Single-cell transcriptome sequencing reveals tumor heterogeneity in family neuroblastoma.

Neuroblastoma(NB) is the most common extracranial solid tumor in childhood, and it is now believed that some patients with NB have an underlying genetic susceptibility, which may be one of the reasons for the multiplicity of NB patients within a family line. Even within the same family, the samples show great variation and can present as ganglioneuroblastoma or even benign ganglioneuroma. The genomics of NB is still unclear and more in-depth studies are needed to reveal its key components. We first performed single-cell RNA sequencing(sc-RNAseq) analysis on clinical specimens of two family neuroblastoma(FNB) and four sporadic NB cases. A complete transcriptional profile of FNB was constructed from 18,394 cells from FNB, and we found that SDHD may be genetically associated with FNB and identified a prognostic related CAF subtype in FNB: Fib-4. Single-cell flux estimation analysis (scFEA) results showed that malignant cells were associated with arginine spermine, oxaloacetate and hypoxanthine, and that malignant cells metabolize lactate at lower levels than T cells. Our study provides new resources and ideas for the development of the genomics of family NB, and the mechanisms of cell-to-cell interactions and communication and the metabolic landscape will provide new therapeutic targets.

Evaluation of Ki-67 as prognostic factor for pediatric neuroblastoma and the possibility of molecular-targeted drugs with VEGF and PDGFR.

BACKGROUND: Neuroblastoma (NB) is a pediatric malignant solid tumor characterized as refractory cancer with poor prognosis. The Mitosis-Karyorrhexis Index (MKI) is a prognostic factor but is prone to observer bias. The usefulness of MKI with Ki-67, as a marker of malignancy, was investigated. The efficacy of molecular-targeted therapeutic agents with fewer side effects in tumors has been studied. Molecular-targeted therapy targets include vascular endothelial growth factor (VEGF), involved in tumor angiogenesis; c-Kit, receptor of Kit/stem cells involved in tumor growth, vasculature, and lymphangiogenesis; platelet-derived growth factor receptor (PDGFR); and B-Raf proto-oncogene, serine/threonine kinase (BRAF), involved in the RAS protein-mediated mitogen-activated protein kinase pathway. Therefore, expression profiles of these factors and growth inhibitory effects of molecular-targeted drugs against NB were investigated. METHODS: Ten frozen NB tissue samples collected from January 1993 to December 2017 were evaluated immunohistochemically for Ki-67 and VEGF. c-Kit, PDGFR, and BRAF expression levels were evaluated using enzyme-linked immunosorbent assays; relationships between these factors and clinicopathological parameters of NB were analyzed. RESULTS: Eight patients with NB showed no amplification of MYCN (MYCN proto-oncogene, bHLH transcription factor). There were two cases of ganglioneuroblastoma (GNB). More NB cells were positive for Ki-67 than for GNB cells. VEGF expression was observed in all NB specimens and was stronger in stage IIB and higher. No BRAF or c-Kit activity was observed; PDGFR activity was greater in NB than in GNB (P=0.02). CONCLUSIONS: Thus, Ki-67 may help evaluate NB malignancy. As the first therapy for NB prevents amplification of MYCN, agents targeting PDGFR as well as VGFG can inhibit NB cell proliferation.

Single-cell profiling of peripheral neuroblastic tumors identifies an aggressive transitional state that bridges an adrenergic-mesenchymal trajectory.

Peripheral neuroblastic tumors (PNTs) represent a spectrum of neural-crest-derived tumors, including neuroblastoma, ganglioneuroblastoma, and ganglioneuroma. Malignant cells in PNTs are theorized to interconvert between adrenergic/noradrenergic and mesenchymal/neural crest cell states. Here, single-cell RNA-sequencing analysis of 10 PNTs demonstrates extensive transcriptomic heterogeneity. Trajectory modeling suggests that malignant neuroblasts move between adrenergic and mesenchymal cell states via an intermediate state that we term "transitional." Transitional cells express programs linked to a sympathoadrenal development and aggressive tumor phenotypes such as rapid proliferation and tumor dissemination. Among primary bulk tumor patient cohorts, high expression of the transitional gene signature is predictive of poor prognosis compared with adrenergic and mesenchymal expression patterns. High transitional gene expression in neuroblastoma cell lines identifies a similar transitional H3K27-acetylation super-enhancer landscape. Collectively, our study supports the concept that PNTs have phenotypic plasticity and uncovers potential biomarkers and therapeutic targets.

