Normal Gland Tissue Research Articles

Early Screening and Identification of an Asymptomatic Pheochromocytoma in a Child with Von Hippel-Lindau: A Case Report

Introduction: Von Hippel-Lindau (VHL) is a diagnosis that leads to increased risk of tumor development over the course of a patient’s lifetime. Patients with VHL undergo screening for multiple tumor types, including pheochromocytomas (PCCs). There is variability among the different international guidelines regarding the age to begin PCC screening, with most suggesting 5 years for screening initiation. Case Presentation: Our patient is a 4-year-old female who underwent screening for PCC at the time of her VHL diagnosis while asymptomatic that identified a unilateral PCC. This was amendable to treatment with laparoscopic partial adrenalectomy. Conclusion: This is the first report of an asymptomatic PCC being identified in a patient under the age of 5. With early identification, this was treated surgically before it caused systemic symptoms with preservation of normal adrenal gland tissue. This supports adhering to the pediatric specific guidelines which recommend earlier initiation of PCC screening in pediatric patients with VHL.

KMT2A and chronic inflammation as potential drivers of sporadic parathyroid adenoma.

Sporadic parathyroid adenoma (PA) is the most common cause of hyperparathyroidism, yet the mechanisms involved in its pathogenesis remain incompletely understood. Surgically removed PA samples, along with normal parathyroid gland (PG) tissues that were incidentally dissected during total thyroidectomy, were analysed using single-cell RNA-sequencing with the 10× Genomics Chromium Droplet platform and Cell Ranger software. Gene set variation analysis was conducted to characterise hallmark pathway gene signatures, and single-cell regulatory network inference and clustering were utilised to analyse transcription factor regulons. Immunohistochemistry and immunofluorescence were performed to validate cellular components of PA tissues. siRNA knockdown and gene overexpression, alongside quantitative polymerase chain reaction, Western blotting and cell proliferation assays, were conducted for functional investigations. There was a pervasive increase in gene transcription in PA cells (PACs) compared with PG cells. This is associated with high expression of histone-lysine N-methyltransferase 2A (KMT2A). High KMT2A levels potentially contribute to promoting PAC proliferation through upregulation of the proto-oncogene CCND2, which is mediated by the transcription factors signal transducer and activator of transcription 3 (STAT3) and GATA binding protein 3 (GATA3). PA tissues are heavily infiltrated with myeloid cells, while fibroblasts, endothelial cells and macrophages in PA tissues are commonly enriched with proinflammatory gene signatures relative to their counterparts in PG tissues. We revealed the previously underappreciated involvement of the KMT2A‒STAT3/GATA3‒CCND2 axis and chronic inflammation in the pathogenesis of PA. These findings underscore the therapeutic promise of KMT2A inhibition and anti-inflammatory strategies, highlighting the need for future investigations to translate these molecular insights into practical applications. Single-cell RNA-sequencing reveals a transcriptome catalogue comparing sporadic parathyroid adenomas (PAs) with normal parathyroid glands. PA cells show a pervasive increase in gene expression linked to KMT2A upregulation. KMT2A-mediated STAT3 and GATA3 upregulation is key to promoting PA cell proliferation via cyclin D2. PAs exhibit a proinflammatory microenvironment, suggesting a potential role of chronic inflammation in PA pathogenesis.

Abstract 1896: Therapeutic potential of TORL-4-500, an antibody-drug conjugate directed against delta like non-canonical notch ligand 1 (DLK1)

