Review of the spectrum of tuberous sclerosis complex: The Saudi Arabian Experience.

OBJECTIVES: To determine the prevalence of tuberous sclerosis complex (TSC) in the paediatric Saudi population and to characterise the range of clinical symptoms, neurocutaneous findings, neuroimaging results, and complications of the disease. METHODS: A total of 61 genetically confirmed TSC patients from the National Guard Health Affairs (NGHA) in Saudi Arabia were the subject of this retrospective descriptive analysis. The data were presented using descriptive measures. RESULTS: The mean age at diagnosis was found to be 4.9 years. Subependymal nodules (86.9%), numerous cortical tubers and/or radial migration lines (63.9%), and hypomelanotic macules (63.9%) were the 3 most common significant criteria. The vast majority (86.9%) of those diagnosed had epilepsy, of which 50% were considered medically intractable. Nearly half of our subjects underwent genetic testing, which revealed that TSC2 predominated over TSC1. Symptoms of Tuberous Sclerosis Complex-Associated Neuropsychiatric Disorders (TAND) were present in 66.7% of TSC1 patients and 73.9% of TSC2 patients. CONCLUSION: The findings of this study demonstrate that the clinical spectrum of TSC among Saudi children is consistent with the body of existing literature. The TSC2 was more prevalent than TSC1. The most frequent signs were cutaneous and neurological. Monitoring TSC patients regularly is crucial to identify any issues as soon as possible.

Sporadic subependymal giant cell astrocytoma with somatic TSC2 mutation: A case report.

Subependymal giant cell astrocytoma (SEGA) is a rare circumscribed astrocytic glioma that occurs in approximately 25% of all tuberous sclerosis (TSC) cases. Herein, we discuss an atypical presentation of SEGA, including the genetic alterations, impact on clinical presentation, and the determinants of each medical and surgical treatment option. A 14-year-old girl presented with intermittent headache and a right intraventricular mass originating near the foramen of Monro. The tumor's proximity to critical structures necessitated maximum safe resection, which improved her symptoms. Histological findings indicated SEGA, and genetic sequencing revealed a TSC2 mutation. However, complete clinical and radiological evaluations failed to reveal TSC. Two months later, a new subependymal nodule was incidentally found. She had a recurrent left occipital horn lesion and diffuse smooth leptomeningeal enhancement with no spine drop metastases. She was administered everolimus as the tumor was considered unresectable. Subsequent imaging revealed a reduction in both residual and new lesions.

Malignant tumors in tuberous sclerosis complex: a case report and review of the literature.

BACKGROUND: Tuberous sclerosis complex (TSC) is a rare, autosomal dominant genetic disease that arises from TSC1 or TSC2 genetic mutations. These genetic mutations can induce the development of benign tumors in any organ system with significant clinical implications in morbidity and mortality. In rare instances, patients with TSC can have malignant tumors, including renal cell carcinoma (RCC) and pancreatic neuroendocrine tumor (PNET). It is considered a hereditary renal cancer syndrome despite the low incidence of RCC in TSC patients. TSC is typically diagnosed in prenatal and pediatric patients and frequently associated with neurocognitive disorders and seizures, which are often experienced early in life. However, penetrance and expressivity of TSC mutations are highly variable. Herein, we present a case report, with associated literature, to highlight that there exist undiagnosed adult patients with less penetrant features, whose clinical presentation may contain non-classical signs and symptoms, who have pathogenic TSC mutations. CASE PRESENTATION: A 31-year-old female with past medical history of leiomyomas status post myomectomy presented to the emergency department for a hemorrhagic adnexal cyst. Imaging incidentally identified a renal mass suspicious for RCC. Out of concern for hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome, the mass was surgically removed and confirmed as RCC. Discussion with medical genetics ascertained a family history of kidney cancer and nephrectomy procedures and a patient history of ungual fibromas on the toes. Genetic testing for hereditary kidney cancer revealed a 5'UTR deletion in the TSC1 gene, leading to a diagnosis of TSC. Following the diagnosis, dermatology found benign skin findings consistent with TSC. About six months after the incidental finding of RCC, a PNET in the pancreatic body/tail was incidentally found on chest CT imaging, which was removed and determined to be a well-differentiated PNET. Later, a brain MRI revealed two small cortical tubers, one in each frontal lobe, that were asymptomatic; the patient's history and family history did not contain seizures or learning delays. The patient presently shows no evidence of recurrence or metastatic disease, and no additional malignant tumors have been identified. CONCLUSIONS: To our knowledge, this is the first report in the literature of a TSC patient without a history of neurocognitive disorders with RCC and PNET, both independently rare occurrences in TSC. The patient had a strong family history of renal disease, including RCC, and had several other clinical manifestations of TSC, including skin and brain findings. The incidental finding and surgical removal of RCC prompted the genetic evaluation and diagnosis of TSC, leading to a comparably late diagnosis for this patient. Reporting the broad spectrum of disease for TSC, including more malignant phenotypes such as the one seen in our patient, can help healthcare providers better identify patients who need genetic evaluation and additional medical care.

