In the latter forms, there may also be a histopathologic continuum with CCD due to dominant RYR1 mutations, reflecting the common genetic background. Pathogenetic mechanisms of RYR1-related MmD are currently not well understood, but likely to involve altered excitability and/or changes in calcium homeoestasis; calcium-binding motifs within the selenoprotein N protein also suggest a possible role in calcium handling. The diagnosis of MmD is based on the presence of suggestive clinical features and multiple cores on muscle biopsy; muscle MRI may aid genetic testing as patterns of selective muscle involvement are distinct depending on the genetic background. Mutational analysis of the RYR1 or the SEPN1 gene may provide genetic confirmation of the diagnosis. Management is mainly supportive and has to address the risk of marked respiratory impairment in SEPN1-related MmD and the possibility of malignant hyperthermia susceptibility in RYR1-related forms. In the majority of patients, weakness is static or only slowly progressive, with the degree of respiratory impairment being the most important prognostic factor.Multi-minicore Disease (MmD) is a recessively inherited neuromuscular disorder characterized by multiple cores on muscle biopsy and clinical features of a congenital myopathy. Prevalence is unknown. Marked clinical variability corresponds to genetic heterogeneity: the most instantly recognizable classic phenotype characterized by spinal rigidity, early scoliosis and respiratory impairment is due to recessive mutations in the selenoprotein N  Multi-minicore Disease; Minicore myopathy; Multicore myopathy; Multiminicore myopathy; Minicore myopathy with external ophthalmoplegia; Multicore myopathy with external ophthalmoplegia; Multiminicore disease with external ophthalmoplegiaMulti-minicore Disease (MmD) is an inherited neuromuscular disorder defined by a) multiple areas with reduced oxidative activity running along an only limited extent of the longitudinal axis of the muscle fibre ("minicores") and b) clinical features of a congenital myopathy.The condition was originally reported in a family with two affected siblings and suggestive histopathological findings ; variousEpidemiological data are only available for the congenital myopathies as a group but not for individual conditions. The incidence of all congenital myopathies is estimated at around 0.06/1,000 live births, or onetenth of all cases of neuromuscular disorders . Regiona(SEPN1) gene are probably rarer than those due to mutations in the skeletal muscle ryanodine receptor (RYR1) gene , as mutations in the latter gene appear to be the most common genetic cause of a wide variety of congenital myopathies including central core disease (CCD)  and is  review,  indicatePN1 gene . AlthougPN1 gene .(RYR1) gene. Homozygous RYR1 mutations have been identified in consanguineous families of different ethnic background with the moderate form (with or without hand involvement) of MmD . MH-relthesis")  or becauthesis") .RYR1 mutations , may al review, ,34,76,78No curative treatment is currently available for MmD and management is mainly supportive based on a multidisciplinary approach.SEPN1 mutations, and exercises promoting truncal stability such as swimming and riding [Regular physiotherapy is aimed at promoting endurance, preservation of muscle function and the prevention of contractures, particularly those of the tendon Achilles. Marked axial involvement is common, particularly in the classic form of MmD secondary to d riding  may be pSEPN1 mutations and has to be anticipated with a high degree of suspicion, as most patients remain independently mobile with only little functional impairment whilst already developing respiratory failure [RYR1 mutations is usually less severe or absent, but considering the not negligible risk monitoring of respiratory function according to the principles outlined above is advisable. In all forms of MmD, respiratory infections should be treated actively.Severe respiratory impairment is almost invariable in the classic phenotype of MmD secondary to  failure ,8. Patie failure . RespiraSEPN or RYR1 genes; however, cores on muscle biopsy are non-specific and may be found as an additional feature in patients with primary cardiomyopathies due to mutations in the DES, TTN, LMNA and ACTA1 genes . Cardiac ultrasound studies therefore ought to be considered in cases where clinical features are unusual and mutations in the SEPN1 and RYR1 genes have been excluded.Right ventricular impairment secondary to respiratory failure has been reported in the classic phenotype of MmD and patients with those clinical features ought to have regular cardiac assessments including cardiac ultrasounds. A primary cardiomyopathy has not yet been reported in patients with confirmed mutations in the RYR1 gene may be at risk of malignant hyperthermia reactions, an abnormal response to muscle relaxants such as succinylcholine and volatile anaesthetics [RYR1-related MH susceptibility but no other clinical features of a congenital myopathy [in vitro contracture testing in some families [Patients with MmD secondary to mutations in the sthetics ,81, as hsthetics ,29; minimyopathy ,31. Althfamilies , precautβ-agonist salbutamol has been recently studied as a pharmacological agent in the treatment of RYR1-related myopathies with encouraging results [The  results , howeverSEPN1-related MmD and the majority of cases associated with mutations in the RYR1 gene are inherited as an autosomal recessive trait; only few families with multi-minicores on muscle biopsy and dominant inheritance have been documented.Genetic counselling should be offered to all families and individuals in whom a diagnosis of MmD has been made. SEPN1 gene is now offered as a diagnostic service in most countries. Diagnostic RYR1 screening of selected exons has been established for malignant hyperthermia patients by a number of laboratories associated