Abstract Delta like non-canonical Notch ligand 1 (DLK1) is a transmembrane protein that belongs to the NOTCH non-canonical ligand family. It has been implicated in adipogenesis, the regulation of stem cell pools, tissue differentiation during development, cancer differentiation, and cancer stem-like cell maintenance. DLK1 is highly expressed in adrenocortical, uterine, and testicular cancers as well as in a large portion of pancreatic, sarcoma, liver and squamous lung cancer patient samples. In contrast there is limited normal tissue expression beyond the normal adrenal gland, pituitary, and ovarian tissue samples. This expression profile makes DLK1 an attractive target for development of a therapeutic antibody-drug conjugate (ADC). This study describes the generation and preclinical characterization of TORL-4-500, an ADC consisting of a humanized anti-DLK1 monoclonal antibody coupled to monomethyl auristatin E (MMAE) via a cleavable linker. TORL-4-500 showed strong binding to DLK1 by flow cytometry in DLK1 native and artificial overexpressing cell lines. In contrast no binding was observed in DLK1 non-expressing cell lines. TORL-4-500 exhibits nanomolar binding affinity for both human and cynomolgus monkey DLK1 and is rapidly internalized in DLK1 expressing cells. TORL-4-500 exhibited selective efficacy in cell line xenograft models of DLK1 positive human cancers. Treatment with TORL-4-500 induced significant regressions in four DLK1 expressing human cancer cell line xenograft studies encompassing liver, small cell lung cancer (SCLC) and sarcoma cancer. Furthermore, anti-tumor responses were sustained in each of the DLK1 expressing models for several weeks post-final dose. In the case of the two SCLC cell lines, complete regressions of xenograft tumors were measured out past 100 days in the TORL-4-500 treated animals. No significant impact on xenograft tumor growth was observed in either a DLK1 non-expressing colon cell line xenograft or in a DLK1 non-expressing melanoma xenograft study. Each of the doses tested in this study was well tolerated in mice with no dose-limiting toxicity observed. The nonclinical pharmacokinetics and toxicokinetics of TORL-4-500 were characterized in mice and monkeys and results support dosing in humans. In summary, TORL-4-500 is a novel therapeutic for DLK1 positive cancers and on the basis of these promising preclinical efficacy results, a first in human trial to evaluate safety, tolerability, pharmacokinetics, and antitumor activity of TORL-4-500 has been launched in patients with advanced cancer and is currently ongoing (NCT06005740). Citation Format: Martina S. McDermott, Neil A. O'Brien, Ming Lu, Jun Zhang, KeWei Gong, Benjamin Hoffstrom, Tong Luo, Min Liang, Weiping Jia, Kevin Chau, Leonard Presta, John Glaspy, Dennis J. Slamon. Therapeutic potential of TORL-4-500, an antibody-drug conjugate directed against delta like non-canonical notch ligand 1 (DLK1) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1896.

PLK4 reflects extrathyroidal invasion, high tumor stage and poor prognosis in papillary thyroid carcinoma patients.

Objective: This study aimed to assess the value of PLK4 as a biomarker in papillary thyroid carcinoma (PTC). Methods: This study reviewed 230 PTC patients receiving surgical resections. PLK4 was detected in tumor tissues and samples of normal thyroid gland tissues by immunohistochemistry. Results: PLK4 was elevated in tumor tissues versus normal thyroid gland tissues (p < 0.001). Tumor PLK4 was linked with extrathyroidal invasion (p = 0.036), higher pathological tumor stage (p = 0.030), node stage (p = 0.045) and tumor/node/metastasis stage (p = 0.022) in PTC patients. Tumor PLK4 immunohistochemistry score >3 was linked with shortened disease-free survival (p = 0.026) and overall survival (p = 0.028) and independently predicted poorer disease-free survival (hazard ratio: 2.797; p = 0.040). Conclusion: Tumor PLK4 reflects extrathyroidal invasion, higher tumor stage and shortened survival in PTC.

E-CADERIN, N-CADERIN, SLUG, SNAIL, and TWIST contribute to epithelial-mesenchymal transition in salivary gland tumors.

Transcription factors are important in the epithelial-mesenchymal transition process and are possibly related to the development of a more invasive tumor phenotype. Thus, the objective of this study was to analyze the expression and identify the localization of cellular markers related to the epithelial-mesenchymal transition process in salivary gland tumors. The expression and localization of E-CADERIN, N-CADERIN, SLUG, SNAIL, and TWIST were evaluated, using immunohistochemistry, in 48 salivary gland tumors, being 17 pleomorphic adenomas (PA), 14 adenoid cystic carcinomas (ACC), and 17 mucoepidermoid carcinomas (MEC). these proteins were compared to clinical and histopathologic parameters. normal gland tissues were included for immunohistochemical comparisons. ACC and MEC cases showed higher expression of SNAIL compared to PA. MEC showed high expression of SLUG and TWIST. Low expression of N-CADHERIN, SNAIL, and TWIST in ACC was frequent in T3 and T4. High expression of TWIST in MEC was more frequent at age ≥ 40 years A positive correlation was only observed between N-cadherin/SNAIL in ACC, between SNAIL/TWIST in MEC, and between SLUG/TWIST in PA. This study provided insight into EMT-related proteins (E-cadherin, N-cadherin, SNAIL, SLUG, and TWIST) and their contribution to the maintenance of morphogenesis and the development of the salivary gland tumors and showed a positive correlation among N-CADHERIN/SNAIL in ACC and SNAIL/TWIST in MEC.

