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.

Comprehensive genetic and phenotype analysis of 95 individuals with mosaic tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a neurogenetic disorder due to loss-of-function TSC1 or TSC2 variants, characterized by tumors affecting multiple organs, including skin, brain, heart, lung, and kidney. Mosaicism for TSC1 or TSC2 variants occurs in 10%-15% of individuals diagnosed with TSC. Here, we report comprehensive characterization of TSC mosaicism by using massively parallel sequencing (MPS) of 330 TSC samples from a variety of tissues and fluids from a cohort of 95 individuals with mosaic TSC. TSC1 variants in individuals with mosaic TSC are much less common (9%) than in germline TSC overall (26%) (p < 0.0001). The mosaic variant allele frequency (VAF) is significantly higher in TSC1 than in TSC2, in both blood and saliva (median VAF: TSC1, 4.91%; TSC2, 1.93%; p = 0.036) and facial angiofibromas (median VAF: TSC1, 7.7%; TSC2 3.7%; p = 0.004), while the number of TSC clinical features in individuals with TSC1 and TSC2 mosaicism was similar. The distribution of mosaic variants across TSC1 and TSC2 is similar to that for pathogenic germline variants in general TSC. The systemic mosaic variant was not present in blood in 14 of 76 (18%) individuals with TSC, highlighting the value of analysis of multiple samples from each individual. A detailed comparison revealed that nearly all TSC clinical features are less common in individuals with mosaic versus germline TSC. A large number of previously unreported TSC1 and TSC2 variants, including intronic and large rearrangements (n = 11), were also identified.

Mosaicism in Tumor Suppressor Gene Syndromes: Prevalence, Diagnostic Strategies, and Transmission Risk.

A mosaic state arises when pathogenic variants are acquired in certain cell lineages during postzygotic development, and mosaic individuals may present with a generalized or localized phenotype. Here, we review the current state of knowledge regarding mosaicism for eight common tumor suppressor genes-NF1, NF2, TSC1, TSC2, PTEN, VHL, RB1, and TP53-and their related genetic syndromes/entities. We compare and discuss approaches for comprehensive diagnostic genetic testing, the spectrum of variant allele frequency, and disease severity. We also review affected individuals who have no mutation identified after conventional genetic analysis, as well as genotype-phenotype correlations and transmission risk for each tumor suppressor gene in full heterozygous and mosaic patients. This review provides new insight into similarities as well as marked differences regarding the appreciation of mosaicism in these tumor suppressor syndromes.

Ultrasensitive profiling of UV-induced mutations identifies thousands of subclinical facial tumors in tuberous sclerosis complex.

BackgroundTuberous sclerosis complex (TSC) is a neurogenetic syndrome due to loss-of-function mutations in TSC2 or TSC1, characterized by tumors at multiple body sites, including facial angiofibroma (FAF). Here, an ultrasensitive assessment of the extent and range of UV-induced mutations in TSC facial skin was performed.MethodsA multiplex high-sensitivity PCR assay (MHPA) was developed, enabling mutation detection at extremely low (<0.1%) variant allele frequencies (VAFs).ResultsMHPA assays were developed for both TSC2 and TP53, and applied to 81 samples, including 66 skin biopsies. UV-induced second-hit mutation causing inactivation of TSC2 was pervasive in TSC facial skin with an average of 4.8 mutations per 2-mm biopsy at median VAF 0.08%, generating more than 150,000 incipient facial tumors (subclinical "micro-FAFs") in the average TSC subject. The MHPA analysis also led to the identification of a refined UV-related indel signature and a recurrent complex mutation pattern, consisting of both a single-nucleotide or dinucleotide variant and a 1- to 9-nucleotide deletion, in cis.ConclusionTSC facial skin can be viewed as harboring a patchwork of clonal fibroblast proliferations (micro-FAFs) with indolent growth, a small proportion of which develop into clinically observable FAF. Our observations also expand the spectrum of UV-related mutation signatures.FundingThis work was supported by the TSC Alliance; the Engles Family Fund for Research in TSC and LAM; and the NIH, National Heart, Lung, and Blood Institute (U01HL131022-04 and Intramural Research Program).

Evidence for shared genetic risk factors between lymphangioleiomyomatosis and pulmonary function.

