Establishing a papillary craniopharyngioma cell line by SV40LT-mediated immortalization.

BACKGROUND: Craniopharyngioma represents a troublesome tumor of the intracranial sellar region. There are currently no available well-characterized craniopharyngioma cell lines. This lack of reliable, immortal cell lines is a major reason for the slow progress in fundamental research related to craniopharyngioma. METHODS: We describe the development of an immortal papillary craniopharyngioma (PCP) cell line by transfecting primary PCP cells with the pLenti-simian virus 40 large T antigen(SV40LT). RESULTS: Three clones have been cultured for more than 14 months so far, while non-transfected cells ceased proliferation within three months of isolation. The established immortal PCP cell lines were identified to have BRAFV600E mutations, while no mutations in tumor suppressor genes were found in primary cells or immortal cells. Immortal cells had higher proliferation rates and formed tumors when implanted in the bran of nude mice. BRAF inhibition in immortal PCP cells altered cell morphology, inhibited cell proliferation and promoted apoptosis. CONCLUSION: We successfully developed PCP cell lines by SV40LT-mediated immortalization. These cell lines represent a powerful tool for fundamental and therapeutical studies on craniopharyngioma.

Feasibility of primary human cell cultures as a model for adamantinomatous craniopharyngioma research: Evidence from RNA-Seq analysis.

Adamantinomatous craniopharyngioma (ACP) is a benign epithelial tumor of the sellar region. Whether primary human cell cultures can be used as a stable research model has yet to be determined. The characteristics of three cultured craniopharyngioma primary cell (CPC) lines were identified using immunofluorescence. The culture duration for each CPC line was 10, 20 and 30 days. Cell lines and paired parental tumor tissues were subsequently analyzed using transcriptome sequencing (RNA-Seq). Transcriptomic differences between ACP tissues and CPC lines were compared. CPCs maintained the original epithelial lineage markers, including pan-cytokeratin and epithelial cell adhesion molecule. However, the Pearson's correlation coefficient of transcriptomes between each pair of CPC lines and ACP tissues decreased from 0.657 (cultured for 10 days) to 0.61 (cultured for 20 days) and further to 0.547 (cultured for 30 days). The number of differentially expressed genes between ACP tissues and CPCs was increased from 1,247 (cultured for 10 days) to 1,643 (cultured for 20 days) and then to 1,949 (cultured for 30 days). The results of Gene Set Enrichment Analysis demonstrated that the diversity of gene sets increased with longer culture time. Significant differences in the majority of signature gene sets were not observed between ACP tissues and CPCs, with the exception of keratinization phenotype [normalized enrichment score (NES)=-2.02, false discovery rate (FDR)=0.0038] and epithelial cell phenotype (NES=-1.82, FDR=0.032). Cell proliferation (NES=1.78, FDR=0.028) and mitosis (NES=1.93, FDR=0.012) were enhanced in CPCs. Therefore, primary human cell cultures can be used as a suitable research platform for ACP, however further experiments are required.

Identification of tumor stem-like cells in admanatimomatous craniopharyngioma and determination of these cells' pathological significance.

OBJECTIVE: Nuclear ß-catenin, a hallmark of active canonical Wnt signaling, can be histologically detected in a subset of cells and cell clusters in up to 94% of adamantinomatous craniopharyngioma (ACP) samples. However, it is unclear whether nuclear ß-catenin-containing cells within human ACPs possess the characteristics of tumor stem cells, and it is unknown what role these cells have in ACP. METHODS: Primary ACP cells were cultured from 12 human ACP samples. Adamantinomatous CP stem cell-like cells (CSLCs) showing CD44 positivity were isolated from the cultured primary ACP cells by performing magnetic-activated cell sorting. The tumor sphere formation, cell cycle distribution, stemness marker expression, and multidifferentiation potential of the CD44- cells and the CSLCs were analyzed. RESULTS: Compared with the CD44- cells, the cultured human CSLCs formed tumor spheres and expressed CD44 and CD133; moreover, these cells demonstrated nuclear translocation of ß-catenin. In addition, the CSLCs demonstrated osteogenic and adipogenic differentiation capacities compared with the CD44- cells. The CSLCs also displayed the capacity for tumor initiation in human-mouse xenografts. CONCLUSIONS: These results indicate that CSLCs play an important role in ACP development, calcification, and cystic degeneration.