Distinct Driver Pathway Enrichments and a High Prevalence of TSC2 Mutations in Right Colon Cancer in Chile: A Preliminary Comparative Analysis.

Colorectal cancer (CRC) is the second leading cause of cancer deaths globally. While ethnic differences in driver gene mutations have been documented, the South American population remains understudied at the genomic level, despite facing a rising burden of CRC. We analyzed tumors of 40 Chilean CRC patients (Chp) using next-generation sequencing and compared them to data from mainly Caucasian cohorts (TCGA and MSK-IMPACT). We identified 388 mutations in 96 out of 135 genes, with TP53 (45%), KRAS (30%), PIK3CA (22.5%), ATM (20%), and POLE (20%) being the most frequently mutated. TSC2 mutations were associated with right colon cancer (44.44% in RCRC vs. 6.45% in LCRC, p-value = 0.016), and overall frequency was higher compared to TCGA (p-value = 1.847 × 10-5) and MSK-IMPACT cohorts (p-value = 3.062 × 10-2). Limited sample size restricts definitive conclusions, but our data suggest potential differences in driver mutations for Chilean patients, being that the RTK-RAS oncogenic pathway is less affected and the PI3K pathway is more altered in Chp compared to TCGA (45% vs. 25.56%, respectively). The prevalence of actionable pathways and driver mutations can guide therapeutic choices, but can also impact treatment effectiveness. Thus, these findings warrant further investigation in larger Chilean cohorts to confirm these initial observations. Understanding population-specific driver mutations can guide the development of precision medicine programs for CRC patients.

Rescue of impaired blood-brain barrier in tuberous sclerosis complex patient derived neurovascular unit.

BACKGROUND: Tuberous sclerosis complex (TSC) is a multi-system genetic disease that causes benign tumors in the brain and other vital organs. The most debilitating symptoms result from involvement of the central nervous system and lead to a multitude of severe symptoms including seizures, intellectual disability, autism, and behavioral problems. TSC is caused by heterozygous mutations of either the TSC1 or TSC2 gene and dysregulation of mTOR kinase with its multifaceted downstream signaling alterations is central to disease pathogenesis. Although the neurological sequelae of the disease are well established, little is known about how these mutations might affect cellular components and the function of the blood-brain barrier (BBB). METHODS: We generated TSC disease-specific cell models of the BBB by leveraging human induced pluripotent stem cell and microfluidic cell culture technologies. RESULTS: Using microphysiological systems, we demonstrate that a BBB generated from TSC2 heterozygous mutant cells shows increased permeability. This can be rescued by wild type astrocytes or by treatment with rapamycin, an mTOR kinase inhibitor. CONCLUSION: Our results demonstrate the utility of microphysiological systems to study human neurological disorders and advance our knowledge of cell lineages contributing to TSC pathogenesis and informs future therapeutics.

Regulation of TSC2 lysosome translocation and mitochondrial turnover by TSC2 acetylation status.

