Completeness of pathology reports in stage II colorectal cancer.

INTRODUCTION: The completeness of the pathological examination of resected colon cancer specimens is important for further clinical management. We reviewed the pathological reports of 356 patients regarding the five factors (pT-stage, tumor differentiation grade, lymphovascular invasion, tumor perforation and lymph node metastasis status) that are used to identify high-risk stage II colon cancers, as well as their impact on overall survival (OS). METHODS: All patients with stage II colon cancer who were included in the first five years of the MATCH study (1 July 2007 to 1 July 2012) were selected (n = 356). The hazard ratios of relevant risk factors were calculated using Cox Proportional Hazards analyses. RESULTS: In as many as 69.1% of the pathology reports, the desired information on one or more risk factors was considered incomplete. In multivariable analysis, age (HR: 1.07, 95%CI 1.04-1.10, p < .001), moderately- (HR: 0.35, 95%CI 0.18-0.70, p = .003) and well (HR 0.11, 95%CI 0.01-0.89, p = .038) differentiated tumors were significantly associated with OS. CONCLUSIONS: Pathology reports should better describe the five high-risk factors, in order to enable proper patient selection for further treatment. Chemotherapy may be offered to stage II patients only in select instances, yet a definitive indication is still unavailable.

A Hirschsprung disease locus at 22q11?

We report a boy with truncus arteriosus, dysmorphic features, developmental delay, passing hypotonia, short segment Hirschsprung disease (HSCR), and paroxysmal hypoventilation. FISH analysis showed an interstitial deletion in chromosome band 22q11.2 coinciding with the deletions found in DiGeorge syndrome and velocardiofacial syndrome. Mutation scanning of RET, GDNF, EDNRB, and EDN3, genes associated with Hirschsprung disease, showed no aberrations. Since we know of two more patients with velocardiofacial syndrome and HSCR, we hypothesise that a gene responsible for proper development of the enteric nervous system may be included in the 22q11.2 region.

Recurrence of DiGeorge syndrome: prenatal detection by FISH of a molecular 22q11 deletion.

We report on a prenatal diagnosis by FISH of a familial 22q11 deletion associated with DiGeorge syndrome (DGS). The deletion was seen in the proband with symptoms of full DGS, in the physically normal father, and in a subsequent pregnancy. After birth this child showed hypocalcaemia, a T cell deficit, and a right sided aortic arch.

A novel member of the transmembrane serine/threonine kinase receptor family is specifically expressed in the gonads and in mesenchymal cells adjacent to the müllerian duct.

The activin and TGF-beta type II receptors are members of a separate subfamily of transmembrane receptors with intrinsic protein kinase activity, which also includes the recently cloned TGF-beta type I receptor. We have isolated and characterized a cDNA clone (C14) encoding a new member of this subfamily. The domain structure of the C14-encoded protein corresponds with the structure of the other known transmembrane serine/threonine kinase receptors. It also contains the two inserts in the kinase domain that are characteristic for this subfamily. Using in situ hybridization, C14 mRNA was detected in the mesenchymal cells located adjacent to the müllerian ducts of males and females at day 15 (E15) of embryonic development. Marked C14 mRNA expression was also detected in the female gonads. In female E16 embryos, the C14 mRNA expression pattern remained similar to that in E15 embryos. However, in male E16 embryos C14 mRNA was detected in a circular area that includes the degenerating müllerian duct. The expression of C14 mRNA was also studied using RNase protection assays. At E15 and E16, C14 mRNA is expressed in the female as well as in the male urogenital ridge. However, at E19, a high C14 mRNA level in the female urogenital ridge contrasts with a lack of C14 mRNA in the male urogenital ridge. This correlates with the almost complete degeneration of the müllerian ducts in male embryos at E19. C14 mRNA expression was also detected in embryonic testes at E15, E16 and E19 using RNase protection assays, but at much lower levels than those found in the developing ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in phosphorylation state of neurofilament proteins in ganglionic and aganglionic bowel segments of children with Hirschsprung's disease.