Genetic and Histopathological Heterogeneity of Neuroblastoma and Precision Therapeutic Approaches for Extremely Unfavorable Histology Subgroups.

Peripheral neuroblastic tumors (neuroblastoma, ganglioneuroblastoma and ganglioneuroma) are heterogeneous and their diverse and wide range of clinical behaviors (spontaneous regression, tumor maturation and aggressive progression) are closely associated with genetic/molecular properties of the individual tumors. The International Neuroblastoma Pathology Classification, a biologically relevant and prognostically significant morphology classification distinguishing the favorable histology (FH) and unfavorable histology (UH) groups in this disease, predicts survival probabilities of the patients with the highest hazard ratio. The recent advance of neuroblastoma research with precision medicine approaches demonstrates that tumors in the UH group are also heterogeneous and four distinct subgroups-MYC, TERT, ALT and null-are identified. Among them, the first three subgroups are collectively named extremely unfavorable histology (EUH) tumors because of their highly aggressive clinical behavior. As indicated by their names, these EUH tumors are individually defined by their potential targets detected molecularly and immunohistochemically, such as MYC-family protein overexpression, TERT overexpression and ATRX (or DAXX) loss. In the latter half on this paper, the current status of therapeutic targeting of these EUH tumors is discussed for the future development of effective treatments of the patients.

Frequency of ALK and GD2 Expression in Neuroblastoma.

Background: The aim of this study was to elucidate the significance of immunohistochemical staining patterns of ALK and GD2 in peripheral neuroblastic tumors with different stages and favorable/unfavorable features. Materials and methods: 32 neuroblastomas, 7 ganglioneuroblastomas, and 1 ganglioneuroma cases were immunohistochemically stained with ALK and GD2, and the expressions were graded and correlated with differentiation, size, and favorable/unfavorable histology. Results: There was no statistically significant correlation between ALK immunopositivity and tumor differentiation or stage. Although there was no statistically significant correlation between GD2 immunopositivity and stage, the intensity and prevalence of GD2 immunostaining were statistically significantly higher in the well differentiated group and in tumors which were smaller than 10 cm. Conclusion: GD2 immunostaining levels correlated with tumor differentiation and size. ALK immunostaining was not related to tumor differentiation or stage.

HuC/D expression in small round cell tumors and neuroendocrine tumors: a useful tool for distinguishing neuroblastoma from childhood small round cell tumors.

The RNA-binding protein HuC/D displays a neuron-specific expression and is involved in neuronal differentiation and the maintenance of the nervous system. Here we investigated the diagnostic value of HuC/D in neuroblastomas. We evaluated 85 neuroblastic tumors: 81 neuroblastomas; 3 ganglioneuroblastomas, intermixed; 1 ganglioneuroma, maturing; and 101 other tumors consisting of 34 Ewing sarcomas, 14 nephroblastomas, 11 rhabdomyosarcomas, 15 pulmonary small cell carcinomas, 18 pancreatic neuroendocrine tumors, and 9 pheochromocytomas. Immunohistochemistry for HuC/D, PHOX2B, and tyrosine hydroxylase was performed. The immunoreactivity for HuC/D was semiquantified using the total score (TS; range, 0-8). HuC/D positivity was defined as a TS ≥6. The TS of the neuroblastic tumors (mean TS, 7.94) was significantly higher than those of the other small round cell tumors and neuroendocrine tumors (P < .001) except for the pheochromocytomas (mean TS, 6.89; P = .074). HuC/D was positive in all 85 neuroblastic tumors, 1 (2.9%) Ewing sarcoma, 1 (6.7%) pulmonary small cell carcinoma, and 8 (89%) pheochromocytomas. PHOX2B was positive in all of the neuroblastic tumors and pheochromocytomas. Tyrosine hydroxylase was positive in 80 (94%) neuroblastic tumors, 1 (9.1%) rhabdomyosarcoma, and all of the pheochromocytomas. Therefore, HuC/D serves as a highly sensitive diagnostic marker to distinguish neuroblastomas from other small round cell tumors. The combination of HuC/D and PHOX2B staining may be valuable for the diagnosis of neuroblastic tumors, especially in the assessment of small sections. HuC/D expression in tumors may be related to catecholamine production or a neural crest-derived cell origin.

