Digital image analysis allows objective stratification of patients with silent PIT1-lineage pituitary neuroendocrine tumors.

Studies describing the clinical presentation and prognosis of patients with silent PIT1 (pituitary specific transcription factor)-lineage pituitary neuroendocrine tumors (PitNETs) are rare. We identified patients with positive PIT1 tumor staining but without evidence of hormone hypersecretion at a tertiary center. Clusters were obtained according to cell morphology and immunostaining from each patient's digitally segmented whole slide image. We compared the clinical presentations, radiological features, and prognoses of the different clusters. We identified 146 patients (68 male, 42.9 ± 14.1 years old) with silent PIT1-lineage PitNETs. Morphology clustering suggested that tumors with large nuclei and apparent eccentricity were associated with a higher proportion of aggressiveness and a higher hazard of recurrence [hazard ratio (HR): 2.64, (95% CI, 1.06-6.55), p = 0.037]. Immunohistochemical clustering suggested that tumors with thyroid stimulating hormone (TSH) staining or all negative PIT1-lineage hormones were associated with a higher proportion of aggressiveness and a higher risk of recurrence [HR: 12.4, (95% CI, 1.60-93.5), p = 0.015]. We obtained three-tier risk profiles by combining morphological and immunohistochemical clustering. Patients with the high-risk profile presented the highest recurrence rate compared with those in the medium-risk and low-risk profiles [HR: 3.54, (95% CI, 1.40-8.93), p = 0.002]. In conclusion, digital image analysis based on cell morphology and immunohistochemical staining allows objective stratification of patients with silent PIT1-lineage tumors. Typical morphological characteristics of high-risk tumors are large tumor nuclei and high eccentricity, and typical immunostaining characteristics are TSH staining or negative staining for all PIT1-lineage hormones.

Characterization of Somatotrope Cell Expansion in Response to GHRH in the Neonatal Mouse Pituitary.

In humans and mice, loss-of-function mutations in growth hormone-releasing hormone receptor (GHRHR) cause isolated GH deficiency. The mutant GHRHR mouse model, GhrhrLit/Lit (LIT), exhibits loss of serum GH, but also fewer somatotropes. However, how loss of GHRH signaling affects expansion of stem and progenitor cells giving rise to GH-producing cells is unknown. LIT mice and wild-type littermates were examined for differences in proliferation and gene expression of pituitary lineage markers by quantitative reverse transcription polymerase chain reaction and immunohistochemistry at postnatal day 5 (p5) and 5 weeks. At p5, the LIT mouse shows a global decrease in pituitary proliferation measured by proliferation marker Ki67 and phospho-histone H3. This proliferative defect is seen in a pituitary cell expressing POU1F1 with or without GH. SOX9-positive progenitors show no changes in proliferation in p5 LIT mice. Additionally, the other POU1F1 lineage cells are not decreased in number; rather, we observe an increase in lactotrope cell population as well as messenger RNA for Tshb and Prl. In the 5-week LIT pituitary, the proliferative deficit in POU1F1-expressing cells observed neonatally persists, while the number and proliferative proportion of SOX9 cells do not appear changed. Treatment of cultured pituitary explants with GHRH promotes proliferation of POU1F1-expressing cells, but not GH-positive cells, in a mitogen-activated protein kinase-dependent manner. These findings indicate that hypothalamic GHRH targets proliferation of a POU1F1-positive cell, targeted to the somatotrope lineage, to fine tune their numbers.

Single-cell sequencing identifies differentiation-related markers for molecular classification and recurrence prediction of PitNET.

Pituitary neuroendocrine tumor (PitNET) is one of the most common intracranial tumors with variable recurrence rate. Currently, the recurrence prediction is unsatisfying and can be improved by understanding the cellular origins and differentiation status. Here, to comprehensively reveal the origin of PitNET, we perform comparative analysis of single-cell RNA sequencing data from 3 anterior pituitary glands and 21 PitNETs. We identify distinct genes representing major subtypes of well and poorly differentiated PitNETs in each lineage. To further verify the predictive value of differentiation biomarkers, we include an independent cohort of 800 patients with an average follow-up of 7.2 years. In both PIT1 and TPIT lineages, poorly differentiated groups show significantly higher recurrence rates while well-differentiated groups show higher recurrence rates in SF1 lineage. Our findings reveal the possible origin and differentiation status of PitNET based on which new differentiation classification is proposed and verified to predict tumor recurrence.

