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PURPOSE: Adamantinomatous craniopharyngiomas (ACP) as benign sellar brain tumors are challenging to treat. In order to develop robust in vivo drug testing methodology, the murine orthotopic craniopharyngioma model (PDX) was characterized by magnetic resonance imaging (MRI) and histology in xenografts from three patients (ACP1-3). METHODS: In ACP PDX, multiparametric MRI was conducted to assess morphologic characteristics such as contrast-enhancing tumor volume (CETV) as well as functional parameters from dynamic contrast-enhanced MRI (DCE-MRI) and diffusion-weighted imaging (DWI) including area-under-the-curve (AUC), peak enhancement (PE), time-to-peak (TTP) and apparent diffusion coefficient (ADC). These MRI parameters evaluated in 27 ACP PDX were correlated to histological features and percentage of vital tumor cell content. RESULTS: Qualitative analysis of MRI and histology from PDX revealed a similar phenotype as seen in patients, although the MRI appearance in mice resulted in a more solid tumor growth than in humans. CETV were significantly higher in ACP2 xenografts relative to ACP1 and ACP3 which correspond to respective average vitality of 41%, <10% and 26% determined histologically. Importantly, CETV prove tumor growth of ACP2 PDX as it significantly increases in longitudinal follow-up of 110 days. Furthermore, xenografts from ACP2 revealed a significantly higher AUC, PE and TTP in comparison to ACP3, and significantly increased ADC relative to ACP1 and ACP3 respectively. Overall, DCE-MRI and DWI can be used to distinguish vital from non-vital grafts, when using a cut off value of 15% for vital tumor cell content. CONCLUSIONS: MRI enables the assessment of craniopharyngioma PDX vitality in vivo as validated histologically.
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Medios de Contraste/metabolismo , Craneofaringioma/patología , Imagen por Resonancia Magnética/métodos , Adulto , Animales , Niño , Preescolar , Craneofaringioma/metabolismo , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Trasplante HeterólogoRESUMEN
Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. ß-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP.
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Craneofaringioma/metabolismo , Sistema de Señalización de MAP Quinasas , Neoplasias Hipofisarias/metabolismo , Transcriptoma , Microambiente Tumoral/fisiología , Animales , Biología Computacional , Craneofaringioma/patología , Craneofaringioma/terapia , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Inflamación/metabolismo , Inflamación/terapia , Captura por Microdisección con Láser , Ratones , Neuroglía/metabolismo , Odontogénesis/fisiología , Hipófisis/embriología , Hipófisis/patología , Neoplasias Hipofisarias/patología , Neoplasias Hipofisarias/terapia , Análisis de Secuencia de ARN , Técnicas de Cultivo de TejidosRESUMEN
Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.
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Neoplasias del Sistema Nervioso Central/diagnóstico , Neoplasias del Sistema Nervioso Central/genética , Metilación de ADN , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias del Sistema Nervioso Central/clasificación , Neoplasias del Sistema Nervioso Central/patología , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Aprendizaje Automático no Supervisado , Adulto JovenRESUMEN
A 6-year old patient presented with an intra and suprasellar cystic lesion accompanied with impairment of the hypothalamic-pituitary axis and partial hypopituitarism. The most likely cause of sellar lesions in this age group are adamantinomatous craniopharyngioma (adaCP) or Rathke´s cleft cysts (RCCs). AdaCP are characterized by CTNNB1 mutations accompanied with aberrant nuclear beta-catenin expression. RCC show neither nuclear beta-catenin expression nor BRAF mutation. The latter is a hallmark of papillary craniopharyngiomas (papCP) that exhibit remarkable histological similarity with metaplasia of RCC. Diagnosis of the patient was elucidated by CTNNB1 and BRAF mutation screening, utilizing different approaches, as well as histological examination of markers, e.g., beta-catenin, claudin-1, EpCAM and the mutated BRAFV600E protein, which are known to be differentially expressed in sellar lesions. The case presented reveals extraordinary aspects for two reasons. Firstly, the lesion appeared clinically, on MRI, intraoperatively and histologically as RCC with prominent squamous metaplasia, but showing an expression pattern of markers also found in papCP, whilst exhibiting a hitherto undescribed BRAFV600E mutation. This important result documents a supposable transition of RCC metaplasia into a papillary craniopharyngioma (papCP). Secondly, this intriguing case shows unexpectedly that although papCP usually occurs almost exclusively in adults, it can also arise in childhood.
