Multiomics integration-based immunological characterizations of adamantinomatous craniopharyngioma in relation to keratinization.

Although adamantinomatous craniopharyngioma (ACP) is a tumour with low histological malignancy, there are very few therapeutic options other than surgery. ACP has high histological complexity, and the unique features of the immunological microenvironment within ACP remain elusive. Further elucidation of the tumour microenvironment is particularly important to expand our knowledge of potential therapeutic targets. Here, we performed integrative analysis of 58,081 nuclei through single-nucleus RNA sequencing and spatial transcriptomics on ACP specimens to characterize the features and intercellular network within the microenvironment. The ACP environment is highly immunosuppressive with low levels of T-cell infiltration/cytotoxicity. Moreover, tumour-associated macrophages (TAMs), which originate from distinct sources, highly infiltrate the microenvironment. Using spatial transcriptomic data, we observed one kind of non-microglial derived TAM that highly expressed GPNMB close to the terminally differentiated epithelial cell characterized by RHCG, and this colocalization was verified by asmFISH. We also found the positive correlation of infiltration between these two cell types in datasets with larger cohort. According to intercellular communication analysis, we report a regulatory network that could facilitate the keratinization of RHCG+ epithelial cells, eventually causing tumour progression. Our findings provide a comprehensive analysis of the ACP immune microenvironment and reveal a potential therapeutic strategy base on interfering with these two types of cells.

Autophagy and immune microenvironment in craniopharyngioma and ameloblastoma.

BACKGROUND: Craniopharyngiomas and ameloblastomas show remarkable histologic and molecular similarities. The immune microenvironment of craniopharyngiomas has been recently studied showing interesting findings, while its composition in ameloblastomas is unknown. Similarly, some evidence of autophagic activity, a process of cellular constituents' degradation has been found in ameloblastomas, but no studies exist in craniopharyngiomas. Thus, the aim of the study is to compare factors of the immune microenvironment and the autophagic apparatus between these two tumor types. METHODS: 26 craniopharyngiomas and 14 ameloblastomas were immunohistochemically studied for PD-L1, CD8, CD20, S100, CD163, MECA-79, LC3B and p62. RESULTS: Craniopharyngiomas showed higher LC3B tumor cell expression, higher CD8+ T cells and higher CD163+ macrophages in comparison to ameloblastomas. LC3B tumor cell expression was associated with overall survival in craniopharyngioma patients and p62 nuclear expression was associated with overall survival in ameloblastoma patients. CONCLUSION: This is the first study showing the presence of autophagic markers in craniopharyngiomas and describing the immune microenvironment of ameloblastomas.

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.

Antibodies Against Hypothalamus and Pituitary Gland in Childhood-Onset Brain Tumors and Pituitary Dysfunction.

Purpose: To detect the presence of antipituitary (APA) and antihypothalamus antibodies (AHA) in subjects treated for brain cancers, and to evaluate their potential association with pituitary dysfunction. Methods: We evaluated 63 patients with craniopharyngioma, glioma, and germinoma treated with surgery and/or radiotherapy and/or chemotherapy at a median age of 13 years. Forty-one had multiple pituitary hormone deficiencies (MPHD), six had a single pituitary defect. GH was the most common defect (65.1%), followed by AVP (61.9%), TSH (57.1%), ACTH (49.2%), and gonadotropin (38.1%). APA and AHA were evaluated by simple indirect immunofluorescence method indirect immunofluorescence in patients and in 50 healthy controls. Results: Circulating APA and/or AHA were found in 31 subjects (49.2%) and in none of the healthy controls. In particular, 25 subjects out of 31 were APA (80.6%), 26 were AHA (83.90%), and 20 were both APA and AHA (64.5%). Nine patients APA and/or AHA have craniopharyngioma (29%), seven (22.6%) have glioma, and 15 (48.4%) have germinoma. Patients with craniopharyngioma were positive for at least one antibody in 39.1% compared to 33.3% of patients with glioma and to 78.9% of those with germinoma with an analogous distribution for APA and AHA between the three tumors. The presence of APA or AHA and of both APA and AHA was significantly increased in patients with germinoma. The presence of APA (P = 0.001) and their titers (P = 0.001) was significantly associated with the type of tumor in the following order: germinomas, craniopharyngiomas, and gliomas; an analogous distribution was observed for the presence of AHA (P = 0.002) and their titers (P = 0.012). In addition, we found a significant association between radiotherapy and APA (P = 0.03). Conclusions: Brain tumors especially germinoma are associated with the development of hypothalamic-pituitary antibodies and pituitary defects. The correct interpretation of APA/AHA antibodies is essential to avoid a misdiagnosis of an autoimmune infundibulo-neurohypophysitis or pituitary hypophysitis in patients with germinoma.

Co-Occurrence of Craniopharyngioma and IgG4-Related Hypophysitis: An Epiphenomenon or a Mere Coincidence?

