=2) genes, and point mtDNA mutations (7%). Three new likely pathogenic mutations were identified in the TWNK and MT-TN genes.ConclusionsPhenotype–genotype correlations cannot be brought in mitochondrial PEO. Muscle biopsy should be the first step in the diagnostic flow of PEO when mitochondrial aetiology is suspected since it also enables the study of mtDNA rearrangements. If no mtDNA deletions are identified, whole mtDNA sequencing should be performed.

DNAJ/HSP40 co-chaperones are integral to the chaperone network, bind client proteins and recruit them to HSP70 for folding. We performed exome sequencing on patients with a presumed hereditary muscle disease and no genetic diagnosis. This identified four individuals from three unrelated families carrying an unreported homozygous stop gain (c.856A>T; p.Lys286Ter), or homozygous missense variants (c.74G>A; p.Arg25Gln and c.785T>C; p.Leu262Ser) in DNAJB4. Affected patients presented with axial rigidity and early respiratory failure requiring ventilator support between the 1st and 4th decade of life. Selective involvement of the semitendinosus and biceps femoris muscles was seen on MRI scans of the thigh. On biopsy, muscle was myopathic with angular fibers, protein inclusions and occasional rimmed vacuoles. DNAJB4 normally localizes to the Z-disc and was absent from muscle and fibroblasts of affected patients supporting a loss of function. Functional studies confirmed that the p.Lys286Ter and p.Leu262Ser mutant proteins are rapidly degraded in cells. In contrast, the p.Arg25Gln mutant protein is stable but failed to complement for DNAJB function in yeast, disaggregate client proteins or protect from heat shock induced cell death consistent with its loss of function. DNAJB4 knockout mice had muscle weakness and fiber atrophy with prominent diaphragm involvement and kyphosis. DNAJB4 knockout muscle and myotubes had myofibrillar disorganization and accumulated Z-disc proteins and protein chaperones. These data demonstrate a novel chaperonopathy associated with DNAJB4 causing a myopathy with early respiratory failure. DNAJB4 loss of function variants may lead to the accumulation of DNAJB4 client proteins resulting in muscle dysfunction and degeneration.

Exercise intolerance is the most common phenotype in McArdle disease. Fixed weakness can be present in advanced ages, mainly affecting the shoulder girdle.

Striated muscle needs to maintain cellular homeostasis in adaptation to increases in physiological and metabolic demands. Failure to do so can result in rhabdomyolysis. The identification of novel genetic conditions associated with rhabdomyolysis helps to shed light on hitherto unrecognized homeostatic mechanisms. Here we report seven individuals in six families from different ethnic backgrounds with biallelic variants in MLIP, which encodes the muscular lamin A/C-interacting protein, MLIP. Patients presented with a consistent phenotype characterized by mild muscle weakness, exercise-induced muscle pain, variable susceptibility to episodes of rhabdomyolysis, and persistent basal elevated serum creatine kinase levels. The biallelic truncating variants were predicted to result in disruption of the nuclear localizing signal of MLIP. Additionally, reduced overall RNA expression levels of the predominant MLIP isoform were observed in patients’ skeletal muscle. Collectively, our data increase the understanding of the genetic landscape of rhabdomyolysis to now include MLIP as a novel disease gene in humans and solidifies MLIP’s role in normal and diseased skeletal muscle homeostasis.

