Creation of an international registry to support discovery in schwannomatosis.

Schwannomatosis is a tumor suppressor syndrome that causes multiple tumors along peripheral nerves. Formal diagnostic criteria were first published in 2005. Variability in clinical presentation and a relative lack of awareness of the syndrome have contributed to difficulty recognizing affected individuals and accurately describing the natural history of the disorder. Many critical questions such as the mutations underlying schwannomatosis, genotype-phenotype correlations, inheritance patterns, pathologic diagnosis of schwannomatosis-associated schwannomas, tumor burden in schwannomatosis, the incidence of malignancy, and the effectiveness of current, or new treatments remain unanswered. A well-curated registry of schwannomatosis patients is needed to facilitate research in field. An international consortium of clinicians and scientists across multiple disciplines with expertise in schwannomatosis was established and charged with the task of designing and populating a schwannomatosis patient registry. The International Schwannomatosis Registry (ISR) was built around key data points that allow confirmation of the diagnosis and identification of potential research subjects to advance research to further the knowledge base for schwannomatosis. A registry with 389 participants enrolled to date has been established. Twenty-three additional subjects are pending review. A formal process has been established for scientific investigators to propose research projects, identify eligible subjects, and seek collaborators from ISR sites. Research collaborations have been created using the information collected by the registry and are currently being conducted. The ISR is a platform from which multiple research endeavors can be launched, facilitating connections between affected individuals interested in participating in research and researchers actively investigating a variety of aspects of schwannomatosis. © 2016 Wiley Periodicals, Inc.

EGFR-STAT3 signaling promotes formation of malignant peripheral nerve sheath tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) develop sporadically or in the context of neurofibromatosis type 1. Epidermal growth factor receptor (EGFR) overexpression has been implicated in MPNST formation, but its precise role and relevant signaling pathways remain unknown. We found that EGFR overexpression promotes mouse neurofibroma transformation to aggressive MPNST (GEM-PNST). Immunohistochemistry demonstrated phosphorylated STAT3 (Tyr705) in both human MPNST and mouse GEM-PNST. A specific JAK2/STAT3 inhibitor FLLL32 delayed MPNST formation in an MPNST xenograft nude mouse model. STAT3 knockdown by shRNA prevented MPNST formation in vivo. Finally, reducing EGFR activity strongly reduced pSTAT3 in vivo. Thus, an EGFR-STAT3 pathway is necessary for MPNST transformation and establishment of MPNST xenografts growth but not for tumor maintenance. Efficacy of the FLLL32 pharmacological inhibitor in delaying MPNST growth suggests that combination therapies targeting JAK/STAT3 might be useful therapeutics.

Meningioma progression in mice triggered by Nf2 and Cdkn2ab inactivation.

Aggressive variants of meningiomas (WHO grade II and III) represent up to 30% of those tumors that are among the most common primary central nervous system tumors in adults. Currently, there is no effective treatment for grade-II and -III meningiomas, the main treatment remaining surgical excision. Genetic studies have highlighted two main events associated with meningioma progression: an increase of chromosomal instability in tumors with NF2 inactivation and homozygous deletions or point mutations of the CDKN2AB locus. In this study we demonstrated that in mice, in addition to bi-allelic Nf2 inactivation, homozygous and heterozygous Adenovirus Cre-mediated Cdkn2ab deletions lead to increased meningioma frequency (72% and 50%, respectively) with a shorter latency (3.5 and 7.8 months, respectively) compared with control cohorts and induce grade II/III meningioma progression with an incidence of 34% and 28%, respectively. Moreover, Cdkn2ab inactivation in arachnoidal cells was associated with decreased senescence compared with Nf2(-/-) and wild-type arachnoidal cells in vitro. We have established three mouse meningioma cell lines and generated a syngenic orthotopic meningioma mouse model with 50-100% grade-II/III meningiomas after reimplantation. Comparative genomic hybridization of four meningiomas from Cdkn2ab homozygous mice and three cell cultures revealed the absence of unbalanced chromosomal segments in tumors and several chromosome imbalances in cell cultures. In addition, we were able to detect meningiomas by using bioluminescence and to evaluate tumor vascular permeability by dynamic magnetic resonance imaging. These results show that Nf2 and Cdkn2ab cooperate to promote meningioma progression in mice. The short latency of tumor development and the ability to derive grade II/III meningioma cell cultures are key aspects of this model to promote its use in pre-clinical drug testing.