Sirtuin1 (SIRT1) activity decreases the tuberous sclerosis complex 2 (TSC2) lysine acetylation status, inhibiting the mechanistic target of rapamycin complex 1 (mTORC1) signalling and concomitantly, activating autophagy. This study analyzes the role of TSC2 acetylation levels in its translocation to the lysosome and the mitochondrial turnover in both mouse embryonic fibroblast (MEF) and in mouse insulinoma cells (MIN6) as a model of pancreatic ß cells. Resveratrol (RESV), an activator of SIRT1 activity, promotes TSC2 deacetylation and its translocation to the lysosome, inhibiting mTORC1 activity. An improvement in mitochondrial turnover was also observed in cells treated with RESV, associated with an increase in the fissioned mitochondria, positive autophagic and mitophagic fluxes and an enhancement of mitochondrial biogenesis. This study proves that TSC2 in its deacetylated form is essential for regulating mTORC1 signalling and the maintenance of the mitochondrial quality control, which is involved in the homeostasis of pancreatic beta cells and prevents from several metabolic disorders such as Type 2 Diabetes Mellitus.

A Case Study of a Rare Undifferentiated Spindle Cell Sarcoma of the Penis: Establishment and Characterization of Patient-Derived Models.

Rare sarcomas present significant treatment challenges compared to more prevalent soft tissue sarcomas due to limited treatment options and a poor understanding of their biology. This study investigates a unique case of penile sarcoma, providing a comprehensive morphological and molecular analysis. Through the creation of experimental patient-derived models-including patient-derived xenograft (PDX), 3D, and monolayer primary cultures-we successfully replicated crucial molecular traits observed in the patient's tumor, such as smooth muscle actin and CD99 expression, along with specific mutations in genes like TSC2 and FGFR4. These models are helpful in assessing the potential for an in-depth exploration of this tumor's biology. This comprehensive approach holds promise in identifying potential therapeutic avenues for managing this exceedingly rare soft tissue sarcoma.

Clinicopathologic and Molecular Characterization of Xanthomatous Giant Cell Renal Cell Carcinomas: Further Support for a Close Morphologic Spectrum to Eosinophilic Solid and Cystic Renal Cell Carcinomas.

A recent study described a rare subtype of tuberous sclerosis complex ( TSC )-mutated renal cell carcinoma primarily characterized by Xanthomatous giant cell morphology. Only 2 cases in young individuals have been reported so far, making the correct diagnosis challenging from a pathological perspective. It remains unknown whether this tumor represents an independent subtype or belongs to other TSC -mutated tumors. We conducted a clinicopathologic evaluation and immunohistochemical profiling of 5 cases of Xanthomatous Giant Cell Renal Cell Carcinoma (XGC RCC) with confirmed TSC2 mutations through targeted DNA sequencing. In addition, we analyzed transcriptomic profiles using RNA-seq for the following samples: XGC RCC, Low-grade Oncocytic tumors (LOT), High-grade Oncocytic tumors/Eosinophilic Vacuolar Tumors (HOT/EVT), Eosinophilic Solid and Cystic Renal Cell Carcinomas (ESC RCC), Chromophobe cell Renal Cell Carcinomas (ChRCC), Renal Oncocytomas (RO), clear cell Renal Cell Carcinomas (ccRCC), and normal renal tissues. There were 2 female and 3 male patients, aged 22 to 58 years, who underwent radical nephrectomy for tumor removal. The tumor sizes ranged from 4.7 to 9.5 cm in diameter. These tumors exhibited ill-defined boundaries, showed an expansive growth pattern, and featured distinctive tumor giant cells with abundant eosinophilic to Xanthomatous cytoplasm and prominent nucleoli. All tumors had low Ki-67 proliferation indices (<1%) and demonstrated immune reactivity for CD10, PAX8, CK20, CathepsinK, and GPNMB. Next-generation sequencing confirmed TSC2 mutations in all cases. RNA sequencing-based clustering indicated a close similarity between the tumor and ESC RCC. One patient (1/5) died of an accident 63 months later, while the remaining patients (4/5) were alive without tumor recurrences or metastases at the time of analysis, with a mean follow-up duration of 43.4 months. Our research supports the concept that Xanthomatous giant cell renal cell carcinoma (XGC RCC) shares clinicopathological and molecular characteristics with ESC RCC and shows a relatively positive prognosis, providing further support for a close morphologic spectrum between the two. We propose considering XGC RCC as a distinct subtype of ESC RCC.

