Interferon-gamma-Jak-Stat signaling in pulmonary lymphangioleiomyomatosis and renal angiomyolipoma: a potential therapeutic target

Pulmonary lymphangioleiomyomatosis (LAM) and renal angiomyolipoma (AML) are proliferative lesions that occur in sporadic patients, and at much higher frequency in patients with tuberous sclerosis (TSC). The TSC1 and TSC2 genes play a critical role in their pathogenesis. Here we report a marked decrease in interferon (IFN)-gamma expression in both sporadic and TSC-associated AML and LAM. A marked increase in Stat1 expression and phosphorylation at Ser 727, and in phospho-Tyr705-Stat3 levels, was also seen in both AML and LAM tissues. Our results demonstrate that the IFN-gamma-Jak-Stat pathway is perturbed in TSC-related and sporadic LAM and AML, and suggest that IFN-gamma has potential therapeutic benefit for treatment of those lesions.     

A cross sectional study of renal involvement in tuberous sclerosis.

Renal disease is a frequent manifestation of tuberous sclerosis (TSC) and yet little is known about its true prevalence or natural history. We reviewed the notes of 139 people with TSC, who had presented without renal symptoms, but who had been investigated by renal ultrasound. Information on the frequency, type, and symptomatology of renal involvement was retrieved. The prevalence of renal involvement was 61%. Angiomyolipomas were detected in 49%, renal cysts in 32%, and renal carcinoma in 2.2%. The prevalence of angiomyolipoma was positively correlated with age, compatible with a two hit aetiology. Renal cysts were the commoner lesion in young children, and their prevalence did not appear to be age related. Renal investigation in people with TSC had been inconsistent and limited. We suggest guidelines for renal investigation in those with TSC.     

Loss of heterozygosity in the tuberous sclerosis (TSC2) region of chromosome band l6p13 occurs in sporadic as well as TSC-associated renal angiomyolipomas

Angiomyolipomas (AMLs) are renal tumors that occur both sporadically and in association with tuberous sclerosis (TSC). TSC is an autosomal dominant disorder characterized by hamartomatous lesions in multiple organs. Two TSC loci are recognized TSCl on 9q34 and TSC2 on 16pl3. Loss of heterozygosity (LOH) at the TSCI and TSC2 loci in lesions from TSC patients has recently been reported. Lesions that are not associated with TSC have not been previously examined for LOH at the TSC loci. We analyzed 29 renal angiomyolipomas from patients without a history of TSC. Three tumors demonstrated LOH on I6p 13. This is the first report indicating that mutations in TSC2 occur in tumors of patients who do not have TSC. We also found LOH on I6p 13 in 5 of 8 TSC-associated AMLs. Two of these tumors were from a single patient and demonstrated different regions of LOH. These findings support the hypothesis that the TSC2 gene functions as a tumor suppressor.     

Multifocal renal cell carcinoma in sibs from a chromosome 9 linked (TSC1) tuberous sclerosis family.

Multifocal renal cell carcinomas (RCCs), together with angiomyolipomas (AMLs) and renal cysts, were identified in early adult life in two sisters with tuberous sclerosis (TSC). They were members of a multigenerational tuberous sclerosis family showing strong evidence for a mutant TSC causing gene on chromosome 9 (TSC1). Previous reports of multifocal RCC in young patients with TSC suggest that constitutional mutations at the TSC loci may predispose to RCC. In the rat a germline mutation affecting the TSC2 gene is associated with transmission of multifocal RCC as an autosomal dominant trait. However, the cases reported here are the first to suggest a similar role for the TSC1 gene in renal cell carcinogenesis.     

Genotype/phenotype correlation in 325 individuals referred for a diagnosis of tuberous sclerosis complex in the United States.