Pancreatic neuroendocrine tumors in patients with tuberous sclerosis complex.

We explored pancreatic neuroendocrine tumors (PanNETs) associated with tuberous sclerosis complex (TSC) to determine their incidence in the TSC population; define their clinical, radiological, and pathological characteristics; and investigate their association with underlying genotypes. Retrospectively reviewed abdominal imaging of 219 patients with TSC, evaluating the incidence, size, and architecture of pancreatic lesions. Pathology records at Massachusetts General Hospital (MGH) were reviewed for all PanNET diagnoses in patients with TSC. Literature was reviewed for TSC-related PanNET cases. Nine patients with TSC were found to have a pancreatic lesion(s) on abdominal imaging and six patients have been diagnosed with a PanNET by pathology at MGH. Twelve cases of TSC-associated PanNETs have been reported in the literature. Of these 18 PanNET cases, one third were cystic, and the average age at resection was 26 years. Germline TSC2 mutations were found in all patients for whom genetic data were available (n = 3). We did not identify pancreatic angiomyolipomas in this series. Our results suggest that PanNETs are the most common pancreatic lesion in patients with TSC. Focal pancreatic mass lesions, solid or cystic, in patients with TSC should be considered possible PanNETs, and resection of the lesion may be clinically indicated.

Identification of a progenitor cell of origin capable of generating diverse meningioma histological subtypes.

Meningiomas are among the most common primary central nervous system tumours in adults. Studies focused on the molecular basis for meningioma development are hampered by a lack of information with regard to the cell of origin for these brain tumours. Herein, we identify a prostaglandin D synthase-positive meningeal precursor as the cell of origin for murine meningioma, and show that neurofibromatosis type 2 (Nf2) inactivation in prostaglandin D2 synthase (PGDS) (+) primordial meningeal cells, before the formation of the three meningeal layers, accounts for the heterogeneity of meningioma histological subtypes. Using a unique PGDSCre strain, we define a critical embryonic and early postnatal developmental window in which biallelic Nf2 inactivation in PGDS (+) progenitor cells results in meningioma formation. Moreover, we identify differentially expressed markers that characterize the two major histological meningioma subtypes both in human and mouse tumours. Collectively, these findings establish the cell of origin for these common brain tumours as well as a susceptible developmental period in which signature genetic mutations culminate in meningioma formation.

Imaging and therapy of experimental schwannomas using HSV amplicon vector-encoding apoptotic protein under Schwann cell promoter.

Schwannomas are benign tumors forming along peripheral nerves that can cause deafness, pain and paralysis. Current treatment involves surgical resection, which can damage associated nerves. To achieve tumor regression without damage to nerve fibers, we generated an HSV amplicon vector in which the apoptosis-inducing enzyme, caspase-1 (ICE), was placed under the Schwann cell-specific P0 promoter. Infection of schwannoma, neuroblastoma and fibroblastic cells in culture with ICE under the P0 promoter showed selective toxicity to schwannoma cells, while ICE under a constitutive promoter was toxic to all cell types. After direct intratumoral injection of the P0-ICE amplicon vector, we achieved marked regression of schwannoma tumors in an experimental xenograft mouse model. Injection of this amplicon vector into the sciatic nerve produced no apparent injury to the associated dorsal root ganglia neurons or myelinated nerve fibers. The P0-ICE amplicon vector provides a potential means of 'knifeless resection' of schwannoma tumors by injection of the vector into the tumor with low risk of damage to associated nerve fibers.

Treatment of implantable NF2 schwannoma tumor models with oncolytic herpes simplex virus G47Delta.

