PHOX2B immunolabeling: a novel tool for the diagnosis of undifferentiated neuroblastomas among childhood small round blue-cell tumors.

Peripheral neuroblastic tumors are the most commonly occurring extracranial tumors in children. Although a reliable diagnosis is achievable in the majority of cases, diagnosis of a minority of peripheral neuroblastic tumor cases (especially undifferentiated neuroblastoma) poses a challenge compared with that of other pediatric small round blue-cell tumors. A panel of immunohistochemical markers and fusion transcripts is available for the diagnosis of such tumors, but the markers for neuroblastoma lack specificity and sensitivity. As the transcription factor PHOX2B is highly specific for the peripheral autonomic nervous system from which peripheral neuroblastic tumors are derived, we have assessed PHOX2B immunolabeling as a diagnostic tool in pediatric small round blue-cell tumors. We observed PHOX2B expression in all peripheral neuroblastic tumors, paragangliomas, and pheochromocytomas tested but in no other pediatric tumors among the 388 cases studied by expression microarray and the 109 cases studied by immunohistochemical analysis. We then assessed the results of PHOX2B immunohistochemistry in 12 cases of undifferentiated pediatric neoplasms: PHOX2B was expressed in 6/6 undifferentiated neuroblastomas and in no other small round blue-cell tumors. Finally, we showed that PHOX2B immunohistochemical analysis improves the diagnosis of undifferentiated neuroblastoma with high specificity and sensitivity.

Abnormal activation of autophagy-induced crinophagy in Paneth cells from patients with Crohn's disease.

Autophagy-related 16 like-1 (ATG16L-1), immunity-related GTPase-M (IRGM), and nucleotide-binding oligomerization domain-containing 2 (NOD2) regulate autophagy, and variants in these genes have been associated with predisposition to Crohn's disease (CD). However, little is known about the role of autophagy in CD. Intestinal biopsies from untreated pediatric patients with CD, celiac disease, or ulcerative colitis were analyzed by immunohistochemistry and electron microscopy. We observed that autophagy was specifically activated in Paneth cells from patients with CD, independently of mucosal inflammation or disease-associated variants of ATG16L1 or IRGM. In these cells, activation of autophagy was associated with a significant decrease in number of secretory granules and features of crinophagy. These observations might account for the disorganization of secretory granules previously reported in Paneth cells from patients with CD.

Mosaicism for oncogenic G12D KRAS mutation associated with epidermal nevus, polycystic kidneys and rhabdomyosarcoma.

BACKGROUND: Epidermal nevus (EN) is a congenital disorder characterised by hyperpigmented epidermal thickening following a Blaschko's line. It is due to somatic mutations in either FGFR3 or PIK3CA in half of the cases, and remains of unknown genetic origin in the other half. EN is also seen as part of complex developmental disorders or in association with bladder carcinomas, also related to FGFR3 and PIK3CA mutations. Mosaic mutations of these genes have been occasionally found in syndromic EN. CASE REPORT: The co-occurrence of EN, rhabdomyosarcoma, polycystic kidneys and growth retardation in an infant is described. RESULTS: An oncogenic G12D KRAS mutation was detected in both the epidermal component of the EN and in the rhabdomyosarcoma but not in the dermal component of the EN lesion or in unaffected tissues, including normal skin or blood. CONCLUSION: This report shows for the first time that a KRAS mutation in epiderma causes EN. Observation of the same G12D KRAS mutation in two distinct regions of the body strongly suggests a somatic mosaicism. Finally, this report highlights the potentially underestimated importance of mosaic oncogene mutations in childhood cancers.

Cholinergic switch associated with morphological differentiation in neuroblastoma.

