ABSTRACT
Background: Primary breast tumors with neuroendocrine (NE) differentiation are a heterogeneous tumor group with diversity of biological behavior, with poorly defined prevalence and prognosis. Objective: To evaluate the chromogranin, synaptophysin, CD56, INSM1 markers expression prevalence and the association between NE differentiation and tumor molecular type. Material and methods: Observational, cross-sectional study which included 110 breast tissue samples with primary invasive carcinoma. Immunohistochemistry was performed for chromogranin, synaptophysin, CD56 and INMS1 markers. NE differentiation was considered with 10-90% positive cells, and NE tumor with > 90% positive cells. Results: 26.3% showed neuroendocrine differentiation. Out of these, 48.2% were luminal-A type, 24.1% luminal-B, 11.5% HER2neu, 17.2% triple-negative; 1.8% were NE tumors. Tumors were marker positive, and out of these to chromogranin in 24.5%, synaptophysin in 28.2%, CD56 in 2.7%, INSM1 in 16.4%. Synaptophysin was expressed in 17.3% luminal-A type, 6.4% luminal-B, 0.9% HER2neu, 3.6% triple-negative. NE differentiation showed association with synaptophysin expression (r = 0.586, p = 0.0001). Conclusion: The NE differentiation prevalence was 26.3% in primary invasive breast cancers, with luminal-A molecular type predominance.
Introducción: los tumores primarios de mama con diferenciación neuroendócrina (NEBC por sus siglas en inglés) son un grupo heterogéneo de tumores con diversidad de comportamiento biológico, con prevalencia y pronóstico poco definido. Objetivo: evaluar la prevalencia de la expresión los marcadores cromogranina, sinaptofisina, CD56, INSM1 y la asociación entre la diferenciación neuroendócrina y el tipo molecular del tumor. Material y métodos: estudio observacional, transversal que incluyó 110 muestras de tejido mamario con carcinoma invasor primario. Se realizó inmunohistoquímica para los marcadores cromogranina, sinaptofisina, CD56 y INMS1. La presencia 10-90% de células positivas se consideró diferenciación neuroendócrina y tumor neuroendócrino con > 90% de células positivas. Resultados: el 26.3% mostró diferenciación neuroendócrina. De estos, 48.2% fueron tipo luminal-A, 24.1% luminal-B, 11.5% HER2neu y 17.2% triple-negativo; 1.8% resultaron tumores neuroendócrinos. Los tumores presentaron marcadores positivos y de estos, 24.5% fueron a cromogranina, 28.2% a sinaptofisina, 2.7% a CD56 y 16.4% a INSM1. La sinaptofisina se expresó en 17.3% del tipo luminal-A, 6.4% luminal-B, 0.9% HER2neu, 3.6% triple-negativo. La diferenciación neuroendócrina mostró asociación con la expresión de sinaptofisina (r = 0.586, p = 0.0001). Conclusión: la prevalencia de la diferenciación neuroendócrina fue del 26.3% en los cánceres invasores primarios de mama, con predominio en el tipo molecular luminal-A.
Subject(s)
Biomarkers, Tumor , Synaptophysin , Humans , Female , Cross-Sectional Studies , Biomarkers, Tumor/metabolism , Middle Aged , Adult , Synaptophysin/metabolism , Aged , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , CD56 Antigen/metabolism , Immunohistochemistry , Repressor Proteins/metabolism , Chromogranins/metabolism , Receptor, ErbB-2/metabolism , Aged, 80 and overABSTRACT
Diagnosis of biliopancreatic cancers by the available serum tumor markers, imaging, and histopathological tissue specimen examination remains a challenge. Circulating cell-free DNA derived from matched pairs of secretin-stimulated duodenal fluid (DF) and plasma from 10 patients with biliopancreatic diseases and 8 control subjects was analyzed using AmpliSeq™ HD technology for Ion Torrent Next-Generation Sequencing to evaluate the potential of liquid biopsy with DF in biliopancreatic cancers. The median cfDNA concentration was greater in DF-derived than in plasma-derived samples. A total of 13 variants were detected: 11 vs. 1 were exclusive for DF relative to the plasma source, and 1 was shared between the two body fluids. According to the four-tier systems, 10 clinical tier-I-II (76.9%), 1 tier-III (7.7%), and 2 tier-IV (15.4%) variants were identified. Notably, the 11 tier-I-III variants were exclusively found in DF-derived cfDNA from five patients with biliopancreatic cancers, and were detected in seven genes (KRAS, TP53, BRAF, CDKN2A, RNF43, GNAS, and PIK3CA); 82% of the tier-I-III variants had a low abundance, with a VAF < 6%. The mutational profiling of DF seems to be a reliable and promising tool for identifying cancer-associated alterations in malignant cancers of the biliopancreatic tract.