Our goal was to analyze postmortem tissues of an adult patient with late-onset thymidine kinase 2 (TK2) deficiency who died of respiratory failure. Compared with control tissues, we found a low mtDNA content in the patient’s skeletal muscle, liver, kidney, small intestine, and particularly in the diaphragm, whereas heart and brain tissue showed normal mtDNA levels. mtDNA deletions were present in skeletal muscle and diaphragm. All tissues showed a low content of OXPHOS subunits, and this was especially evident in diaphragm, which also exhibited an abnormal protein profile, expression of non-muscular β-actin and loss of GAPDH and α-actin. MALDI-TOF/TOF mass spectrometry analysis demonstrated the loss of the enzyme fructose-bisphosphate aldolase, and enrichment for serum albumin in the patient’s diaphragm tissue. The TK2-deficient patient’s diaphragm showed a more profound loss of OXPHOS proteins, with lower levels of catalase, peroxiredoxin 6, cytosolic superoxide dismutase, p62 and the catalytic subunits of proteasome than diaphragms of ventilated controls. Strong overexpression of TK1 was observed in all tissues of the patient with diaphragm showing the highest levels. TK2 deficiency induces a more profound dysfunction of the diaphragm than of other tissues, which manifests as loss of OXPHOS and glycolytic proteins, sarcomeric components, antioxidants and overactivation of the TK1 salvage pathway that is not attributed to mechanical ventilation.

Some phase 2 trials had reported encouraging progression-free survival with Bevacizumab in monotherapy or combined with chemotherapy in glioblastoma. However, phase 3 trials showed a significant improvement in progression free survival without a benefit in overall survival. To date, there are no predictive biomarker of response for Bevacizumab in glioblastoma. We used Immunochemical analysis on tumor samples and pretreatment and post-treatment perfusion-MRI to try to identify possible predictive angiogenesis-related biomarkers of response and survival in patients with glioblastoma treated with bevacizumab in the first recurrence. We analyzed histological parameters: vascular proliferation, mitotic number and Ki-67 index; molecular factors: MGMT promoter methylation, EGFR amplification and EGFR variant III; immunohistochemical: MET, Midkine, HIF1, VEGFA, VEGF-R2, CD44, Olig2, microvascular area and microvascular density; and radiological: rCBV. In the statistical analysis, no significant correlation of any histological, molecular, microvascular or radiological parameters could be demonstrated with the response rate, PFS or OS with bevacizumab treatment. Unfortunately, in this histopathological, molecular, immunohistochemical and neuroradiological study we did not find any predictive biomarker of response or survival benefit for Bevacizumab in glioblastoma.

In a previous report of a new phenotype with predominant scapulo-humeral-peroneal-distal myopathy associated with the Glu197Asp mutation in ACTA1, muscle biopsies did not show nemaline rods, nor could nemaline rods formation be demonstrated in an exhaustive functional in vivo or in vitro study. However, muscle biopsy in members of our family, carrying a similar clinical phenotype of some members of the original family and the same ACTA1 mutation, revealed the presence of numerous nemaline rods, suggesting that there must be other factors that explain the absence of nemaline rods.

Background and purpose Our objectives were: (1) compare dynamic susceptibility-weighted (DSC) and dynamic contrast-enhanced (DCE) permeability parameters, (2) evaluate diagnostic accuracy of DSC and DCE discriminating high- and low-grade tumors, (3) analyze relationship of permeability parameters with overall (OS) and progression-free survival (PFS) and (4) assess differences in high-grade tumors classified according to molecular biomarkers. Materials and methods 49 patients with histologically proved diffuse gliomas underwent DSC and DCE imaging. Parametric maps of cerebral blood volume (CBV), CBV-leakage corrected, volume transfer coefficient (Ktrans), fractional volume of the extravascular extracellular space (EES) (Ve), fractional blood plasma volume (Vp) and rate constant between EES and blood plasma (Kep) were calculated. High-grade gliomas were also classified according to isocitrate dehydrogenase (IDH), alpha-thalassemia/mental retardation syndrome X-linked (ATRX) and O6-methylguanine-dna-methyltransferase promoter methylation (MGMT) status. Results There is correlation between parameters leakage, Ktrans and Vp. ROC curve analysis showed significance in both Ktrans and Ve for glioma grading. Threshold value of 0.075 for Ve generated the best combination of sensitivity (80%) and specificity (75%) in tumor gradation. Leakage was the only permeability parameter related to OS (P = 0.006) and PFS (0.012); with prolonged survival for leakage values lower than 1.2. IDH-mutated high-grade tumors showed lower leakage and Ktrans values. High-grade tumors with loss of ATRX presented lower leakage and Vp values. Conclusions Both DSC and DCE permeability parameters serve as non-invasive method for glioma grading. Leakage was the unique permeability parameter related to survival and the best discriminating high-grade gliomas classified according to IDH and ATRX status.