Analysis of High-Risk Neuroblastoma Transcriptome Reveals Gene Co-Expression Signatures and Functional Features.

Neuroblastoma represents a neoplastic expansion of neural crest cells in the developing sympathetic nervous system and is childhood's most common extracranial solid tumor. The heterogeneity of gene expression in different types of cancer is well-documented, and genetic features of neuroblastoma have been described by classification, development stage, malignancy, and progression of tumors. Here, we aim to analyze RNA sequencing datasets, publicly available in the GDC data portal, of neuroblastoma tumor samples from various patients and compare them with normal adrenal gland tissue from the GTEx data portal to elucidate the gene expression profile and regulation networks they share. Our results from the differential expression, weighted correlation network, and functional enrichment analyses that we performed with the count data from neuroblastoma and standard normal gland samples indicate that the analysis of transcriptome data from 58 patients diagnosed with high-risk neuroblastoma shares the expression pattern of 104 genes. More importantly, our analyses identify the co-expression relationship and the role of these genes in multiple biological processes and signaling pathways strongly associated with this disease phenotype. Our approach proposes a group of genes and their biological functions to be further investigated as essential molecules and possible therapeutic targets of neuroblastoma regardless of the etiology of individual tumors.

Functioning Endocrine Outcome after Endoscopic Endonasal Transsellar Approach for Pituitary Neuroendocrine Tumors.

The endoscopic endonasal approach (EEA) is a well-established technique for the treatment of pituitary neuroendocrine tumor Preservation of normal gland tissue is crucial to retain effective neuroendocrine pituitary function. The aim of this paper is to analyze pituitary endocrine secretion after EEA for pituitary neuroendocrine tumor to identify potential predictors of functioning gland recovery. Patients who underwent an exclusive EEA for pituitary neuroendocrine tumors between October 2014 and November 2019 were reviewed. Patients were divided into groups according to postoperative pituitary function (Group 1, unchanged; group 2, recovering; group 3, worsening). Among the 45 patients enrolled, 15 presented a silent tumor and showed no hormonal impairment, and 30 patients presented pituitary dysfunction. A total of 19 patients (42.2%) were included in group 1, 12 (26.7%) patients showed pituitary function recovery after surgery (group 2), and 14 patients (31.1%) exhibited the onset of new pituitary deficiency postoperatively (group 3). Younger patients and those with functioning tumor were more likely to have complete pituitary hormonal recovery (p = 0.0297 and p = 0.007, respectively). No predictors of functional gland worsening were identified. EEA for pituitary neuroendocrine tumor is a reliable and safe technique regarding postoperative hormonal function. Preserving pituitary function after tumor resection should be a primary goal in a minimally invasive approach.

Abstract 2437: HMOX1 modulates tumor stemness and metastatic properties in prostate cancer

Abstract Eighty percent of prostate cancers (PCa) metastasize to bone, showing a significant source of patient morbidity. PCa stem-like cells (PCSCs) are capable to quiescently outlast retaining the ability to proliferate and regenerate, consequently remaining able to develop therapy-resistant tumors and metastatic lesions. We have previously reported that the overexpression of heme oxygenase 1 (HO-1), the rate limiting enzyme in heme degradation, leads to a less aggressive PCa phenotype. However, its effect on metastasis-stemness (MS) remains unknown. In this work, we address the biological significance of HO-1 in association with relevant MS genes for PCa progression. Clonogenic assays performed in PCa cells (PC3 and C4-2B) to assess colony formation, evidenced a reduction on the stem-like properties of tumor cells treated with hemin (FDA approved drug and specific HO-1 inducer). RNA-seq analysis to identify MS genes that could be modulated by HO-1 induction, revealed 32 MS-genes that were modulated in PC3-hemin vs. PC3-control cells. We then mined the Oncomine database (n = 1,128) and found that 15/32 HO-1-modulated MS genes were significantly dysregulated in PCa compared with normal gland. To extend our findings, further computational analyses were conducted, and a custom-made bioinformatics tool (Gene Hunter) was created to analyze gene expression and clinicopathological profiles across multiple publicly available PCa datasets with normal, adjacent, tumoral and metastatic samples (n = 1,629). We found that ADAM15, BCL2L1, LTBR, MBNL2 and SPINT1 are consistently dysregulated across different comparisons, with expression profiles in tumors that could be reverted by hemin treatment, according to our RNA-seq results. We constructed a risk score that efficiently stratified patients in high, intermediate and low-risk of biochemical-relapse groups (p&amp;lt;0.01). We also performed multiple survival analyses including, overall, progression-free, relapse-free and metastasis-free survival data. We found that high MBNL2, a gene previously reported to be related to embryonic stem cell differentiation and to antimetastatic effects in other tumors, was associated with lower risk of relapse, progression and metastasis (p&amp;lt;0.05). Of note, the expression of MBNL2 was downregulated in PCa samples compared with normal gland or normal adjacent tissue. Interestingly MBNL2 expression can be reverted by HO-1 induction in PCa cells, as validated by RT-qPCR. Accordingly, those patients with high expression of MBNL2 showed a decreased risk of biochemical-relapse when they presented high HMOX1 levels (HR = 0.4615, p = 0.0186). Altogether, we highlight the relevance of HO-1 in modulating MS-associated genes in PCa and point out to MBNL2 as a potential druggable target for disease intervention. Citation Format: Agustina A. Sabater, Ayelen R. Toro, Pablo A. Sanchis, Gaston M. Pascual, Rocio A. Seniuk, Elba S. Vazquez, Javier Cotignola, Juan A. Bizzotto, Sofia Lage-Vickers, Geraldine Gueron. HMOX1 modulates tumor stemness and metastatic properties in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2437.