Schwannomas are benign tumors composed of dedifferentiated Schwann cells that form along peripheral nerves causing nerve compression often associated with pain and loss of function. Current surgical therapy involves total or subtotal surgical removal of the tumor, which may cause permanent nerve damage. In the present study, we explore an alternate means of therapy in which schwannomas are injected with a replication-conditional herpes simplex virus (HSV) vector to shrink the tumor through cell lysis during virus propagation. The oncolytic vector used, G47Delta, has deletions in HSV genes, which allow it to replicate selectively in dividing cells, sparing neurons. Two schwannoma cell lines were used to generate subcutaneous tumors in nude mice: HEI193, an immortalized human line previously established from an NF2 patient and NF2S-1, a newly generated spontaneous mouse line. Subcutaneous HEI193 tumors grew about ten times as fast as NF2S-1 tumors, and both regressed substantially following injection of G47Delta. Complete regression of HEI193 tumors was achieved in most animals, whereas all NF2S-1 tumors resumed growth within 2 weeks after vector injection. These studies provide a new schwannoma model for testing therapeutic strategies and demonstrate that oncolytic HSV vectors can be successfully used to shrink growing schwannomas.

Diagnostic criteria for schwannomatosis.

The neurofibromatoses are a diverse group of genetic conditions that share a predisposition to the development of tumors of the nerve sheath. Schwannomatosis is a recently recognized third major form of neurofibromatosis (NF) that causes multiple schwannomas without vestibular tumors diagnostic of NF2. Patients with schwannomatosis represent 2.4 to 5% of all patients requiring schwannoma resection and approximately one third of patients with schwannomatosis have anatomically localized disease with tumors limited to a single limb or segment of spine. Epidemiologic studies suggest that schwannomatosis is as common as NF2, but that familial occurrence is inexplicably rare. Patients with schwannomatosis overwhelmingly present with pain, and pain remains the primary clinical problem and indication for surgery. Diagnostic criteria for schwannomatosis are needed for both clinicians and researchers, but final diagnostic certainly will await the identification of the schwannomatosis locus itself.

Survey of somatic mutations in tuberous sclerosis complex (TSC) hamartomas suggests different genetic mechanisms for pathogenesis of TSC lesions.

Tuberous sclerosis complex (TSC), an autosomal dominant disease caused by mutations in either TSC1 or TSC2, is characterized by the development of hamartomas in a variety of organs. Concordant with the tumor-suppressor model, loss of heterozygosity (LOH) is known to occur in these hamartomas at loci of both TSC1 and TSC2. LOH has been documented in renal angiomyolipomas (AMLs), but loss of the wild-type allele in cortical tubers appears to be very uncommon. Analysis of second, somatic events in tumors for which the status of both TSC1 and TSC2 is known is essential for exploration of the pathogenesis of TSC-lesion development. We analyzed 24 hamartomas from 10 patients for second-hit mutations, by several methods, including LOH, scanning of all exons of both TSC1 and TSC2, promoter methylation of TSC2, and clonality analysis. Our results document loss of the wild-type allele in six of seven AMLs, without evidence of the inactivation of the second allele in many of the other lesions, including tumors that appear to be clonally derived. Laser-capture microdissection further demonstrated loss of the second allele in all three cellular components of an AML. This study thus provides evidence that, in both TSC1 and TSC2, somatic mutations resulting in the loss of wild-type alleles may not be necessary in some tumor types-and that other mechanisms may contribute to tumorigenesis in this setting.

Pattern of self-organization in tumour systems: complex growth dynamics in a novel brain tumour spheroid model.