Tuberous sclerosis complex is an autosomal dominant neurocutaneous disorder marked by hamartoma growth in multiple organ systems. We performed mutational analyses on 325 individuals with definite tuberous sclerosis complex diagnostic status. We identified mutations in 72% (199/257) of de novo and 77% (53/68) of familial cases, with 17% of mutations in the TSC1 gene and 50% in the TSC2 gene. There were 4% unclassified variants and 29% with no mutation identified. Genotype/phenotype analyses of all observed tuberous sclerosis complex findings in probands were performed, including several clinical features not analyzed in two previous large studies. We showed that patients with TSC2 mutations have significantly more hypomelanotic macules and learning disability in contrast to those with TSC1 mutations, findings not noted in previous studies. We also observed results consistent with two similar studies suggesting that individuals with mutations in TSC2 have more severe symptoms. On performing meta-analyses of our data and the other two largest studies in the literature, we found significant correlations for several features that individual studies did not have sufficient power to conclude. Male patients showed more frequent neurologic and eye symptoms, renal cysts, and ungual fibromas. Correlating genotypes with phenotypes should facilitate the disease management of tuberous sclerosis complex.     

Leiomyomatosis-like lymphangioleiomyomatosis of the colon in a female with tuberous sclerosis.

Smooth muscle lesions of the large bowel, excluding the rectum, are generally rare, and diffuse smooth muscle lesions, termed leiomyomatosis, are even rarer. In this report, we document, for the first time, leiomyomatosis-like lymphangioleiomyomatosis (LAM) of the ascending, transverse, and descending colon in association with bilateral renal angiomyolipoma (AML) in a 30-year-old Chinese female with tuberous sclerosis complex (TSC). She presented with protracted constipation for which a colectomy was performed. Histology disclosed multiple confluent nodular CD34 and CD117 negative smooth muscle proliferation within the large bowel wall, whereas the renal biopsy revealed typical features of AML. Interestingly, the epithelioid smooth muscle cells of both the colonic and renal lesions were HMB45 positive, suggesting that leiomyomatosis-like LAM of the colon, pulmonary LAM and AML are closely related entities. The patient remained free of complications for the last five years after surgery. Leiomyomatosis-like LAM of the large bowel probably represents another manifestation of the tendency of TSC to be associated with proliferative lesions.     

Bilateral multiple renal oncocytomas and cysts associated with a constitutional translocation (8;9)(q24.1;q34.3) and a rare constitutional VHL missense substitution

We report here on a patient with bilateral multifocal renal oncocytomas and cysts. Cytogenetic analysis of the patient's lymphocytes revealed a constitutional 46,XY,add (9)(q34.3) karyotype. The rearrangement was further resolved as a constitutional reciprocal t(8;9)(q24.1;q34.3) by microdissection and FISH. Because the 9q breakpoint was located in the same region as the tuberous sclerosis type 1 locus (TSC1), which is associated with renal tumors, we performed FISH with two TSC1 flanking cosmids that were mapped proximal to the 9q breakpoint, thus excluding its involvement. Loss of heterozygosity (LOH) studies of the tumors revealed LOH in chromosome 1, further strengthening the molecular diagnosis of oncocytoma. A previously unreported germline missense substitution, Pro40Arg, in exon 1 of the VHL gene was also found in the patient's constitutional DNA, adding to the complexity of the genetic profile. Genes Chromosomes Cancer 21:260–264, 1998. © 1998 Wiley-Liss, Inc.     

Subependymal astrocytic hamartomas in the Eker rat model of tuberous sclerosis.

Tuberous sclerosis (TSC) is an autosomal dominant syndrome that is linked to two genetic loci: TSC1 (9q34) and TSC2 (16p13). Brain manifestations such as cortical tubers and subependymal hamartoma/giant cell astrocytomas are major causes of TSC-related morbidity. In this study, we describe the central nervous system involvement in a unique rodent model of tuberous sclerosis. The Eker rat carries a spontaneous germline mutation of the TSC2 gene and is predisposed to multiple neoplasia. In a series of 45 adult Eker carriers (TSC2 +/-), three types of focal intracranial lesions were found, of which the subependymal and subcortical hamartomas were most prevalent (65%). There exist remarkable phenotypic similarities between the Eker rat and human subependymal lesions. Our study indicates that the predominant cellular phenotype of the subependymal hamartomas is astroglial and suggests that the neuronal contribution within these lesions is, in part, the result of pre-existing myelinated axons. The hamartomas did not show evidence of loss of the wild-type TSC2 allele; it remains to be determined whether TSC2 inactivation is necessary for their pathogenesis. This genetically-defined rodent model may be useful in elucidating the molecular and developmental basis of the subependymal giant cell astrocytoma in humans.     