Immunolabelling of SCF and c-KIT in canine perianal gland tumours

c-KIT and its ligand stem cell factor (SCF) play a direct role in the oncogenesis of various cancers by regulating the cell fate. Recent evidence indicates that an increased expression of c-KIT/SCF, driven by hormonal imbalances, is an important step in the development of hormone-dependent cancers. We investigated the possible role of the c-KIT/SCF system in the carcinogenesis in 44 perianal gland tumours (16 adenomas, 15 epitheliomas and 13 carcinomas) and 10 normal perianal gland tissues by assessing the percentage and type of cells that expressed c-KIT and SCF as well as the cellular localization of immunoreactivity. No differences in immunolabelling of SCF were found between normal glands and neoplastic cells of any histotype. The highest expression of c-KIT was seen in carcinomas and a positive correlation was found between c-KIT labelling score and mitotic index (R = 0.876; P <0.01). c-KIT labelling patterns in hepatoid cells varied among the tumour histotypes with adenomas having only membranous labelling. Three labelling patterns (membranous only, membranous and cytosolic, and cytosolic only) were seen in the other tumour histotypes. Cytosolic labelling was statistically more frequent in carcinomas than in adenomas (P <0.001). These findings suggest that c-KIT expression and its cellular localization may play a role in the development and progression of perianal gland tumours by influencing cell proliferation.

AHNAK2 is a biomarker and a potential therapeutic target of adenocarcinomas.

Adenocarcinoma is the second largest histological type of cervical cancer, second only to cervical squamous cell carcinoma. At present, despite the clinical treatment strategies of cervical adenocarcinoma and cervical squamous cell carcinoma being similar, the outcome and prognosis of cervical adenocarcinoma are significantly poor. Therefore, it is urgent to find specific biomarker and therapeutic target for cervical adenocarcinoma. In this study, we aim to reveal and verify the potential biomarkers and therapeutic targets of cervical adenocarcinoma. Weighted correlation network analysis (WGCNA) reveals the differentially-expressed genes significantly related to the histological characteristics of the two cervical cancer subtypes. We select the genes with the top 20 significance for further investigation. Through microarray and immunohistochemical (IHC) analyses of a variety of tumor tissues, we find that among these 20 genes, AHNAK2 is highly expressed not only in cervical adenocarcinoma, but also in multiple of adenocarcinoma tissues, including esophagus, breast and colon, while not in normal gland tissues. In vitro, AHNAK2 knockdown significantly inhibits cell proliferation and migration of adenocarcinoma cell lines. In vivo, AHNAK2 knockdown significantly inhibits tumor progression and metastasis of various adenocarcinomas. RNA-sequencing and bioinformatics analyses suggest that the inhibitory effect of AHNAK2 knockdown on tumor progression is achieved by regulating DNA replication and upregulating Bim expression. Together, we demonstrate that AHNAK2 is a biomarker and a potential therapeutic target for adenocarcinomas.

Carcinoma Epitelial - Mioepitelial

Two epithelial - muoepithelial carcinoma of parotid gland from two female patients of 48 and 80 years old, and normal gland tissue, are studied with Del Río Hortega -Polak silver impregnation technique. It is a selective and alternative method to mark myoepithelial cells in selections of paraffin embedding material of in frozen sections.