We propose that a highly malignant brain tumour is an opportunistic, self-organizing and adaptive complex dynamic biosystem rather than an unorganized cell mass. To test the hypothesis of related key behaviour such as cell proliferation and invasion, we have developed a new in vitro assay capable of displaying several of the dynamic features of this multiparameter system in the same experimental setting. This assay investigates the development of multicellular U87MGmEGFR spheroids in a specific extracellular matrix gel over time. The results show that key features such as volumetric growth and cell invasion can be analysed in the same setting over 144 h without continuously supplementing additional nutrition. Moreover, tumour proliferation and invasion are closely correlated and both key features establish a distinct ratio over time to achieve maximum cell velocity and to maintain the system's temporo-spatial expansion dynamics. Single cell invasion follows a chain-like pattern leading to the new concept of a intrabranch homotype attraction. Since preliminary studies demonstrate that heterotype attraction can specifically direct and accelerate the emerging invasive network, we further introduce the concept of least resistance, most permission and highest attraction as an essential principle for tumour invasion. Together, these results support the hypothesis of a self-organizing adaptive biosystem.

Developmental expression of the tuberous sclerosis proteins tuberin and hamartin.

Tuberous sclerosis complex is an autosomal dominant multisystem disorder, characterized by the development of hamartomas in multiple organs, primarily the skin, heart, kidney, and brain. The tuberous sclerosis genes, TSC1 and TSC2, encode hamartin and tuberin, respectively. Employing specific antibodies for hamartin and tuberin, we analyzed the expression of these two proteins by Western blot analyses in normal developing human and rat tissues. Both proteins are expressed ubiquitously in human fetal tissues and placenta, but are expressed at relatively low levels in human adult tissues, except brain. Similarly, high expression of these two proteins is observed in rat embryonic tissues, with a progressive decline after birth. To better characterize the developmental expression of tuberin and hamartin, we conducted a detailed study in rat tissues from embryonic day 13 to adult by Western blot analysis and immunohistochemistry. Immunohistochemical staining of rat tissues for tuberin and hamartin revealed tissue-specific expression patterns throughout development. Both tuberin and hamartin are expressed in epithelia, muscle (smooth, cardiac and skeletal muscle) and the nervous system (neurons, glia, choroid plexus and arachnoid). Except for the central nervous system, immunostaining intensity declines with age, confirming the protein blot analysis. These results indicate that tuberin and hamartin may play a critical role in development, and thus provide a framework for understanding the developmental and hamartomatous manifestations of tuberous sclerosis. These findings also suggest that tuberin and hamartin have additional functions in the adult brain, consistent with the marked neurological problems that afflict many patients with tuberous sclerosis.

Molecular subtypes of anaplastic oligodendroglioma: implications for patient management at diagnosis.

PURPOSE: In a prior study of anaplastic oligodendrogliomas treated with chemotherapy at diagnosis or at recurrence after radiotherapy, allelic loss of chromosome 1p correlated with better chemotherapeutic response and overall survival. However, in this group of patients in whom therapeutic management was not uniform, loss of 1p did not identify all chemosensitive tumors, nor did all patients whose tumors harbor a 1p loss have long survival. EXPERIMENTAL DESIGN: To clarify the clinical relevance of molecular genetic testing at the time of diagnosis for patients with anaplastic oligodendrogliomas, we studied a larger, more homogeneous group of 50 patients with histologically defined anaplastic oligodendrogliomas treated with a chemotherapeutic regimen as the principal initial therapy. RESULTS: We demonstrate that these tumors can be divided genetically into four therapeutically and prognostically relevant subgroups. Patients whose tumors have combined but isolated losses of 1p and 19q have marked and durable responses to chemotherapy associated with long survival, with or without postoperative radiation therapy. Other tumors with chromosome 1p alterations also respond to chemotherapy, but with shorter duration of response and patient survival. Tumors lacking 1p loss can also be divided into two subgroups: those with TP53 mutations, which may also respond to chemotherapy but recur quickly, and those without TP53 mutations, which are poorly responsive, aggressive tumors that are clinically and genotypically similar to glioblastomas. CONCLUSIONS: These data raise the possibility, for the first time, that therapeutic decisions at the time of diagnosis might be tailored to particular genetic subtypes of anaplastic oligodendroglioma.

NHE-RF, a merlin-interacting protein, is primarily expressed in luminal epithelia, proliferative endometrium, and estrogen receptor-positive breast carcinomas.

