Clinical Classifications Of Suprasellar Invasion Of Pituitary Adenomas And Basic Research On The Regional Heterogeneity Of Invasive Pituitary Adenomas

Part Ⅰ The classifications of pituitary adenoma with suprasellar extension: surgical strategies and outcomesObjective: Suprasellar pituitary adenomas（PAs）can be located in either extradural or intradural spaces,which impacts surgical strategies and outcomes.This study determined how to distinguish these two different types of PAs and analyzed their corresponding surgical strategies and outcomes.Methods: We retrospectively analyzed 389 patients who underwent surgery for PAs with suprasellar extension between 2016 to 2020 at our center.PAs were classified into three grades according to tumor topography and their relationships to the diaphragm sellae（DS）and DS-attached residual pituitary gland（PG）.Grade 1a:PAs presented as an “inflated balloon” with expansive growth toward the suprasellar region.The suprasellar aspects of the tumors were regularly shaped and smooth with clear borders.The arteries of the circle of Willis were located at the edge of these tumors and still outside the DS.Grade 1b: The suprasellar portion of the adenomas had an asymmetrically lobulated appearance,with clear borders.Blood vessels were also often located at the edges of the tumor and still outside the DS.Grade 2: The tumor broke through the DS growing into the suprasellar region.These tumors grew along the suprasellar cistern and encircled the neurovascular structures.In these tumors,the shape of the suprasellar adenoma was irregular and matched the suprasellar cistern morphology.Tumor characteristics,surgical strategies,and outcomes were analyzed.Results: Of 389 PAs,292（75.1%）were surrounded by a bilayer structure formed by the DS and the residual PG and classified as grade 1a,63（16.2%）had lobulated or daughter tumors resulting from the thinning or absence of the residual PG and subsequently rendering the bilayer weaker were classified as Grade 1b,and the remaining 34（8.7%）PAs that broke through the DS or traversed the diaphragmic opening and encased suprasellar neurovascular structures were classified as Grade 2.We found that the gross total removal of the suprasellar part of grade 1a,1b,and 2PAs decreased with grading（88.4%,71.4%,and 61.8%,respectively）.The rate of major operative complications,including cerebrospinal fluid leakage,hemorrhage,and death,increased with grading.Conclusions: It is essential to identify whether PAs with suprasellar extension are located extradurally or intradurally,which depends on whether the bilayer structure is intact.PAs with an intact bilayer structure were classified as grade 1.These were extradural and usually had good surgical outcomes and lower complications.PAs with no bilayer structure surrounding them were classified as grade 2.These were intradural,connected to the cranial cavity,and had increased surgical complications and a lower rate of gross total removal.Different surgical strategies should be adopted for extradural and intradural PAs.Part Ⅱ Basic research on the regional heterogeneity of invasive pituitary adenomas by single-cell transcriptome sequencingObjective: The mechanism by which PAS develop invasiveness remains poorly understood,and there are currently no biomarkers that reliably predict the aggressive behavior of PAS.Moreover,it is still unclear whether there are different microenvironments in different parts of the tumor,such as intrasellar and suprasellar tumors,in the process of invasive pituitary adenoma invasion into the perisellar region.In recent years,with the development of technologies,single-cell transcriptome sequencing（sc RNA-seq）has enabled the characterization of molecular features within individual cells,provided high-resolution data to study cellular heterogeneity,and provided new opportunities to understand the dynamic tumor microenvironment system composed of tumor cells,stromal cells,and immune cells.The use of sc RNA-seq technology for tumor heterogeneity is overwhelming,but less is known about single cells in aggressive pituitary adenomas.Therefore,in this study,scRNA-seq was used to investigate the internal cellular composition and gene expression of invasive PAS at the single-cell level,to investigate the differences in the presence of tumors at different sites,and to find the mechanism of invasiveness of invasive pas.Methods: Fresh pituitary tumor tissue samples（Knosp 4 in Knosp grading system）resected intraoperatively from 1 patient（55 years old,female）,who was clinically diagnosed as a nonfunctioning pituitary adenoma（silent ACTH adenoma）.Two tumor samples,one located intrasellar（P1₁）and one located cavernous sinus（P1₂）,were retained,followed by single-cell suspension preparation,experimental procedures such as library construction,and post sequencing data quality control and analysis.Results: After quality control of the raw data obtained by sequencing,17091 high-quality cells were obtained,and a total of 11 cell subgroups were obtained after the annotation of the dimensional reduction clusters: epithelial cells,macrophages,monocytes,T lymphocytes,NK cells,B lymphocytes,plasma cells,neutrophils,cycling cells,endothelial cells,and fibroblasts.All epithelial cells expressed the pituitary ACTH cell lineage signature genes TBX19 and NEUROD1,and postoperative pathology confirmed a silent ACTH adenoma in this patient.The percentage of epithelial cells was more than 60% in both samples,while the percentage of stromal and immune cells was relatively lower.Moreover,the percentage of tumor epithelial cells was higher in the cavernous sinus,while the percentage of immune cells was decreased.Comparison of differentially expressed genes in tumor epithelial cells between the intrasellar and cavernous sinus revealed that these differentially expressed genes were mainly enriched in endoplasmic reticulum stress and hypoxia-related pathways.Further analysis of subsets of tumor-associated macrophages（TAMs）showed that TAMs could be divided into three subsets,C1Q+ TAMs,FCN1+ TAMs and SPP1+ TAMs,with the three subsets accounting for different proportions in the two samples and no SPP1+ TAMs3 subset in the intracavernous sinus tumors.Cell communication analysis suggested that PAs tumor cells were closely associated with stromal cells and less associated with other immune cells except TAMs,perhaps as one of the potential mechanisms of immune escape of PAs.These results suggest the complexity and heterogeneity of the immune microenvironment of PAs.Copy number variation analysis of epithelial cells revealed heterogeneity within the tumor tumor.Conclusions: In this study,we have mapped the single cell landscape of an aggressive silent ACTH pituitary adenoma at the single-cell level,characterized the composition of the immune microenvironment,and revealed heterogeneity among different sites of the pituitary adenoma,these results provide a rationale and new ideas for the study of invasive pituitary adenomas.However,the molecular mechanism and clinical significance of the heterogeneous formation of PAs remain unclear,and the remodelling effect of PAs cells on the immune microenvironment and the molecular mechanisms remain unknown for further study.