INTRODUCTION: Lymphangioleiomyomatosis (LAM) is a rare low-grade metastasising disease characterised by cystic lung destruction. The genetic basis of LAM remains incompletely determined, and the disease cell-of-origin is uncertain. We analysed the possibility of a shared genetic basis between LAM and cancer, and LAM and pulmonary function. METHODS: The results of genome-wide association studies of LAM, 17 cancer types and spirometry measures (forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), FEV1/FVC ratio and peak expiratory flow (PEF)) were analysed for genetic correlations, shared genetic variants and causality. Genomic and transcriptomic data were examined, and immunodetection assays were performed to evaluate pleiotropic genes. RESULTS: There were no significant overall genetic correlations between LAM and cancer, but LAM correlated negatively with FVC and PEF, and a trend in the same direction was observed for FEV1. 22 shared genetic variants were uncovered between LAM and pulmonary function, while seven shared variants were identified between LAM and cancer. The LAM-pulmonary function shared genetics identified four pleiotropic genes previously recognised in LAM single-cell transcriptomes: ADAM12, BNC2, NR2F2 and SP5. We had previously associated NR2F2 variants with LAM, and we identified its functional partner NR3C1 as another pleotropic factor. NR3C1 expression was confirmed in LAM lung lesions. Another candidate pleiotropic factor, CNTN2, was found more abundant in plasma of LAM patients than that of healthy women. CONCLUSIONS: This study suggests the existence of a common genetic aetiology between LAM and pulmonary function.

Spectrum of germline and somatic mitochondrial DNA variants in Tuberous Sclerosis Complex.

Tuberous Sclerosis Complex (TSC) is caused by loss of function variants in either TSC1 or TSC2 and is characterized by broad phenotypic heterogeneity. Currently, there is limited knowledge regarding the role of the mitochondrial genome (mtDNA) in TSC pathogenesis. In this study, we aimed to determine the prevalence and spectrum of germline and somatic mtDNA variants in TSC and identify potential disease modifiers. Analysis of mtDNA amplicon massively parallel sequencing (aMPS) data, off-target mtDNA from whole-exome sequencing (WES), and/or qPCR, revealed mtDNA alterations in 270 diverse tissues (139 TSC-associated tumors and 131 normal tissue samples) from 199 patients and six healthy individuals. Correlation of clinical features to mtDNA variants and haplogroup analysis was done in 102 buccal swabs (age: 20-71 years). No correlation was found between clinical features and either mtDNA variants or haplogroups. No pathogenic variants were identified in the buccal swab samples. Using in silico analysis, we identified three predicted pathogenic variants in tumor samples: MT-ND4 (m.11742G>A, p. Cys328Tyr, VAF: 43%, kidney angiomyolipoma), MT-CYB (m.14775T>C, p. Leu10Pro, VAF: 43%, LAM abdominal tumor) and MT-CYB (m.15555C>T, p. Pro270Leu, VAF: 7%, renal cell carcinoma). Large deletions of the mitochondrial genome were not detected. Analysis of tumors from 23 patients with corresponding normal tissue did not reveal any recurrent tumor-associated somatic variants. The mtDNA/gDNA ratio between tumors and corresponding normal tissue was also unchanged. Overall, our findings demonstrate that the mitochondrial genome is highly stable across tissues and within TSC-associated tumors.

Differential and Common Signatures of miRNA Expression and Methylation in Childhood Central Nervous System Malignancies: An Experimental and Computational Approach.

Epigenetic modifications are considered of utmost significance for tumor ontogenesis and progression. Especially, it has been found that miRNA expression, as well as DNA methylation plays a significant role in central nervous system tumors during childhood. A total of 49 resected brain tumors from children were used for further analysis. DNA methylation was identified with methylation-specific MLPA and, in particular, for the tumor suppressor genes CASP8, RASSF1, MGMT, MSH6, GATA5, ATM1, TP53, and CADM1. miRNAs were identified with microarray screening, as well as selected samples, were tested for their mRNA expression levels. CASP8, RASSF1 were the most frequently methylated genes in all tumor samples. Simultaneous methylation of genes manifested significant results with respect to tumor staging, tumor type, and the differentiation of tumor and control samples. There was no significant dependence observed with the methylation of one gene promoter, rather with the simultaneous presence of all detected methylated genes' promoters. miRNA expression was found to be correlated to gene methylation. Epigenetic regulation appears to be of major importance in tumor progression and pathophysiology, making it an imperative field of study.

Phase II Clinical Trial of Everolimus in a Pan-Cancer Cohort of Patients with mTOR Pathway Alterations.