Subject(s)
High-Throughput Nucleotide Sequencing , Mutation , Pancreatic Neoplasms , Humans , Male , Female , Middle Aged , Aged , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/diagnosis , Pancreatic Neoplasms/pathology , High-Throughput Nucleotide Sequencing/methods , Duodenum/metabolism , Duodenum/pathology , Biomarkers, Tumor/genetics , Liquid Biopsy/methods , Adult , Cell-Free Nucleic Acids/genetics , Biliary Tract Neoplasms/genetics , GTP-Binding Protein alpha Subunits, Gs/genetics , GTP-Binding Protein alpha Subunits, Gs/metabolism , ChromograninsABSTRACT
OBJECTIVES: Pseudohypoparathyroidism (PHP) comprises a cluster of heterogeneous diseases characterized by hypocalcemia and hyperphosphatemia due to parathyroid hormone (PTH) resistance. PHP type 1B (PHP1B) is caused by heterozygous maternal deletions within GNAS or STX16. STX16 exon 2-6 deletion is commonly observed in autosomal dominant (AD)-PHP1B, while sporadic PHP1B commonly results from methylation abnormalities of maternal differentially methylated regions and remains unclear at the molecular level. CASE PRESENTATION: A 39-year-old male patient with PHP1B, who had his first seizure at 15 years of age, presented to our hospital. The methylation-specific multiplex ligation-dependent probe amplification results showed a half-reduced copy number of STX16 exon 5-7 and loss of methylation at GNAS exon A/B. His mother also had a half-reduced copy number of STX16 exon 5-7 but with normal methylation of GNAS. His father has a normal copy number of STX16 and normal methylation of GNAS. CONCLUSIONS: For the recognition and early diagnosis of this kind of disease, here we report the clinical symptoms, auxiliary examinations, genetic testing characteristics, and treatment of the patient.
Subject(s)
Exons , Pseudohypoparathyroidism , Syntaxin 16 , Humans , Male , Pseudohypoparathyroidism/genetics , Pseudohypoparathyroidism/complications , Adult , Syntaxin 16/genetics , Exons/genetics , Sequence Deletion , GTP-Binding Protein alpha Subunits, Gs/genetics , Prognosis , Chromogranins/geneticsABSTRACT
Approximately 30%-40% of growth hormone-secreting pituitary adenomas (GHPAs) harbor somatic activating mutations in GNAS (α subunit of stimulatory G protein). Mutations in GNAS are associated with clinical features of smaller and less invasive tumors. However, the role of GNAS mutations in the invasiveness of GHPAs is unclear. GNAS mutations were detected in GHPAs using a standard polymerase chain reaction (PCR) sequencing procedure. The expression of mutation-associated maternally expressed gene 3 (MEG3) was evaluated with RT-qPCR. MEG3 was manipulated in GH3 cells using a lentiviral expression system. Cell invasion ability was measured using a Transwell assay, and epithelial-mesenchymal transition (EMT)-associated proteins were quantified by immunofluorescence and western blotting. Finally, a tumor cell xenograft mouse model was used to verify the effect of MEG3 on tumor growth and invasiveness. The invasiveness of GHPAs was significantly decreased in mice with mutated GNAS compared with that in mice with wild-type GNAS. Consistently, the invasiveness of mutant GNAS-expressing GH3 cells decreased. MEG3 is uniquely expressed at high levels in GHPAs harboring mutated GNAS. Accordingly, MEG3 upregulation inhibited tumor cell invasion, and conversely, MEG3 downregulation increased tumor cell invasion. Mechanistically, GNAS mutations inhibit EMT in GHPAs. MEG3 in mutated GNAS cells prevented cell invasion through the inactivation of the Wnt/ß-catenin signaling pathway, which was further validated in vivo. Our data suggest that GNAS mutations may suppress cell invasion in GHPAs by regulating EMT through the activation of the MEG3/Wnt/ß-catenin signaling pathway.
Subject(s)
Chromogranins , Epithelial-Mesenchymal Transition , GTP-Binding Protein alpha Subunits, Gs , Growth Hormone-Secreting Pituitary Adenoma , Mutation , Neoplasm Invasiveness , RNA, Long Noncoding , GTP-Binding Protein alpha Subunits, Gs/genetics , GTP-Binding Protein alpha Subunits, Gs/metabolism , Animals , Humans , Growth Hormone-Secreting Pituitary Adenoma/genetics , Growth Hormone-Secreting Pituitary Adenoma/pathology , Growth Hormone-Secreting Pituitary Adenoma/metabolism , Mice , Chromogranins/genetics , Chromogranins/metabolism , Epithelial-Mesenchymal Transition/genetics , RNA, Long Noncoding/genetics , Female , Male , Cell Line, Tumor , Adenoma/genetics , Adenoma/pathology , Adenoma/metabolism , Middle Aged , Adult , Cell Proliferation/genetics , Xenograft Model Antitumor Assays , Wnt Signaling Pathway/genetics , Gene Expression Regulation, NeoplasticABSTRACT
Introduction: The dysregulation of cell fate toward osteoprecursor cells associated with most GNAS-based disorders may lead to episodic de novo extraskeletal or ectopic bone formation in subcutaneous tissues. The bony lesion distribution suggests the involvement of abnormal differentiation of mesenchymal stem cells (MSCs) and/or more committed precursor cells. Data from transgenic mice support the concept that GNAS is a crucial factor in regulating lineage switching between osteoblasts (OBs) and adipocyte fates. The mosaic nature of heterotopic bone lesions suggests that GNAS genetic defects provide a sensitized background for ectopic osteodifferentiation, but the underlying molecular mechanism remains largely unknown. Methods: The effect of GNAS silencing in the presence and/or absence of osteoblastic stimuli was evaluated in the human L88/5 MSC line during osteodifferentiation. A comparison of the data obtained with data coming from a bony lesion from a GNAS-mutated patient was also provided. Results: Our study adds some dowels to the current fragmented notions about the role of GNAS during osteoblastic differentiation, such as the premature transition of immature OBs into osteocytes and the characterization of the differences in the deposed bone matrix. Conclusion: We demonstrated that our cell model partially replicates the in vivo behavior results, resulting in an applicable human model to elucidate the pathophysiology of ectopic bone formation in GNAS-based disorders.