The deubiquitinating enzyme UCHL1 is a favorable prognostic marker in neuroblastoma as it promotes neuronal differentiation.

BACKGROUND: Neuroblastoma (NB) is the most common pediatric solid tumor that originates from neural crest-derived sympathoadrenal precursor cells that are committed to development of sympathetic nervous system. The well differentiated histological phenotype of NB tumor cells has been reportedly associated with favorable patient outcome. Retinoic acid (RA) can effectively induce NB cell differentiation, thereby being used in the clinic as a treatment agent for inducing the differentiation of high-risk NB. However, the underlying molecular mechanisms of regulating differentiation remain elusive. METHODS: The correlation between clinical characteristics, survival and the deubiquitinating enzyme ubiquitin C-terminal hydrolase 1 (UCHL1) expression were assessed using a neuroblastic tumor tissue microarray, and then validated in three independent patient datasets. The different expression of UCHL1 in ganglioneuroblastoma, ganglioneuroma and NB was detected by immunohistochemistry, mass spectra and immunoblotting analysis, and the correlation between UCHL1 expression and the differentiated histology was analyzed, which was also validated in three independent patient datasets. Furthermore, the roles of UCHL1 in NB cell differentiation and proliferation and the underlying mechanisms were studied by using short hairpin RNA and its inhibitor LDN57444 in vitro. RESULTS: Based on our neuroblastic tumor tissue microarrays and three independent validation datasets (Oberthuer, Versteeg and Seeger), we identified that UCHL1 served as a prognostic marker for better clinical outcome in NB. We further demonstrated that high UCHL1 expression was associated with NB differentiation, indicated by higher UCHL1 expression in ganglioneuroblastomas/ganglioneuromas and well-differentiated NB than poorly differentiated NB, and the positive correlation between UCHL1 and differentiation markers. As expected, inhibiting UCHL1 by knockdown or LDN57444 could significantly inhibit RA-induced neural differentiation of NB tumor cells, characterized by decreased neurite outgrowth and neural differentiation markers. This effect of UCHL1 was associated with positively regulating RA-induced AKT and ERK1/2 signaling activation. What's more, knockdown of UCHL1 conferred resistance to RA-induced growth arrest. CONCLUSION: Our findings identify a pivotal role of UCHL1 in NB cell differentiation and as a prognostic marker for survival in patients with NB, potentially providing a novel therapeutic target for NB.

Discrimination of histopathologic types of childhood peripheral neuroblastic tumors based on clinical and biological factors.

The aim of this study was to discriminate the children malignant peripheral neuroblastic tumors (PNTs) from those with benign histotype ganglioneuroma (GN) based on clinical and biological characteristics in all PNTs. Four hundred and seventy-six patients were included in this study, containing 345 patients for model development and 131 patients for external validation. Multivariate logistic regression analysis was conducted to select potentially useful characteristics for discrimination of histopathology. External validation was performed for model evaluation. Compared with the main characteristics of GN (85/345, 24.6%), those of malignant PNTs (260/345, 75.4%) showed significant differences. Multivariate analysis was performed to further find the characteristics linked to histopathology. The results indicated that for the patients younger than 49 months, the primary site of adrenal and thoracic, the level of serum neuron-specific enolase (NSE) > 33 ng/mL, and tumor encasing blood vessels were the extremely important discrimination factors of malignant PNTs. The area under the receiver-operating-characteristic of the discrimination model was 0.96. The accuracy rate, sensitivity and specificity were 93.4%, 96.3% and 83.8%, respectively. Meanwhile, the accuracy rate of the external validation from the 131 patients was 97.0%. Overall, histopathologic type of childhood malignant PNTs can be discriminated based on age, primary site, NSE level and the relationship between primary tumor and blood vessels.