Hirschsprung's disease is characterized by the absence of enteric neurons in the myenteric and submucosal plexus and the presence of many unmyelinated axons, visible in ganglion like structures, in the aganglionic part of the bowel. In previous studies we showed that the immunoreactivity of a monoclonal antibody (2F11) specific for neurofilament proteins is increased in aganglionic bowel segments. We now investigated whether the increased neurofilament protein staining results from an increase in neurofilament protein immunoreactivity in the aganglionic segment or if it is also related to differences in the phosphorylation state of neurofilament proteins. Bowel resection specimens of patients with Hirschsprung's disease and control patients were investigated by immunohistochemical techniques using a panel of different monoclonal antibodies that are specific for neurofilament proteins and have well known reaction patterns against different phosphorylated epitopes present on two neurofilament proteins, the middle (NF-M) and the high (NF-H) molecular weight subunit. For comparison the specimens were also stained for acetylcholinesterase, neuron-specific enolase (NSE), S-100, and glial fibrillary acidic protein (GFAP). Immunostaining with this panel of antineurofilament-antibodies showed differences in the phosphorylation state of neurofilament proteins in the aganglionic and the ganglionic bowel segments of patients with Hirschsprung's disease. These changes involved the phosphorylation state of these proteins and the ratio of NF-H and NF-M in neurofilament proteins. Staining with NSE and S-100 showed no significant differences between Hirschsprung's disease patients and control patients. We surmise that during the ingrowth and differentiation of hypertrophic axons the composition of neurofilament proteins formed in the aganglionic bowel segment differs from the neurofilament proteins formed in the ganglionic and control bowel segments.(ABSTRACT TRUNCATED AT 250 WORDS)

Colonization characteristics of enteric neural crest cells: embryological aspects of Hirschsprung's disease.

This study explores the development of the enteric nervous system in avian embryos. Particular emphasis was given to colonization characteristics of neural crest cells present in primitive enteric ganglia. By coculturing neuronal and aneuronal gut of quail and chicken embryos, we investigated if and when neural crest cells in primitive enteric ganglia could detach from these ganglia, migrate, and colonize adjacent chicken gut. Quail neural crest cells were identified using the quail nucleolar marker and the HNK-1 antibody. Enteric neurons were identified using three monoclonal antibodies directed against neurofilament proteins. We found that neural crest cells detached from primitive ganglia in neuronal quail gut from E6 till E9, whereas neural crest cells did not leave enteric ganglia from E10 gut. These observations show that there is a transient phase during which enteric neural crest cells can leave the gut. To determine whether neural crest cells could colonize neuronal gut we cocultured neuronal gut or the neural primordium and neuronal chicken gut (E11). We found that quail neural crest cells do not colonize neuronal E11 gut, whereas they do colonize aneuronal gut of the same age. We suggest that aneuronal gut attracts neural crest cells by diffusing factors.

History of body weight and physical activity of elderly women differing in current physical activity.

Development of overweight and physical activity during life was studied retrospectively in a group of physically active and a group of sedentary elderly women. The two groups of elderly women were selected based on a validated physical activity questionnaire. A previous study on their current dietary intake and nutritional status showed a 12 kg higher body weight in the sedentary group compared to the physically active group, whereas body height did not differ. In order to study the relationship between development of overweight and physical activity in these elderly women, a retrospective study was carried out in 45 subjects. Information about former physical activity was collected by means of a detailed structured interview. Information about body shape and fatness, expressed as 'weight index', was obtained using silhouettes and subjective rating of obesity of subjects compared with peers. Classification of obesity was checked by old photographs rated by interviewers, sizes of clothing and recalled body weight and height. Information was collected about the situation at age 12, 25, 40 and 55 years whereas the mean current age is 71. Weight index was statistically significantly different between the active and sedentary group from age 25 onwards (P less than 0.05). Photographs proved to be useful for a valid and objective categorization. No differences were found in physical activity scores between the groups at age 12, 25, 40 and 55 years. It is concluded that the current difference in body shape and fatness between physically active and inactive elderly women was already present at age 25 and persisted throughout their adult life.(ABSTRACT TRUNCATED AT 250 WORDS)

Congenital malformation of the enteric nervous system: history, immunohistodiagnosis and experimental approaches.