Subject(s)
Cell Differentiation , Chromogranins , GTP-Binding Protein alpha Subunits, Gs , Mesenchymal Stem Cells , Osteoblasts , Osteogenesis , Humans , GTP-Binding Protein alpha Subunits, Gs/genetics , GTP-Binding Protein alpha Subunits, Gs/metabolism , Chromogranins/genetics , Cell Differentiation/genetics , Osteogenesis/genetics , Osteoblasts/metabolism , Osteoblasts/cytology , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Gene Silencing , Cell LineABSTRACT
Keloids are pathological scar tissue resulting from skin trauma or spontaneous formation, often accompanied by itching and pain. Although GNAS antisense RNA 1 (GNAS-AS1) shows abnormal upregulation in keloids, the underlying molecular mechanism is unclear. The levels of genes and proteins in clinical tissues from patients with keloids and human keloid fibroblasts (HKFs) were measured using quantitative reverse transcription PCR, western blot and enzyme-linked immunosorbent assay. The features of HKFs, including proliferation and migration, were evaluated using cell counting kit 8 and a wound healing assay. The colocalization of GNAS-AS1 and miR-196a-5p in HKFs was measured using fluorescence in situ hybridization. The relationships among GNAS-AS1, miR-196a-5p and C-X-C motif chemokine ligand 12 (CXCL12) in samples from patients with keloids were analysed by Pearson correlation analysis. Gene interactions were validated by chromatin immunoprecipitation and luciferase reporter assays. GNAS-AS1 and CXCL12 expression were upregulated and miR-196a-5p expression was downregulated in clinical tissues from patients with keloids. GNAS-AS1 knockdown inhibited proliferation, migration, and extracellular matrix (ECM) accumulation of HKFs, all of which were reversed by miR-196a-5p downregulation. Signal transducer and activator of transcription 3 (STAT3) induced GNAS-AS1 transcription through GNAS-AS1 promoter interaction, and niclosamide, a STAT3 inhibitor, decreased GNAS-AS1 expression. GNAS-AS1 positively regulated CXCL12 by sponging miR-196-5p. Furthermore, CXCL12 knockdown restrained STAT3 phosphorylation in HKFs. Our findings revealed a feedback loop of STAT3/GNAS-AS1/miR-196a-5p/CXCL12/STAT3 that promoted HKF proliferation, migration and ECM accumulation and affected keloid progression.
Subject(s)
Cell Proliferation , Chemokine CXCL12 , Fibroblasts , Keloid , MicroRNAs , RNA, Long Noncoding , STAT3 Transcription Factor , Keloid/metabolism , Keloid/genetics , Keloid/pathology , Humans , MicroRNAs/metabolism , MicroRNAs/genetics , STAT3 Transcription Factor/metabolism , STAT3 Transcription Factor/genetics , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Chemokine CXCL12/metabolism , Chemokine CXCL12/genetics , Fibroblasts/metabolism , Cell Movement , Feedback, Physiological , Chromogranins/genetics , Chromogranins/metabolism , Male , Female , GTP-Binding Protein alpha Subunits, Gs/genetics , GTP-Binding Protein alpha Subunits, Gs/metabolism , Signal Transduction , Adult , Cells, Cultured , Up-RegulationSubject(s)
Cognitive Dysfunction , Mutation , Humans , Cognitive Dysfunction/genetics , Chromogranins/genetics , Dystonic Disorders/genetics , Dystonic Disorders/diagnosis , Dystonic Disorders/physiopathology , Dystonia/genetics , Male , Female , Child , Heterozygote , GTP-Binding Protein alpha SubunitsABSTRACT
The study of circulating tumor DNA (ctDNA) plays a pivotal role in advancing precision oncology, providing valuable information for individualized patient care and contributing to the ongoing effort to improve cancer diagnosis, treatment, and management. However, its applicability in pseudomyxoma peritonei (PMP) remains unexplored. In this multicenter retrospective study involving 21 PMP patients, we investigated ctDNA presence in peripheral blood using three distinct methodologies. Despite mucinous tumor tissues exhibiting KRAS and GNAS mutations, ctDNA for these mutations was undetectable in blood samples. In this pilot study, circulating tumor DNA was not detected in blood when the tumor harbored mutations of known significance. In the future, a study with a larger sample size is needed to confirm these findings and to determine whether ctDNA could identify patients at risk for early recurrence and/or systemic metastases.