Pathological Relationship Between Adamantinomatous Craniopharyngioma and Adjacent Structures Based on QST Classification.

The aim of this study was to clarify pathological and anatomical relationships between adamantinomatous craniopharyngiomas (ACP) and their surrounding structures. We previously established a QST classification scheme based on the apparent anatomic origin of the tumors. According to this classification, 13 type Q tumors, 6 type S tumors, and 42 type T ACPs were analyzed. Type Q tumors, which are most likely to involve the pituitary gland, did not invade the area of contact with the adenohypophysis. Instead, tumor invasion was observed in areas where the tumor contacted the neurohypophysis. Type S tumors primarily involved the pituitary stalk; the arachnoid remained present between these tumors and normal structures. Type T tumors were located beneath the basal arachnoid membrane and outside the pia mater. The pia mater was disrupted and finger-like invasions were found in the neural layer of the third ventricle floor along the invasive front. Tumors were never observed to break through the ependymal layer of the third ventricle. The QST classification has important implications for understanding the growth pattern of tumors and can be used to guide surgical procedures.

TREM-1 expression in craniopharyngioma and Rathke's cleft cyst: its possible implication for controversial pathology.

Whether a mixed type of craniopharyngioma (CP) exists and whether papillary craniopharyngioma (pCP) is on a histopathological continuum with Rathke's cleft cyst (RCC) remain controversial. Herein, we examined the expression and localization of ß-catenin, BRAF p.V600E (V600E), and triggering receptor expressed on myeloid cells-1 (TREM-1) in 58 samples including 20 pCPs, 26 adamantinomatous craniopharyngiomas (aCP), and 12 RCCs. Five aCPs were diagnosed with mixed type CPs and the remaining 21 cases were pure aCPs. Four of the 12 RCCs presented with significant squamous epithelium (SE). V600E immunoreactivity was observed in all pCPs in the cytoplasm, but not in the nuclei. aCPs and RCCs, including mixed type CP, did not express V600E. Nuclear ß-catenin translocation was detected exclusively in aCPs. TREM-1 was expressed in pCPs. Additionally, TREM-1 expression was detected in the SE of 5 "mixed type" CPs, while it was absent in pure aCPs. TREM-1 was expressed in 4 RCCs with SE, but not in the remaining 8 RCCs. TREM-1 mRNA levels were compared in cultured pCP and aCP cells. TREM-1 mRNA level was significantly (p < 0.001; up to 4.045 fold) higher in pCPs than in aCPs. Western blotting revealed a significantly (p < 0.001; up to 7.19 fold) lower level of TREM-1 expression in aCP cells compared to that in pCP cells. Our findings further supported that RCC and pCP may represent two ends of a morphological spectrum. A variant showing overlapping histological features of aCP and pCP should not be considered as a mixed type.

Origin of craniopharyngiomas: implications for growth pattern, clinical characteristics, and outcomes of tumor recurrence.

OBJECT Craniopharyngiomas are associated with a high rate of recurrence. The surgical management of recurrent lesions has been among the most challenging neurosurgical procedures because of the craniopharyngioma's complex topographical relationship with surrounding structures. The aim of this study was to define the determinative role of the site of origin on the growth pattern and clinical features of recurrent craniopharyngiomas. METHODS The authors performed a retrospective analysis of 52 patients who had undergone uniform treatment by a single surgeon. For each patient, data concerning symptoms and signs, imaging features, hypothalamic-pituitary function, and recurrence-free survival rate were collected. RESULTS For children, delayed puberty was more frequent in the group with Type I (infradiaphragmatic) craniopharyngioma than in the group with Type TS (tuberoinfundibular and suprasellar extraventricular) lesions (p < 0.05). For adults, blindness was more frequent in the Type I group than in the Type TS group (p < 0.05). Nausea or vomiting, delayed puberty, and growth retardation were more frequent in children than in adults (p < 0.05). Overall clinical outcome was good in 48.07% of the patients and poor in 51.92%. Patients with Type TS recurrent tumors had significantly worse functional outcomes and hypothalamic function than patients with the Type I recurrent tumors but better pituitary function especially in children. CONCLUSIONS The origin of recurrent craniopharyngiomas significantly affected the symptoms, signs, functional outcomes, and hypothalamic-pituitary functions of patients undergoing repeated surgery. Differences in tumor growth patterns and site of origin should be considered when one is comparing outcomes and survival across treatment paradigms in patients with recurrent craniopharyngiomas.