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To assess the clinical relevance of transgenic and patient-derived xenograft models of adamantinomatous craniopharyngioma (ACP) using serial magnetic resonance imaging (MRI) and high resolution post-mortem microcomputed tomography (µ-CT), with correlation with histology and human ACP imaging. The growth patterns and radiological features of tumors arising in Hesx1Cre/+ ;Ctnnb1lox(ex3)/+ transgenic mice, and of patient-derived ACP xenografts implanted in the cerebral cortex, were monitored longitudinally in vivo with anatomical and functional MRI, and by ex vivo µ-CT at study end. Pathological correlates with hematoxylin and eosin stained sections were investigated. Early enlargement and heterogeneity of Hesx1Cre/+ ;Ctnnb1lox(ex3)/+ mouse pituitaries was evident at initial imaging at 8 weeks, which was followed by enlargement of a solid tumor, and development of cysts and hemorrhage. Tumors demonstrated MRI features that recapitulated those of human ACP, specifically, T1 -weighted signal enhancement in the solid tumor component following Gd-DTPA administration, and in some animals, hyperintense cysts on FLAIR and T1 -weighted images. Ex vivo µ-CT correlated with MRI findings and identified smaller cysts, which were confirmed by histology. Characteristic histological features, including wet keratin and calcification, were visible on µ-CT and verified by histological sections of patient-derived ACP xenografts. The Hesx1Cre/+ ;Ctnnb1lox(ex3)/+ transgenic mouse model and cerebral patient-derived ACP xenografts recapitulate a number of the key radiological features of the human disease and provide promising foundations for in vivo trials of novel therapeutics for the treatment of these tumors.
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Craneofaringioma/diagnóstico por imagen , Craneofaringioma/patología , Modelos Animales de Enfermedad , Animales , Craneofaringioma/genética , Xenoinjertos/diagnóstico por imagen , Xenoinjertos/patología , Proteínas de Homeodominio/genética , Humanos , Imagen por Resonancia Magnética , Masculino , Ratones Transgénicos , Persona de Mediana Edad , Proteínas Represoras/genética , Microtomografía por Rayos X , beta Catenina/genéticaRESUMEN
Stem cells have been discovered as key players in the genesis of different neoplasms including craniopharyngioma (CP), a rare tumour entity in the sellar region. Sox2 and Sox9 are well-known stem cell markers involved in pituitary development. In this study we analysed the expression of both transcription factors using immunohistochemistry in a large cohort of 64 adamantinomatous (aCP) and 9 papillary CP (pCP) and quantitative PCR in 26 aCP and 7 pCP. Whereas immunohistochemically Sox2+ cells were verifiable in only five aCP (7.8%) and in 39.1% of the respective surrounding cerebral tissue, pCP specimens appeared always negative. In contrast, Sox9 was detectable in all tumours with a significantly higher expression in aCP compared to pCP (protein, p < 0.0001; mRNA p = 0.0484) This was also true for the respective tumour adjacent CNS where 63 aCP (98.4%) and six pCP (66.7%) showed Sox9+ cells. We further confirmed absence of Sox9 expression in nuclear ß-catenin accumulating cells of aCP. Our results point to the conclusion that Sox2 and Sox9, seem to play essential roles not only in the specific formation of aCP, but also in processes involving the cerebral tumour environment, which needs to be illuminated in the future.
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Craneofaringioma/genética , Factor de Transcripción SOX9/genética , Factores de Transcripción SOXB1/genética , beta Catenina/genética , Adolescente , Adulto , Anciano , Biomarcadores de Tumor/genética , Encéfalo/metabolismo , Encéfalo/patología , Niño , Preescolar , Craneofaringioma/patología , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Lactante , Masculino , Persona de Mediana Edad , Células Madre Neoplásicas/metabolismo , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/patología , ARN Mensajero/genética , Microambiente Tumoral/genética , Adulto JovenRESUMEN
Even though ACP is a benign tumor, treatment is challenging because of the tumor's eloquent location. Today, with the exception of surgical intervention and irradiation, further treatment options are limited. However, ongoing molecular research in this field provides insights into the pathways involved in ACP pathogenesis and reveal a plethora of druggable targets. In the next step, appropriate models are essential to identify the most suitable and effective substances for clinical practice. Primary cell cultures in low passages provide a proper and rapid tool for initial drug potency testing. The patient-derived xenograft (PDX) model accommodates ACP complexity in that it shows respect to the preserved architecture and similar histological appearance to human tumors and therefore provides the most appropriate means for analyzing pharmacological efficacy. Nevertheless, further research is needed to understand in more detail the biological background of ACP pathogenesis, which provides the identification of the best targets in the hierarchy of signaling cascades. ACP models are also important for the continuous testing of new targeting drugs, to establish precision medicine.