STUDY OF IMMUNOHISTOCHEMICAL MARKERS RELATED TO EPITHELIAL-MESENCHYMAL TRANSITION IN SALIVARY GLAND TUMORS

Objectives To analyze the protein expression and identify the localization of cellular markers related to the epithelial-mesenchymal transition process in salivary gland tumors. Study Design The expression and localization of E-cadherin, N-cadherin, SLUG, SNAIL, and TWIST were evaluated using immunohistochemistry in 48 salivary gland tumors: 17 pleomorphic adenomas, 14 cystic adenoid carcinomas (CAC), 17 mucoepidermoid carcinomas (MEC), as well as in 7 samples of normal gland tissue. Results A low expression of E-cadherin was observed in most of the evaluated tumors and a high expression of N-cadherin in malignant tumors but without statistical significance. In addition, there were statistically significant differences in the expression of SNAIL (P = .03), SLUG (P = .03), and TWIST (P = .01) in the tumor parenchyma of the MEC, as well as a high expression of the SNAIL (P = .02) in the parenchyma cases of CAC. Conclusions We observed changes in the expression of protein markers, especially SNAIL, SLUG, and TWIST, in the evaluated malignant tumors. These transcription factors are important in the epithelial-mesenchymal transition process and are possibly related to the development of a more invasive tumor phenotype. To analyze the protein expression and identify the localization of cellular markers related to the epithelial-mesenchymal transition process in salivary gland tumors. The expression and localization of E-cadherin, N-cadherin, SLUG, SNAIL, and TWIST were evaluated using immunohistochemistry in 48 salivary gland tumors: 17 pleomorphic adenomas, 14 cystic adenoid carcinomas (CAC), 17 mucoepidermoid carcinomas (MEC), as well as in 7 samples of normal gland tissue. A low expression of E-cadherin was observed in most of the evaluated tumors and a high expression of N-cadherin in malignant tumors but without statistical significance. In addition, there were statistically significant differences in the expression of SNAIL (P = .03), SLUG (P = .03), and TWIST (P = .01) in the tumor parenchyma of the MEC, as well as a high expression of the SNAIL (P = .02) in the parenchyma cases of CAC. We observed changes in the expression of protein markers, especially SNAIL, SLUG, and TWIST, in the evaluated malignant tumors. These transcription factors are important in the epithelial-mesenchymal transition process and are possibly related to the development of a more invasive tumor phenotype.

Sprouting Angiogenesis in Human Pituitary Adenomas.

IntroductionAngiogenesis in pituitary tumors is not fully understood, and a better understanding could help inform new pharmacologic therapies, particularly for aggressive pituitary tumors.Materials and Methods219 human pituitary tumors and 12 normal pituitary glands were studied. Angiogenic genes were quantified by an angiogenesis qPCR array and a TaqMan probe-based absolute qPCR. Angiogenesis inhibition in pituitary tumors was evaluated in vitro with the endothelial tube formation assay and in vivo in RbΔ19 mice.Results71 angiogenic genes, 40 of which are known to be involved in sprouting angiogenesis, were differentially expressed in pituitary tumors. Expression of endothelial markers CD31, CD34, and ENG was significantly higher in pituitary tumors, by 5.6, 22.3, and 8.2-fold, respectively, compared to in normal pituitary tissue. There was no significant difference in levels of the lymphatic endothelial marker LYVE1 in pituitary tumors compared with normal pituitary gland tissue. Pituitary tumors also expressed significantly higher levels of angiogenesis growth factors, including VEGFA (4.2-fold), VEGFB (2.2), VEGFC (19.3), PGF (13.4), ANGPT2 (9.2), PDGFA (2.7), PDGFB (10.5) and TGFB1 (3.8) compared to normal pituitary tissue. Expression of VEGFC and PGF was highly correlated with the expression of endothelial markers in tumor samples, including CD31, CD34, and ENG (endoglin, a co-receptor for TGFβ). Furthermore, VEGFR inhibitors inhibited angiogenesis induced by human pituitary tumors and prolonged survival of RbΔ19 mice.ConclusionHuman pituitary tumors are characterized by more active angiogenesis than normal pituitary gland tissue in a manner consistent with sprouting angiogenesis. Angiogenesis in pituitary tumors is regulated mainly by PGF and VEGFC, not VEGFA and VEGFB. Angiogenesis inhibitors, such as the VEGFR2 inhibitor cabozantinib, may merit further investigation as therapies for aggressive human pituitary tumors.