Arachnoid membranes in the posterior half of the incisural space: an inverted Liliequist membrane-like arachnoid complex.

OBJECT: The aim of this study was to describe the similarity of configuration between the arachnoid complex in the posterior half of the incisural space and the Liliequist membrane. METHODS: Microsurgical dissection and anatomical observation were performed in 20 formalin-fixed adult cadaver heads. The origin, distribution, and configuration of the arachnoid membranes and their relationships with the vascular structures in the posterior half of the incisural space were examined. RESULTS: The posterior perimesencephalic membrane and the cerebellar precentral membrane have a common origin at the tentorial edge and form an arachnoid complex strikingly resembling an inverted Liliequist membrane. Asymmetry between sides is not uncommon. If the cerebellar precentral membrane is hypoplastic on one side or both, the well-developed quadrigeminal membrane plays a prominent part in partitioning the subarachnoid space in the posterior half of the incisural space. CONCLUSIONS: The arachnoid complex in the posterior half of the incisural space can be regarded as an inverted Liliequist membrane. This concept can help neurosurgeons to gain better understanding of the surgical anatomy at the level of the tentorial incisura.

Weighted Youden index and its two-independent-sample comparison based on weighted sensitivity and specificity.

BACKGROUND: Most indices for evaluating a diagnostic test can be expressed as functions of sensitivity (SEN) and specificity (SPE). Practically, all existing methods suffer from the inability to weight sensitivity and specificity relative to their importance. In this paper, we developed a novel index, the weighted Youden index, that allows Youden index to be a combination of sensitivity and specificity with user-defined weights. METHODS: The weighted Youden index Jw is defined as Jw = 2(w×SEN + (1-w)SPE)-1 (0 ≤ w ≤ 1). It has three properties: (1) the sum of the weights which are attached to sensitivity and specificity should be equal to 1; (2) the range of Jw should be within [-1, 1], which is the range of the Youden index J; (3) Jw should be equal to J when sensitivity and specificity have equal weights. According to the central limit theorem, we obtain the standard error of Jw, and propose a statistical inference method to compare two weighted Youden indices. The monotonicity of the test statistic was discussed. RESULTS: An example of comparing two diagnostic tests for pheochromocytoma was used to demonstrate the weighted Youden index method. Weighted Youden index, the confidence interval for each test and the hypothesis test of comparing two independent diagnostic tests were presented. Assigning the weights is essential to the weighted Youden index approach. CONCLUSION: The weighted Youden index can broaden its applications in diagnostic test development and motivate further research in weighting sensitivity and specificity explicitly.

Anatomical and histological study of Liliequist's membrane: with emphasis on its nature and lateral attachments.

PURPOSE: In previous studies, some disagreements regarding the nature (inner or outer arachnoid membrane) and lateral boundaries (temporal uncus or tentorial edge) of Liliequist's membrane remain. The aim was to clarify whether Liliequist's membrane is an inner or outer arachnoid membrane, and the distribution of Liliequist's membrane with emphasis on its lateral attachments. METHODS: Liliequist's membrane was investigated by microsurgical dissection in 24 formalin-fixed adult cadaver heads and by histological sections of sellar-suprasellar specimens from another four formalin-fixed adult cadaver heads. RESULTS: The results obtained in the present study indicated that 1) Liliequist's membrane arises from the basal arachnoid membrane and has two components: a basal part comprising a folding inner layer of the arachnoid mater and an attaching part consisting of accumulated arachnoid trabeculae; 2) similar histological features are also present in other inner arachnoid membranes with attachments on basal arachnoid membrane, demonstrating Liliequist's membrane is an inner arachnoid membrane; 3) laterally, Liliequist's membrane attaches to the anterior tentorial edge constantly and to the mesial temporal uncus in more than half; 4) the oculomotor nerve courses above Liliequist's membrane and is fixed on Liliequist's membrane by the oculomotor membrane, which can also attach on temporal uncus and should be differentiated from the true temporal attachments of Liliequist's membrane. CONCLUSION: Liliequist's membrane is an inner rather than outer arachnoid membrane. Understanding of its individual variation and topographic relationships with surrounding neurovascular and arachnoid structures is important for neurosurgical practice.