Subject(s)
Circulating Tumor DNA , Peritoneal Neoplasms , Pseudomyxoma Peritonei , Humans , Pseudomyxoma Peritonei/genetics , Pseudomyxoma Peritonei/blood , Pseudomyxoma Peritonei/pathology , Peritoneal Neoplasms/genetics , Peritoneal Neoplasms/secondary , Peritoneal Neoplasms/blood , Circulating Tumor DNA/genetics , Circulating Tumor DNA/blood , Retrospective Studies , Female , Middle Aged , Male , Aged , GTP-Binding Protein alpha Subunits, Gs/genetics , Chromogranins/genetics , Mutation , Proto-Oncogene Proteins p21(ras)/genetics , Pilot Projects , AdultABSTRACT
Cellular myxoma is a benign soft tissue tumor frequently associated with GNAS mutation that may morphologically resemble low-grade myxofibrosarcoma. This study aimed to identify the undescribed methylation profile of cellular myxoma and compare it to myxofibrosarcoma. We performed molecular analysis on twenty cellular myxomas and nine myxofibrosarcomas and analyzed the results using the methylation-based DKFZ sarcoma classifier. A total of 90% of the cellular myxomas had GNAS mutations (four loci had not been previously described). Copy number variations were found in all myxofibrosarcomas but in none of the cellular myxomas. In the classifier, none of the cellular myxomas reached the 0.9 threshold. Unsupervised t-SNE analysis demonstrated that cellular myxomas form their own clusters, distinct from myxofibrosarcomas. Our study shows the diagnostic potential and the limitations of molecular analysis in cases where morphology and immunohistochemistry are not sufficient to distinguish cellular myxoma from myxofibrosarcoma, particularly regarding GNAS wild-type tumors. The DKFZ sarcoma classifier only provided a valid prediction for one myxofibrosarcoma case; this limitation could be improved by training the tool with a more considerable number of cases. Additionally, the classifier should be introduced to a broader spectrum of mesenchymal neoplasms, including benign tumors like cellular myxoma, whose distinct methylation pattern we demonstrated.
Subject(s)
DNA Copy Number Variations , DNA Methylation , Fibrosarcoma , Myxoma , Humans , Myxoma/genetics , Myxoma/diagnosis , Myxoma/pathology , Fibrosarcoma/genetics , Fibrosarcoma/pathology , Fibrosarcoma/diagnosis , Fibrosarcoma/metabolism , Middle Aged , Female , Aged , Male , Adult , Mutation , Diagnosis, Differential , GTP-Binding Protein alpha Subunits, Gs/genetics , Chromogranins/genetics , Aged, 80 and over , Soft Tissue Neoplasms/genetics , Soft Tissue Neoplasms/diagnosis , Soft Tissue Neoplasms/pathologyABSTRACT
PURPOSE: To summarize the clinical manifestations of craniofacial fibrous dysplasia (CFD) patients with ocular complications, and find effective methods to diagnose early. METHODS: Nine CFD patients with ocular complications, and their parents were recruited in this study. All patients underwent ocular and systemic examinations. Bone lesions from all patients and peripheral blood from patients and their parents were collected for whole exome sequencing (WES). According to the screening for low-frequency deleterious variants, and bioinformatics variants prediction software, possible disease-causing variants were found in multiple CFD patients. The variants were validated by Sanger sequencing. Trio analysis was performed to verify the genetic patterns of CFD. RESULTS: All patients were diagnosed with CFD, according to the clinical manifestations, classic radiographic appearance, and pathological biopsy. The main symptoms of the 9 CFD patients, included visual decline (9/9), craniofacial deformity (3/9) and strabismus (2/9), with few extraocular manifestations. The family backgrounds of all the CFD patients indicated that only the patient was affected, and their immediate family members were normal. GNAS variants were identified in all bone lesions from CFD patients, including two variant types: c.601C > T:p.R201C(6/9) and c.602G > A:p.R201H (3/9) in exon 8. The detection rate reached 100% by WES, but only 77.8% by Sanger sequencing. Interestingly, we found GNAS variants could not be detected in peripheral blood samples from CFD patients or their parents, and other potentially disease-causing gene variants related to CFD were not found. CONCLUSIONS: For CFD patients with bone lesions involving the optic canal or sphenoid sinus regions, ocular symptoms should also be considered. Furthermore, we confirmed that CFD is not inherited, somatic variants in the GNAS gene are the main pathogenic gene causing CFD. Compared to the traditional methods in molecular genetic diagnosis of CFD, WES is more feasible and effective but limited in the type of samples.