BACKGROUND: Craniopharyngiomas are sellar-suprasellar tumors that commonly present in children, with nonspecific symptoms of increased intracranial pressure, visual disturbances, and pituitary insufficiencies. Rarely has secondary hypophysitis (lymphocytic and xanthogranulomatous) been reported in association with craniopharyngioma. CASE DESCRIPTION: We have reported the case of a 16-year-old boy who had presented with gradually progressive diminution of vision in the right eye, intermittent headache, deceleration in growth velocity, and the lack of development of secondary sexual characteristics. Imaging revealed a sellar-suprasellar cystic lesion (3.8 × 3.1 × 3.5 cm) with calcifications. Laboratory tests revealed hypothyroidism, hypocortisolism, hypogonadism, and growth hormone deficiency. Craniopharyngioma was provisionally diagnosed. He underwent pterional craniotomy and gross total excision of the lesion. The excised tissue showed features of adamantinomatous craniopharyngioma, with a dense lymphoplasmacytic infiltrate and fibrosis involving the pituitary and dura mater. The lymphocytes were CD20-positive, and the plasma cells stained positive for IgG4 (50-60 IgG4-positive plasma cells/high power field). Consequently, the possibility of IgG4-related hypophysitis was considered in our patient. His serum IgG4 level was not elevated. Systemic involvement by IgG4-related disease was thoroughly ruled out. Fluorodeoxyglucose positron emission tomography/computed tomography did not show any clinically significant hypermetabolism anywhere in the body. At the 3-month follow-up examination, his headache had resolved. However, he had not regained vision in his right eye. Repeat imaging studies showed no residual tumor tissue. CONCLUSIONS: The present case might represent the first ever report, to the best of our knowledge, of secondary IgG4-related hypophysitis due to craniopharyngioma, or it might, perhaps, be a chance association of these 2 entirely different disease entities.

Immune Microenvironment of Primary and Recurrent Craniopharyngiomas: A Study of the Differences and Clinical Significance.

OBJECTIVE: This study explored the differences between the immune microenvironments of primary and recurrent craniopharyngiomas (CPs). In addition, we investigated the relationship between the immune microenvironment and clinical characteristics of CP. METHODS: We collected 52 specimens from 26 patients with CPs. For each patient, specimens for both primary and recurrent CPs were obtained. We performed an immunohistochemical analysis of these specimens to determine the distributions of M2 macrophages, CD8+ T cells, programmed cell death 1 ligand 1 (PD-L1), and Ki67. RESULTS: In recurrent CP specimens, the distributions of M2 macrophages, Ki67, and PD-L1 increased compared with primary CP specimens (P = 0.019, P = 0.0084, and P = 0.0319, respectively). Moreover, the distributions of M2 macrophages, CD8+ T cells, and PD-L1 in papillary CPs were higher than those observed in adamantinomatous craniopharyngiomas (ACPs) (P = 0.0317, P = 0.0359, and P < 0.0001, respectively). In the adult ACP group, M2 macrophages, CD8+ T cells, and PD-L1 were more abundant/expressed than in the child ACP group (P = 0.0159, P = 0.0215, and P < 0.0088, respectively). A positive correlation was found between M2 macrophages and CD8+ T cells (r = 0.4079; P = 0.0027). Correspondingly, M2 macrophages and CD8+ T cells were both positively correlated with PD-L1 (r = 0.4564; P = 0.0007 and r = 0.3987; P = 0.0034, respectively). The observed high expression of M2 macrophages in primary CPs suggests a shortened time for tumor recurrence (P = 0.0131). CONCLUSIONS: The microenvironment of recurrent CP varies from that of primary CP. The abundance of M2 macrophages in primary CP may indicate a risk of early recurrence. Therefore, it is recommended to increase the frequency of follow-up examinations in these patients.

The role of inflammation in the genesis of the cystic component of craniopharyngiomas.

BACKGROUND: Craniopharyngioma accounts for 5-10% of childhood tumors and, despite of the benign histological features, its clinical course can be malignant because of critical anatomical relationships with neural and vascular structures and the possible morbidity associated to resection. Only a few studies have addressed the molecular characterization of the cyst fluid so far and the mechanisms of action of intracystic agents are not clearly understood yet. METHODS: The acidic soluble proteins contained in the cystic fluid of six patients with cystic craniopharyngioma, three of them treated with intratumoral interferon-α, were analyzed. A high performance liquid chromatography electrospray ionization mass spectrometry analysis was performed. FINDINGS: The antimicrobial peptides α-defensins 1-3 relevant for innate immunity were detected in the cystic fluid before the intratumoral treatment. Amount of peptides significantly decreased in cystic fluid during pharmacological treatment. INTERPRETATION: Detection of α-defensins 1-3 excludes that cyst fluid formation can derive from disruption of blood-brain barrier and suggests the involvement of innate immune response in pathology of craniopharyngioma cyst formation. The reduction of α-defensins could derive both from direct antitumoral effect of interferon-α on squamous epithelial cells of craniopharyngioma cyst and from its immuno-modulatory effects on the recruitment of cells of innate immune systems. Interestingly, the clinical patient outcome well correlates with the gradual reduction of α-defensins 1-3 amount. Additional studies will be necessary to establish the role of these molecules in the pathogenesis of craniopharyngioma, and further investigations will be necessary to confirm the efficacy of the antitumoral activity of interferon-α.