NHE-RF, a regulatory cofactor for NHE (Na(+)-H(+) exchanger) type 3, interacts with ion transporters and receptors through its PDZ domains and with the MERM proteins (merlin, ezrin, radixin and moesin) via its carboxyl terminus. Thus, NHE-RF may act as a multifunctional adaptor protein and play a role in the assembly of signal transduction complexes, linking ion channels and receptors to the actin cytoskeleton. NHE-RF expression is up-regulated in response to estrogen in estrogen receptor-positive breast carcinoma cell lines, suggesting that it may be involved in estrogen signaling. To further understand NHE-RF function and its possible role in estrogen signaling, we analyzed NHE-RF expression in normal human tissues, including cycling endometrium, and in breast carcinomas, tissues in which estrogen plays an important role in regulating cell growth and proliferation. NHE-RF is expressed in many epithelia, especially in cells specialized in ion transport or absorption, and is often localized to apical (luminal) membranes. NHE-RF expression varies markedly in proliferative versus secretory endometrium, with high expression in proliferative (estrogen-stimulated) endometrium. Furthermore, estrogen receptor status and NHE-RF expression correlate closely in breast carcinoma specimens. These findings support a role for NHE-RF in estrogen signaling.

Long survival and therapeutic responses in patients with histologically disparate high-grade gliomas demonstrating chromosome 1p loss.

OBJECT: Allelic loss of chromosome 1p is a powerful predictor of tumor chemosensitivity and prolonged survival in patients with anaplastic oligodendrogliomas. Chromosome 1p loss also occurs in astrocytic and oligoastrocytic gliomas, although less commonly than in pure oligodendroglial tumors. This observation raises the possibility investigated in this study that chromosome 1p loss might also provide prognostic information for patients with high-grade gliomas with astrocytic components. METHODS: The authors report on seven patients with high-grade gliomas composed of either pure astrocytic or mixed astrocytic-oligodendroglial phenotypes, who had remarkable neuroradiological responses to therapy or unexpectedly long survivals. All of the tumors from these seven patients demonstrated chromosome 1p loss, whereas other genetic alterations characteristic of high-grade gliomas (p53 gene mutations, EGFR gene amplification, chromosome 10 loss, chromosome 19q loss, or CDKN2A/p16 deletions) were only found in occasional cases. The authors also assessed the frequency of chromosome 1p loss in a series of anonymous high-grade astrocytoma samples obtained from a tumor bank and demonstrate that this genetic change is uncommon, occurring in only 10% of cases. CONCLUSIONS: Although any prognostic importance of chromosome 1p loss in astrocytic or mixed astrocytic-oligodendroglial gliomas can only be determined in larger and prospective series, these findings raise the possibility that some high-grade gliomas with chromosome 1p loss, in addition to pure anaplastic oligodendrogliomas, may follow a more favorable clinical course.

Malignant transformation of neurofibromas in neurofibromatosis 1 is associated with CDKN2A/p16 inactivation.

Patients with neurofibromatosis 1 (NF1) are predisposed to develop multiple neurofibromas (NFs) and are at risk for transformation of NFs to malignant peripheral nerve sheath tumors (MPNSTs). Little is known, however, about the biological events involved in the malignant transformation of NFs. We examined the CDKN2A/p16 gene and p16 protein in NFs and MPNSTs from patients with NF1. On immunohistochemical analysis, all NFs expressed p16 protein. The MPNSTs, however, were essentially immunonegative for p16, with striking transitions in cases that contained both benign and malignant elements. None of the benign tumors had CDKN2A/p16 deletions, whereas three of six MPNSTs appeared to have homozygous CDKN2A/p16 deletions. Methylation analysis and mutation analysis of CDKN2A/p16 in MPNSTs did not reveal any abnormalities. These results show that malignant transformation of NF is associated with loss of p16 expression, which is often secondary to homozygous deletion of the CDKN2A/p16 gene. The findings suggest that CDKN2A/p16 inactivation occurs during the malignant transformation of NFs in NF1 patients and raises the possibility that p16 immunohistochemistry may provide ancillary information in the distinction of NF from MPNST.