A 29-year-old man developed, since the age of 18, exercise intolerance and exercise-induced rhabdomyolysis, with myoglobinuria. Muscle biopsy showed ragged-red fibers. Multiple mitochondrial DNA deletions were detected. The previously reported pathogenic homozygous mutation c.323C>T (p.Thr108Met) in TK2 was identified. This case expands the phenotypic spectrum of TK2 deficiency and indicates that it should be considered in the differential diagnosis of episodic rhabdomyolysis and exercise intolerance, along with other metabolic and mitochondrial myopathies. Since a new treatment is under development, it is essential improving knowledge of the natural history of TK2 deficiency.

The extraordinary plasticity of glioma cells allows them to contribute to different cellular compartments in tumor vessels, reinforcing the vascular architecture. It was recently revealed that targeting glioma-derived pericytes, which represent a big percentage of the mural cell population in aggressive tumors, increases the permeability of the vessels and improves the efficiency of chemotherapy. However, the molecular determinants of this transdifferentiation process have not been elucidated. Here we show that mutations in EGFR stimulate the capacity of glioma cells to function as pericytes in a BMX- (bone marrow and X-linked) and SOX9-dependent manner. Subsequent activation of platelet-derived growth factor receptor beta in the vessel walls of EGFR-mutant gliomas stabilized the vasculature and facilitated the recruitment of immune cells. These changes in the tumor microenvironment conferred a growth advantage to the tumors but also rendered them sensitive to pericyte-targeting molecules such as ibrutinib or sunitinib. In the absence of EGFR mutations, high-grade gliomas were enriched in blood vessels, but showed a highly disrupted blood–brain barrier due to the decreased BMX/SOX9 activation and pericyte coverage, which led to poor oxygenation, necrosis, and hypoxia. Overall, these findings identify EGFR mutations as key regulators of the glioma-to-pericyte transdifferentiation, highlighting the intricate relationship between the tumor cells and their vascular and immune milieu. Our results lay the foundations for a vascular-dependent stratification of gliomas and suggest different therapeutic vulnerabilities determined by the genetic status of EGFR., This work was supported by FONDECYT grant (1140697 to V. Palma), CONICYT Fellowship (to B.S. Casas), by Ministerio de Economía y Competitividad and FEDER funds (PI13/01258 to A. Hernandez-Laín; PI17/01621 to J.M. Sepulveda-S anchez; and PI16/01580 and DTS18/00181 to A. Matheu), by Young Employment Initiative (Comunidad de Madrid) to M. Garranzo-Asensio, by “Asociacion Espanola contra ~ el Cancer” (AECC) grants (INVES192GARG to R. Gargini; GCTRA16015SEDA to J.M. Sepulveda-S anchez); and by Ministerio de Ciencia, Innovacion y Universidades and FEDER funds (RTI2018-093596 to P. Sanchez-Gomez).

Craniopharyngiomas (CPs) are histologically benign tumors that are associated with high levels of morbidity. Two clinicopathological variants - adamantinomatous (ACP) and papillary (PCP) - have been described. They differ in their molecular features, whereby activating mutations in BRAF (V600E) and CTNNB1 genes characterize PCP and ACP, respectively. Recently, both variants have been shown to express elevated PD-L1 protein expression, but ACP also exhibited tumor cell-intrinsic PD-1 expression. In this study we analyze these molecular alterations in 52 cases with a long follow-up and examine their associations with immunohistochemical and clinical characteristics. ACPs comprise 73.1% of cases, while 21.2% are PCPs. Aberrant nuclear immunoreactivity for β-catenin was observed in all ACPs. BRAF p.V600E mutations were observed in 90.9% of PCPs. Only one ACP case featured both alterations. Both types of CP exhibited strong nuclear staining for p63 with diffuse and basal distribution. ACP and PCP consistently expressed PD-L1, most in a substantial percentage of tumor cells, with a distinctive spatial distribution of expression in each subtype; only ACP demonstrated PD-1 expression. There was no evidence of differences in clinical prognosis between ACPs and PCPs. The identification of hallmark molecular signatures in the two CP variants is useful for sub-categorization in routine histopathology reporting. It is also pertinent to personalized therapy and for the development of improved non-invasive therapeutic strategies in this disease.

