Composite Pheochromocytoma/Paraganglioma-Ganglioneuroma: A Clinicopathologic Study of Eight Cases with Analysis of Succinate Dehydrogenase.

Ganglioneuromas represent the most well-differentiated spectrum of neoplasia arising from the sympathetic nervous system, while neuroblastomas represent the most poorly differentiated counterpart, and ganglioneuroblastomas represent intermediate stages of differentiation. Small series of cases have documented the co-occurrence of ganglioneuroma with a pheochromocytoma (Pheo)/paraganglioma (PGL) component. We report the clinicopathologic features of eight such cases, diagnosed between 2003 and 2015 with a mean follow-up of 22 months (1-47), which were evaluated for syndrome associations, SDHB expression, and clinical outcome. Mutations of the succinate dehydrogenase (SDH) complex subunits (A, B, C, D, and SDHAF2) have been implicated in predicting metastatic behavior and in identifying possible paraganglioma syndromes. The proliferative index was calculated by manual quantification of Ki-67-positive cells at selected hot-spots using ImageJ (NIH). In our series, composite Pheo/PGL-ganglioneuromas predominantly involved the adrenal gland (Pheo 7, PGL 1). The cases had an equal gender distribution (males 4, females 4), with a mean age at diagnosis of 67 years (range 53 to 86 years), an average size of 5.2 cm (range 2 to 8.2 cm), an average weight of 49.3 g (7.8 to 144.7 g, n = 6), and the majority were functionally active (7 of 8, 88%). The mean Ki67 proliferation rate was 2% (range 0.3 to 3%), and all cases retained SDHB expression (8/8, 100%). No patient (0/8, 0%) developed metastatic disease on follow-up. One patient had a retroperitoneal composite PGL-ganglioneuroma in the setting of neurofibromatosis type 1. No recurrent disease or other associations were identified. In our study, composite Pheo/PGL-ganglioneuromas predominantly affected the adrenal gland in older patients, showed no loss of SDHB, and no disease recurrence was identified.

Exome Sequencing Reveals Germline SMAD9 Mutation That Reduces Phosphatase and Tensin Homolog Expression and Is Associated With Hamartomatous Polyposis and Gastrointestinal Ganglioneuromas.

Hamartomatous polyposis syndromes (HPS) account for a small but appreciable proportion of inherited gastrointestinal cancer predisposition syndromes; patients with HPS have an increased risk for colon and extracolonic malignancies. We present a unique case of familial juvenile polyposis syndrome associated with gastrointestinal ganglioneuromas of unknown etiology. The patient was tested for HPS-associated genes, but no mutation was detected. Exome sequencing identified a germline heterozygous mutation in SMAD9 (SMAD9(V90M)). This mutation was predicted to be an activating mutation. HEK cells transfected to express SMAD9(V90M) had reduced expression of phosphatase and tensin homolog; this reduction was also observed in a polyp from the patient. We have therefore identified a new susceptibility locus for HPS. Patients with hamartomatous polyposis in the colon associated with ganglioneuromatosis should be referred for genetic assessments.

Loss of mitochondria in ganglioneuromas.

A shift in cellular energy production from oxidative phosphorylation (OXPHOS) to glycolysis, even under aerobic conditions, is called the Warburg effect. To elucidate changes of the mitochondrial energy metabolism in ganglioneuroma (GN) individual OXPHOS enzymes were analyzed by activity assays and by immunohistochemical staining methods. GN (n=7) showed a significant reduction in the activity and content of OXPHOS enzymes. Citrate synthase activity was also severely diminished in GN compared to normal cortical kidney (p=0.0002) and adrenal (p=0.0024) tissues. Furthermore, the mitochondrial membrane protein porin was undetectable or significantly reduced. Accordingly, a reduction of the copy number of mitochondrial DNA was observed in GN compared to cortical kidney tissue. The striking decline of mitochondrial mass is specific for GN but not for neuroblastoma, in which a reduction of the OXPHOS complexes without reduction of mitochondrial mass was reported. Knowledge of the mechanism by which tumor cells achieve the Warburg effect will provide a starting point for functional studies aimed at restoring aerobic energy metabolism as a potential new therapeutic strategy to treat malignancies.

Expression of the reversion-inducing cysteine-rich protein with Kazal motifs and matrix metalloproteinase-14 in neuroblastoma and the role in tumour metastasis.