Identification of a nonsense mutation at the 5'' end of the TSC2 gene in a family with a presumptive diagnosis of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is an autosomal dominantly inherited disease with a high mutation rate. It is clinically a very variable disorder and hamartomas can occur in many different organs. TSC shows genetic heterogeneity; one gene, TSC1, is on chromosome 9q34, and the second gene, TSC2, on chromosome 16p13.3. Clinical criteria for diagnosis have been established, but diagnosis of patients with minimal expression of the disease can be very difficult. In children the phenotype is often incomplete or not fully assessable. Hence mildly affected subjects, at risk for severely affected offspring, may remain undiagnosed. The detection of (small) mutations in the tuberous sclerosis gene located on chromosome 16 (TSC2) has recently become possible and may be helpful in the diagnosis of ambiguous cases. To our knowledge, this is the first report of a point mutation in the TSC2 gene in a familial case of tuberous sclerosis. A nonsense mutation was detected in a family in which the father had only minor signs hinting at tuberous sclerosis. The son had multiple cardiac tumours and white patches, but full clinical investigation was impossible in this child. This case illustrates that mutation analysis can contribute to a diagnosis of tuberous sclerosis in families with an incomplete phenotype.     

A human PKD1 transgene generates functional polycystin-1 in mice and is associated with a cystic phenotype

Three founder transgenic mice were generated with a 108 kb human genomic fragment containing the entire autosomal dominant polycystic kidney disease (ADPKD) gene, PKD1, plus the tuberous sclerosis gene, TSC2. Two lines were established (TPK1 and TPK3) each with approximately 30 copies of the transgene. Both lines produced full-length PKD1 mRNA and polycystin-1 protein that was developmentally regulated, similar to the endogenous pattern, with expression during renal embryogenesis and neonatal life, markedly reduced at the conclusion of renal development. Tuberin expression was limited to the brain. Transgenic animals from both lines (and the TPK2 founder animal) often displayed a renal cystic phenotype, typically consisting of multiple microcysts, mainly of glomerular origin. Hepatic cysts and bile duct proliferation, characteristic of ADPKD, were also seen. All animals with two copies of the transgenic chromosome developed cysts and, in total, 48 of the 100 transgenic animals displayed a cystic phenotype. To test the functionality of the transgene, animals were bred with the Pkd1(del34) knockout mouse. Both transgenic lines rescued the embryonically lethal Pkd1(del34/del34) phenotype, demonstrating that human polycystin-1 can complement for loss of the endogenous protein. The rescued animals were viable into adulthood, although more than half developed hepatic cystic disease in later life, similar to the phenotype of older Pkd1(del34/+) animals. The TPK mice have defined a minimal area that appropriately expresses human PKD1. Furthermore, this model indicates that over-expression of normal PKD1 can elicit a disease phenotype, suggesting that the level of polycystin-1 expression may be relevant in the human disease.     

9q34 loss of heterozygosity in a tuberous sclerosis astrocytoma suggests a growth suppressor-like activity also for the TSC1 gene

Tuberous sclerosis is an autosomal dominant disease whose characteristicfeature is the development of multiple hamartomas in a varietyof organs and tissues. Two major loci have been identified sofar: TSC1 on chromosome 9q34 and TSC2 on chromosome 16p13.3.Loss of heterozygosity at 16p13.3-associated markers has beenrecently observed in hamartomatous lesions of some tuberoussclerosis patients. Here we report the first evidence of lossof heterozygosity at the TSC1 critical region in a giant cellastrocytoma of a familial tuberous sclerosis case. Segregationanalysis showed that the 9q34 haplotype lost carried the putativenormal TSC1 gene. These data support the hypothesis of botha germline and somatic loss-of-function mutation for the developmentof tuberous sclerosis hamartomas and suggest a tumor-suppressor-likeactivity also for the TSC1 gene product. Finally, the possiblesignificance of a second small region of loss of heterozygosityat 9p21, found in the same astrocytoma, is discussed.     