Immunohistochemical survey of p16INK4A expression in normal human adult and infant tissues.

p16INK4A is a cell cycle inhibitor that is commonly inactivated in human tumors and tumor cell lines. Despite its importance in human neoplasia, the normal pattern of p16 expression remains largely unknown. Therefore, we analyzed the immunohistochemical localization of p16 in all human organs and demonstrated that cellular p16 expression is highly selective. In adults, proliferative endometrium, breast ductal epithelium, squamous and tubal metaplastic epithelium of the uterine cervix, esophageal squamous epithelium, salivary glands, and antral gastric glands all strongly express the protein. p16 is also widely expressed in endocrine glands, including Langerhans cells in the pancreas and anterior pituicytes and Leydig and Sertoli cells in testis. Within each tissue, however, p16 expression does not correlate with cellular proliferation or maturation. In infants, p16 staining was limited to thymic Hassall's corpuscles, occasional thymic lymphocytes, and only rare pancreatic epithelial cells. Therefore, increased expression of p16 in adult tissues, as in mouse tissues, may reflect a role of p16 in cellular senescence. Restriction of p16 expression in infants to the thymus, the only organ committed to early senescence, is also consistent with such a role. Documentation of the pattern of p16 expression in normal tissues will contribute to our understanding of the normal function of this protein and to interpretation of potentially altered p16 expression in human tumors.

The hPMS2 exon 5 mutation and malignant glioma. Case report.

Patients with Turcot syndrome (TS) are predisposed to colon tumors and primary brain tumors, typically glioblastomas or medulloblastomas. The authors describe a patient with TS featuring a known germline mutation of exon 5 of the hPMS2 mismatch repair gene who developed two metachronous glioblastomas, both with distinct oligodendroglial features. Molecular genetic analysis revealed allelic loss of chromosome 19q in the patient's second tumor but no allelic loss of chromosome 1p. Prominent microsatellite instability was also found in this tumor, consistent with a germline mismatch repair defect. Because this patient had an unusual underlying condition and his tumor had a unique histological appearance for TS, it was hypothesized that this genetic defect may predispose to malignant gliomas with oligodendroglial features. The authors therefore evaluated whether sporadic glioblastomas and oligodendrogliomas undergo mutations of this region of the hPMS2 gene. However, single-strand conformation polymorphism analysis of hPMS2 exon 5 failed to reveal mutations in 20 sporadic glioblastomas and 16 sporadic oligodendroglial gliomas. Thus, although it is possible that the germline hPMS2 exon 5 mutation may predispose to glioblastomas with an oligodendroglial component, the same genetic defect is not commonly involved in sporadic oligodendrogliomas or glioblastomas.

Loss of the NF2 gene and merlin occur by the tumorlet stage of schwannoma development in neurofibromatosis 2.

Loss of the neurofibromatosis 2 (NF2) gene-encoded protein merlin is a universal finding in sporadic and NF2-associated schwannomas. Certain NF2 patients may develop numerous minute Schwann cell tumorlets of the spinal nerve roots in addition to larger, frank schwannomas and thereby provide an opportunity to investigate the timing of NF2 gene/merlin loss in Schwann cell tumorigenesis. We studied an NF2 patient with a germline NF2 gene frameshift mutation who had many Schwann cell tumorlets and schwannomas. Loss of heterozygosity studies of DNA from microdissected specimens showed allelic loss of the NF2 region of chromosome 22q in tumorlets as well as schwannomas. Immunohistochemistry further demonstrated loss of merlin expression in tumorlets as well as schwannomas, with intact expression in adjacent nerve. Thus, loss of both NF2 alleles and merlin occur early in Schwann cell tumorigenesis, before the tumorlet stage. The study of tumorlets and schwannomas in such patients may also provide an opportunity to elucidate mechanisms responsible for the subsequent growth of Schwann cell lesions into symptomatic tumors.