Neuroblastoma is the most common malignant tumour in infancy; the reversion-inducing cysteine-rich protein with Kazal motifs gene (RECK) is a tumour suppressor gene. Previous studies show that RECK inhibits tumour invasion and metastasis through negative regulation of the matrix metalloproteinase (MMP)-2, MMP-9 and MMP-14. Therefore, we wanted to detect the expression of RECK and MMP-14 in neuroblastomas to assess the correlation between the expression levels of these proteins, and to investigate the roles in the metastasis and development of the tumour. PV-6000 immunohistochemistry method was used to detect the expression levels of RECK and MMP-14 in 36 samples of neuroblastoma tissue. Samples from paraffin wax-embedded specimens and the complete clinicopathological data of 36 neuroblastoma and 10 ganglioneuroma patients were collected. The rate of expression of the RECK protein in the neuroblastoma was low (16.7%). Furthermore, it reduced with the increase in the invasive depth and distant metastasis (P = 0.015; P < 0.05). The rate of expression of the MMP-14 protein in the neuroblastoma was high (58.3%) and increased with the increase in the extent of invasive depth and distant metastasis (P = 0.002; P < 0.05). The expression of the RECK protein correlated negatively with that of MMP-14 (r = -0.418; P < 0.05). Low levels of the RECK protein are expressed in the neuroblastoma, while the MMP-14 protein is expressed at high levels. The RECK and MMP-14 proteins may serve as markers in the estimation of the extent of metastasis and dissemination of the neuroblastoma.

Germline inactivation of PTEN and dysregulation of the phosphoinositol-3-kinase/Akt pathway cause human Lhermitte-Duclos disease in adults.

Lhermitte-Duclos disease (LDD), or dysplastic gangliocytoma of the cerebellum, is an unusual hamartomatous overgrowth disorder. LDD can be familial or, more commonly, sporadic. It has been only recently recognized that LDD may be associated with Cowden syndrome (CS). Over 80% of patients with CS carry germline mutations in PTEN. It remains unclear whether all cases of LDD, even without features of CS, are caused by germline PTEN mutation and whether somatic PTEN mutation occurs in sporadic LDD. We obtained paraffin-embedded LDD lesions from 18 unselected, unrelated patients and performed mutational analysis of PTEN. Overall, 15 (83%) of 18 samples were found to carry a PTEN mutation. All individuals with mutations were adult-onset patients, but the three without mutations were diagnosed at the ages of 1, 3, and 11 years. Germline DNA was available from six adult-onset cases, and all had germline PTEN mutations. Of these six, two had CS features, one did not have CS features, and three were of unknown CS status. Immunohistochemistry revealed that 75% of the LDD samples had complete or partial loss of PTEN expression accompanied by elevated phosphorylated Akt, specifically in the dysplastic gangliocytoma cells. These data suggest that the loss of PTEN function is sufficient to cause LDD. The high frequency and spectrum of germline PTEN mutations in patients ascertaining by LDD alone confirm that LDD is an important defining feature of CS. Individuals with LDD, even without apparent CS features, should be counseled as in CS.

Loss of caspase-8 expression in highly malignant human neuroblastoma cells correlates with resistance to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis.

Human neuroblastoma (NB) is a highly heterogeneous childhood cancer that is aggressively malignant or can undergo spontaneous regression that may involve apoptosis. NB-derived cell lines were tested for their sensitivity to apoptosis induced by the tumor-selective ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Noninvasive S-type cell lines (NB cell lines of substrate adherent phenotype) are highly sensitive to TRAIL, whereas invasive N-type cell lines (NB cell lines of neuronal phenotype) are resistant. Whereas both S- and N-type cell lines express TRAIL-R2, FADD, and caspase-3 and -10, only S-type cells express caspase-8. Reduced levels of caspase-8 protein were also observed in a malignant stage IV NB tumor when compared with a benign ganglioneuroma. The caspase-8 gene is not deleted in either N-type NB cell lines or high-stage NB tumors. Caspase-8 expression can be induced by demethylation with 5-aza-2'deoxycytidine, which enhances sensitivity to TRAIL. Therefore, caspase-8 expression is silenced in malignant NB, which correlates to tumor severity and resistance to TRAIL-induced apoptosis.

Telomerase activity distinguishes between neuroblastomas with good and poor prognosis.