Multiple angiomyolipomata of the liver: a case report.

Angiomyolipoma (AML) is a rare benign tumor that occurs most commonly in the kidney. Occasionally it may be found in the liver. Lesions in the liver are usually solitary. Multiple AMLs are extremely rare and are typically seen in patients with tuberous sclerosis. We now report an unusual case of a 46-year-old woman with multiple hepatic AMLs. There were more than 15 lesions distributed predominantly in the right hepatic lobe. The tumors ranged from 0.2 to 6 cm in size and consisted of a variable admixture of proliferating blood vessels, adipose tissue, and smooth muscle. There was no clinical evidence of tuberous sclerosis in this patient. Polymerase chain reaction amplification of the highly polymorphic human androgen receptor gene (HUMARA) was performed and the pattern of X chromosome inactivation was analyzed. Three of the five representative AML nodules showed a preferential loss of one of the two HUMARA alleles indicating a clonal proliferation with involvement of different alleles. Histologic examination of the corresponding lesions showed clonal lesions to be predominantly composed of epithelioid myoid cells while the polyclonal lesions were predominantly composed of adipose tissue. While the histologic diagnosis of AML in a surgical resection specimen is often straightforward, the radiographic, cytologic and intra-operative interpretation of a case with multiple lesions presents a considerable challenge.     

Multifocal angiomyolipoma affecting the liver and lung without tuberous sclerosis

The occurrence of angiomyolipoma (AML) in tissue other than the kidney is uncommon, as is multiple AML developing exclusively in organs other than the kidney. This report describes a case in which AML occurred multifocally in the liver and lung, but spared the kidney, and which might have been associated with tuberous sclerosis complex (TSC). A Japanese woman underwent a partial hepatectomy for a suspected malignant liver tumour at the age of 57. The tumour consisted predominantly of a trabecular arrangement of myoid cells with a sinusoidal pattern and inflammatory cell infiltration, and was diagnosed as a primary liver AML by HMB-45 immunoreactivity. Five years later, multiple nodules were found in both lungs, for which video assisted thoracic surgery was performed. The tumour showed a mixture of epithelioid cells containing HMB-45 positive material and mature lipocytes, and was subsequently diagnosed as AML. Molecular analysis of both lesions showed no allelic loss of the TSC1 and TSC2 regions. Molecular analysis of the tumours ruled out an association with TSC, and both liver and lung lesions displayed benign histological features, so that these were probably multifocal lesions of AML without TSC.     

Tuberous sclerosis-associated renal cell carcinoma. Clinical, pathological, and genetic features.

The tuberous sclerosis complex (TSC) is a multisystem autosomal dominant disorder characterized by seizures, mental retardation, and hamartomas. Patients with TSC have been reported to develop renal cell carcinomas (RCC) with increased frequency, an observation that is supported by the Eker rat model. To address the role of the tuberous sclerosis tumor suppressor genes in the pathogenesis of RCC, we studied six TSC-associated RCCs. Our findings suggest that some TSC-associated RCCs have clinical, pathological, or genetic features distinguishing them from sporadic RCC. Clinically, the TSC-associated tumors occurred at a younger age (mean, 36 years) than sporadic tumors and occurred primarily in women. Four of the six patients died of metastatic disease. Pathologically, five tumors displayed clear cell morphology. Of those five, two had high-grade spindle cell areas and one had granular cell histology in addition to the clear cell areas. A sixth tumor was anaplastic throughout. Four of the six tumors immunostained positively for a melanocyte-associated marker, HMB-45. HMB-45 positivity has been seen in two other TSC lesions: renal angiomyolipomas and pulmonary lymphangiomyomatosis. Five tumors were analyzed for loss of heterozygosity. Two had loss of heterozygosity on chromosome 9q34 and one had loss of heterozygosity on chromosome 16p13. We conclude that TSC-associated RCCs occur at an earlier age than sporadic RCCs, that some TSC-associated renal carcinomas have a different immunophenotype than sporadic RCCs, and that the TSC tumor suppressor genes may play a specific pathogenic role in these tumors.     