Subject(s)
Craniofacial Fibrous Dysplasia , Exome Sequencing , Humans , Male , Female , Child , Adolescent , Craniofacial Fibrous Dysplasia/genetics , Craniofacial Fibrous Dysplasia/diagnosis , Adult , Young Adult , Mutation , GTP-Binding Protein alpha Subunits, Gs/genetics , Chromogranins/genetics , DNA Mutational Analysis , Child, Preschool , Pedigree , Molecular Diagnostic Techniques/methods , Strabismus/genetics , Strabismus/diagnosisABSTRACT
The crosstalk between the chromaffin and adrenocortical cells is essential for the endocrine activity of the adrenal glands. This interaction is also likely important for tumorigenesis and progression of adrenocortical cancer and pheochromocytoma. We developed a unique in vitro 3D model of the whole adrenal gland called Adrenoid consisting in adrenocortical carcinoma H295R and pheochromocytoma MTT cell lines. Adrenoids showed a round compact morphology with a growth rate significantly higher compared to MTT-spheroids. Confocal analysis of differential fluorescence staining of H295R and MTT cells demonstrated that H295R organized into small clusters inside Adrenoids dispersed in a core of MTT cells. Transmission electron microscopy confirmed the strict cell-cell interaction occurring between H295R and MTT cells in Adrenoids, which displayed ultrastructural features of more functional cells compared to the single cell type monolayer cultures. Adrenoid maintenance of the dual endocrine activity was demonstrated by the expression not only of cortical and chromaffin markers (steroidogenic factor 1, and chromogranin) but also by protein detection of the main enzymes involved in steroidogenesis (steroidogenic acute regulatory protein, and CYP11B1) and in catecholamine production (tyrosine hydroxylase and phenylethanolamine N-methyltransferase). Mass spectrometry detection of steroid hormones and liquid chromatography measurement of catecholamines confirmed Adrenoid functional activity. In conclusion, Adrenoids represent an innovative in vitro 3D-model that mimics the spatial and functional complexity of the adrenal gland, thus being a useful tool to investigate the crosstalk between the two endocrine components in the pathophysiology of this endocrine organ.
Subject(s)
Adrenal Gland Neoplasms , Pheochromocytoma , Humans , Adrenal Glands/metabolism , Catecholamines/metabolism , Chromogranins/metabolismABSTRACT
BACKGROUND: Pseudohypoparathyroidism (PHP) is caused by loss-of-function mutations at the GNAS gene (as in the PHP type 1A; PHP1A), de novo or inherited at heterozygous state, or by epigenetic alterations at the GNAS locus (as in the PHP1B). The condition of PHP refers to a heterogeneous group of disorders that share common clinical and biological features of PTH resistance. Manifestations related to resistance to other hormones are also reported in many patients with PHP, in association with the phenotypic picture of Albright hereditary osteodystrophy characterized by short stature, round facies, subcutaneous ossifications, brachydactyly, mental retardation and, in some subtypes, obesity. The purpose of our study is to report a new mutation in the GNAS gene and to describe the significant phenotypic variability of three sisters with PHP1A bearing the same mutation. CASE PRESENTATION: We describe the cases of three sisters with PHP1A bearing the same mutation but characterized by a significantly different phenotypic picture at onset and during follow-up in terms of clinical features, auxological pattern and biochemical changes. Clinical exome sequencing revealed a never before described heterozygote mutation in the GNAS gene (NM_000516.5 c.118_139 + 51del) of autosomal dominant maternal transmission in the three siblings, confirming the diagnosis of PHP1A. CONCLUSIONS: This study reported on a novel mutation of GNAS gene and highlighted the clinical heterogeneity of PHP1A characterized by wide genotype-phenotype variability. The appropriate diagnosis has crucial implications for patient care and long-term multidisciplinary follow-up.
Subject(s)
Chromogranins , GTP-Binding Protein alpha Subunits, Gs , Pseudohypoparathyroidism , Humans , GTP-Binding Protein alpha Subunits, Gs/genetics , Pseudohypoparathyroidism/genetics , Pseudohypoparathyroidism/diagnosis , Chromogranins/genetics , Female , Child , Phenotype , Pedigree , Mutation , Adolescent , Child, PreschoolABSTRACT
OBJECTIVES: Inactivating GNAS mutations result in varied phenotypes depending on parental origin. Maternally inherited mutations typically lead to hormone resistance and Albright's hereditary osteodystrophy (AHO), characterised by short stature, round facies, brachydactyly and subcutaneous ossifications. Paternal inheritance presents with features of AHO or ectopic ossification without hormone resistance. This report describes the case of a child with osteoma cutis and medulloblastoma. The objective of this report is to highlight the emerging association between inactivating germline GNAS mutations and medulloblastoma, aiming to shed light on its implications for tumor biology and promote future development of targeted surveillance strategies to improve outcomes in paediatric patients with these mutations. CASE PRESENTATION: A 12-month-old boy presented with multiple plaque-like skin lesions. Biopsy confirmed osteoma cutis, prompting genetic testing which confirmed a heterozygous inactivating GNAS mutation. At 2.5 years of age, he developed neurological symptoms and was diagnosed with a desmoplastic nodular medulloblastoma, SHH molecular group, confirmed by MRI and histology. Further analysis indicated a biallelic loss of GNAS in the tumor. CONCLUSIONS: This case provides important insights into the role of GNAS as a tumor suppressor and the emerging association between inactivating GNAS variants and the development of medulloblastoma. The case underscores the importance of careful neurological assessment and ongoing vigilance in children with known inactivating GNAS variants or associated phenotypes. Further work to establish genotype-phenotype correlations is needed to inform optimal management of these patients.