BACKGROUND: Treatment of neuroblastoma has remained a major challenge in pediatric oncology because the assessment of the individual prognosis, particularly in disseminated disease is still obscure. Previous studies have correlated clinical outcome with activity levels of telomerase, a cellular reverse transcriptase which has been detected in the majority of human malignant tumors. PATIENTS AND METHODS: In this blind-trial study, a non-radioactive telomeric repeat amplification protocol (TRAP) with an internal telomerase-assay standard was used on an automated laser fluorescence sequencer for the detection and semiquantitative analysis of telomerase activity (TA) in 67 neuroblastomas of all clinical stages from the German Neuroblastoma Trial and 2 ganglioneuromas. TA levels were correlated with event-free and overall survival rates and established prognostic markers such as MYCN. RESULTS: TA was present in 14 of 69 (20%) samples, including 3 of 22 stage IVS, 8 of 14 stage IV, 1 of 10 stage III, 1 of 7 stage II and 1 of 14 stage I neuroblastomas and 0 of 2 ganglioneuromas. We found a strong statistical correlation between the presence of TA and poor clinical prognosis with regard to all tumor stages. Multivariate analysis revealed TA as an independent prognostic marker. In particular, the analysis of TA in IVS neuroblastomas distinguished two different prognostic groups. CONCLUSIONS: Our data suggest that TA is an independent prognostic marker in neuroblastoma which, in combination with other markers such as MYCN, may proof useful in assessing the individual patient's prognosis.

Correlating telomerase activity levels with human neuroblastoma outcomes.

Telomerase activity was analysed in 100 neuroblastoma cases. Although telomerase activity was not detected in normal adrenal tissues or benign ganglioneuromas, almost all neuroblastomas (94%) did express it, suggesting an important role for telomerase in neuroblastoma development. Neuroblastomas with high telomerase activity had other genetic changes (for example, N-myc amplification) and an unfavourable prognosis, whereas tumours with low telomerase activity were devoid of such genetic alterations and were associated with a favourable prognosis. Three neuroblastomas lacking telomerase activity regressed (stage IVS). Thus telomerase expression may be required as a critical step in the multigenetic process of tumorigenesis, and two different pathways may exist for the development of neuroblastoma.

Localization of neuron-specific (gamma gamma) enolase in proliferating (supportive and neoplastic) Schwann cells. An immunohisto- and electron-immunocyto-chemical study of ganglioneuroblastoma and schwannomas.

Neuron-specific (gamma gamma) enolase, a glycolytic enzyme used as a relatively specific marker for normal neurons and neuroendocrine cells, has recently been found in a variety of neoplastic cells and in reactive astrocytes. Its localization was investigated by immunohisto- and electron-immunocyto-chemistry, in the proliferating supportive Schwann cells of a peripheral ganglioneuroblastoma and in the neoplastic Schwann cells of four acoustic tumours. By light microscopy, the neoplastic Schwann cells showed moderate uneven diffuse immunopositivity for enolase. By electron-immunocytochemistry, both types of Schwann cells demonstrated immunopositivity discretely limited to their cell surface membranes. The neoplastic ganglion cells and axons of the ganglioneuroblastoma and the normal neurons and axons included in the schwannomas were, as expected, intensely immunopositive. The visualization of gamma gamma enolase on the cell surface membranes of both neoplastic and non-neoplastic proliferating Schwann cells suggests that increased glycolytic activity may occur on the surface of these proliferating cells irrespective of the nature of the proliferation.

Distinct isozyme patterns of cyclic nucleotide phosphodiesterase in human neuroblastoma and ganglioneuroma; a possible marker of differentiation of neural crest-derived tumors and Schwann cells.

By DEAE-cellulose chromatography, the 30,000g supernatants of human neuroblastoma (n = 7), ganglioneuroma (n = 5), sympathetic ganglia (n = 3), and Schwannoma (n = 2) were found to contain three major peaks of adenosine 3',5'-cyclic monophosphate (cAMP) phosphodiesterase (PDE) activity, which were termed peaks I, II, and III in the order of their elution from the column. Peak I isozyme was calmodulin-dependent, and had two different Km values for cAMP (32 and 2.3 microM) and a low Km for guanosine 3',5'-cyclic monophosphate (cGMP) (2.9 microM). Peak II isozyme had a high Km for both cAMP, 76 microM, and cGMP, 32 microM, and peak III isozyme was a cAMP-PDE with Km of 1.8 microM. The peak II and III isozymes were calmodulin-independent. The activity ratio of peak I isozyme to peak III isozyme (I/III isozyme ratio) was significantly different (P less than 0.001) in neuroblastoma and in ganglioneuroma/sympathetic ganglia, i.e., 0.23 +/- 0.11 for neuroblastoma vs. 0.79 +/- 0.20 for ganglioneuroma and 0.51 +/- 0.08 for sympathetic ganglia. Schwannoma showed the highest value of 1.05 (P less than 0.05). These results suggest that the I/III isozyme ratio of cAMP-PDE could be a useful marker in studies on the differentiation of neural crest-derived tumors and Schwann cells.