Integrated proteogenomic characterization across major histological types of pituitary neuroendocrine tumors.

Pituitary neuroendocrine tumor (PitNET) is one of the most common intracranial tumors. Due to its extensive tumor heterogeneity and the lack of high-quality tissues for biomarker discovery, the causative molecular mechanisms are far from being fully defined. Therefore, more studies are needed to improve the current clinicopathological classification system, and advanced treatment strategies such as targeted therapy and immunotherapy are yet to be explored. Here, we performed the largest integrative genomics, transcriptomics, proteomics, and phosphoproteomics analysis reported to date for a cohort of 200 PitNET patients. Genomics data indicate that GNAS copy number gain can serve as a reliable diagnostic marker for hyperproliferation of the PIT1 lineage. Proteomics-based classification of PitNETs identified 7 clusters, among which, tumors overexpressing epithelial-mesenchymal transition (EMT) markers clustered into a more invasive subgroup. Further analysis identified potential therapeutic targets, including CDK6, TWIST1, EGFR, and VEGFR2, for different clusters. Immune subtyping to explore the potential for application of immunotherapy in PitNET identified an association between alterations in the JAK1-STAT1-PDL1 axis and immune exhaustion, and between changes in the JAK3-STAT6-FOS/JUN axis and immune infiltration. These identified molecular markers and alternations in various clusters/subtypes were further confirmed in an independent cohort of 750 PitNET patients. This proteogenomic analysis across traditional histological boundaries improves our current understanding of PitNET pathophysiology and suggests novel therapeutic targets and strategies.

Single-cell transcriptome reveals cellular hierarchies and guides p-EMT-targeted trial in skull base chordoma.

Skull base chordoma (SBC) is a bone cancer with a high recurrence rate, high radioresistance rate, and poorly understood mechanism. Here, we profiled the transcriptomes of 90,691 single cells, revealed the SBC cellular hierarchies, and explored novel treatment targets. We identified a cluster of stem-like SBC cells that tended to be distributed in the inferior part of the tumor. Combining radiated UM-Chor1 RNA-seq data and in vitro validation, we further found that this stem-like cell cluster is marked by cathepsin L (CTSL), a gene involved in the packaging of telomere ends, and may be responsible for radioresistance. Moreover, signatures related to partial epithelial-mesenchymal transition (p-EMT) were found to be significant in malignant cells and were related to the invasion and poor prognosis of SBC. Furthermore, YL-13027, a p-EMT inhibitor that acts through the TGF-ß signaling pathway, demonstrated remarkable potency in inhibiting the invasiveness of SBC in preclinical models and was subsequently applied in a phase I clinical trial that enrolled three SBC patients. Encouragingly, YL-13027 attenuated the growth of SBC and achieved stable disease with no serious adverse events, underscoring the clinical potential for the precision treatment of SBC with this therapy. In summary, we conducted the first single-cell RNA sequencing of SBC and identified several targets that could be translated to the treatment of SBC.

Low-rank fusion convolutional neural network for prediction of remission after stereotactic radiosurgery in patients with acromegaly: a proof-of-concept study.

Artificial intelligence approaches to analyze pathological images (pathomic) for outcome prediction have not been sufficiently considered in the field of pituitary research. A total of 5,504 hematoxylin & eosin-stained pathology image tiles from 58 acromegalic patients with a good or poor outcome were integrated with other clinical and genetic information to train a low-rank fusion convolutional neural network (LFCNN). The model was externally validated in 1,536 patches from an external cohort. The primary outcome was the time to the first endocrine remission after stereotactic radiosurgery (SRS). The median time of initial endocrine remission was 43 months (interquartile range [IQR]: 13-60 months) after SRS, and the 24-month initial cumulative remission rate was 57.9% (IQR: 46.4-72.3%). The patient-wise accuracy of the LFCNN model in predicting the primary outcome was 92.9% in the internal test dataset, and the sensitivity and specificity were 87.5 and 100.0%, respectively. The LFCNN model was a strong predictor of initial cumulative remission in the training cohort (hazard ratio [HR] 9.58, 95% confidence interval [CI] 3.89-23.59; p < 0.001) and was higher than that of established prognostic markers. The predictive value of the LFCNN model was further validated in an external cohort (HR 9.06, 95% CI 1.14-72.25; p = 0.012). In this proof-of-concept study, clinically and genetically useful prognostic markers were integrated with digital images to predict endocrine outcomes after SRS in patients with active acromegaly. The model considerably outperformed established prognostic markers and can potentially be used by clinicians to improve decision-making regarding adjuvant treatment choices. © 2022 The Pathological Society of Great Britain and Ireland.