Expression of Extracellular Signal-regulated Kinase 5 and Ankyrin Repeat Domain 1 in Composite Pheochromocytoma and Ganglioneuroblastoma Detected Incidentally in the Adult Adrenal Gland.

Composite pheochromocytoma (cPC) is extremely rare, arising in the adrenal medulla as a mixture of PC and other tumors of neural origin. We herein report on a case of adrenal incidentaloma post-operatively diagnosed as cPC with ganglioneuroblastoma (GNBL). The PC component had 7 points on the PASS, a Ki-67 index of 5.1%, a focal absence of sustentacular cells, and no genetic aberrations in succinate dehydrogenase subunit B. The GNBL component exhibited no N-myc amplification. Tumor cells of both components were stained positively for extracellular signal-regulated kinase 5 and ankyrin repeat domain 1. The aberrant activation of growth signaling may play a role in the marginal malignancy of cPC.

Collaboration of cancer-associated fibroblasts and tumour-associated macrophages for neuroblastoma development.

Neuroblastoma is the most common extracranial solid tumour in children and is histologically classified by its Schwannian stromal cells. Although having fewer Schwannian stromal cells is generally associated with more aggressive phenotypes, the exact roles of other stromal cells (mainly macrophages and fibroblasts) are unclear. Here, we examined 41 cases of neuroblastoma using immunohistochemistry for the tumour-associated macrophage (TAM) markers CD68, CD163, and CD204, and a cancer-associated fibroblast (CAF) marker, alpha smooth muscle actin (αSMA). Each case was assigned to low/high groups on the basis of the number of TAMs or three groups on the basis of the αSMA-staining area for CAFs. Both the number of TAMs and the area of CAFs were significantly correlated with clinical stage, MYCN amplification, bone marrow metastasis, histological classification, histological type, and risk classification. Furthermore, TAM settled in the vicinity of the CAF area, suggesting their close interaction within the tumour microenvironment. We next determined the effects of conditioned medium of a neuroblastoma cell line (NBCM) on bone marrow-derived mesenchymal stem cells (BM-MSCs) and peripheral blood mononuclear cell (PBMC)-derived macrophages in vitro. The TAM markers CD163 and CD204 were significantly up-regulated in PBMC-derived macrophages treated with NBCM. The expression of αSMA by BM-MSCs was increased in NBCM-treated cells. Co-culturing with CAF-like BM-MSCs did not enhance the invasive ability but supported the proliferation of tumour cells, whereas tumour cells co-cultured with TAM-like macrophages had the opposite effect. Intriguingly, TAM-like macrophages enhanced not only the invasive abilities of tumour cells and BM-MSCs but also the proliferation of BM-MSCs. CXCL2 secreted from TAM-like macrophages plays an important role in tumour invasiveness. Taken together, these results indicate that PBMC-derived macrophages and BM-MSCs are recruited to a tumour site and activated into TAMs and CAFs, respectively, followed by the formation of favourable environments for neuroblastoma progression. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Cyclin D1 in pediatric neuroblastic tumors: A microarray analysis.