Glycoprotein non-metastatic melanoma protein B promotes tumor growth and is a biomarker for lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare, progressive cystic lung disease affecting almost exclusively female-sexed individuals. The cysts represent regions of lung destruction caused by smooth muscle tumors containing mutations in one of the two tuberous sclerosis (TSC) genes. mTORC1 inhibition slows but does not stop LAM advancement. Furthermore, monitoring disease progression is hindered by insufficient biomarkers. Therefore, new treatment options and biomarkers are needed. LAM cells express melanocytic markers, including glycoprotein non-metastatic melanoma protein B (GPNMB). The function of GPNMB in LAM is currently unknown; however, GPNMB's unique cell surface expression on tumor versus benign cells makes GPNMB a potential therapeutic target, and persistent release of its extracellular ectodomain suggests potential as a serum biomarker. Here, we establish that GPNMB expression is dependent on mTORC1 signaling, and that GPNMB regulates TSC2-null tumor cell invasion in vitro. Further, we demonstrate that GPNMB enhances TSC2-null xenograft tumor growth in vivo, and that ectodomain release is required for this xenograft growth. We also show that GPNMB's ectodomain is released from the cell surface of TSC2-null cells by proteases ADAM10 and 17, and we identify the protease target sequence on GPNMB. Finally, we demonstrate that GPNMB's ectodomain is present at higher levels in LAM patient serum compared to healthy controls and that ectodomain levels decrease with mTORC1 inhibition, making it a potential LAM biomarker.

Generation of a TSC2 knockout embryonic stem cell line by CRISPR/Cas9 editing.

Tuberous Sclerosis Complex (TSC) is a severe developmental disorder with various clinical effects, primarily caused by TSC2 gene mutations, often involving loss of function(Henske,et al., 2016).To explore role of TSC2 in human heart development, we successfully developed a TSC2 knockout (TSC2-/-) human embryonic stem cells (hESCs) line using CRISPR/Cas9 gene editing. This TSC2-/- hESC line maintained a normal karyotype, expressed pluripotency markers strongly, and could differentiate into all three germ layers in vivo. This cell line will be a valuable tool for future research on the role of TSC2 in heart development.

The Genetics of Tuberous Sclerosis Complex and Related mTORopathies: Current Understanding and Future Directions.

The mechanistic target of rapamycin (mTOR) pathway serves as a master regulator of cell growth, proliferation, and survival. Upregulation of the mTOR pathway has been shown to cause malformations of cortical development, medically refractory epilepsies, and neurodevelopmental disorders, collectively described as mTORopathies. Tuberous sclerosis complex (TSC) serves as the prototypical mTORopathy. Characterized by the development of benign tumors in multiple organs, pathogenic variants in TSC1 or TSC2 disrupt the TSC protein complex, a negative regulator of the mTOR pathway. Variants in critical domains of the TSC complex, especially in the catalytic TSC2 subunit, correlate with increased disease severity. Variants in less crucial exons and non-coding regions, as well as those undetectable with conventional testing, may lead to milder phenotypes. Despite the assumption of complete penetrance, expressivity varies within families, and certain variants delay disease onset with milder neurological effects. Understanding these genotype-phenotype correlations is crucial for effective clinical management. Notably, 15% of patients have no mutation identified by conventional genetic testing, with the majority of cases postulated to be caused by somatic TSC1/TSC2 variants which present complex diagnostic challenges. Advancements in genetic testing, prenatal screening, and precision medicine hold promise for changing the diagnostic and treatment paradigm for TSC and related mTORopathies. Herein, we explore the genetic and molecular mechanisms of TSC and other mTORopathies, emphasizing contemporary genetic methods in understanding and diagnosing the condition.

A large deletion in TSC2 causes tuberous sclerosis complex by dysregulating PI3K/AKT/mTOR signaling pathway.