Transcription Factor ASCL1 Acts as a Novel Potential Therapeutic Target for the Treatment of the Cushing's Disease.

BACKGROUND: The pathogenesis of Cushing's disease (CD) is still not adequately understood despite the identification of somatic driver mutations in USP8, BRAF, and USP48. In this multiomics study, we combined RNA sequencing (RNA-seq) with Sanger sequencing to depict transcriptional dysregulation under different gene mutation backgrounds. Furthermore, we evaluated the potential of achaete-scute complex homolog 1 (ASCL1), a pioneer transcription factor, as a novel therapeutic target for treatment of CD and its possible downstream pathway. METHODS: RNA-seq was adopted to investigate the gene expression profile of CD, and Sanger sequencing was adopted to detect gene mutations. Bioinformatics analysis was used to depict transcriptional dysregulation under different gene mutation backgrounds. The function of ASCL1 in hormone secretion, cell proliferation, and apoptosis were studied in vitro. The effectiveness of an ASCL1 inhibitor was evaluated in primary CD cells, and the clinical relevance of ASCL1 was examined in 68 patients with CD. RNA-seq in AtT-20 cells on Ascl1 knockdown combined with published chromatin immunoprecipitation sequencing data and dual luciferase assays were used to explore downstream pathways. RESULTS: ASCL1 was exclusively overexpressed in USP8-mutant and wild-type tumors. Ascl1 promoted adrenocorticotrophin hormone overproduction and tumorigenesis and directly regulated Pomc in AtT-20 cells. An ASCL1 inhibitor presented promising efficacy in both AtT-20 and primary CD cells. ASCL1 overexpression was associated with a larger tumor volume and higher adrenocorticotrophin secretion in patients with CD. CONCLUSION: Our findings help to clarify the pathogenesis of CD and suggest that ASCL1 is a potential therapeutic target the treatment of CD. SUMMARY: The pathogenesis of Cushing's disease (CD) is still not adequately understood despite the identification of somatic driver mutations in USP8, BRAF, and USP48. Moreover, few effective medical therapies are currently available for the treatment of CD. Here, using a multiomics approach, we first report the aberrant overexpression of the transcription factor gene ASCL1 in USP8-mutant and wild-type tumors of CD. Ascl1 promoted adrenocorticotrophin hormone overproduction and tumorigenesis and directly regulated Pomc in mouse AtT-20 cells. Notably, an ASCL1 inhibitor presented promising efficacy in both AtT-20 and primary CD cells. Importantly, ASCL1 overexpression was associated with a larger tumor volume and higher adrenocorticotrophin secretion in patients with CD. Thus, our findings improve understanding of CD pathogenesis and suggest that ASCL1 is a potential therapeutic target the treatment of CD.

The role of bone morphogenetic protein 4 in corneal injury repair.