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Craneofaringioma/patología , Neoplasias Hipofisarias/patología , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Quimioterapia Adyuvante , Craneofaringioma/diagnóstico por imagen , Craneofaringioma/tratamiento farmacológico , Descubrimiento de Drogas/métodos , Humanos , Ratones , Trasplante de Neoplasias , Neoplasias Hipofisarias/diagnóstico por imagen , Neoplasias Hipofisarias/tratamiento farmacológico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE In this study, the authors investigated the underlying mechanisms responsible for high tumor recurrence rates of adamantinomatous craniopharyngioma (ACP) after radiotherapy and developed new targeted treatment protocols to minimize recurrence. ACPs are characterized by the activation of the receptor tyrosine kinase epidermal growth factor receptor (EGFR), known to mediate radioresistance in various tumor entities. The impact of tyrosine kinase inhibitors (TKIs) gefitinib or CUDC-101 on radiation-induced cell death and associated regulation of survivin gene expression was evaluated. METHODS The hypothesis that activated EGFR promotes radioresistance in ACP was investigated in vitro using human primary cell cultures of ACP (n = 10). The effects of radiation (12 Gy) and combined radiochemotherapy on radiosensitivity were assessed via cell death analysis using flow cytometry. Changes in target gene expression were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Survivin, identified in qRT-PCR to be involved in radioresistance of ACP, was manipulated by small interfering RNA (siRNA), followed by proliferation and vitality assays to further clarify its role in ACP biology. Immunohistochemically, survivin expression was assessed in patient tumors used for primary cell cultures. RESULTS In primary human ACP cultures, activation of EGFR resulted in significantly reduced cell death levels after radiotherapy. Treatment with TKIs alone and in combination with radiotherapy increased cell death response remarkably, assessed by flow cytometry. CUDC-101 was significantly more effective than gefitinib. The authors identified regulation of survivin expression after therapeutic intervention as the underlying molecular mechanism of radioresistance in ACP. EGFR activation promoting ACP cell survival and proliferation in vitro is consistent with enhanced survivin gene expression shown by qRT-PCR. TKI treatment, as well as the combination with radiotherapy, reduced survivin levels in vitro. Accordingly, ACP showed reduced cell viability and proliferation after survivin downregulation by siRNA. CONCLUSIONS These results indicate an impact of EGFR signaling on radioresistance in ACP. Inhibition of EGFR activity by means of TKI treatment acts as a radiosensitizer on ACP tumor cells, leading to increased cell death. Additionally, the results emphasize the antiapoptotic and pro-proliferative role of survivin in ACP biology and its regulation by EGFR signaling. The suppression of survivin by treatment with TKI and combined radiotherapy represents a new promising treatment strategy that will be further assessed in in vivo models of ACP.
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Craneofaringioma/metabolismo , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/metabolismo , Neoplasias Hipofisarias/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Tolerancia a Radiación/efectos de los fármacos , Adolescente , Adulto , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Supervivencia Celular/efectos de la radiación , Niño , Craneofaringioma/patología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Regulación hacia Abajo/efectos de la radiación , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis/efectos de la radiación , Masculino , Persona de Mediana Edad , Neoplasias Hipofisarias/patología , Tolerancia a Radiación/fisiología , Survivin , Células Tumorales CultivadasRESUMEN
The epithelial cell adhesion molecule (EpCAM) is a type I glycoprotein located on the surface of epithelial cells. It is strongly expressed in many neoplasms and already used in the diagnosis and distinction of various tumour subtypes. Comparative studies about EpCAM expression in cystic sellar lesions are lacking. Therefore, we analysed its distribution pattern in adamantinomatous (aCP) and papillary (pCP) craniopharyngiomas (CP) and Rathke's Cleft Cysts (RCC) using immunohistochemistry and gene expression profiling. Whereas the protein was not detectable in pCP (n = 10), all aCP (n = 64) showed distinct staining patterns. The vast majority of RCC (n = 10) also appeared positive, but these displayed notably lower labeling scores. Additionally, significantly higher mRNA levels were detectable in aCP (n = 19) when compared to pCP (n = 10) (p = 9.985(-8)). Furthermore, pediatric aCP cases, in general, exhibited stronger EpCAM staining levels compared to adult ones (p = 0.015). However, we were not able to verify this result on mRNA level. In summary, our findings demonstrate that EpCAM can be used as an additional distinction-marker for cystic lesions of the sellar region. Its unknown function in aCP and the presence of an approved monoclonal bispecific trifunctional antibody for cancer therapy are interesting starting points for further studies.