Hirschsprung's disease is characterized by the absence of enteric neurons from the distal colon and rectum. We reviewed the history of Hirschsprung's disease from its first description till the experimental approaches of its pathogenesis today. In our laboratory we introduced the use of monoclonal antibodies directed against neurofilament epitopes as useful tools in the diagnosis of Hirschsprung's disease and allied disorders. One particular monoclonal antibody (2FII) enables to distinguish between classical Hirschsprung's disease, long segment aganglionosis, hypoganglionosis, hyperganglionosis and chronic constipation. We also used monoclonal antibodies in experimental studies concerning the formation and malformation of the enteric nervous system in murine and chicken embryos. One particular antibody (HNK-I) was found to be a marker for very early precursors of enteric neurons in chicken (and human) embryos. In chicken embryos HNK-I visualizes cephalic neural crest cells, the area in the embryo that gives rise to all neurons in the gut. Using a microsurgical technique, we developed a model for Hirschsprung's disease in the chicken embryo.

The cellular retinoic-acid-binding protein is expressed in tissues associated with retinoic-acid-induced malformations.

Retinoic acid (RA) is thought to play a role in embryonic pattern formation in vertebrates. A naturally occurring gradient of endogenous RA has been demonstrated in the developing chick limb bud, while local application of RA leads to the formation of additional digits. In mammals, a well-defined spectrum of birth defects has been reported as a result of fetal exposure to excess RA. In analogy to the chick limb bud, it may be speculated that these malformations are the result of disturbance of morphogenetic RA concentration gradients. A candidate gene involved in the regulation of endogenous RA concentrations is the gene encoding cellular RA binding protein (CRABP). We have isolated a partial cDNA clone corresponding to the chicken homolog of CRABP, and performed in situ hybridization experiments on sections of embryos at various stages of development. CRABP expression was detected in the CNS, the craniofacial mesenchyme, ganglia of the peripheral nervous system, the limb bud, and the visceral arch area. Our results indicate that the spatiotemporally specified expression pattern displayed by the CRABP gene exhibits a striking correspondence to the tissues that are affected by exposure of avian or mammalian embryos to RA. We hypothesize that CRABP plays an important role in normal embryogenesis and that embryonic tissues showing high CRABP expression are susceptible to the adverse effects of excess RA.

Formation and malformation of the enteric nervous system in mice: an organ culture study.

Despite some progress in the treatment of congenital malformations of the enteric nervous system, there is no knowledge about the pathogenesis. The study of the normal formation of the enteric nervous system is hampered by the difficulty of manipulating and culturing mammalian embryos and their organs. Three methods to culture bowel explants of murine embryos, (chorioallantoic membrane grafting, organotypic tissue culture, and renal subcapsular space grafting) were compared. The three-dimensional cytoarchitecture of the bowel developed almost normally in the renal subcapsular space cultures. Using this culture system, it was found that neural crest cells colonize the murine bowel in distinct phases. The distal bowel was colonized at the 13th day of development. In a spontaneous mouse mutant model for intestinal aganglionosis, the lethal spotted mouse, the colonization of the distal 2 mm of the bowel did not occur at E13.

A model for aganglionosis in the chicken embryo.

We investigated the ability of neural crest (NC) cells to colonize hindgut, which had remained aneuronal due to bowel transection in ovo at an early stage. The fact that the bowel remained aneuronal proved that the "sacral" NC does not provide precursor cells for enteric neurons in the hindgut. HNK-1 immunostaining of aneuronal hindgut revealed cell-free, ganglionic structures at the site of the myenteric plexus and a sub-population of mesenchymal cells in the submucosa. We cocultured this particular type of aneuronal bowel with the vagal neural anlage of either quail or chick embryos on the chick chorioallantoic membrane. After 1 week coculture, it appeared that NC cell colonisation of the hindgut had taken place, although there was a difference between the quail-chick and chick-chick model. Quail NC cells had given rise to submucous plexuses and some myenteric plexuses. Chick NC cells had only colonised the submucous region. These findings indicate that the cell-free ganglionic structures hamper neural crest cell colonization in the myenteric region.

Diagnosis of innervation-related motility disorders of the gut and basic aspects of enteric nervous system development.