The morphology of malignant cells distinguishes between undifferentiated, poorly differentiated and differentiating neuroblastomas and constitutes a strong prognostic factor. Spontaneous or treatment-induced maturation characterizes a subset of neuroblastomas. It constitutes the basis of retinoic acid treatment to improve survival in aggressive neuroblastomas. However, the molecular events that drive differentiation are poorly understood. In the present study we have investigated the relationships between gene expression profiles and differentiation criteria in stroma-poor neuroblastomas. This study included three undifferentiated (UN), 20 poorly differentiated (PDN) and 11 differentiating (DN) neuroblastomas. These groups could be clearly separated using unsupervised clustering methods, which further enabled a major classification impact of genes involved in neural development, differentiation and function to be identified. UNs are characterized by high ASCL1, high PHOX2B, low GATA2, low TH and low DBH expressions. Most PDNs harbour a clear adrenergic phenotype, even in the presence of missense PHOX2B mutations. Finally, all DN tumours demonstrate cholinergic features. Depending upon their association with adrenergic characteristics, this enables dual 'cholinergic/adrenergic' and 'fully cholinergic' neuroblastomas to be defined. This suggests that the cholinergic switch, a final specification process that occurs physiologically in a minority of sympathetic neurons, is a critical step of differentiation in some neuroblastic tumours. This switch is associated with a down regulation of DBH that is apparently not strictly dependent upon PHOX2B. Conversely, GATA2 and TFAP2B may play critical roles in maintaining adrenergic features in poorly differentiated tumours.

Aquaporin-2 in the human endolymphatic sac.

OBJECTIVE: To detect and localize aquaporin-2 (AQP-2), a water channel regulated by the antidiuretic hormone, in human endolymphatic sac. MATERIAL AND METHODS: Human endolymphatic sacs were sampled during removal of vestibular schwannomas via a translabyrinthine approach. Samples were immediately fixed in 10% formalin (24 h) and embedded in paraffin; in situ hybridization and immunohistochemistry were performed with an AQP-2-specific probe and a polyclonal antibody. RESULTS: Both AQP-2 mRNA and protein were detected in the epithelium of the endolymphatic sac. AQP-2 immunostaining was mainly cytoplasmic, suggesting that most AQP-2 was located in intracellular pools. CONCLUSIONS: In the endolymphatic sac, AQP-2 probably participates in the homeostasis of endolymph; the possibility of reducing the volume of endolymph by inhibiting its expression and membranous insertion using an antidiuretic hormone inhibitor represents a new therapeutic approach for the treatment of Ménière's disease.

Glial cell line derived neurotrophic factor is expressed by epithelia of human renal dysplasia.

PURPOSE: Differentiation of the metanephros is abnormal in cases of renal dysplasia, resulting in abnormal kidney organization. In vitro and in vivo studies indicate that glial cell line derived neurotrophic factor (GDNF) is a major regulator of kidney development and ureteral arborization. Therefore, we investigated the pattern of GDNF gene expression in human dysplastic kidneys. MATERIALS AND METHODS: Specimens of whole tissues of human normal and dysplastic kidneys associated with obstructive uropathy were analyzed for GDNF mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). Immunohistochemistry with GDNF antibody and laser capture microdissection plus RT-PCR were done to identify cells producing GDNF. Apoptosis, BCL-2 and Ki67 were also studied. RESULTS: There were few if any GDNF transcripts in normal kidneys, whereas GDNF was over expressed in renal dysplasia specimens. Strong GDNF expression was found in the dysplastic tubules of dysplastic kidneys, whereas peritubular mesenchyma expressed no GDNF protein. Laser capture microdissection/RT-PCR detected GDNF mRNA in epithelial cells isolated from dysplastic tubules but not in cells from the surrounding mesenchyma, which was confirmed by sequence analysis. GDNF expression by epithelial cells was associated with high proliferation, BCL-2 expression and rare apoptosis. CONCLUSIONS: GDNF gene expression is restricted to the tubular epithelium of dysplastic human kidneys. Our results strongly suggest that GDNF not only influences kidney morphogenesis, but is also implicated in abnormal kidney development.