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Quistes del Sistema Nervioso Central/genética , Craneofaringioma/genética , Molécula de Adhesión Celular Epitelial/genética , Perfilación de la Expresión Génica , Neoplasias Hipofisarias/genética , Adolescente , Adulto , Anciano , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Quistes del Sistema Nervioso Central/metabolismo , Niño , Preescolar , Craneofaringioma/metabolismo , Molécula de Adhesión Celular Epitelial/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Hipofisarias/metabolismo , Adulto JovenRESUMEN
Hedgehog (HH) signaling is known to be essential during the embryonal development of the pituitary gland but the knowledge about its role in the adult pituitary and in associated tumors is sparse. In this report we investigated the effect of excess Hh signaling activation in murine pituitary explants and analyzed the HH signaling status of human adenopituitary lobes and a large cohort of pituitary adenomas. Our data show that excess Hh signaling led to increased proliferation of Sox2(+) and Sox9(+) adult pituitary stem cells and to elevated expression levels of adrenocorticotropic hormone (Acth), growth hormone (Gh) and prolactin (Prl) in the adult gland. Inhibition of the pathway by cyclopamine reversed these effects indicating that active Hh signaling positively regulates proliferative processes of adult pituitary stem cells and hormone production in the anterior pituitary. Since hormone producing cells of the adenohypophysis as well as ACTH-, GH- and PRL-immunopositive adenomas express SHH and its target GLI1, we furthermore propose that excess HH signaling is involved in the development/maintenance of hormone-producing pituitary adenomas. These findings advance the understanding of physiological hormone regulation and may open new treatment options for pituitary tumors.
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Hormona Adrenocorticotrópica/biosíntesis , Proliferación Celular , Hormona del Crecimiento/biosíntesis , Proteínas Hedgehog/metabolismo , Hipófisis/fisiología , Prolactina/biosíntesis , Transducción de Señal , Células Madre/fisiología , Adulto , Animales , Humanos , RatonesRESUMEN
The original version of this article unfortunately contained a mistake in the author list. The name of one co-author is written wrong in the final version of the article; Dr Hans Ulrich Knappe should be Ulrich Johannes Knappe. The updated author list is provided below: Christian P. Miermeister, Stephan Petersenn, Michael Buchfelder, Rudolf Fahlbusch, Dieter K.Lüdecke, Annett Hölsken, Markus Bergmann, Ulrich Johannes Knappe, Volkmar H. Hans, Jörg Flitsch, Wolfgang Saeger and Rolf Buslei.
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INTRODUCTION: Craniopharyngiomas (CP) are rare epithelial tumors of the sellar region. Two subtypes, adamantinomatous (adaCP) and papillary CP (papCP), were previously identified based on histomorphological and epidemiological aspects. Recent data indicates that both variants are defined by specific genetic alterations, and influenced by distinct molecular pathways and particular origins. The fact that CP is an uncommon tumor entity renders studies on large cohorts difficult and exceptional. In order to achieve further insights distinguishing CP variants, we conducted whole genome methylation (450 k array) and microarray-based gene expression studies in addition to CTNNB1 and BRAF mutation analysis using a comprehensive cohort of 80 adaCP and 35 papCP. RESULTS: BRAF V600E mutations were solely found in the papCP subgroup and were not detectable in adaCP samples. In contrast, CTNNB1 mutations were exclusively detected in adaCP. The methylome fingerprints assigned DNA specimens to entity-specific groups (papCP (n = 18); adaCP (n = 25)) matching perfectly with histology-based diagnosis, suggesting that they represent truly distinct biological entities. However, we were not able to detect within the adaCP group (including 11 pediatric and 14 adult cases) a significant difference in methylation signature by age. Integrative comparison of the papCP with the adaCP group based on differential gene expression and methylation revealed a distinct upregulation of Wnt- and SHH signaling pathway genes in adaCP. CONCLUSIONS: AdaCP and papCP thus represent distinct tumor subtypes that harbor mutually exclusive gene mutations and methylation patterns, further reflected in differences in gene expression. This study demonstrates that DNA methylation analyses are an additional method to classify CP into subtypes, and implicates a role of epigenetic mechanisms in the genesis of the respective CP variants. Detection of tumor-specific signaling pathway activation enables the possibility of target-oriented intervention.