Subject(s)
Cerebellar Neoplasms , Chromogranins , GTP-Binding Protein alpha Subunits, Gs , Medulloblastoma , Ossification, Heterotopic , Skin Diseases, Genetic , Humans , GTP-Binding Protein alpha Subunits, Gs/genetics , Male , Chromogranins/genetics , Medulloblastoma/genetics , Medulloblastoma/pathology , Ossification, Heterotopic/genetics , Ossification, Heterotopic/pathology , Skin Diseases, Genetic/genetics , Skin Diseases, Genetic/pathology , Skin Diseases, Genetic/complications , Infant , Cerebellar Neoplasms/genetics , Cerebellar Neoplasms/pathology , Cerebellar Neoplasms/complications , Prognosis , Bone Diseases, Metabolic/genetics , Bone Diseases, Metabolic/pathology , MutationABSTRACT
PURPOSE: Cardiac myxomas (CMs) are the second most common benign primary cardiac tumors, mainly originating within the left atrium. Approximately 5% of CM cases are associated with Carney Complex (CNC), an autosomal dominant multiple neoplasia syndrome often caused by germline mutations in the protein kinase A regulatory subunit 1A (PRKAR1A). Data concerning PRKAR1A alterations in sporadic myxomas are variable and sparse, with PRKAR1A mutations reported to range from 0% to 87%. Therefore, we investigated the frequency of PRKAR1A mutations in sporadic CM using next-generation sequencing (NGS). Additionally, we explored mutations in the catalytic domain of the Protein Kinase A complex (PRKACA) and examined the presence of GNAS mutations as another potential driver. METHODS AND RESULTS: This study retrospectively collected histological and clinical data from 27 patients with CM. First, we ruled out the possibility of underlying CNC through clinical evaluations and standardized interviews for each patient. Second, we performed PRKAR1A immunohistochemistry (IHC) analysis and graded the reactivity of myxoma cells semi-quantitatively. NGS was then applied to analyze the coding regions of PRKAR1A, PRKACA, and GNAS in all 27 cases. Of the 27 sporadic CM cases, 13 (48%) harbored mutations in PRKAR1A. Among these 13 mutant cases, six displayed more than one mutation in PRKAR1A. Most of the identified mutations resulted in premature stop codons or affected splicing. In PRKAR1A mutant CM cases, the loss of PRKAR1A protein expression was significantly more common. In two cases with missense mutations, protein expression remained preserved. Furthermore, a single mutation was detected in the catalytic domain of the protein kinase A complex, while no GNAS mutations were found. CONCLUSION: We identified a relatively high frequency of PRKAR1A mutations in sporadic CM. These PRKAR1A mutations may also represent an important oncogenic mechanism in sporadic myxomas, as already known in CM cases associated with CNC.
Subject(s)
Chromogranins , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit , GTP-Binding Protein alpha Subunits, Gs , Heart Neoplasms , Myxoma , Humans , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/genetics , GTP-Binding Protein alpha Subunits, Gs/genetics , Chromogranins/genetics , Heart Neoplasms/genetics , Heart Neoplasms/pathology , Heart Neoplasms/enzymology , Middle Aged , Female , Male , Myxoma/genetics , Myxoma/pathology , Myxoma/enzymology , Adult , Aged , Retrospective Studies , DNA Mutational Analysis , Genetic Predisposition to Disease , Mutation , Young Adult , Phenotype , High-Throughput Nucleotide Sequencing , Adolescent , Carney Complex/genetics , Carney Complex/enzymology , Carney Complex/pathology , Biomarkers, Tumor/genetics , Cyclic AMP-Dependent Protein Kinase Catalytic SubunitsABSTRACT
OBJECTIVES: Pseudohypoparathyroidism type 1A (PHP1A) encompasses the association of resistance to multiple hormones, features of Albright hereditary osteodystrophy and decreased Gsα activity. Little is known about the early signs of PHP1A, with a delay in diagnosis. We report two PHP1A cases and their clinical and biochemical findings during a 20-year follow-up. CASE PRESENTATION: Clinical suspicion was based on obesity, TSH resistance and ectopic ossifications which appeared several months before PTH resistance, at almost 3â¯years of age. Treatment with levothyroxine, calcitriol and calcium was required in both patients. DNA sequencing of GNAS gene detected a heterozygous pathogenic variant within exon 7 (c.569_570delAT) in patient one and a deletion from XLAS to GNAS-exon 5 on the maternal allele in patient 2. In patient 1, ectopic ossifications that required surgical excision were found. Noticeably, patient 2 displayed adult short stature, intracranial calcifications and psychomotor delay. In terms of weight, despite early diagnosis of obesity, dietary measures were established successfully in both cases. CONCLUSIONS: GNAS mutations should be considered in patients with obesity, ectopic ossifications and TSH resistance presented in early infancy. These cases emphasize the highly heterogeneous clinical picture PHP1A patients may present, especially in terms of final height and cognitive impairment.
