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Summary Several unique features of neuroblastoma (NB), including the capacity for spontaneous regression and maturation to benign pathology, suggest that genes that regulate cellular proliferation, survival and differentiation may be involved in directing clinical tumour aggressiveness. The in situ expression of Bcl-2, Rb, p21, p53 and Bax proteins, as well as the proliferation marker proliferating cell nuclear antigen (PCNA) were examined immunocytochemically in a selection of 38 stage-and outcome-identified NB tumours. Apoptotic cells were identified morphologically and by a DNA fragmentation labelling technique (TUNEL). Although the tumour cell density of Bcl-2, p53, Bax, PCNA and TUNEL positivity correlated with patient survival, a spatially organized expression pattern was further recognized in stroma-poor differentiating tumours. Immature tumour cells adjacent to thin fibrovascular stroma are proliferating, as evidenced by PCNA positivity, and often express Bcl-2. At increasing distance from this fibrovascular stroma, intermediately differentiated tumour cells express Rb, while with more advanced differentiation, proliferation ceases and Bcl-2 immunoreactivity is lost. The most differentiated tumour cells, which often express p53, and occasionally p21 and Bax, lie adjacent to TUNEL-positive, morphologically apoptotic cells. This spatial organization in favourable outcome NB tumours suggests that physiological regulation of differentiation and apoptosis may be involved in tumour regression.

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The tyrosine kinase receptor Ret is expressed in the ureteric bud and is required for normal renal development. Constitutive loss of Ret, its co-receptor gfr␣-1, or the ligand glial cell line-derived neurotrophic factor results in renal agenesis. Transgenic embryos that express a constitutively active form of Ret (Ret MEN2B) under the control of the dopamine-␤-hydroxylase (D␤H) promoter develop profound neuroglial hyperplasia of their sympathetic ganglia and adrenal medullae. Embryos from two independent D␤H-Ret MEN2B-transgenic lines exhibit renal malformations. In contrast with ret؊/؊ embryos, renal maldevelopment in D␤H-Ret MEN2B-transgenic embryos results from primary changes in sympathoadrenal organs extrinsic to the kidney. The ureteric bud invades the metanephric mesenchyme normally, but subsequent bud branching and nephrogenesis are retarded, resulting in severe renal hypoplasia. Ablation of sympathoadrenal precursors restores normal renal growth in vivo and in vitro. We postulate that disruption of renal development results because Ret MEN2B derived from the hyperplastic nervous tissue competes with endogenous renal Ret for gfr␣-1 or other signaling components. This hypothesis is supported by the observation that renal malformations, which do not normally occur in a transgenic line with low levels of D␤H-Ret MEN2B expression, arise in a gdnf؉/؊ background. However, renal maldevelopment was not recapitulated in kidneys that were co-cultured with explanted transgenic ganglia in vitro. Our observations illustrate a novel pathogenic mechanism for renal dysgenesis that may explain how putative activating mutations of the RET gene can produce a phenotype usually associated with RET deficiency. (Am J Pathol 1999, 155:2167-2179)The tyrosine kinase receptor RET is a central component in the pathogenesis of several human diseases. Translocations that involve RET are frequently associated with papillary carcinoma of the thyroid.1-3 In addition, the inherited cancer syndromes multiple endocrine neoplasia (MEN) types 2A and 2B and familial medullary thyroid carcinoma are caused by missense mutations in the RET gene that confer constitutive activity. 4 -9 In contrast, loss of function mutations of RET produce Hirschsprung disease and/or congenital central hypoventilation syndrome. 10 -15 Mice homozygous for loss-of-function mutations in ret have neuronal deficiencies, including intestinal aganglionosis, and agenic/dysgenic kidneys. 16 It is likely that altered RET activity underlies some cases of human renal agenesis/dysgenesis as well, but no examples have been reported.Ret functions as one component of a more complex receptor system for members of the glial cell line-derived neurotrophic factor (gdnf) family. 17 Ligands that activate ret include gdnf, neurturin, artemin, and persephin. The ligands bind to one or more gdnf family receptors (gfr␣-1 to -4), which are glycosyl-phosphatidyl inositol-linked proteins. Ligand binding to gfr␣ probably elicits a conformational change in the latter that affects its interact...

Multiple endocrine neoplasia type 2B (MEN2B) syndrome is caused by a missense mutation in the RET gene, which replaces Met918 by Thr in the intracellular kinase domain of the protein. This single amino acid substitution transforms the receptor into a constitutively active monomeric kinase (RET(Men2B)) and produces an autosomal dominant syndrome characterized by medullary thyroid carcinoma, pheochromocytomas, musculoskeletal anomalies, and mucosal ganglioneuromas. The ligand, GDNF, stimulates RET activity through a co-receptor, GFR alpha-1. In vitro studies have shown that the kinase and mitogenic properties of RET(Men2B) are enhanced by GDNF/GFR alpha-1 stimulation. A relevant clinical question is whether ablation of either GDNF or GFR alpha-1 could alter penetrance or severity of the MEN2B syndrome. We report that ganglioneuromatous tumors caused by a RET(Men2B) transgene in mice are not affected grossly or microscopically by the absence of gdnf or gfr alpha-1. Loss-of-function mutations in ret, gdnf, or gfr alpha-1 cause pan-intestinal aganglionosis in mice. We find that expression of the RET(Men2B) transgene in enteric neural progenitors, after they colonize the gut, does not prevent intestinal aganglionosis associated with gdnf or gfr alpha-1 deficiency.