Exclusion of brain metastases from clinical trials is a major cause of the limited therapeutic options for this growing population of cancer patients. Here, we report a medium-throughput drug-screening platform (METPlatform) based on organotypic cultures that allows to evaluate inhibitors against metastases growing in situ. By applying this approach to brain metastasis, we identified several hits from a library of FDA approved inhibitors and others being tested in clinical trials. A blood-brain barrier permeable HSP90 inhibitor showed high potency against mouse and human brain metastases at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis applied to organotypic cultures with metastases treated with the chaperone inhibitor revealed novel biomarkers in human brain metastasis and actionable mechanisms of resistance. Our work validates METPlatform as a potent resource for metastasis research integrating drug-screening and unbiased omic approaches that is fully compatible with human samples. We envision that METPlatform could be established as a clinically relevant strategy to personalize the management of metastatic disease in the brain and elsewhere.SummarySystemic spread of cancer continues to be the key aspect associated with lethality. In this publication, Zhu et al. describes a drug-screening platform specifically designed to study vulnerabilities of metastasis when colonizing secondary organs and demonstrates its value in difficult-to-treat brain metastasis using new models and patient-derived samples.

RationaleGlioma progression is driven by the induction of vascular alterations but how the tumor genotype influence these changes is still a pending issue. We propose to study the underlying mechanisms by which the genetic changes in isocitrate dehydrogenase 1/2 (IDH1/2) and epidermal growth factor receptor (EGFR) genes establish the different vascular profiles of gliomas.MethodsWe stratified gliomas based on the genetic status of IDH1/2 and EGFR genes. For that we used in silico data and a cohort of 93 glioma patients, where we analyzed the expression of several transcripts and proteins. For the in vitro and in vivo studies, we used a battery of primary glioblastoma cells derived from patients, as well as novel murine glioma cell lines expressing wild-type or mutant EGFR. In these models, the effect of the small molecule ibrutinib or the downregulation of CD248 and SOX9 was evaluated to establish a molecular mechanism.ResultsWe show that IDH1/2 mutations associate with a normalized vasculature. By contrast, EGFR mutations stimulate the plasticity of glioma cells and their capacity to function as pericytes in a bone-marrow and X-linked (BMX)/SOX9 dependent manner. The presence of tumor-derived pericytes stabilize the profuse vasculature and confers a growth advantage to these tumors, although they render them sensitive to pericyte-targeted molecules. Wild-type/amplified EGFR gliomas are enriched in blood vessels too, but they show a highly disrupted blood-brain-barrier due to a decreased BMX/SOX9 activation and pericyte coverage. This leads to poor nutrient supply, necrosis and hypoxia.ConclusionsThe function of tumor-derived pericytes delimitates two distinct and aggressive vascular phenotypes in IDH1/2 wild-type gliomas. Our results lay the foundations for a vascular dependent stratification of gliomas and suggest different therapeutic vulnerabilities depending on the genetic status of EGFR.Graphical Abstract. Schematic view of IDH and EGFR function in the regulation of glioma microenvironment.Mutant IDH gliomas express low levels of angiogenic molecules and have a vasculature reminiscent of normal tissue. EGFR mutations drive glioma growth by promoting tumor-to-pericyte transdifferentiation and vascular stabilization in a BMX-SOX9 dependent way. Leaky vessels with hypoxia and necrosis characterize tumors overexpressing the wild-type isoform of the receptor. These phenotypes determine the response to therapy of the different IDH wild-type gliomas.