PURPOSE: Corneal injury may cause neovascularization and lymphangiogenesis in cornea which have a detrimental effect to vision and even lead to blindness. Bone morphogenetic protein 4 (BMP4) regulates a variety of biological processes, which is closely relevant to the regulation of corneal epithelium and angiogenesis. Herein, we aimed to evaluate the effect of BMP4 on corneal neovascularization (CNV), corneal lymphangiogenesis (CL), corneal epithelial repair, and the role of BMP4/Smad pathway in these processes. METHODS: We used MTT assay to determine the optimal concentration of BMP4. The suture method was performed to induce rat CNV and CL. We used ink perfusion and HE staining to visualize the morphological change of CNV, and utilized RT-qPCR and ELISA to investigate the expression of angiogenic factors and lymphangiogenic factors. The effects of BMP4 and anti-VEGF antibody on migration, proliferation and adhesion of corneal epithelium were determined by scratch test, MTT assay and cell adhesion test. RESULTS: BMP4 significantly inhibited CNV and possibly CL. Topical BMP4 resulted in increased expression of endogenous BMP4, and decreased expression of angiogenic factors and lymphangiogenic factors. Compared with anti-VEGF antibody, BMP4 enhanced corneal epithelium migration, proliferation and adhesion, which facilitated corneal epithelial injury repair. Simultaneously, these processes could be regulated by BMP4/Smad pathway. CONCLUSIONS: Our results demonstrated unreported effects of BMP4 on CNV, CL, and corneal epithelial repair, suggesting that BMP4 may represent a potential therapeutic target in corneal injury repair.

The best optical zone for small-incision lenticule extraction in high myopic patients.

Small-incision lenticule extraction (SMILE) is an effective and safe procedure for the correction of myopia due to minimally invasive and noncorneal flap surgery. However, the SMILE procedure has certain requirements for corneal cap thickness, attempted refractive correction, residual stromal bed thickness, and optical zone diameter, which sometimes make surgeons hesitant to choose SMILE or other refractive surgeries. The requirements limit its use in patients with high myopia. The purpose of this review was to find the optimal parameters of SMILE through discussing the best optical zone for high myopic patients, the visual quality of different optical zones, the choice of corneal cap thickness, and their effects on corneal biomechanical parameters, so surgeons can provide reference recommendations for patients with high myopia in choosing a reasonable and safe procedure.

Regulation of bone morphogenetic protein 4 on epithelial tissue.

Epithelial tissues provide tissue barriers and specialize in organs and glands. When epithelial homeostasis is physiologically or pathologically stimulated, epithelial cells produce mesenchymal cells through the epithelial-mesenchymal transition, forming new tissues, promoting the cure of diseases or leading to illness. A variety of cytokines are involved in the regulation of epithelial cell differentiation. Bone morphogenetic proteins (BMPs), especially the bone morphogenetic protein 4 (BMP4) has a variety of biological functions and plays a prominent role in the regulation of epithelial cell differentiation. BMP4 is an important regulatory factor of a series of life activities in vertebrates, which is also related to cell proliferation, differentiation and mobility, it also has relation with tumor development. This paper mainly reviews the mechanism of BMP4's regulation on epithelial tissues, as well as its effect on the growth, differentiation, benign lesions and malignant lesions of epithelial tissues, and expounds the function of BMP4 in epithelial tissues, to provide theoretical support for the research on reducing epithelial diseases.

Regulation of matrix metalloproteinases 2 and 9 in corneal neovascularization.

Corneal neovascularization (CNV), a pathological process of angiogenesis, can lead to serious consequences in the cornea. CNV is generally proved to associate with inflammation in the cornea closely, which is mainly elicited by the disruption of equilibrium between angiogenic and antiangiogenic factors. Angiogenic factors including vascular endothelial growth factors (VEGFs), basic fibroblast growth factors (bFGFs), and matrix metalloproteinases (MMPs) are vital factors in the formation of CNV. Especially VEGFs are convinced to be the core angiogenic factors in CNV, and MMPs are proved to exert dual effects on the process. Strikingly, matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) are determined to play key roles in the formation of CNV, while the mechanism is still vague. In this review, the latest researches are reviewed to discuss the role of MMP-2 and MMP-9 in CNV, respectively, and some inhibitors of them are presented. We hope to provide a new direction of drug research for CNV.

MAPK signaling pathways in eye wounds: Multifunction and cooperation.

As is widely distributed in eukaryotic cells, the mitogen-activated protein kinase (MAPK) signaling pathway family plays an inevitable role in diverse cellular processes, being capable of responding to particular physiological reactions induced by multiple extracellular signals or stimuli, such as protean concentrations, ischemia/reperfusion, and inflammation. The physiological reactions mediated by the MAPK signaling pathway contribute to the progression and healing of eye wounds. Meanwhile, several pathways in the MAPK family can cooperate with each other and establish distinct responses to different, or even the same, stimuli and, thus, more attention may be paid to the pathway in future research.