Prediction Of The Relationship Between Craniopharyngioma And Contiguous Structures With Exploring Its Origin

Background and objective Craniopharyngioma(CP)is a benign tumor,which grows in cranial cavity and is thought to originate from the remnants of Rathke’s capsule.Surgical resection is currently the mainstream treatment.The relationships between CP and surrounding structures are complex and it is traditionally considered that CP can extensively invade surrounding structures,which is at odds with the definition of CP as a benign tumor.When CP invades the hypothalamus,it often leading to hypothalamuic damage,which seriously affects the patient’s quality of life,or even lead to patient death.Treatment remains extremely challenging.In addition,some CPs with a high recurrence rate even after total resection,which may be due to the special original site of CP,making it difficult to truly achieve total resection of this tumor.Therefore,it is important to preoperatively predict the relationships between CP and surrounding structures,especially with the hypothalamus,and to confirm the origin of CP.This study aims to explore a tool to preoperatively predict the pathological relationships between CP and the hypothalamus,and with other surrounding structures,and to explore the origin of CP with its molecular characteristics.Materials and methods1.First,in daily clinical work,we have noted a sign on preoperative sagittal MRI,consisting of the third ventricle floor(TVF),mamillary bodies(MBs)and cerebral peduncle(Ped),which is alike an eagle and named “eagle sign” here.The morphology of the "eagle sign" may vary under the influence of the different relationships between CP and hypothalamus.The previous CP cases that undergone endoscopic endonasal resection were enrolled.Tthe pathological relationships between CP and hypothalamus was analysed firstly through analyzing the tissues that collected form the area of previous CP attaching hypothalamus with H&E and immunofluorescence.The morphology of "Eagle sign",the intraoperative video records,the histopathological detection and the prognosis of CPs were analyzed to access the associateion of the different morphologies of "Eagle sign" with the different relationships between CP and hypothalamus.2.In the second part,CPs treated by transnasal endoscopic approach between May2020 and July 2021 in our center were prospectively enrolled.Firstly,“Eagle sigh” of all CPs were evaluated preoperatively.Secondly,the interface of CP with hypothalamus,pituitary stalk(PS),pituitary gland(PG),optic chaism(OC)and other brain tissues(BT)were observed,and the interfacial specimens of CPs attaching these structures were collected for histopathological detection.The interfacial tissues of CPs attaching the hypothalamus were examined for the presence of finger-like protrusion(FP)to validate the prediction accuracy of “Eagle sigh”.Meanwhile,all the CPs were divided into two groups according to the presence or absence of FP(group H: FP existed;group S: no FP).The demarcation of all CPs with these structures and the presence of FP were determined by H&E staining and immunofluorescence detection.The prognosis of all CP cases was evaluated.Finally,the origin of CP was explored by analyzing the distribution of nuclear β-catenin and stem cell markers(CD44,CD133)in the tumor.3.In the third part,one CP of group H was enrolled.The interfacial tissue of CP attaching the hypothalamus was analyzed using spatial transcriptome sequencing.After sequencing and library construction,quality control was conducted using Space Range,the official software of 10×Genomics.Principal Components Analysis(PCA)was conducted to reduce the dimension.Graph based were used for spots clustering.The results of reductional dimension and clustering was visualized using UMAP and t-SNE methods.Secondary Analysis of Gene Expression is Seurat.The Seurat package was used to normalise data,imensionality reduction,clustering and differential expression.The top10 highly variable genes(highly expressed in each cluster)were selected as the markers of each Cluster.Then,these markers were applied for cluster annotation using public databases(Panglao DB,Cell Marker).The top 50 differentially expressed genes of each Cluster were input to DAVID for GO and KEGG analysis,and visualized by R for graphing.Results1.A total of 146 CPs patients,who undergone endoscopic endonasal procedure were divided into four groups based on the variants of “eagle sign”.Group A: 24 patients with the upward sign;group B: 81 with the downward sign;group C: 21 with the anterior TVF upward sign and group D: 20 with the unidentifiable sign.Pathological examination was conducted in 38 previously collected CP tissues at the site attaching the hypothalamus.It was suggested that the relationships between CPs and hypothalamus could be defined as adherence or invasion.When FP were present locally,the relationship was invasion,when FP was absent,the relationship could be defined as aadherence.Surgical–pathological combination analysis showed significant relationships between 95.8% CPs in group A and 95.2% in group C with tumor topography and tumor adherence to the hypothalamus.In contrast,groups B and D showed hypothalamic infiltration by tumor in 97.5% and 95% of cases in groups B and D,respectively.Outcomes of groups A and C were relatively better than groups B and D.Predictive sensitivity and specificity of “eagle sign” were more than 90%.2.In the second part of the study,a total of 34 CP patients were enrolled.Among them,there were 9 CPs with the upward “Eagle sigh”(the former group A pattern),13 CPs with the downward “Eagle sigh”(the former group B pattern),8 CPs with the anterior TVF upward “Eagle sigh”(the former group C pattern)and 4 CPs with “Eagle sigh” unidentifiable.Combining the intraoperative video observation and postoperative histopathological examination of the CP tissue attaching the hypothalamus,two CPs with the upward “Eagle sigh” were found the tumoral capsule merged with the hypothalamus and FP was present in the two tissues.Howecer,the remaining seven CPs with this sigh were found clearly demarcated from the hypothalamus and FP was absent in these tissues.11 CPs with the downward “Eagle sigh” were found their capsules