PURPOSE: This was a multicenter, histology-agnostic, single-arm prospective phase II trial of therapeutic activity of everolimus, an oral mTORC1 inhibitor, in patients with advanced solid tumors that harbored TSC1/TSC2 or MTOR mutations. PATIENTS AND METHODS: Patients with tumors with inactivating TSC1/TSC2 or activating MTOR mutations identified in any Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory were eligible. Patients were treated with everolimus 10 mg once daily until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR). Whole-exome sequencing was performed to identify co-occurring genomic alterations. RESULTS: Between November 2015 and October 2018, 30 patients were enrolled at Dana-Farber Cancer Institute and Memorial Sloan Kettering Cancer Center. Tumors harbored TSC1 (13/30), TSC2 (15/30), concurrent TSC1 and TSC2 (1/30), or MTOR (1/30) mutations. The most common treatment-related adverse event of any grade was mucositis (8/30, 27%); 1 patient had fatal pneumonitis. Partial responses were seen in 2 patients [7%; 95% confidence interval (CI), 1%-22%]. Median progression-free survival was 2.3 months (95% CI, 1.8-3.7 months) and median overall survival (OS) was 7.3 months (95% CI, 4.5-12.7 months). There was no clear association between other genomic alterations and response. Of the 2 patients with objective response, 1 had upper tract urothelial carcinoma with biallelic inactivation of TSC1 and high tumor mutation burden, and the other had uterine carcinoma with biallelic TSC2-inactivating mutations and PEComa-like pathologic features. CONCLUSIONS: Everolimus therapy had a disappointing ORR (7%) in this pan-cancer, mutation-selected, basket study.See related commentary by Kato and Cohen, p. 3807.

Therapeutic Targeting of DGKA-Mediated Macropinocytosis Leads to Phospholipid Reprogramming in Tuberous Sclerosis Complex.

Lymphangioleiomyomatosis is a rare destructive lung disease affecting primarily women and is the primary lung manifestation of tuberous sclerosis complex (TSC). In lymphangioleiomyomatosis, biallelic loss of TSC1/2 leads to hyperactivation of mTORC1 and inhibition of autophagy. To determine how the metabolic vulnerabilities of TSC2-deficient cells can be targeted, we performed a high-throughput screen utilizing the "Repurposing" library at the Broad Institute of MIT and Harvard (Cambridge, MA), with or without the autophagy inhibitor chloroquine. Ritanserin, an inhibitor of diacylglycerol kinase alpha (DGKA), was identified as a selective inhibitor of proliferation of Tsc2-/- mouse embryonic fibroblasts (MEF), with no impact on Tsc2+/+ MEFs. DGKA is a lipid kinase that metabolizes diacylglycerol to phosphatidic acid, a key component of plasma membranes. Phosphatidic acid levels were increased 5-fold in Tsc2-/- MEFs compared with Tsc2+/+ MEFs, and treatment of Tsc2-/- MEFs with ritanserin led to depletion of phosphatidic acid as well as rewiring of phospholipid metabolism. Macropinocytosis is known to be upregulated in TSC2-deficient cells. Ritanserin decreased macropinocytic uptake of albumin, limited the number of lysosomes, and reduced lysosomal activity in Tsc2-/- MEFs. In a mouse model of TSC, ritanserin treatment decreased cyst frequency and volume, and in a mouse model of lymphangioleiomyomatosis, genetic downregulation of DGKA prevented alveolar destruction and airspace enlargement. Collectively, these data indicate that DGKA supports macropinocytosis in TSC2-deficient cells to maintain phospholipid homeostasis and promote proliferation. Targeting macropinocytosis with ritanserin may represent a novel therapeutic approach for the treatment of TSC and lymphangioleiomyomatosis. SIGNIFICANCE: This study identifies macropinocytosis and phospholipid metabolism as novel mechanisms of metabolic homeostasis in mTORC1-hyperactive cells and suggest ritanserin as a novel therapeutic strategy for use in mTORC1-hyperactive tumors, including pancreatic cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2086/F1.large.jpg.

Subependymal giant cell astrocytomas are characterized by mTORC1 hyperactivation, a very low somatic mutation rate, and a unique gene expression profile.