with the European Malignant Hyperthermia Group (EMHG) [RYR1 coding sequence currently not widely available. Unlike mutations associated with a CCD phenotype, MmD-related RYR1 mutations appear to be distributed throughout the RYR1 coding sequence [RYR1-related phenotypes [Mutational analysis of the p (EMHG)  however,sequence ,32 and aenotypes  poses a SEPN1-related MmD and has to be managed pro-actively; patients can usually be maintained on nocturnal non-invasive ventilation for many years without major functional deterioration.Respiratory impairment is the main prognostic factor in RYR1 mutations are usually associated with a mild to moderate degree of disability and carry an overall favourable prognosis, and, with the exception of the most severely affected neonatal cases, almost all patients achieve the ability to walk independently. The course of RYR1-related MmD in childhood and adolescence is static or only slowly progressive [The forms of MmD due to recessive gressive ,32, but RYR1 mutations may affect function of the tetrameric RyR1 protein only in the homozygous state; this may be modulated through the effect on the binding of accessory and regulatory proteins involved in channel kinetics. More extensive functional studies are required to further delineate specific genotype-phenotype correlations in MmD and are likely to advance our understanding of excitation-contraction (E-C) coupling as a potential basis for future rational therapeutic approaches.The precise molecular mechanisms underlying MmD are currently only partially understood. Recent limited functional studies suggest that recessive RYR1 mutations associated with CCD, MHS and MmD are at least partially understood, the precise molecular mechanisms in the pathogenesis of SEPN1-related MmD remains unclear; a calcium-binding motif within the selenoprotein N protein suggests that intracellular Ca2+ handling may also be affected in this subgroup of MmD and points at a common pathway in the pathogenesis of core formation.While the functional effects of RYR1 or SEPN1 genes and this group is still awaiting genetic resolution.A proportion of patients with typical features of MmD do not harbour mutations in the


Many people living with HIV/AIDS (PLWHA) suffer from stigma and discrimination. There is an ongoing debate, however, about whether stigma, fear and discrimination actually fuel the persisting spread of HIV, or slow it down by reducing contacts between the whole population and high-risk minorities. To contribute to this debate, we analysed the relationship between perceived discrimination and unsafe sex in a large sample of French PLWHAs.In 2003, we conducted a national cross-sectional survey among a random sample of HIV-infected patients. The analysis was restricted to sexually active respondents . Unsafe sex was defined as sexual intercourse without a condom with a seronegative/unknown serostatus partner during the prior 12 months. Separate analyses were performed for each transmission group (injecting drug use (IDU), homosexual contact, heterosexual contact). Overall, 24% of respondents reported experiences of discrimination in their close social environment  and 18% reported unsafe sex during the previous 12 months. Both prevalences were higher in the IDU group . In multivariate analyses, experience of discrimination in the close social environment was associated with an increase in unsafe sex for both PLWHAs infected through IDU and heterosexual contact (OR = 1.65 and 1.80 respectively).Our study clearly confirms a relationship between discrimination and unsafe sex among PLWHAs infected through either IDU or heterosexual contact. This relationship was especially strong in the heterosexual group that has become the main vector of HIV transmission in France, and who is the more likely of sexual mixing with the general population. These results seriously question the hypothesis that HIV-stigma has no effect or could even reduce the infection spread of HIV. It is unfortunately a well-established fact that many people living with HIV/AIDS (PLWHA) suffer from stigma and discrimination, especially those already marginalised by gender, race and socio-economic status The first national representative survey conducted among a large sample of HIV-infected outpatients attending French hospitals (VESPA/ANRS 2003) gave us the opportunity to analyze the relationship between PLWHAS' experience of discrimination by their social environment and their sexual risk behaviours. Such analysis may contribute to the ongoing debate about the extent to which stigma, fear and discrimination indeed fuel the persisting spread of HIV.In 2003, the French National Agency of AIDS Research (ANRS) supported a national cross-sectional survey conducted among a random sample of 4,963 HIV-infected patients, recruited in 102 French hospital departments delivering HIV care. The methodology of this survey has been detailed elsewhere The survey design has been approved by both the French Data Protection Authority (CNIL) and the National Council for Statistical Information (CNIS).The questionnaire comprised about 400 questions, but only a subset of them have been used in the present article. Respondents were asked about their number of sexual partners during the prior 12 months and condom use with occasional and/or regular sexual partner(s). Unsafe sex was defined as reporting at least one sexual intercourse without condom with a seronegative/unknown serostatus partner during the prior 12 months.versus those who never experienced such discrimination). An alternative “quantitative” indicator was obtained by summing the three items (with a score ranging from 0 to 3). Respondents were also asked whether they have ever experienced discriminative attitudes from care providers .Regarding AIDS-related stigma, participants were asked whether they have ever felt discriminated against by relatives, friends, or colleagues, due to their