Immunohistochemical localisation of cytokeratins in craniopharyngioma.

BACKGROUND: Although craniopharyngiomas have been examined in several microscopical studies to date, immunohistochemical analysis has not been sufficient. METHOD: In addition to the routine haematoxylin and eosin staining, 38 cases of intra- and/or supra-sellar craniopharyngioma, including 34 adamatinomatous and 4 squamous papillary types, were studied using immunohistochemistry for expression of four types of cytokeratin. FINDINGS: Histological examination found epithelial cells in 26 of 38 (68.40%) cases. However, cytokeratins were demonstrated in 35 of 38 (92.1%) cases. The remaining 3 cases without demonstration of epithelial cell nests were supposed to be adamantinomatous craniopharyngiomas based on the findings in the stroma. In 31 of 34 adamantinomatous craniopharyngioma cases, the epithelium was detected by immunostaining for cytokeratins. The epithlieum expressed 56 kDa (KL-1) and 40 kDa (cytokeratin 19) cytokeratins with similar staining patterns and intensities. The staining intensity of 54 kDa cytokeratin (cytokeratin 7) was similar to that of the high molecular weight cytokeratin (keratin M-903). However, in many cases (15 of 27), immunoreactivity of cytokeratin 7 was not demonstrated in an outer palisaded basal layer. In all 4 squamous papillary craniopharyngiomas, moderate staining with cytokeratin 7 appeared in the superficial layer, whereas basal or mid-zone epithelial cells were negative for cytokeratin 7. The basal layer stained negatively for KL-1, as well as cytokeratin 7. INTERPRETATION: Immunostaining for cytokeratin is valuable in the investigation of craniopharyngioma, especially when specimens contain only a small or questionable part of epithelium. Most notably, KL-1 or cytokeratin 7 stainings are suitable for analyzing these tumours, with special reference to histological subtypes.

Inflammatory infiltrates and natural killer cell presence in human brain tumors.

Immunohistochemical analysis of subpopulations of inflammatory cells in 81 primary and secondary human brain tumors was done. Natural killer (NK) cells, representing non-major histocompatibility complex-restricted, spontaneous cytotoxicity and monocytic cells are virtually absent in infiltrates of gliomas and account only for a minor percentage of inflammatory cells in brain metastases of carcinoma and in craniopharyngeomas. Infiltrates in gliomas consist almost exclusively of T-cells of the suppressor/cytotoxic type whereas infiltrates in carcinoma metastases and craniopharyngeomas contain considerable numbers of T-helper/inducer cells and B-cells. From this the authors conclude (1) that NK cells do not play a major role in tumor rejection, and (2) that the kind of inflammatory reaction does not depend upon the tumor site but more likely on the tumor type. No correlation between tumor differentiation and infiltrate composition is evident.

[A case of prolactinoma in close association with Rathke's cleft cyst].

We report a case of Rathke's cleft cyst in pituitary tumor. A 31-year-old woman admitted to our hospital complained of visual disturbance, headache, amenorrhea and galactorrhea. Serum prolactin level was 3,060 ng/ml. By means of CT scan and MRI, we found suprasellar-extending tumor to have cystic component in the center of the tumor. Transsphenoidal surgery revealed grayish jelly-like content in the cyst. Histologically tumor cells were composed of chromophobic, basophilic and eosinophilic cells. The cyst wall in the pituitary adenoma was composed of ciliated or non-ciliated columnar epithelium intermingled with goblet cells, being similar to Rathke's cleft cyst. Except for the cells of the cyst wall, most of the tumor cells were immunoreactive for prolactin. As for intermediate filament, tumor cells in both solid and cystic portions showed positive immunoreactivity for cytokeratin and GFAP, and not for vimentin and neurofilament. These results suggest that the nature of the intracytoplasmic filament in the pituitary tumor with Rathke's cleft cyst may be not only keratin but also GFAP.

Alterations in the cellular immune response of patients with cerebral glioma, benign intracranial tumour, and spontaneous subarachnoid haemorrhage measured in vitro by the leucocyte migration inhibition test.

Leucocyte migration inhibition in response to ubiquitous antigens was studied in 104 patients as an in vitro indicator of cell-mediated immunity. Patients with cerebral glioma, benign intracranial tumours, and subarachnoid haemorrhage demonstrated impaired inhibition of leucocyte migration compared with control subjects. The greatest impairment occurred in patients with subarachnoid haemorrhage, while the least impairment was seen in patients with glioma. Significant rises in inhibition of leucocyte migration in response to antigen preparations from glioma and normal brain were seen in the early post-operative period in patients with glioma and subarachnoid haemorrhage. Impaired cellular immunity, together with sensitivity of lymphocytes to brain-derived antigens, are features of cerebral disease in general and not specific for glioma.