Neuroblastoma is the most common extracranial solid childhood tumor, which is believed to originate from primitive neuroblasts giving rise to the sympathetic nervous system. It was previously shown that cyclin D1 (CCDN1) in pediatric neuroblastic tumors (neuroblastoma, ganglioneuroblastoma, and ganglioneuroma) recapitulates its expression during the development of peripheral sympathetic nervous system (PSNS). In the present study, we performed a microarray analysis in order to evaluate the expression of cyclin D1 in neuroblastoma as compared to ganglioneuroma and ganglioneuroblastoma. We first confirmed that comparable levels of cyclin D1 are present in neuroblastoma and fetal neuroblasts. In addition, we observed that neuroblastoma is associated to significantly higher levels of cyclin D1 as compared to both ganglioneuroma and ganglioneuroblastoma. No differences are instead observable between ganglioneuroblastoma and ganglioneuroma. Finally, bioinformatic analysis of cyclin D1-functionally related genes, identified cyclin D2 as an additional marker/etiopathogenic factor in the development of neuroblastoma.

Composite pheochromocytoma of the adrenal gland: a case series.

BACKGROUND: Composite pheochromocytoma is a rare pathological condition characterized by elements of both pheochromocytoma and neurogenic tumors. However, detailed clinical outcomes of this tumor have not been fully shown. From 2007 to 2013, we experienced three cases of adrenal composite pheochromocytoma. In this report, we investigate the clinicopathological features of these three cases of composite pheochromocytoma and compare them with previously reported cases. CASE PRESENTATIONS: Cases 1 and 2 were a 29-year-old Japanese woman and a 59-year-old Japanese man, respectively. They underwent laparoscopic left adrenalectomy, and pathological examination revealed composite pheochromocytoma-ganglioneuroma. Case 3 was a 53-year-old Japanese man who had been receiving hemodialysis for 17 years. He underwent laparoscopic right adrenalectomy, and pathological examination revealed composite pheochromocytoma-ganglioneuroblastoma. Although the Ki67-positive rates varied from 1.0 to 6.2% among the three cases, no clinical recurrences occurred. Despite the relatively high rate of Ki67 positivity, complete tumor resection resulted in favorable clinical outcomes. CONCLUSION: We experienced three cases of adrenal composite pheochromocytoma. Although the clinical findings and treatment outcomes of composite pheochromocytoma were similar to those of ordinary pheochromocytoma, further studies of the biological behavior and genetic profiles of composite pheochromocytoma are necessary to achieve a better understanding of this tumor.

Galectin-3 is a marker of favorable prognosis and a biologically relevant molecule in neuroblastic tumors.

Childhood neuroblastic tumors are characterized by heterogeneous clinical courses, ranging from benign ganglioneuroma (GN) to highly lethal neuroblastoma (NB). Although a refined prognostic evaluation and risk stratification of each tumor patient is becoming increasingly essential to personalize treatment options, currently only few biomolecular markers (essentially MYCN amplification, chromosome 11q status and DNA ploidy) are validated for this purpose in neuroblastic tumors. Here we report that Galectin-3 (Gal-3), a ß-galactoside-binding lectin involved in multiple biological functions that has already acquired diagnostic relevance in specific clinical settings, is variably expressed in most differentiated and less aggressive neuroblastic tumors, such as GN and ganglioneuroblastoma, as well as in a subset of NB cases. Gal-3 expression is associated with the INPC histopathological categorization (P<0.001) and Shimada favorable phenotype (P=0.001), but not with other prognostically relevant features. Importantly, Gal-3 expression was associated with a better 5-year overall survival (P=0.003), and with improved cumulative survival in patient subsets at worse prognosis, such as older age at diagnosis, advanced stages or NB histopathological classification. In vitro, Gal-3 expression and nuclear accumulation accompanied retinoic acid-induced cell differentiation in NB cell lines. Forced Gal-3 overexpression increased phenotypic differentiation and substrate adherence, while inhibiting proliferation. Altogether, these findings suggest that Gal-3 is a biologically relevant player for neuroblastic tumors, whose determination by conventional immunohistochemistry might be used for outcome assessment and patient's risk stratification in the clinical setting.

[Clinicopathologic features of peripheral neuroblastic tumors].