Motility disorders of the gut in children have become a matter of increasing concern for the pediatric surgeon. Infantile hypertrophic pyloric stenosis is the most common disease requiring surgery in early infancy. While this entity was first described as early as 1888 by Hirschsprung, its etiology and pathogenesis are still an enigma. Fortunately, its surgical treatment is simple and safe, which cannot be said of all other motility disorders of the infantile gut. Dysmotility in small bowel atresia and in gastroschisis is related to damaged smooth muscle cells caused by concomitant ischemia of the bowel wall. In contrast, the temporarily adynamic bowel of the prematurely born child, as well as Hirschsprung's disease and related disorders, is the result of anomalies of the intestinal innervation. The pathogenesis of congenital malformations of the enteric nervous system is still a mystery to surgeons and physicians alike. With his pressure studies of the colon, Swenson first recognized Hirschsprung's disease for what it was. This led to the resection of the manometrically diagnosed abnormal colon, which was found to be aganglionic. Histological investigation of the bowel wall became the decisive tool, replacing manometry, in the diagnosis of Hirschsprung's disease. Histochemical investigation of the bowel wall is not conclusive in other malformations of the enteric nervous system, since the presence or absence of enteric neurons is not the definitive factor discriminating between normally and abnormally functioning bowel. Monoclonal antibodies raised against neuron-specific markers may become important tools for differentiation within the spectrum of congenital malformations of the enteric nervous system. The immunocytochemical technique, however, does not provide sufficient information to explain the cause of innervation disorders of the gut in infancy and childhood. Primary migration disturbances or selective disappearance of enteric neurons following ischemia are highly unlikely to cause aganglionosis of the gut. With respect to the pathogenesis and etiology of Hirschsprung's disease, current research is focused on the embryonic bowel (target organ) in plexus formation. The enteric nervous system is still an enigma, although its origin is known, at least in birds. Why neural crest cells travel, along what paths, how they reach their destination, and what may go wrong during the migratory process, are questions that must be answered. There is increasing knowledge concerning the way in which neural crest cells aggregate and form plexuses in the gut. It is largely unknown why neural crest cells settle, in the bowel, at the sites of the myenteric and submucous plexus.(ABSTRACT TRUNCATED AT 400 WORDS)

The influence of the stage of differentiation of the gut on the migration of neural cells: an experimental study of Hirschsprung's disease.

Based on experimental studies in mutant mouse strains, an imbalance between the rate of migration of neural crest cells and the rate of differentiation of the mesenchyme of the distal gut has been proposed as an etiological factor in Hirschsprung's disease. We studied the influence of the stage of differentiation of embryonal chick gut on the migration of neural crest cells in an in vivo culture system: the chorioallantoic membrane. Neural crest cells in cultured gut were demonstrated with antibodies directed against the HNK-1 epitope. Enteric neurons were demonstrated with neurofilament immunoreactivity. By culturing isolated gut segments of E4 embryos, we obtained aneuronal (neurofilament-negative) embryonal chick gut up to 25 days of development. In cocultures of aneuronal gut and the neural anlage (neural tube and neural crest) neural crest cell colonization was observed, even in advanced stages of differentiation. The significance of the results is discussed in terms of the etiology of Hirschsprung's disease.

Cell division in migratory and aggregated neural crest cells in the developing gut: an experimental approach to innervation-related motility disorders of the gut.

Extensive studies in the chicken embryo have recently supplied more insights into the development of the enteric nervous system, which mainly derives from the vagal neural crest (i.e., the neural crest opposite somites 1 to 7). Crest cells migrate from this region to and via the developing gut. By means of a double labeling technique of both neural crest cells and cells in the S-phase of the cell cycle, we found that these migrating crest cells still proliferate in the gut. Some cells even go through cell division after the formation of a nerve plexus. Some implications for the pathogenesis of congenital innervation abnormalities such as hyperganglionosis and the aganglionosis of Hirschsprung's disease are discussed.

Monoclonal antibodies for diagnosis and research in enteric nervous system pathology. A review.

Monoclonal antibodies (MAb) directed against neuron-specific epitopes are valuable tools in the diagnosis of congenital and acquired enteric nervous system anomalies. MAb raised against cytoskeleton proteins (neurofilaments) revealed a characteristic staining pattern in patients with various motility disorders of the gut. Application of MAb in the study of the development of the enteric nervous system in the chicken embryo provided new insights into the fate of migrating neural crest cells. The relationship between mesenchymal target cells in the gut and proliferating neural crest cells was studied by means of MAb raised against cell surface markers (HNK-1) in combination with characterization of the microenvironment using monoclonal antibodies raised against cell adhesion molecules (N-CAM).