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Craneofaringioma , Mutación/genética , Neoplasias Hipofisarias/genética , Proteínas Proto-Oncogénicas B-raf/genética , beta Catenina/genética , Adolescente , Adulto , Anciano , Niño , Preescolar , Estudios de Cohortes , Craneofaringioma/clasificación , Craneofaringioma/genética , Craneofaringioma/metabolismo , Metilación de ADN , Análisis Mutacional de ADN , Epigenómica , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Hipofisarias/metabolismo , Neoplasias Hipofisarias/patología , Transcriptoma/genética , Adulto JovenRESUMEN
Enhancer of zeste homolog 2 (EZH2) is a core epigenetic regulator, playing a crucial role in cell cycle regulation. The protein is known to be associated with proliferation and worse outcome in several tumor entities. In this study, we immunohistochemically investigated the expression pattern of EZH2 in a large cohort of pituitary tumors. These results were correlated with clinical features and double immunofluorescence stainings (DIS) were conducted to evaluate co-expression of EZH2 and proliferation marker Ki-67. Furthermore, we analyzed the effect of EZH2 inhibition on cell proliferation in vitro using the pituitary cell line AtT-20. While in the normal anterior pituitary EZH2 was almost absent, the cohort of tumors showed enhanced expression levels (p ≤ 0.0005). This was positively associated with Ki-67 indices (r = 0.834, p ≤ 0.0005) and DIF confirmed a predominant co-expression of both markers. In vitro experiments revealed a significant (p ≤ 0.05) decrease of tumor cell proliferation using the EZH2 inhibitor GSK126. Our results further support that epigenetic events are involved in the pathogenesis and biology of pituitary adenomas (PA). Therefore, EZH2 may function as a new potential target for therapeutic interventions in PA.
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Adenoma/genética , Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica , Indoles/farmacología , Neoplasias Hipofisarias/genética , Complejo Represivo Polycomb 2/genética , Piridonas/farmacología , Adenoma/diagnóstico , Adenoma/metabolismo , Adenoma/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Estudios de Cohortes , Proteína Potenciadora del Homólogo Zeste 2 , Epigénesis Genética , Femenino , Histonas/genética , Histonas/metabolismo , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Masculino , Metilación/efectos de los fármacos , Persona de Mediana Edad , Hipófisis/efectos de los fármacos , Hipófisis/metabolismo , Hipófisis/patología , Neoplasias Hipofisarias/diagnóstico , Neoplasias Hipofisarias/metabolismo , Neoplasias Hipofisarias/patología , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Complejo Represivo Polycomb 2/metabolismo , Pronóstico , Transducción de SeñalRESUMEN
INTRODUCTION: The term atypical pituitary adenoma (APA) was revised in the 2004 World Health Organization (WHO) classification of pituitary tumors. However, two of the four parameters required for the diagnosis of APAs were formulated rather vaguely (i.e., "extensive" nuclear staining for p53; "elevated" mitotic index). Based on a case-control study using a representative cohort of typical pituitary adenomas and APAs selected from the German Pituitary Tumor Registry, we aimed to obtain reliable cut-off values for both p53 and the mitotic index. In addition, we analyzed the impact of all four individual parameters (invasiveness, Ki67-index, p53, mitotic index) on the selectivity for differentiating both adenoma subtypes. METHODS: Of the 308 patients included in the study, 98 were diagnosed as APAs (incidence 2.9 %) and 10 patients suffered from a pituitary carcinoma (incidence 0.2 %). As a control group, we selected 200 group matched patients with typical pituitary adenomas (TPAs). Cut-off values were attained using ROC analysis. RESULTS: We determined significant threshold values for p53 (≥2 %; AUC: 0.94) and the mitotic index (≥2 mitosis within 10 high power fields; AUC: 0.89). The most reliable individual marker for differentiating TPAs and APAs was a Ki-67-labeling index ≥ 4 % (AUC: 0.98). Using logistic regression analysis (LRA) we were able to show that all four criteria (Ki-67 (p < 0.001); OR 5.2// p53 (p < 0.001); OR 3.1// mitotic index (p < 0.001); OR 2.1// invasiveness (p < 0.001); OR 8.2)) were significant for the group of APAs. Furthermore, we describe the presence of nucleoli as a new favorable parameter for TPAs (p = 0.008; OR: 0.4; CI95 %: 0.18; 0.77). CONCLUSIONS: Here we present a proposed rectification of the current WHO classification of pituitary tumors describing an additional marker for TPA and specific threshold values for p53 and the mitotic index. This will greatly help in the reliable diagnosis of APAs and facilitate further studies to ascertain the prognostic relevance of this categorization.