Ultra-High-Field 7 T Magnetic Resonance Imaging Including Dynamic and Static Contrast-Enhanced T1-Weighted Imaging Improves Detection of Secreting Pituitary Microadenomas.

A prospective preoperative evaluation of 7 T ultra-high-field magnetic resonance imaging (MRI) in patients with suspected pituitary microadenomas for both adenoma detection and intrasellar localization compared with 3 T MRI was carried out. Patients underwent prospective preoperative standardized 3 and 7 T MRI. A distinct qualitative (lesion detection, intrasellar lesion location) and quantitative (lesion diameters, T1/T2 signal intensity ratio of the lesion to normal pituitary gland tissue) analysis was performed, along with an evaluation of image quality (IQ) regarding overall IQ, anatomical parameters, and artifacts; the findings of the qualitative analysis were compared with intraoperative findings and endocrinological outcomes. Sixteen patients (mean age, 43 ± 16 years; 13 women) with pituitary microadenomas were included. Using 7 T MRI allowed the detection of 15 microadenomas-3 more than 3 T MRI. In addition, 7 T MRI allowed more precise lesion localization with 93.75% (15/16) agreement with intraoperative findings, compared with 75% (12/16) agreement using 3 T MRI. Lesion diameters showed no significant difference between 3 and 7 T MRI. T1 and T2 signal intensity ratio between microadenomas and normal pituitary gland tissue were higher in 7 T MRI than in 3 T MRI. The overall IQ and the IQ of each anatomical parameter of 7 T MRI were rated higher than those of 3 T MRI. No significant differences in susceptibility or head motion artifacts were observed between 3 and 7 T MRI; however, 7 T MRI was more susceptible to pulsation artifacts. Ultra-high-field MRI surpasses 3 T MRI in pituitary microadenoma detection and enables more precise delineation with higher correlation with intraoperative findings. Thus, 7 T sellar imaging is a promising option-especially in previously magnetic resonance-negative patients with endocrinologically confirmed hormone oversecretion-and helps reduce the need for invasive diagnostics.

ErbB4 Is a Potential Key Regulator of the Pathways Activated by NTRK-Fusions in Thyroid Cancer

NTRK gene fusions are drivers of tumorigenesis events that specific Trk-inhibitors can target. Current knowledge of the downstream pathways activated has been previously limited to the pathways of regulator proteins phosphorylated directly by Trk receptors. Here, we aimed to detect genes whose expression is increased in response to the activation of these pathways. We identified and analyzed differentially expressed genes in thyroid cancer samples with NTRK1 or NTRK3 gene fusions, and without any NTRK fusions, versus normal thyroid gland tissues, using data from the Cancer Genome Atlas, the DESeq2 tool, and the Genome Enhancer and geneXplain platforms. Searching for the genes activated only in samples with an NTRK fusion as opposed to those without NTRK fusions, we identified 29 genes involved in nervous system development, including AUTS2, DTNA, ERBB4, FLRT2, FLRT3, RPH3A, and SCN4A. We found that genes regulating the expression of the upregulated genes (i.e., upstream regulators) were enriched in the “signaling by ERBB4” pathway. ERBB4 was also one of three genes encoding master regulators whose expression was increased only in samples with an NTRK fusion. Moreover, the algorithm searching for positive feedback loops for gene promoters and transcription factors (a so-called “walking pathways” algorithm) identified the ErbB4 protein as the key master regulator. ERBB4 upregulation (p-value = 0.004) was confirmed in an independent sample of ETV6-NTRK3-positive FFPE specimens. Thus, ErbB4 is the potential key regulator of the pathways activated by NTRK gene fusions in thyroid cancer. These results are preliminary and require additional biochemical validation.

Identification of Early Diagnostic and Prognostic Biomarkers via WGCNA in Stomach Adenocarcinoma.