Distribution of neurofilament protein and neuron-specific enolase in peripheral neuronal tumours.

Peripheral neuronal tumours were studied by the peroxidase-antiperoxidase (PAP) method for the presence of the neurofilament protein (NFP) and neuron-specific enolase (NSE). All cases of ganglioneuromas and ganglioneuroblastomas were positive for NFP and NSE. Both markers were observed only in tumour cells showing differentiation towards ganglion cells. Of the 14 cases of neuroblastoma, 8 were positive for NFP and 12 were positive for NSE. NSE was detected in most neuroblastic tumour cells. However, NFP was found in neuroblasts with signs of differentiation, such as nuclear enlargement, but not in immature, small round cells. NFP was present in cell bodies as well as in cytoplasmic processes of partially differentiated neuroblasts. The majority of pseudorosettes showed no NFP stain. Thus, antibodies against both NFP and NSE are useful in the diagnosis of peripheral neuronal tumours. Moreover, the presence of NFP seemed to be related to the degree of tumour cell differentiation.

[Intestinal ganglioneuromatosis: histochemical, histoenzymological and ultrastructural study of a case]. / La ganglioneuromatose intestinale: étude histochimique, histo-enzymologique et ultrastructurale d'une observation.

Report of a child with disseminated ganglioneuromatosis of the gut. The complexity of the intestinal nervous system malformation is proved by histochemical, histoenzymological and ultrastructural studies. The malformation is characterized by: hyperplasia and hypertrophy of enteric plexus and nerves bundles in the meso, high acetylcholinesterase activity, aplasia of the sympathetic innervation with the exception of perivascular plexus, qualitative and likely quantitative integrity of the endocrine digestive system. These data are compared with similar observations in the literature.

High levels of immunoreactive nervous system-specific enolase in sera of patients with neuroblastoma.

Serum levels of nervous system-specific enolase (NSE, gamma gamma form plus alpha gamma form) in patients with neuroblastoma and in control subjects were determined with a sensitive solid-phase sandwich enzyme immunoassay system. Serum levels of NSE in healthy adults ranged from 1.4-5.7 ng/ml (2.87 +/- 1.18 ng/ml, n = 20), and in control children (1-7 years old) from 2.6 to 10.8 ng/ml (5.76 +/- 2.42 ng/ml, n = 20). Serum samples (n = 13) from patients with neuroblastoma contained high levels of NSE, range 13.6 to 330 ng/ml (mean 96 ng/ml); however those (n = 7) from ganglioneuroblastoma patients were within a normal range (3.0-25.0 ng/ml; mean 8.3 ng/ml). These results suggested that the NSE in serum might be a valuable marker substance for screening and therapeutic monitoring of neuroblastoma.

Neuron-specific enolase in relation to differentiation in human neuroblastoma.

The presence of the two forms of enolase, neuron-specific enolase (NSE) and non-neuronal enolase (NNE), have been examined in biopsy material of human neuroblastoma, ganglioneuroblastoma, ganglioneuroma and cultured neuroblastoma cells, after separation with ion exchange chromatography. The enolase activities were inhibited in the presence of NaCl but remained active in KCl, which were used in the chromatographic step. The relative NSE levels in the neuroblastoma tissues were found to be lower than in the histopathologically more differentiated forms of the tumour, i.e. ganglioneuroblastoma and ganglioneuroma. The human neuroblastoma in vitro cell lines SK-N-SH, SH-SY5Y, SK-N-MC and IMR-32 contained considerably lower relative levels of NSE compared to the levels in the neuroblastoma biopsies. After treatment of the cultured cells with nerve growth factor or dibutyryl-cAMP some cells showed morphological differentiation and concomitantly an increase in the NSE levels. The results indicate that NSE might be useful as a marker for differentiation in human neuroblastoma.