BACKGROUND/AIM: Tuberous sclerosis complex (TSC) is a multi-system syndrome caused by loss-of-function mutation in TSC1 or TSC2. Most TSC patients present with cardiac rhabdomyoma or cortical tubers during fetal life, and the symptoms are not uniform as their age. The gene products of TSC1/2 are components of the TSC protein complex and are important role in the PI3K/AKT/mTOR (PAM) signaling pathway. Based on three members of a family with variable expressivity, the purpose of this study was to clarify the clinical features of TSC in different age groups and to analyze the genetic characteristics of TSC2 gene. METHODS: Clinical exome sequencing and co-segregation were used to identify a three-generation family with four affected individuals. HEK-293T cell model was constructed for subsequent experiments. Quantitative RT-PCR, western blotting, and subcellular localization were used to analyze the expression effect of TSC2 mutation. CCK-8 assay, wound healing assay, and cell cycle analysis were used to analyze the function effect of TSC2 mutation. RESULT: We identified a TSC family with heterozygous deletion of exon 4 in TSC2 by clinical exon sequencing. Sanger sequencing indicated that the affected individuals have 2541-bp deletion that encompassed exon 4 and adjacent introns. Deletion of exon 4 decreased the TSC2 mRNA and protein levels in HEK-293T cells, and activated the PI3K/AKT/mTOR pathway, thereby altering the cell cycle and promoting cell proliferation and migration. CONCLUSION: We confirmed the pathogenicity of the large deletion in TSC2 in a three- generations family.. Deletion of exon 4 of TSC2 affected cell proliferation, migration, and cell cycle via abnormal activation of the PAM pathway. This study evaluated the pathogenic effect of deletion of exon 4 of TSC2 and investigated the underlying mechanism.

Identification of a complex intrachromosomal inverted insertion in the long arm of chromosome 9 as a cause of tuberous sclerosis complex in a Korean family.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant multisystem disorder, caused by a loss-of-function of either TSC1 or TSC2 gene. However, in 10%-15% TSC patients there is no pathogenic variant identified in either TSC1 or TSC2 genes based on standard clinical testing. METHODS: In this study, genome sequencing was performed for families with clinical diagnosis of TSC with negative results from TSC1 and TSC2 single-gene tests. RESULTS: Herein, we report a family presenting a classical TSC phenotype with an unusual, complex structural variant involving the TSC1 gene: an intrachromosomal inverted insertion in the long arm of chromosome 9. We speculate that the inverted 9q33.3q34.13 region was inserted into the q31.2 region with the 3'-end of the breakpoint of the inversion being located within the TSC1 gene, resulting in premature termination of TSC1. CONCLUSIONS: In this study, we demonstrate the utility of genome sequencing for the identification of complex chromosomal rearrangement. Because the breakpoints are located within the deep intronic/intergenic region, this copy-neutral variant was missed by the TSC1 and TSC2 single-gene tests and contributed to an unknown etiology. Together, this finding suggests that complex structural variants may be underestimated causes for the etiology of TSC.

Renal Cell Carcinoma Associated With TSC/MTOR Genomic Alterations: An Update on its Expanding Spectrum and an Approach to Clinicopathologic Work-up.

Renal cell carcinoma (RCC) with tuberous sclerosis complex (TSC)/mammalian target of rapamycin (MTOR) pathway-related genomic alterations have been classically described in hereditary TSC syndrome setting involving germline mutations, whereby cells with a bi-allelic inactivation of genes originate tumors in a classic tumor-suppressor "two-hit" Knudson paradigm. Initial studies of TSC-associated RCC categorized tumors into 3 broad heterogeneous morphologic groups: RCC with smooth muscle stroma, chromophobe-like, and eosinophilic-macrocytic. Recently, a similar morphologic spectrum has been increasingly recognized in novel and emerging entities characterized by somatic mutations in the TSC1/2 and MTOR in patients who do not suffer from the TSC. Correct recognition of RCC with TSC / MTOR mutations is critical for accurate prognostication because such tumors with aggressive behavior have the potential to be tailored to mTOR inhibitors. Whether TSC/MTOR mutated renal epithelial neoplasms represent a distinct molecular class has been confounded by the fact that TSC1/2 , and the gene encoding the downstream protein MTOR, are mutated secondarily in ∼5% of the more common subtypes of RCC, including the commonest subtype of clear cell RCC. This review summarizes the expanding morphologic spectrum of renal tumors with TSC/mTOR pathway alterations, specifically for sporadically occurring tumors where these genomic alterations likely are primary pathologic events. Finally, a practical surgical pathology approach to handling these tumors, and a conceptual framework of renal epithelial tumors with TSC/MTOR mutations as a "family of tumors", is presented.