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Adenoma/patología , Neoplasias Hipofisarias/patología , Sistema de Registros , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Alemania , Humanos , Antígeno Ki-67/metabolismo , Modelos Logísticos , Masculino , Persona de Mediana Edad , Curva ROC , Sistema de Registros/estadística & datos numéricos , Estudios Retrospectivos , Proteína p53 Supresora de Tumor/metabolismo , Adulto JovenRESUMEN
Adamantinomatous craniopharyngiomas (adaCP) cause hypothalamic pituitary dysfunction. Elucidation of pathomechanisms underlying tumor progression is essential for the development of targeted chemotherapeutic treatment options. In order to study the mechanisms of tumor outgrowth, we implanted human primary adaCP tissue from three different surgical specimens stereotactically into the brain of immunodeficient mice (n = 20). Three months after tumor inoculation, magnetic resonance imaging and histology confirmed tumor engraftment in all 20 mice (100%) that obtained tissue transplants. The lesions invaded adjoining brain tissue with micro finger-shaped protrusions. Immunohistochemical comparison of the primary tumor and xenotransplants revealed a similar amount of proliferation (Mib-1) and cytokeratin expression pattern (KL-1). Whole tumor reconstruction using serial sections confirmed whirl-like cell clusters with nuclear ß-catenin accumulations at the tumor brain border. These whirls were surrounded by a belt of Claudin-1 expressing cells, showed an activated epidermal growth factor receptor (EGFR) and distinct CD133 as well as p21(WAF1/Cip1) positivity, indicating a tumor stem cell phenotype. Consistent with our previous in vitro studies, intracranial xenotransplants of adaCP confirmed cells with nuclear ß-catenin and activated EGFR being the driving force of tumor outgrowth. This model provides the possibility to study in vivo tumor cell migration and to test novel treatment regimens targeting this tumor stem cell niche.
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Neoplasias Encefálicas/fisiopatología , Encéfalo/fisiopatología , Craneofaringioma/fisiopatología , Adolescente , Adulto , Animales , Encéfalo/patología , Neoplasias Encefálicas/patología , Claudina-1/metabolismo , Craneofaringioma/patología , Progresión de la Enfermedad , Receptores ErbB/metabolismo , Femenino , Humanos , Inmunohistoquímica , Queratina-1 , Queratinas Específicas del Pelo/metabolismo , Imagen por Resonancia Magnética , Masculino , Ratones Mutantes , Persona de Mediana Edad , Trasplante de Neoplasias , Ubiquitina-Proteína Ligasas/metabolismo , beta Catenina/metabolismoRESUMEN
AIMS: The differential diagnosis of cystic epithelial masses of the sellar region, especially the histopathological differentiation of craniopharyngiomas and Rathke's cleft cysts, poses a challenge even to experienced diagnosticians. Recently, BRAF V600E mutations have been described as a genetic hallmark of papillary craniopharyngiomas. We investigated a series of 33 Rathke's cleft cysts to determine the frequency of BRAF V600E mutations and its suitability as an additional diagnostic marker for the differentiation of cystic lesions of the sellar region. METHODS: Thirty-three Rathke's cleft cysts and 18 papillary craniopharyngiomas were analysed for BRAF mutational status by immunohistochemistry using a monoclonal antibody (VE1) that selectively recognizes the BRAF V600E mutant epitope and additional BRAF pyrosequencing in a subset of samples. RESULTS: Thirty of 33 specimens diagnosed as Rathke's cleft cysts were negative by VE1 immunohistochemistry and pyrosequencing, whereas in three cysts and in all the 18 papillary craniopharyngiomas, a BRAF V600E mutation was detected. Clinical and histological re-evaluation of the three BRAF V600E mutated cases formerly diagnosed as Rathke's cleft cysts revealed unusual presentations. Two of them were rediagnosed as papillary craniopharyngiomas. The patient of the third case had a history of craniopharyngioma operated 14 years before, and reoperation showed a cystic epithelial lesion with unclear histology. CONCLUSIONS: The determination of BRAF mutational status is recommended in any cystic sellar lesion and can in most cases be provided by VE1 immunohistochemistry even in specimens of low cellularity. Confirmation by (pyro-)sequencing should be attempted whenever sufficient epithelium is available due to variable staining results.