Subependymal giant-cell astrocytomas (SEGAs) are slow-growing brain tumors that are a hallmark feature seen in 5-10% of patients with Tuberous Sclerosis Complex (TSC). Though histologically benign, they can cause serious neurologic symptoms, leading to death if untreated. SEGAs consistently show biallelic loss of TSC1 or TSC2. Herein, we aimed to define other somatic events beyond TSC1/TSC2 loss and identify potential transcriptional drivers that contribute to SEGA formation. Paired tumor-normal whole-exome sequencing was performed on 21 resected SEGAs from 20 TSC patients. Pathogenic variants in TSC1/TSC2 were identified in 19/21 (90%) SEGAs. Copy neutral loss of heterozygosity (size range: 2.2-46 Mb) was seen in 76% (16/21) of SEGAs (44% chr9q and 56% chr16p). An average of 1.4 other somatic variants (range 0-7) per tumor were identified, unlikely of pathogenic significance. Whole transcriptome RNA-sequencing analyses revealed 190 common differentially expressed genes in SEGA (n = 16, 13 from a prior study) in pairwise comparison to each of: low grade diffuse gliomas (n = 530) and glioblastoma (n = 171) from The Cancer Genome Atlas (TCGA) consortium, ganglioglioma (n = 10), TSC cortical tubers (n = 15), and multiple normal tissues. Among these, homeobox transcription factors (TFs) HMX3, HMX2, VAX1, SIX3; and TFs IRF6 and EOMES were all expressed >12-fold higher in SEGAs (FDR/q-value < 0.05). Immunohistochemistry supported the specificity of IRF6, VAX1, SIX3 for SEGAs in comparison to other tumor entities and normal brain. We conclude that SEGAs have an extremely low somatic mutation rate, suggesting that TSC1/TSC2 loss is sufficient to drive tumor growth. The unique and highly expressed SEGA-specific TFs likely reflect the neuroepithelial cell of origin, and may also contribute to the transcriptional and epigenetic state that enables SEGA growth following two-hit loss of TSC1 or TSC2 and mTORC1 activation.

MITF is a driver oncogene and potential therapeutic target in kidney angiomyolipoma tumors through transcriptional regulation of CYR61.

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor syndrome, characterized by tumor development in multiple organs, including renal angiomyolipoma. Biallelic loss of TSC1 or TSC2 is a known genetic driver of angiomyolipoma development, however, whether an altered transcriptional repertoire contributes to TSC-associated tumorigenesis is unknown. RNA-seq analyses showed that MITF A isoform (MITF-A) was consistently highly expressed in angiomyolipoma, immunohistochemistry showed microphthalmia-associated transcription factor nuclear localization, and Chromatin immuno-Precipitation Sequencing analysis showed that the MITF-A transcriptional start site was highly enriched with H3K27ac marks. Using the angiomyolipoma cell line 621-101, MITF knockout (MITF.KO) and MITF-A overexpressing (MITF.OE) cell lines were generated. MITF.KO cells showed markedly reduced growth and invasion in vitro, and were unable to form xenografted tumors. In contrast, MITF.OE cells grew faster in vitro and as xenografted tumors compared to control cells. RNA-Seq analysis showed that both ID2 and Cysteine-rich angiogenic inducer 61 (CYR61) expression levels were increased in the MITF.OE cells and reduced in the MITF.KO cells, and luciferase assays showed this was due to transcriptional effects. Importantly, CYR61 overexpression rescued MITF.KO cell growth in vitro and tumor growth in vivo. These findings suggest that MITF-A is a transcriptional oncogenic driver of angiomyolipoma tumor development, acting through regulation of CYR61.

Single-Cell Transcriptomic Analysis Identifies a Unique Pulmonary Lymphangioleiomyomatosis Cell.

Rationale: Lymphangioleiomyomatosis (LAM) is a metastatic neoplasm of reproductive-age women associated with mutations in tuberous sclerosis complex genes. LAM causes cystic remodeling of the lung and progressive respiratory failure. The sources and cellular characteristics of LAM cells underlying disease pathogenesis remain elusive.Objectives: Identification and characterization of LAM cells in human lung and uterus using a single-cell approach.Methods: Single-cell and single-nuclei RNA sequencing on LAM (n = 4) and control (n = 7) lungs, immunofluorescence confocal microscopy, ELISA, and aptamer proteomics were used to identify and validate LAMCORE cells and secreted biomarkers, predict cellular origins, and define molecular and cellular networks in LAM.Measurements and Main Results: A unique cell type termed LAMCORE was identified, which was distinct from, but closely related to, lung mesenchymal cells. LAMCORE cells expressing signature genes included known LAM markers such as PMEL, FIGF, CTSK, and MLANA and novel biomarkers validated by aptamer screening, ELISA, and immunofluorescence microscopy. LAM cells in lung and uterus are morphologically indistinguishable and share similar gene expression profiles and biallelic TSC2 mutations, supporting a potential uterine origin for the LAMCORE cell. Effects of LAM on resident pulmonary cell types indicated recruitment and activation of lymphatic endothelial cells.Conclusions: A unique population of LAMCORE cells was identified in lung and uterus of patients with LAM, sharing close transcriptomic identity. LAM cell selective markers, secreted biomarkers, and the predicted cellular molecular features provide new insights into the signaling and transcriptional programs that may serve as diagnostic markers and therapeutic targets to influence the pathogenesis of LAM.