merged with the hypothalamus and FP was present in the merged areas of these CPs.The remaining 2 CPs with this sigh were clearly demarcated with the hypothalamus,and no FP was seen in the local tissues.7 CPs with the anterior TVF upward “Eagle sigh” were clearly demarcated with the hypothalamus,and no FP were seen in the local tissues.the remaining 1 CP with this sigh was found merged with the hypothalamus and FP was present.Four CPs with “Eagle sigh” unidentifiable,were found their capsules merged with the hypothalamus,and FP was present in these merged tissues.The sensitivity and specificity of the "Eagle sigh" in predicting the relationship between CP and hypothalamus exceeded 77.8% in the prospective study.Based on the above findings,all the 34 CPs were divided into two groups.Group H comprised 18 CPs whereby the pathological staining of tumoral specimens attaching to the hypothalamus exhibited FP.Cleavage of these CPs merged with the hypothalamus.Tumoral specimens attaching to the PS,PG,OC,and BT showed clear demarcations with these structures and intact cleavage of CPs,and no FP.Group S comprised 14 CPs whereby the staining of tumoral specimens attaching to the hypothalamus,PG,OC and BT exhibited no FP.Staining of the tumoral specimens attaching to the PS exhibited FP.Clear demarcations were observed between CPs and hypothalamus,PG,OC,and BT,but the cleavage of these CPs merged with the PS.FP in PS were relatively larger,less numerous and shorter than in hypothalamus.Assessment of hypothalamic function revealed a better prognosis of CP cases in group S than in group H.Taken together,these findings suggested that CP only invades a specific site on the hypothalamicpituitary axis.The site of invasion was found consistent with the location of Rathke’s pouch precursor cells,which was considered to be the CP origin.In additional,β-catenin and stem cells markers(CD44,CD133)showed more in FP regions of both groups compared with the rest of other portions.It is suggested that FP may be the origin of CP.CP in group H may originate from the hypothalamus and CP in group S may originate from the PS.Meanwhile,it also suggested that the "Eagle sigh" can predict the relationships between the CP and surrounding structures,and the origin of CP.3.In the third part,spatial transcriptome sequencing was performed on one FP tissue of group H.After quality control,the sequencing data were divided into seven clusters.Differentially expressed genes and marker genes of each cluster was screened out.Cellular annotation of the 7 clusters based on marker genes revealed that cluster 1 was PE region;cluster 2 was glial region;cluster 3 was cystic region;cluster 4 was wet keratins region;cluster 5 was possible tumor mesenchymal region;cluster 6 was SR region;cluster 7 was Whorl-like cell cluster region.CTNNB1(encoding β-catenin),CD44 and PROM1(encoding CD133)were expressed highly in cluster 7.GO and KEGG analysis showed that cells in the PE region was mainly involved in DNA replication,cell proliferation(GO: 0003677,GO: 0006270,has03030),with the possibility of being regulated by signals from other sites.The glial region not only had its own neurological metabolic activities,but also an inflammatory response occurring(has04620)in this area,and there was also a possible signaling dialogue with the PE region.There was no KEGG pathways enriched in cystic region.The genes of this area mostly defined as cell surfaces,cell membranes,etc.,which was consistent with the behavior of secreting inflammatory factors.In the wet keratin region,genes mainly localized extracellularly(GO: 0005576),and also enriched to the formation activity of teeth containing dentin(GO: 0031424),structural components of enamel(GO:0030345),etc.,which suggested that wet keratin formation may be similar to tooth development.Cluster 5 may be a tumor stromal region,with gene enrichment analysis revealed the presence of protein binding(GO: 005515),protein regulation(GO:0032880)and pulmonary vascular development(GO: 0060426),which was consistent with the behavioral activity of stromal structures.The presence of PI3 K signaling pathway(hsa04151),keratinization formation(GO: 0031424),and positive regulation of osteoblast differentiation were enriched in the SR region(GO.0045669),suggesting that this region may be a differentiatial transit station of wholrs-like cell clusters into wet keratin.Whorl-like cell cluster was very active,and various types of tumor signaling pathways were present,such as hsa05200,hsa05226,hsa05224,hsa04010,and the classical WNT signaling pathway,Ras signaling pathway and inflammatory responses were also present,which further suggested that the FP may be the original portion of CPs.Conclusions1.“Eagle sigh” is an effective and accurate tool for prediction the relationship between CP and hypothalamus.If the preoperative MRI of CP shows upward “eagle sign”,it implies that the relationship between the CP and hypothalamus is adherence.If the preoperative MRI of CP shows non-upward “eagle sign”,it implies that the relationship of them is infiltration.2.The CP only invades a specific site of hypothalamic-pituitary axis with FP structure,and other structures,other than this specific site,would only be adherent by the CP.The FP that invade the hypothalamus is more,longer,invading the hypothalamus deeply,and smaller than the FP that invade the pituitary stalk.The latter would be relatively less,shorter and lager,and invade the PS shallowly.The “Eagle sigh” can predict both the relationship between the CP and the surrounding structures and the origin of the CP.3.Whorl-like cell cluster is the core site of CP.In addition to the presence of moreβ-catenin and stem cell marker expression,there are several tumoral signaling pathways,which may be the main target of tumor therapy and further confirm that the FP is the portion of tumor origin.PE area proliferates significantly,which may be derived from the paracrine signals of the whorl-like cell cluster and may have a signaling dialogue with its adjacent glia.SR may be a transit point for differentiation of stem cells from whorl-like cell clusters to keratin.There is an extensive immunoinflammatory response in ACP,which may attribute to the continuous keratinocyte deposition.