Subject(s)
GTP-Binding Protein alpha Subunits, Gs , Pseudohypoparathyroidism , Adult , Humans , GTP-Binding Protein alpha Subunits, Gs/genetics , Pseudohypoparathyroidism/diagnosis , Pseudohypoparathyroidism/genetics , Mutation , Obesity , Thyrotropin , Chromogranins/geneticsABSTRACT
Approximately 5% of colorectal cancers (CRCs) have a gain-of-function mutation in the GNAS gene, which leads to the activation of cAMP-dependent signaling pathways and associates with poor prognosis. We investigated the effect of an activating GNAS mutation in CRC cell lines on gene expression and cell proliferation in vitro, and tumor growth in vivo. GNAS-mutated (GNASmt) HCT116 cells showed stimulated synthesis of cAMP as compared to parental (Par) cells. The most upregulated gene in the GNASmt cells was cAMP-hydrolyzing phosphodiesterase 4D (PDE4D) as detected by RNA sequencing. To further validate our finding, we analyzed PDE4D expression in a set of human CRC tumors (n = 35) and demonstrated overexpression in GNAS mutant CRC tumors as compared to GNAS wild-type tumors. The GNASmt HCT116 cells proliferated more slowly than the Par cells. PDE4 inhibitor Ro 20-1724 and PDE4D subtype selective inhibitor GEBR-7b further suppressed the proliferation of GNASmt cells without an effect on Par cells. The growth inhibitory effect of these inhibitors was also seen in the intrinsically GNAS-mutated SK-CO-1 CRC cell line having high levels of cAMP synthesis and PDE4D expression. In vivo, GNASmt HCT116 cells formed smaller tumors than the Par cells in nude mice. In conclusion, our findings demonstrate that GNAS mutation results in the growth suppression of CRC cells. Moreover, the GNAS mutation-induced overexpression of PDE4D provides a potential avenue to impede the proliferation of CRC cells through the use of PDE4 inhibitors.
Subject(s)
Chromogranins , Colorectal Neoplasms , Cyclic Nucleotide Phosphodiesterases, Type 4 , GTP-Binding Protein alpha Subunits, Gs , Animals , Humans , Mice , Chromogranins/genetics , Chromogranins/metabolism , Colorectal Neoplasms/genetics , Cyclic Nucleotide Phosphodiesterases, Type 4/genetics , Cyclic Nucleotide Phosphodiesterases, Type 4/metabolism , GTP-Binding Protein alpha Subunits, Gs/genetics , GTP-Binding Protein alpha Subunits, Gs/metabolism , HCT116 Cells , Mice, Nude , Mutation , Phosphodiesterase 4 Inhibitors/pharmacologyABSTRACT
Primary pulmonary myxoid sarcoma (PPMS) and thoracic angiomatoid fibrous histiocytoma (AFH) are rare neoplasms with EWSR1 fusions and overlapping morphology. Both tumor types often show epithelial membrane antigen expression, but AFH characteristically co-expresses desmin. We encountered a case of PPMS with the unexpected finding of patchy, strong anaplastic lymphoma kinase (ALK) (previously reported in AFH) and synaptophysin expression. We evaluated a cohort of PPMS and thoracic AFH with systematic morphologic comparison and surveyed for aberrant expression of ALK and synaptophysin. Medical records and slides were reviewed for 16 molecularly confirmed cases of PPMS (n=5) and thoracic AFH (n=11). Each case was scored for morphologic characteristics typical of PPMS and/or AFH. ALK, synaptophysin, chromogranin, desmin, and epithelial membrane antigen immunostains were performed on cases with available tissue. AFH and PPMS cases showed similar age at presentation and long-term tumor behavior. Almost all cases of PPMS and AFH had a fibrous pseudocapsule and lymphoid rim. All PPMS had myxoid stroma and reticular growth pattern, but these features were also present in a subset of AFH. Synaptophysin expression was present in 6 of 11 AFH and 1 of 5 PPMS; all tested cases were negative for chromogranin (n=15). One case of AFH and 1 case of PPMS showed focally strong coexpression of synaptophysin and ALK. AFH and PPMS show considerable clinicopathologic overlap. When supportive, the immunohistochemical findings described may aid in diagnosis before molecular confirmation. PPMS and AFH may be morphologic variants of the same clinicopathologic entity, which can show more immunophenotypic variability than previously reported.
Subject(s)
Histiocytoma, Benign Fibrous , Histiocytoma, Malignant Fibrous , Humans , Synaptophysin , Mucin-1 , Desmin , Chromogranins , Histiocytoma, Malignant Fibrous/genetics , Histiocytoma, Malignant Fibrous/surgery , Histiocytoma, Malignant Fibrous/diagnosis , Receptor Protein-Tyrosine KinasesABSTRACT
IMPLICATIONS: The study identifies an opportunity to discover a PKA-independent pathway downstream of oncogene GNAS for managing IPMN lesions and their progression to PDAC.