OBJECTIVE: To study the clinicopathologic characteristics of peripheral neuroblastic tumors and to evaluate the prognostic significance of these features. METHODS: The clinical and pathologic findings were retrospectively reviewed in 121 cases of peripheral neuroblastic tumor. The clinical outcomes of patients were evaluated. The three-year event-free survival rate was analyzed, with respect to age of patients, Evan's staging, International Neuroblastoma Pathology Classification and mitosis-karyorrhexis index. RESULTS: The median age at diagnosis was 2.7 years; and 96 cases (79.3%) occurred in patients younger than 5 years old. The number of cases in Evan's staging I, II, III, IV and IVs was 24, 39, 24, 29 and 5, respectively. There were 82 cases of neuroblastoma (NB) (including 2 cases of undifferentiated NB, 52 cases of poorly differentiated NB and 28 cases of differentiating NB), 9 cases of ganglioneuroblastoma, intermixed type (GNBi), 19 cases of ganglioneuroma, maturing type (GN) and 11 cases of ganglioneuroblastoma, nodular type (GNBn). Forty-nine cases were in the favorable histology subgroup and 72 cases in the unfavorable histology subgroup. The overall three-year event-free survival rate of the 121 cases was 73.0% ± 4.3%. The three-year event-free survival rates were associated with age (P = 0.002), Evan's staging (P = 0.000), histologic category (P = 0.000), mitosis-karyorrhexis index (P = 0.043), prognostic subgroup (P = 0.000). CONCLUSIONS: Most of the peripheral neuroblastic tumors occur in the children younger than 5 years old. It is composed of NB, GNBi, GN and GNBn. The three-year event-free survival rate is approximately 70%. Significant prognostic parameters include age of patients, Evan's staging, International Neuroblastoma Pathology Classification and mitosis-karyorrhexis index.

ERBB3 is a marker of a ganglioneuroblastoma/ganglioneuroma-like expression profile in neuroblastic tumours.

BACKGROUND: Neuroblastoma (NB) tumours are commonly divided into three cytogenetic subgroups. However, by unsupervised principal components analysis of gene expression profiles we recently identified four distinct subgroups, r1-r4. In the current study we characterized these different subgroups in more detail, with a specific focus on the fourth divergent tumour subgroup (r4). METHODS: Expression microarray data from four international studies corresponding to 148 neuroblastic tumour cases were subject to division into four expression subgroups using a previously described 6-gene signature. Differentially expressed genes between groups were identified using Significance Analysis of Microarray (SAM). Next, gene expression network modelling was performed to map signalling pathways and cellular processes representing each subgroup. Findings were validated at the protein level by immunohistochemistry and immunoblot analyses. RESULTS: We identified several significantly up-regulated genes in the r4 subgroup of which the tyrosine kinase receptor ERBB3 was most prominent (fold change: 132-240). By gene set enrichment analysis (GSEA) the constructed gene network of ERBB3 (n = 38 network partners) was significantly enriched in the r4 subgroup in all four independent data sets. ERBB3 was also positively correlated to the ErbB family members EGFR and ERBB2 in all data sets, and a concurrent overexpression was seen in the r4 subgroup. Further studies of histopathology categories using a fifth data set of 110 neuroblastic tumours, showed a striking similarity between the expression profile of r4 to ganglioneuroblastoma (GNB) and ganglioneuroma (GN) tumours. In contrast, the NB histopathological subtype was dominated by mitotic regulating genes, characterizing unfavourable NB subgroups in particular. The high ErbB3 expression in GN tumour types was verified at the protein level, and showed mainly expression in the mature ganglion cells. CONCLUSIONS: Conclusively, this study demonstrates the importance of performing unsupervised clustering and subtype discovery of data sets prior to analyses to avoid a mixture of tumour subtypes, which may otherwise give distorted results and lead to incorrect conclusions. The current study identifies ERBB3 as a clear-cut marker of a GNB/GN-like expression profile, and we suggest a 7-gene expression signature (including ERBB3) as a complement to histopathology analysis of neuroblastic tumours. Further studies of ErbB3 and other ErbB family members and their role in neuroblastic differentiation and pathogenesis are warranted.

GLUT1 protein expression correlates with unfavourable histologic category and high risk in patients with neuroblastic tumours.