Tumors with TSC mutations are sensitive to CDK7 inhibition through NRF2 and glutathione depletion.

Tuberous sclerosis complex (TSC) is characterized by tumor development in the brain, heart, kidney, and lungs. In TSC tumors, loss of the TSC1/TSC2 protein complex leads to activation of mTORC1 with downstream effects on anabolism and cell growth. Because mTORC1 activation enhances mRNA transcription, we hypothesized that aberrant mTORC1 activation might confer TSC-null cell dependence on transcriptional regulation. We demonstrate that TSC1- or TSC2-null cells, in contrast to their wild-type counterparts, are sensitive to pharmacological inhibition of CDK7. Mechanistic studies revealed that CDK7 inhibition markedly reduces glutathione levels and increases reactive oxygen species due to reduced expression of NRF2 and glutathione biosynthesis genes. Treatment of both Tsc2+/ - mice and a TSC1-null bladder cancer xenograft model with a CDK7 inhibitor showed marked reduction in tumor volume and absence of regrowth in the xenograft model. These results suggest that CDK7 inhibition is a promising therapeutic approach for treatment of TSC-associated tumors and cancers with mutations in either TSC1 or TSC2.

Low-level mosaicism in tuberous sclerosis complex: prevalence, clinical features, and risk of disease transmission.

PURPOSE: To examine the prevalence and spectrum of mosaic variant allele frequency (MVAF) in tuberous sclerosis complex (TSC) patients with low-level mosaicism and correlate genetic findings with clinical features and transmission risk. METHODS: Massively parallel sequencing was performed on 39 mosaic TSC patients with 170 different tissue samples. RESULTS: TSC mosaic patients (MVAF: 0-10%, median 1.7% in blood DNA) had a milder and distinct clinical phenotype in comparison with other TSC series, with similar facial angiofibromas (92%) and kidney angiomyolipomas (83%), and fewer seizures, cortical tubers, and multiple other manifestations (p < 0.0001 for six features). MVAF of TSC1/TSC2 pathogenic variants was highly variable in different tissue samples. Remarkably, skin lesions were the most reliable tissue for variant identification, and 6 of 39 (15%) patients showed no evidence of the variant in blood. Semen analysis showed absence of the variant in 3 of 5 mosaic men. The expected distribution of MVAF in comparison with that observed here suggests that there is a considerable number of individuals with low-level mosaicism for a TSC2 pathogenic variant who are not recognized clinically. CONCLUSION: Our findings provide information on variability in MVAF and risk of transmission that has broad implications for other mosaic genetic disorders.

A genome-wide association study implicates NR2F2 in lymphangioleiomyomatosis pathogenesis.

INTRODUCTION: Lymphangioleiomyomatosis (LAM) occurs either associated with tuberous sclerosis complex (TSC) or as sporadic disease (S-LAM). Risk factors for development of S-LAM are unknown. We hypothesised that DNA sequence variants outside of TSC2/TSC1 might be associated with susceptibility for S-LAM and performed a genome-wide association study (GWAS). METHODS: Genotyped and imputed data on 5 426 936 single nucleotide polymorphisms (SNPs) in 426 S-LAM subjects were compared, using conditional logistic regression, with similar data from 852 females from COPDGene in a matched case-control design. For replication studies, genotypes for 196 non-Hispanic White female S-LAM subjects were compared with three different sets of controls. RNA sequencing and immunohistochemistry analyses were also performed. RESULTS: Two noncoding genotyped SNPs met genome-wide significance: rs4544201 and rs2006950 (p=4.2×10-8 and 6.1×10-9, respectively), which are in the same 35 kb linkage disequilibrium block on chromosome 15q26.2. This association was replicated in an independent cohort. NR2F2 (nuclear receptor subfamily 2 group F member 2), a nuclear receptor and transcription factor, was the only nearby protein-coding gene. NR2F2 expression was higher by RNA sequencing in one abdominal LAM tumour and four kidney angiomyolipomas, a LAM-related tumour, compared with all cancers from The Cancer Genome Atlas. Immunohistochemistry showed strong nuclear expression in both LAM and angiomyolipoma tumours. CONCLUSIONS: SNPs on chromosome 15q26.2 are associated with S-LAM, and chromatin and expression data suggest that this association may occur through effects on NR2F2 expression, which potentially plays an important role in S-LAM development.