CBAP regulates the function of Akt-associated TSC protein complexes to modulate mTORC1 signaling.

The Akt-Rheb-mTORC1 pathway plays a crucial role in regulating cell growth, but the mechanisms underlying the activation of Rheb-mTORC1 by Akt remain unclear. In our previous study, we found that CBAP was highly expressed in human T-ALL cells and primary tumors, and its deficiency led to reduced phosphorylation of TSC2/S6K1 signaling proteins as well as impaired cell proliferation and leukemogenicity. We also demonstrated that CBAP was required for Akt-mediated TSC2 phosphorylation in vitro. In response to insulin, CBAP was also necessary for the phosphorylation of TSC2/S6K1 and the dissociation of TSC2 from the lysosomal membrane. Here we report that CBAP interacts with AKT and TSC2, and knockout of CBAP or serum starvation leads to an increase in TSC1 in the Akt/TSC2 immunoprecipitation complexes. Lysosomal-anchored CBAP was found to override serum starvation and promote S6K1 and 4EBP1 phosphorylation and c-Myc expression in a TSC2-dependent manner. Additionally, recombinant CBAP inhibited the GAP activity of TSC2 complexes in vitro, leading to increased Rheb-GTP loading, likely due to the competition between TSC1 and CBAP for binding to the HBD domain of TSC2. Overexpression of the N26 region of CBAP, which is crucial for binding to TSC2, resulted in a decrease in mTORC1 signaling and an increase in TSC1 association with the TSC2/AKT complex, ultimately leading to increased GAP activity toward Rheb and impaired cell proliferation. Thus, we propose that CBAP can modulate the stability of TSC1-TSC2 as well as promote the translocation of TSC1/TSC2 complexes away from lysosomes to regulate Rheb-mTORC1 signaling.

Identification of a Novel TSC2 c.170G>A Missense Variant: A Case Report and Elaboration on the Yield of Targeted Options against Tuberous Sclerosis Complex Manifestations.

BACKGROUND: Tuberous sclerosis complex (TSC) is a rare genetic disease that affects multiple organs and affects the quality of life. Mutations in TSC1 and TSC2 genes are causing dysregulations in the mammalian target of the rapamycin (mTOR) pathway, inducing mostly benign but also malignant tumors, including renal cell carcinoma (RCC). The diagnosis of TSC, based on established clinical and genetic criteria, is essential for the optimal surveillance and management of patients. CASE PRESENTATION: With the current report, we present the case of two sisters who were consequently diagnosed with early-stage chromophobe-like RCC, possibly familial given their young age. The younger sister also had a previous diagnosis of differentiated thyroid carcinoma, for which she had been treated properly. Genetic testing of both revealed the same heterozygous TSC2 variant that is currently regarded as a variant of unknown significance, while both patients did not fulfill the clinical criteria for the diagnosis of TSC. Owing to these data, we opted to manage and surveil both sisters as TSC patients, while we also considered the specific TSC2 variant to be pathogenic - but of low penetrance - based on clinical judgment and functional analyses. Furthermore, we discussed the implementation of mTOR inhibitors for the treatment of TSC complications. CONCLUSION: As novel pathogenic variants of TSC genes are constantly being explored, the identification of TSC variants of unknown significance in combination with absent clinical diagnostic criteria cannot exclude a TSC diagnosis. We support the implementation of clinical judgment in assisting the diagnosis of TSC, as well as the enrollment of patients in clinical trials due to the rarity of the disease.

Non-canonical functions of a mutant TSC2 protein in mitotic division.

Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. TSC is caused by mutations in the TSC1 or TSC2 genes, which encode the hamartin/tuberin proteins respectively. These proteins function as a heterodimer that negatively regulates the mechanistic Target of Rapamycin Complex 1 (mTORC1). TSC research has focused on the effects of mTORC1, a critical signaling hub, on regulation of diverse cell processes including metabolism, cell growth, translation, and neurogenesis. However, non-canonical functions of TSC2 are not well studied, and the potential disease-relevant biological mechanisms of mutations affecting these functions are not well understood. We observed aberrant multipolar mitotic division, a novel phenotype, in TSC2 mutant iPSCs. The multipolar phenotype is not meaningfully affected by treatment with the inhibitor rapamycin. We further observed dominant negative activity of the mutant form of TSC2 in producing the multipolar division phenotype. These data expand the knowledge of TSC2 function and pathophysiology which will be highly relevant to future treatments for patients with TSC.

TSC2 inactivation, low mutation burden and high macrophage infiltration characterise hepatic angiomyolipomas.

AIMS: Although TSC1 or TSC2 inactivating mutations that lead to mTORC1 hyperactivation have been reported in hepatic angiomyolipomas (hAML), the role of other somatic genetic events that may contribute to hAML development is unknown. There are also limited data regarding the tumour microenvironment (TME) of hAML. The aim of the present study was to identify other somatic events in genomic level and changes in TME that contribute to tumorigenesis in hAML. METHODS AND RESULTS: In this study, we performed exome sequencing in nine sporadic hAML tumours and deep-coverage targeted sequencing for TSC2 in three additional hAML. Immunohistochemistry and multiplex immunofluorescence were carried out for 15 proteins to characterise the tumour microenvironment and assess immune cell infiltration. Inactivating somatic variants in TSC2 were identified in 10 of 12 (83%) cases, with a median allele frequency of 13.6%. Five to 18 somatic variants (median number: nine, median allele frequency 21%) not in TSC1 or TSC2 were also identified, mostly of uncertain clinical significance. Copy number changes were rare, but detection was impaired by low tumour purity. Immunohistochemistry demonstrated numerous CD68+ macrophages of distinct appearance from Küpffer cells. Multiplex immunofluorescence revealed low numbers of exhausted PD-1+/PD-L1+, FOXP3+ and CD8+ T cells. CONCLUSION: hAML tumours have consistent inactivating mutations in TSC2 and have a low somatic mutation rate, similar to other TSC-associated tumours. Careful histological review, standard IHC and multiplex immunofluorescence demonstrated marked infiltration by non-neoplastic inflammatory cells, mostly macrophages.

An mTORC1-Dependent Mouse Model for Cardiac Sarcoidosis.

Background Sarcoidosis is an inflammatory, granulomatous disease of unknown cause affecting multiple organs, including the heart. Untreated, unresolved granulomatous inflammation can lead to cardiac fibrosis, arrhythmias, and eventually heart failure. Here we characterize the cardiac phenotype of mice with chronic activation of mammalian target of rapamycin (mTOR) complex 1 signaling in myeloid cells known to cause spontaneous pulmonary sarcoid-like granulomas. Methods and Results The cardiac phenotype of mice with conditional deletion of the tuberous sclerosis 2 (TSC2) gene in CD11c+ cells (TSC2fl/flCD11c-Cre; termed TSC2KO) and controls (TSC2fl/fl) was determined by histological and immunological stains. Transthoracic echocardiography and invasive hemodynamic measurements were performed to assess myocardial function. TSC2KO animals were treated with either everolimus, an mTOR inhibitor, or Bay11-7082, a nuclear factor-kB inhibitor. Activation of mTOR signaling was evaluated on myocardial samples from sudden cardiac death victims with a postmortem diagnosis of cardiac sarcoidosis. Chronic activation of mTORC1 signaling in CD11c+ cells was sufficient to initiate progressive accumulation of granulomatous infiltrates in the heart, which was associated with increased fibrosis, impaired cardiac function, decreased plakoglobin expression, and abnormal connexin 43 distribution, a substrate for life-threatening arrhythmias. Mice treated with the mTOR inhibitor everolimus resolved granulomatous infiltrates, prevented fibrosis, and improved cardiac dysfunction. In line, activation of mTOR signaling in CD68+ macrophages was detected in the hearts of sudden cardiac death victims who suffered from cardiac sarcoidosis. Conclusions To our best knowledge this is the first animal model of cardiac sarcoidosis that recapitulates major pathological hallmarks of human disease. mTOR inhibition may be a therapeutic option for patients with cardiac sarcoidosis.