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Quistes del Sistema Nervioso Central/genética , Craneofaringioma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Adulto , Anciano , Anticuerpos Monoclonales , Quistes del Sistema Nervioso Central/patología , Craneofaringioma/patología , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Proteínas Proto-Oncogénicas B-raf/inmunología , Adulto JovenRESUMEN
INTRODUCTION: Early disease onset, clinical manifestation, histomorphology, and increased tendency to relapse distinguish the adamantinomatous craniopharyngioma (adaCP) from the more favorable papillary variant (papCP). A molecular hallmark of adaCP is the activated Wnt signaling pathway indicated by nuclear ß-catenin accumulation in a subset of tumor cells. A mouse model recently illustrated that these cells are the driving force in tumorigenesis of adaCP. This observation and the peculiar growth pattern points to the existence of a specific tumor stem cell (TSC) population in human CP. MATERIALS AND METHODS: To prove this hypothesis, the TSC markers CD133 (Prominin1) and CD44 were examined in papCP (n = 8) and adaCP (n = 25) on mRNA level using quantitative real time PCR of total tumor RNA. Furthermore, we investigated protein expression performing immunohistochemical analyses of formalin-fixed paraffin embedded tumor samples. RESULTS: PapCP revealed a homogenous CD44 expression pattern predominantly at the cell membrane, whereas CD133 labeling was hardly detectable. In adaCP, on the other hand all markers were consistently and predominantly co-expressed in nuclear ß-catenin accumulating cell clusters, which was confirmed by double immunofluorescence staining. Overall expression of CD44 was significantly decreased in adaCP versus papCP, whereas CD133 showed significantly higher protein and mRNA levels in adaCP. CONCLUSIONS: Our results indicate tumor stem cell-like characteristics of ß-catenin accumulating cell clusters in adaCP, which may represent a tumor stem cell niche and might contribute to tumor recurrence. The potential impact of these special cell groups in regard to future CP management, including postoperative follow-up and additional treatment remains to be explored.
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Biomarcadores de Tumor , Craneofaringioma/patología , Células Madre Neoplásicas/patología , Neoplasias Hipofisarias/patología , Vía de Señalización Wnt , Antígeno AC133 , Adolescente , Adulto , Antígenos CD/metabolismo , Niño , Preescolar , Receptores ErbB/metabolismo , Exones , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Glicoproteínas/metabolismo , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Masculino , Persona de Mediana Edad , Osteopontina/metabolismo , Péptidos/metabolismo , Transducción de Señal , Adulto Joven , beta Catenina/metabolismoRESUMEN
BACKGROUND: Claudins are tight junction proteins expressed in epithelial tissues that play important roles in cell polarity and adhesion. Altered distribution of claudin-1(CLDN1) affects cell mobility and tumor invasiveness. Craniopharyngiomas (CPs) represent epithelial tumors of the sellar region, consisting of adamantinomatous (adaCP) and papillary (papCP) variants. Their tendency to infiltrate surrounding brain structures complicates successful surgery. Reliable markers are required to predict tumor behavior and to establish individualized treatment protocols. METHODS: We describe the distribution pattern of CLDN1 in a large cohort of 66 adaCPs, 21 papCPs, and 24 Rathke`s cleft cyst (RCC) cases using immunohistochemistry. CLDN1 mRNA levels were analyzed with qRT-PCR in 33 CP samples. The impact on the migration potential was studied in primary adaCP cell cultures (n = 11) treated with small interfering RNA (siRNA) for CLDN1. Furthermore, CLDN1 distribution patterns and expression levels were compared between invasive (n = 16) and noninvasive (n = 17) tumor groups. RESULTS: PapCPs and RCCs exhibited a distinct homogenous and membranous expression pattern, whereas CLDN1 immunoreactivity appeared weaker and more heterogeneous in adaCPs. In the latter cases, whirl-like cell clusters showed complete absence of CLDN1. mRNA analysis confirmed reduced CLDN1 levels in adaCPs versus papCPs. Interestingly, invasive tumors exhibited significantly lower CLDN1 expression compared with noninvasive counterparts regardless of CP subtype. Accordingly, siRNA treatment for CLDN1 altered tumor cell migration in vitro. CONCLUSION: CLDN1 represents a novel marker in the differential diagnosis of CP variants and RCCs. Low CLDN1 expression levels correlate with an invasive CP growth pattern and may serve as a prognostic marker.