Expanding the histomorphologic spectrum of TFE3-rearranged perivascular epithelioid cell tumors.

Perivascular epithelioid cell tumors (PEComas) are a family of mesenchymal neoplasms that have smooth muscle and melanocytic differentiation. They can be sporadic or associated with tuberous sclerosis complex and commonly present in the kidney as angiomyolipoma or in the lung as pulmonary clear cell sugar tumors or lymphangioleiomyomatosis. However, they can present at any visceral or soft tissue site. They usually have a benign clinical course, but rarely can behave in a malignant fashion. Most PEComas demonstrate abnormalities of TSC2, but a recently described subset harbor TFE3 rearrangements that seem to be mutually exclusive of TSC2 alterations. TFE3-rearranged PEComas demonstrate a distinct alveolar morphology that lacks spindle cells and smooth muscle differentiation. Distinction between these may have important therapeutic consequences. Herein, we present a case of a TFE3-rearranged PEComa without the customary morphology that required ancillary investigation with TFE3 immunohistochemistry and break-apart fluorescence in situ hybridization for proper categorization.

Subependymal giant cell astrocytomas in Tuberous Sclerosis Complex have consistent TSC1/TSC2 biallelic inactivation, and no BRAF mutations.

Subependymal giant cell astrocytomas (SEGAs) are rare, low-grade glioneuronal brain tumors that occur almost exclusively in patients with tuberous sclerosis complex (TSC). Though histologically benign, SEGAs can lead to serious neurological complications, including hydrocephalus, intractable seizures and death. Previous studies in a limited number of SEGAs have provided evidence for a biallelic two-hit inactivation of either TSC1 or TSC2, resulting in constitutive activation of the mechanistic target of rapamycin complex 1 pathway. The activating BRAF V600E mutation is a common genetic alteration in low grade gliomas and glioneuronal tumors, and has been reported in SEGAs as well. In the present study, we assessed the prevalence of the BRAF V600E mutation in a large cohort of TSC related SEGAs (n=58 patients including 56 with clinical TSC) and found no evidence of either BRAF V600E or other mutations in BRAF. To confirm that these SEGAs fit the classic model of two hit TSC1 or TSC2 inactivation, we also performed massively parallel sequencing of these loci. Nineteen (19) of 34 (56%) samples had mutations in TSC2, 10 (29%) had mutations in TSC1, while 5 (15%) had no mutation identified in TSC1/TSC2. The majority of these samples had loss of heterozygosity in the same gene in which the mutation was identified. These results significantly extend previous studies, and in agreement with the Knudson two hit mechanism indicate that biallelic alterations in TSC2 and less commonly, TSC1 are consistently seen in SEGAs.

Notch transactivates Rheb to maintain the multipotency of TSC-null cells.

Differentiation abnormalities are a hallmark of tuberous sclerosis complex (TSC) manifestations; however, the genesis of these abnormalities remains unclear. Here we report on mechanisms controlling the multi-lineage, early neuronal progenitor and neural stem-like cell characteristics of lymphangioleiomyomatosis (LAM) and angiomyolipoma cells. These mechanisms include the activation of a previously unreported Rheb-Notch-Rheb regulatory loop, in which the cyclic binding of Notch1 to the Notch-responsive elements (NREs) on the Rheb promoter is a key event. This binding induces the transactivation of Rheb. The identified NRE2 and NRE3 on the Rheb promoter are important to Notch-dependent promoter activity. Notch cooperates with Rheb to block cell differentiation via similar mechanisms in mouse models of TSC. Cell-specific loss of Tsc1 within nestin-expressing cells in adult mice leads to the formation of kidney cysts, renal intraepithelial neoplasia, and invasive papillary renal carcinoma.