Stomach adenocarcinoma (STAD) is a leading cause of cancer deaths, and the outcome of the patients remains dismal for the lack of effective biomarkers of early detection. Recent studies have elucidated the landscape of genomic alterations of gastric cancer and reveal some biomarkers of advanced-stage gastric cancer, however, information about early-stage biomarkers is limited. Here, we adopt Weighted Gene Co-expression Network Analysis (WGCNA) to screen potential biomarkers for early-stage STAD using RNA-Seq and clinical data from TCGA database. We find six gene clusters (or modules) are significantly correlated with the stage-I STADs. Among these, five hub genes, i.e., MS4A1, THBS2, VCAN, PDGFRB, and KCNA3 are identified and significantly de-regulated in the stage-I STADs compared with the normal stomach gland tissues, which suggests they can serve as potential early diagnostic biomarkers. Moreover, we show that high expression of VCAN and PDGFRB is associated with poor prognosis of STAD. VCAN encodes a large chondroitin sulfate proteoglycan that is the main component of the extracellular matrix, and PDGFRB encodes a cell surface tyrosine kinase receptor for members of the platelet-derived growth factor (PDGF) family. Consistently, Gene Ontology (GO) analysis of differentially expressed genes in the STADs indicates terms associated with extracellular matrix and receptor ligand activity are significantly enriched. Protein-protein network interaction analysis (PPI) and Gene Set Enrichment Analysis (GSEA) further support the core role of VCAN and PDGFRB in the tumorigenesis. Collectively, our study identifies the potential biomarkers for early detection and prognosis of STAD.

Ultrasound-Guided Biopsy of Suspected Salivary Gland Lymphoma in Sjögren's Syndrome.

To evaluate the safety and utility of core needle biopsy (CNB) for diagnosis of salivary gland lymphoma in Sjögren's syndrome (SS). We analyzed data from consecutive SS patients who underwent ultrasound-guided major salivary gland CNB for lymphoma diagnosis and determined whether CNB yielded an actionable diagnosis without need for further intervention. CNBs were performed in 24 patients to evaluate discrete parotid (n = 6) or submandibular (n = 2) gland masses or diffuse enlargement (n = 16; 15 parotid). One patient had 3 CNBs of the same mass. Of the 26 CNBs, 24 included flow cytometry, using CNB and/or fine needle aspirate material, and 14 targeted sonographically identified focal lesions. No patient reported complications. In the 23 patients with 1 CNB, final diagnoses were marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT; n = 6), atypical lymphoid infiltration (n = 3), benign lymphoepithelial sialadenitis (n = 9), normal gland tissue (n = 4), and lymphoepithelial cyst (n = 1). In the patient with serial CNBs, the initial one without flow cytometry was benign, but the next 2 showed atypical lymphoid infiltration. Monoclonal lymphoid infiltration was detected in 12 patients: 6 with MALT lymphoma, 3 were benign, and 3 with atypical lymphoid infiltration. Of the latter 3, 1 was treated with rituximab and 2 with expectant observation. The diagnosis changed from atypical lymphoid infiltration to MALT lymphoma in 1 patient following biopsy of inguinal adenopathy 6 months post-CNB. CNB provided actionable results and avoided open excisional biopsies in all cases. CNB is safe and useful in the evaluation of suspected salivary gland lymphoma in SS.

A potential impact of SARS-CoV-2 on pituitary glands and pituitary neuroendocrine tumors

IntroductionAngiotensin-converting enzyme 2 (ACE2) is the receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The effects of SARS-CoV-2 on normal pituitary glands function or pituitary neuroendocrine tumors (PitNETs) have not yet been elucidated. Thus, the present study aimed to investigate the potential risks of SARS-CoV-2 infection on the impairment of pituitary glands and the development of PitNETs.MethodsPitNETs tissues were obtained from 114 patients, and normal pituitary gland tissues were obtained from the autopsy. The mRNA levels of ACE2 and angiotensin II receptor type 1 (AGTR1) were examined by quantitative real-time PCR. Immunohistochemical staining was performed for ACE2 in 69 PitNETs and 3 normal pituitary glands. The primary tumor cells and pituitary cell lines (MMQ, GH3 and AtT-20/D16v-F2) were treated with diminazene aceturate (DIZE), an ACE2 agonist, with various dose regimens. The pituitary hormones between 43 patients with SARS-CoV-2 infection were compared with 45 healthy controls.ResultsPituitary glands and the majority of PitNET tissues showed low/negative ACE2 expression at both the mRNA and protein levels, while AGTR1 showed high expression in normal pituitary and corticotroph adenomas. ACE2 agonist increased the secretion of ACTH in AtT-20/D16v-F2 cells through downregulating AGTR1. The level of serum adrenocorticotropic hormone (ACTH) was significantly increased in COVID-19 patients compared to normal controls (p < 0.001), but was dramatically decreased in critical cases compared to non-critical patients (p = 0.003).ConclusionsThis study revealed a potential impact of SARS-CoV-2 infection on corticotroph cells and adenomas.

