DNA methylation-based classification of central nervous system tumours.

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

Rathke's Cleft Cyst as Origin of a Pediatric Papillary Craniopharyngioma

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

Tight junction protein claudin-1 is differentially expressed in craniopharyngioma subtypes and indicates invasive tumor growth

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

EZH2 is highly expressed in pituitary adenomas and associated with proliferation

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 coexpression 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

Expression of SRY-related HMG Box Transcription Factors (Sox) 2 and 9 in Craniopharyngioma Subtypes and Surrounding Brain Tissue

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 β-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

EpCAM (CD326) is differentially expressed in craniopharyngioma subtypes and Rathke’s cleft cysts

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

Histological criteria for atypical pituitary adenomas – data from the German pituitary adenoma registry suggests modifications

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

Comparison of primary human MRI and histology with the induced model.

<p>MR images and corresponding histology of an ACP patient (ACP2) showing the primary tumor and relapse after 145 days are illustrated in comparison with an example of a direct vital descendent of the surgically removed primary tumor specimen implanted into the nude mouse number one out of ten (ACP2 M1). T2 weighted images show a cystic component within the craniopharyngioma in the human patient (a and b; arrow) with contrast enhancement in the rim of the lesion (e and f; arrow) at baseline (a and e) as compared to 145 days later (b and f) with an increase in size over time. The craniopharyngioma xenograft in the mouse is inhomogeneous in T2 (c and d) and post contrast T1 (g and h) weighted images. The T2w hyperintense and T1 contrast enhancing areas in the tumor increase over 110 days as well (c and d; g and h). Histology of the primary tumor (i) shows similar characteristics to the descendent tumors in human (j) and in mice (k and k`) in terms of vital tumor proportions (arrow), wet keratin (asterisk) and calcification (arrowhead). Vital tumor areas (arrow, k´) in the murine graft were also identified on MRI (arrows, d and h) by T2 hyperintensity and contrast enhancing areas in T1. However, calcified regions appear hypointense (arrowhead, k´, d and h). PDX tumor volume (n = 5) defined 28 and 138 days post transplantation (dpt) in T2 weighted as well as post contrast T1 weighted images revealed a significant increase over time (p<0.05) (l).</p

Characterization of the murine orthotopic adamantinomatous craniopharyngioma PDX model by MRI in correlation with histology

<div><p>Purpose</p><p>Adamantinomatous craniopharyngiomas (ACP) as benign sellar brain tumors are challenging to treat. In order to develop robust <i>in vivo</i> 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).</p><p>Methods</p><p>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.</p><p>Results</p><p>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.</p><p>Conclusions</p><p>MRI enables the assessment of craniopharyngioma PDX vitality <i>in vivo</i> as validated histologically.</p></div

Comparison of patient specific engrafted tumors.

<p>Primary (a, e and i) and descended transplanted ACP tissue (b, c, f, g, j and k) show comparable histological appearance with regressive changes e.g. wet keratin (*) and calcifications (arrowhead) with estimated vitality indicated in the magnified images. Associated T1 weighted MR images after contrast medium application (d, h and l) are shown for comparison with histological appearance.</p