Subject(s)
Cell Proliferation , Chromogranins , Cyclic AMP-Dependent Protein Kinases , GTP-Binding Protein alpha Subunits, Gs , Organoids , Pancreatic Neoplasms , Humans , GTP-Binding Protein alpha Subunits, Gs/metabolism , GTP-Binding Protein alpha Subunits, Gs/genetics , Organoids/metabolism , Organoids/pathology , Chromogranins/genetics , Chromogranins/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Pancreatic Ducts/metabolism , Pancreatic Ducts/pathology , Pancreatic Ducts/cytology , Acinar Cells/metabolism , Acinar Cells/pathologyABSTRACT
BACKGROUND: Indistinct and analogous histopathological features of various fibro-osseous lesions make establishing a definitive diagnosis a challenge. There is a need for additional molecular and histochemical tools to support and differentiate these lesions in order to establish a concrete diagnosis. MATERIALS AND METHODS: A retrospective analysis of biopsied lesions in formalin-fixed paraffin-embedded sections (10 cases each of fibrous dysplasia, ossifying fibroma, and cement-osseous dysplasia) retrieved from the archives was studied for immunoexpression of osteocalcin (quantitative analysis in osteocytes), collagen characterization using Azan, Picrosirus, and Toluidine blue stain for evaluating intensity and localization of collagen fibers, and morphometric analysis of vasculature (for evaluating mean vessel density as square microns). RESULTS: Positive immunostaining of osteocalcin suggested mutations of the GNAS-1 gene found in fibrous dysplasia indirectly, as it is a negative regulator of bone formation. Osteocalcin immunopositivity was quantitatively measured in the fibro-osseous lesions, with fibrous dysplasia measuring 14.47 ± 3.628 as compared to ossifying fibroma measuring 5.23 ± 1.33, followed by cemento-osseous dysplasia measuring 2.30 ± 1.409. Toluidine blue suggests the presence of oxytalan fibers (resistant to acid hydrolysis) in ossifying fibroma and cemento-osseous dysplasia, pointing toward the pathogenesis of the lesion. Azan stain and Picrosirus (under a polarizing microscope) helped in distinguishing hard tissue characteristics (70% of cases of fibrous dysplasia showed only a magenta component followed by intermixed magenta with a blue component in 20% of cases and only 10% of cases showed magenta with blue borders whereas for ossifying fibroma, 40% of cases depicted magenta with blue borders along with the other 40% with intermixed magenta with blue component). The mean vessel density was also highest in fibrous dysplasia measuring 7.90 ± 1.079 (in Sq. micron area), followed by ossifying fibroma and cemento-osseous dysplasia. CONCLUSION: The diagnosis of fibro-osseous lesions by hematoxylin and eosin alone is confusing and thus should be supported by relatively simple histomorphometric analysis for better treatment outcomes. At the diagnostic stage of fibro-osseous lesions, evaluation of intralesional vessel size, reliable molecular marker, and histochemical nature can aid in differentiating fibrous dysplasia from central ossifying fibroma and cemento-osseous dysplasia alongside, other clinical, radiographic and pathological criteria. These parameters help in the diagnostic decision-making of fibro-osseous lesions.
Subject(s)
Fibroma, Ossifying , Fibrous Dysplasia of Bone , Immunohistochemistry , Osteocalcin , Humans , Retrospective Studies , Fibroma, Ossifying/pathology , Fibroma, Ossifying/genetics , Fibroma, Ossifying/diagnosis , Fibrous Dysplasia of Bone/genetics , Fibrous Dysplasia of Bone/pathology , Fibrous Dysplasia of Bone/diagnosis , Osteocalcin/genetics , GTP-Binding Protein alpha Subunits, Gs/genetics , Chromogranins/genetics , Cementoma/pathology , Cementoma/diagnosis , Cementoma/genetics , Collagen , Male , Female , BiopsyABSTRACT
Pseudohypoparathyroidism type 1B (PHP1B) results from aberrant genomic imprinting at the GNAS gene. Defining the underlying genetic cause in new patients is challenging because various genetic alterations (e.g., deletions, insertions) within the GNAS genomic region, including the neighboring STX16 gene, can cause PHP1B, and the genotype-epigenotype correlation has not been clearly established. Here, by analyzing patients with PHP1B with a wide variety of genotypes and epigenotypes, we identified a GNAS differentially methylated region (DMR) of distinct diagnostic value. This region, GNAS AS2, was hypomethylated in patients with genetic alterations located centromeric but not telomeric of this DMR. The AS2 methylation status was captured by a single probe of the methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA) assay utilized to diagnose PHP1B. In human embryonic stem cells, where NESP55 transcription regulates GNAS methylation status on the maternal allele, AS2 methylation depended on 2 imprinting control regions (STX16-ICR and NESP-ICR) essential for NESP55 transcription. These results suggest that the AS2 methylation status in patients with PHP1B reflects the position at which the genetic alteration affects NESP55 transcription during an early embryonic period. Therefore, AS2 methylation levels can enable mechanistic PHP1B categorization based on genotype-epigenotype correlation and, thus, help identify the underlying molecular defect in patients.