Retrospective Analysis of Clinicopathological Characteristics of Lacrimal Gland Pleomorphic Adenoma and Mechanism of Tumorigenesis by the Imbalance Between Apoptosis and Proliferation.

BackgroundLacrimal gland pleomorphic adenoma (LGPA) is the most common clinically benign epithelial tumor of the lacrimal gland and is predominantly comprised of epithelial cells and interstitial components. At present, the exact pathogenesis of LGPA remains unclear. Previous research has indicated that the occurrence of LGPA may be related to excessive cell proliferation.Material/MethodsThis study observed the clinicopathological characteristics of LGPA and investigated the tumorigenesis mechanism of cell over-proliferation caused by the imbalance between apoptosis and proliferation. A total of 27 cases were collected from the Department of Ophthalmology of the Affiliated Hospital of Chengde Medical University and the Third Medical Center of Chinese PLA General Hospital from April 2017 to November 2019. Hematoxylin-eosin (HE) staining and immunohistochemical staining were used to observe the pathological characteristics and analyze the expression of bcl-2 and bax in the lacrimal gland.ResultsCompared with normal lacrimal gland tissues, LGPA tumor tissues had obvious changes in pathological morphology. The expression of bcl-2 in LGPA lesion tissues was dramatically higher (P<0.001), the expression of bax was not significantly different between groups (P=0.25), but the ratio of bcl-2/bax was significantly higher in tumor tissues (P=0.01).ConclusionsWe found that the lacrimal gland tumor tissues had obvious excessive proliferation in pathomorphology, which revealed the necessity of complete surgical removal of the capsule from the perspective of pathological morphology and provided a theoretical basis for the hypothesis that the imbalance between apoptosis and proliferation could lead to cell hyperproliferation.

Pituitary Adenomas Evade Apoptosis via Noxa Deregulation in Cushing's Disease

Introduction: Sporadic pituitary adenomas are common (>10%) in the general population. Microadenomas (< 1 cm across) can become symptomatic with adrenocorticotropin oversecretion causing Cushing’s disease (CD), leading to high morbidity and mortility. Pathogenic mechanisms in a majority of adenomas including wildtype CD adenomas remain unknown. Mechanistic studies are hindered by a lack of in-vitro models and normal human pituitary gland controls. We hypothesized that comparative analysis of human adenomas with syngeneic normal pituitary glands could reveal pathogenic mechanisms driving CD. Methods: We surgically annotated adenomas and en-route adjacent normal glands in 34 patients. We performed single cell RNAseq and captured 28,724 cells in 6 patients (3 CD and 3 non-CD; 5 pairwise), bulk RNAseq in 7 patients (5 CD and 2 non-CD; 5 pairwise) and DNA methylation analysis in 6 patients (3 CD and 3 non-CD). We confirmed the mechanisms of sporadic CD adenoma pathogenesis in 2 additional syngeneic pairs of human cell lines and using a novel murine normal corticotroph-enriched cell line. Results: Using marker-based classification, we confirmed canonical resident cell classes including corticotrophs in the human pituitary gland. We found SOX2+/S100B+ folliculostellate cells which play a role in pituitary cell replenishment and homeostasis in the normal gland but not in the adenoma compartment. Within the terminally differentiated corticotrophs in the adenoma compartment, we discovered a subset of proliferating cells with upregulation of canonical oncogenes, histone modifiers and DNA repair genes. We also found that consensus dominant genes including PMAIP1 (encoding the pro-apoptotic BH3 only Bcl-2 protein noxa) sharply demarcated adenoma cells from normal gland. The PMAIP1 locus was hypomethylated in CD adenomas compared to non-CD samples. However, despite epigenetic and transcriptional upregulation, we found proteasomal degradation of noxa protein in CD adenomas. Using the selective proteasomal inhibitor bortezomib, we were able to rescue noxa ex-vivo and in-vitro, induce apoptosis, and reduce cell survival of adenoma cell lines. Conclusions: We defined the transcriptionl and DNA methylation landscape of human pituitary adenomas compared to adjacent normal gland tissue. We identified proteasomal-mediated noxa degradation despite PMAIP1 overexpression as an escape mechanism from apoptosis in adenomatous corticotrophs. The proteasomal inhibitor bortezomib rescued noxa and induced apoptosis in adenoma cells, highlighting its potential as a novel therapeutic strategy in CD.