GLUT1 is a hypoxia-induced gene that has many biologically important functions, and the overexpression of the GLUT1 protein correlates with poor prognosis in several adult cancers. The clinical significance of the GLUT1 protein in peripheral neuroblastic tumours (NTs) has not been comprehensively documented. In the present retrospective study, immunohistochemical analyses revealed the presence of GLUT1 in 44/96 (46 %) NTs. Membranous GLUT1 was present in neuroblasts of 44/87 neuroblastomas (NBs) and nodular ganglioneuroblastomas (nGNBs) but was absent in ganglion cells. The presence of GLUT1 was significantly increased in NBs and nGNBs compared with maturing ganglioneuromas and intermixed ganglioneuroblastomas (P < 0.001). The proportion of NBs and nGNBs expressing GLUT1 was significantly increased in the high-risk and low/intermediate-risk groups compared with the very-low-risk group (P = 0.022) and the unfavourable compared with the favourable pathology prognostic group (P = 0.027). In the Cox regression analyses, GLUT1 expression indicated a worse overall survival (OS; hazard rate ratio (HR) 2.29, P = 0.053) and event-free survival (EFS; HR 1.68, P = 0.181) which was not attenuated by adjustment for the mitosis-karyorrhexis index and MYCN amplification (OS: adjusted HR 2.44, P = 0.053 and EFS: adjusted HR 1.63, P = 0.244). This indicated that GLUT1 protein expression was independent of mitosis-karyorrhexis index and MYCN amplification as a prognostic factor. Our data may have clinical significance because GLUT1 was also present in a higher proportion of high-risk NTs.

High anaplastic lymphoma kinase immunohistochemical staining in neuroblastoma and ganglioneuroblastoma is an independent predictor of poor outcome.

Anaplastic lymphoma kinase (ALK) mutations occur in 3% to 11% of neuroblastoma (NBL) cases and are associated with high ALK levels. However, high ALK levels appear to be a mutation-independent hallmark of NBL. Evidence about the prognostic relevance of ALK mutations and ALK tumor positivity in patients with NBL has been inconclusive. In this study, we investigated the prognostic relevance of ALK positivity by IHC and ALK mutation status by PCR sequencing in 71 NBL, 12 ganglioneuroblastoma (GNBL), and 20 ganglioneuroma samples in a multivariate model. ALK mutations were present in 2 of 72 NBL and 2 of 12 GNBL samples, which all contained many ALK-positive cells (>50%). In addition, half of all NBL samples showed ALK positivity in most (>50%) of tumor cells, whereas half of the GNBL showed staining in <20% of the tumor cells. In most ganglioneuroma samples, a low percentage of tumor cells stained positive for ALK, which mainly involved ganglion cells. Higher percentages of ALK-positive cells in NBL and GNBL patient samples correlated with inferior survival in univariate and multivariate analyses with established prognostic factors, such as stage, age, and MYCN status. In conclusion, ALK positivity by IHC is an independent, poor prognostic factor in patients with GNBL and NBL. ALK IHC is an easy test suitable for future risk stratification in patients with NBL and GNBL.

Mediastinal ganglioneuroblastoma-secreting vasoactive intestinal peptide causing secretory diarrhoea.

In this case report we describe a case of mediastinal ganglioneuroblastoma-secreting vasoactive intestinal peptide (VIP), causing secretory diarrhoea in an 18-month-old child. An 18-month-old girl presented with a 2-month history of diarrhoea, abdominal distension and weight loss. Investigations revealed secretory diarrhoea with hypokalaemia, hyponatraemia and hypochloraemia and metabolic acidosis. Her stool output was 2.5-3.lday(-1) with increased stool sodium. VIP levels were strikingly high with normal glucagon and gastrin levels. X-ray of the chest revealed a well-defined mass in the right upper zone with tracheal shift, which was confirmed with computed tomography (CT) of the chest. The mass was resected and the patient became asymptomatic. This case shows that secretory diarrhoea caused by VIP and produced by ganglioneuroblastoma indicates a favourable prognosis, provided it is resectable.