Phenotypic and genotypic characterization of Chinese children diagnosed with tuberous sclerosis complex

We investigated the clinical phenotypes and genetic mutations in Chinese children diagnosed with tuberous sclerosis complex (TSC). Sequencing of TSC1 and TSC2 genes was performed in 117 children with TSC and their parents. Association of TSC gene mutations with clinical manifestations was investigated. All gene mutations were heterozygous including in 16 patients (13.7%) with mutations in TSC1 gene and 101 patients (86.3%) with mutations in TSC2 gene. Among the 16 patients with TSC1 gene mutations, 15 different types of mutations were found, which included 5 novel mutations; all patients had skin manifestations and epilepsy. Among the 101 patients with TSC2 mutations, 85 different types of mutations were found, which included 25 novel mutations; 97 patients (96.0%) had skin manifestations; 97 (96.0%) had epilepsy; 74 (73.3%) had intellectual disability and 25 patients (24.8%) were autistic. The clinical phenotype of the 14 children with familial TSC was more severe than that of their parents.     

The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis

Tuberous sclerosis is an autosomal dominant trait in which the dysregulation of cellular proliferation and differentiation results in the development of hamartomatous growths in many organs. The TSC2 gene is one of two genes determining tuberous sclerosis. Inactivating germline mutations of TSC2 in patients with tuberous sclerosis and somatic loss of heterozygosity at the TSC2 locus in the associated hamartomas indicate that TSC2 functions as a tumour suppressor gene and that loss of function is critical to expression of the tuberous sclerosis phenotype. The TSC2 product, tuberin, has a region of homology with the GTPase activating protein rap1GAP and stimulates the GTPase activity of rap1a and rab5a in vitro. Here we show that the region of homology between tuberin and human rap1GAP and the murine GAP mSpa1 is more extensive than previously reported and spans approximately 160 amino acid residues encoded within exons 34-38 of the TSC2 gene. Single strand conformation polymorphism analysis of these exons in 173 unrelated patients with tuberous sclerosis and direct sequencing of variant conformers together with study of additional family members enabled characterisation of disease associated mutations in 14 cases. Missense mutations, which occurred in exons 36, 37 and 38 were identified in eight cases, four of whom shared the same recurrent change P1675L. Each of the five different missense mutations identified was shown to occur de novo in at least one sporadic case of tuberous sclerosis. The high proportion of missense mutations detected in the region of the TSC2 gene encoding the GAP-related domain supports its key role in the regulation of cellular growth.      

Uterine angiosarcoma associated with lymphangioleiomyomatosis in a patient with tuberous sclerosis complex: an autopsy case report with immunohistochemical and genetic analysis

A 41-year-old woman carrying a germline tuberous sclerosis complex 2 (TSC2) mutation, whose regular medical follow-up for tuberous sclerosis complex and tuberous sclerosis complex-associated lymphangioleiomyomatosis had continued for 2 years, had uterine angiosarcoma concomitant with uterine lymphangioleiomyomatosis. Immunohistochemically, the uterine angiosarcoma cells showed an extremely skewed lymphatic differentiation; they were diffusely immunopositive for CD31 but negative for other vascular endothelial markers including factor VIII and CD34 yet strongly immunopositive for lymphatic endothelial markers including D2-40 and Prox-1. Loss of heterozygosity analysis demonstrated that not only lymphangioleiomyomatosis and renal angiomyolipoma but also the uterine angiosarcoma had loss of heterozygosity on TSC2. Furthermore, direct sequencing revealed a TP53 mutation in the uterine angiosarcoma. Collectively, the findings suggest that combined dysfunction of the p53 and TSC2 tumor suppressor proteins may contribute to the development of uterine angiosarcoma in this rare clinical setting.     