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Carcinoma Papilar/patología , Claudina-1/metabolismo , Craneofaringioma/patología , Neoplasias Hipofisarias/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis , Western Blotting , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Movimiento Celular , Proliferación Celular , Niño , Preescolar , Claudina-1/antagonistas & inhibidores , Claudina-1/genética , Estudios de Cohortes , Craneofaringioma/genética , Craneofaringioma/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Neoplasias Hipofisarias/genética , Neoplasias Hipofisarias/metabolismo , Pronóstico , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Adulto JovenRESUMEN
PURPOSE: Constitutive Wnt signaling caused by mutations in the ß-catenin gene is a molecular hallmark of adamantinomatous craniopharyngiomas (adaCP) and promotes infiltration into adjacent brain tissue. Herein, we studied the pathogenic role of epidermal growth factor receptor (EGFR) activation in adaCP and whether tumor cell migration can be inhibited by the tyrosine kinase inhibitor gefitinib. EXPERIMENTAL DESIGN: EGFR expression and activation [phosphorylated EGFR (EGFR-P)] was examined in a cohort of 25 surgical adaCP samples by PCR and Western blotting. Regional and cellular localization patterns of EGFR-P, ß-catenin, and its target gene product Fascin were determined by immunofluorescence microscopy. Mutation analysis and gene copy number assay were carried out to examine genetic alterations in the EGFR gene. The impact of EGFR signaling on tumor cell migration was studied in vitro by using 11 primary human adaCP cultures treated with the EGFR ligand EGF and its inhibitor gefitinib. RESULTS: Neither mutations nor amplifications in the EGFR gene were detected in our adaCP series. However, EGFR-P was detectable in tumor cell clusters located at the brain infiltration border and colocalized with nuclear ß-catenin and Fascin. Activated EGFR significantly promoted tumor cell migration in vitro, whereas gefitinib reduced both tumor cell motility and Fascin expression. CONCLUSION: Our data suggest EGFR signaling to play a role in cell migration and brain infiltration of adaCP. Targeting the EGFR signaling pathway by gefitinib may present a promising pharmacologic option in the treatment of this challenging tumor disease.
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Craneofaringioma/genética , Craneofaringioma/patología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Transducción de Señal/genética , Adolescente , Adulto , Anciano , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Movimiento Celular , Células Cultivadas , Niño , Preescolar , Variaciones en el Número de Copia de ADN/genética , Femenino , Humanos , Masculino , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Persona de Mediana Edad , Mutación/genética , Fosforilación , Adulto Joven , beta Catenina/genéticaRESUMEN
Activating beta-catenin mutations with aberrant cytoplasmic and nuclear protein accumulation are hallmarks of adamantinomatous craniopharyngiomas (adaCP). These tumours tend to be associated with unfavourable and occasionally disastrous sequelae, as they invade adjacent brain structures such as the hypothalamus. The peculiar digitate growth pattern does not always allow gross surgical removal often leading to recurrence. The tips of invading adaCP epithelium harbour cell clusters with nuclear beta-catenin accumulations, suggesting an influence of beta-catenin-dependent signal transduction on the tumour migratory capacity. This hypothesis was tested by suppressing beta-catenin expression in six primary human adaCP cell cultures using small interfering RNA (siRNA) directed against the beta-catenin gene (CTNNB1). Tumour cell migration was significantly reduced in Boyden chamber and wound-healing experiments following siRNA treatment. We further showed that fascin, a target gene of beta-catenin TCF signalling in colorectal cancer cells and a key component of filopodia, is also involved in this process. beta-Catenin accumulating tumour cells co-express fascin and fascin mRNA levels can be significantly down-regulated in adaCP cultures treated with CTNNB1 siRNA. Furthermore, migration experiments showed a significantly lower cell motility of adaCP tumour cells in vitro when transfected with fascin siRNA. This suggests that activated Wnt-signalling serves as a promoter of the epithelial migration machinery by regulating target molecules such as fascin in adaCP tumour cells.