Genotype/phenotype correlation in 123 Chinese patients with Tuberous Sclerosis Complex

Tuberous Sclerosis Complex (TSC) is a multisystemic neurocutaneous disorder with autosomal dominant inheritance. We performed mutation analyses on 123 Chinese patients with “definite TSC” according to the latest diagnostic criteria. Pathogenic / likely-pathogenic variants were identified in 72.2% of all index patients (70/97), in which 35.7% (25/70) had TSC1 variants and 64.3% (45/70) had TSC2 variants. 84.5% (82/97) cases were sporadic and 15.5% (15/97) cases were familial. 62 unique variants were reported, in which 41.9% (26/62) were novel. Male patients had significantly more subependymal nodules (p=0.029) than females, whereas renal angiomyolipoma (p=0.032) occurred predominantly in females. Sporadic cases also had more renal angiomyolipoma (p=0.004), cortical tubers (p=0.008), hypopigmented macules (p=0.018) and fibrous cephalic plaques (p=0.028) than cases with known inheritance. Patients with TSC2 pathogenic variants were more likely to have mental retardation (p<0.001), cardiac rhabdomyoma (p=0.004), renal angiomyolipoma (p=0.006) and facial angiofibromas (p=0.026) than those with TSC1 pathogenic variants, while mutation-negative cases showed a mixed phenotype between those with TSC1 and TSC2 variants. There were no significant phenotypic differences between patients with and without TSC1/TSC2 variants, but TSC2 missense and in-frame variants were associated with higher frequencies of mental retardation (P<0.001), renal angiomyolipoma (p=0.001), cardiac rhabdomyoma (p=0.012) and facial angiofibroma (p=0.021) than those with TSC1 frameshift and splice site variants. Furthermore, a higher frequency of mental retardation (p=0.013) was observed in patients with TSC2 missense and in-frame variants than those with frameshift and splice site variants. All 14 antenatal-onset patients had cardiac rhabdomyoma. They had fewer seizures (p=0.028) than patients with paediatric-onset, but were more likely to have mental retardation (p=0.035) than individuals with adult-onset disease. Generally, paediatric-onset patients had more neurological manifestations, while initial presentations of adult-onset TSC were more diverse.     

Sclerosing TSC1 mutated renal cell carcinoma: An unusual pattern mimicking MITF family translocation renal cell carcinoma

The tuberous sclerosis genes and MTOR are increasingly being found to have important roles in novel subtypes of renal cancer, particularly emerging entities eosinophilic solid and cystic renal cell carcinoma (RCC) and high‐grade oncocytic renal tumor (HOT)/RCC with eosinophilic and vacuolated cytoplasm. We report a unique renal neoplasm in a 66‐year‐old woman that initially mimicked MITF family translocation RCC due to mixed clear and eosinophilic cells, extensive stromal hyalinization, and psammoma bodies, yet which was negative for TFE3 and TFEB fluorescence in situ hybridization and a next generation sequencing (NGS) gene fusion assay. Cytoplasmic stippling triggered consideration of TSC‐associated neoplasms, and a targeted NGS assay revealed a variant in exon 21 of TSC1 resulting in c.2626G>T p.(Glu876*) truncating mutation. This report adds to the morphologic spectrum of TSC‐related renal neoplasms, including prominent stromal hyalinization as a potentially deceptive pattern. Due to the overlap in cytoplasmic stippling between eosinophilic solid and cystic RCC and HOT/RCC with eosinophilic and vacuolated cytoplasm, it is debatable which category this example would best fit. Further understanding of these entities and other renal neoplasms with alterations in the TSC genes will elucidate whether they should be considered a family of tumors.