serostatus. Responses to these three items were collapsed into a “qualitative” binary indicator of reported discrimination in the social environment , homosexual contact, heterosexual contact and other), being currently treated with highly active antiretroviral therapy (HAART), CD4 cell count  and symptoms of alcohol abuse during the previous 12 months , living in couple, and precarity of living conditions. Living conditions were considered precarious for respondents who reported financial difficulties in their household  or food privation .2 to compare the reported experiences of discrimination between the three transmission groups.We restricted the present analysis to respondents who reported at least one sexual intercourse during the prior 12 months, and whose diagnosis of HIV infection dated back at least for one year (then unsafe sex during the previous 12 months could not have occurred before diagnosis). We used Pearson's χ2 and Student's t-test), and dichotomous logistic models were computed for each transmission group with a stepwise selection method (entry threshold p<0.10).Then we investigated the factors associated with unsafe sex among each transmission groups separately. Variables initially introduced in the analysis included socio-demographic background, medical information and indicators of perceived discrimination. Bivariate analysis was conducted . Patients most frequently explained their refusal by lack of time. Non-respondents were not significantly different from respondents for gender, age, viral load or CD4 lymphocyte count at time of the survey. Among participants, 2,136 reported at least one sexual intercourse during the prior 12 months and had been diagnosed HIV-infected for at least one year. Among them, 345 have been infected through injecting drug use (IDUs), 983 through homosexual contacts, and 740 through heterosexual contacts.Among responding PLWHAs, 12% reported experiences of discrimination from relatives ; 12% have already felt discriminated against by a friend ; and 7% by a colleague in their working environment . Overall, 24% of respondents reported experiences of discrimination in their close social environment with a higher prevalence in the IDU group (32%) than in the two other groups . Moreover, 27% of respondents reported attitudes of discrimination during interactions with health care providers .Among responding PLWHAs, 18% reported unsafe sex during the previous 12 months  see . In the Moreover, results of multivariate analysis presented in Since the advent of the HIV epidemic, French public health authorities have adopted policies explicitly linking HIV prevention, free of charge access to care for all those who are HIV-positive and protection of PLWHAs against stigma and discrimination Before discussing our results, we must acknowledge several limitations in the present study. First, 41% of solicited patients refused to participate. Such rate of non-response is understandable given the length of the questionnaire (40 minutes for completion on average). However, we collected several characteristics of non-respondents, who were not different from respondents for several key variables . It should also be noted that the VESPA survey was only representative of PLWHAs who are aware of their serostatus and who benefit from care in French hospitals: other groups who may contribute to the epidemiological transmission of HIV include those who have not yet been diagnosed for their HIV infection Since the advent of HAART, concerns have been raised that improvements in treatment may result in an increase of unsafe sex among PLWHAs Among the homosexual transmission group, unsafe sex was more prevalent among the less educated ones and among those who reported a greater number of sexual partners during the previous 12 months. This is consistent with previous research which found a strong correlation between sexual activity and unsafe sex in this population Some authors, apparently influenced by approaches borrowed from the field of socio-biology, have recently argued that HIV-related stigma does not contribute to the spread of HIV. They even proposed the following paradox that stigma and discrimination could produce positive outcomes for public health, as it would reduce the opportunities that marginalised groups have to transmit HIV to the broader population, and would consequently slow down the spread of infection in the general population As in many other countries, an increased incidence of sexually transmitted diseases has been reported among homosexual men in France Indeed, both the results of the VESPA study and French epidemiological data seriously question the hypothesis that HIV-stigma has no effect or could even reduce the infection spread within the general population. Heterosexual transmission has become the major contributor to the HIV epidemics in France: migrants originating from sub-Saharan Africa accounted for 27% of HIV new diagnoses in France in 2004; whereas the majority of these infections has been acquired in their country of origin before they moved to France, there is some evidence that an increasing number of HIV infections among migrants now occur after they arrived in France Results of the VESPA Study are in line with previous research that has suggested various pathways through which stigma can contribute to the spread of HIV. First, in other cultural contexts, stigma leads some HIV-infected mothers to opt for breast-feeding instead of formula feeding because the later one would raise suspicion about their serostatus Of course, the VESPA study was focused on the French population who is already HIV-positive. However, to our knowledge, there is no empirical evidence to support the hypothesis that persistence of discrimination against PLWHAs may actually decrease the likelihood of sexual mixing with those already infected in the HIV-negative population. Since the early years of the HIV epidemic, the highest frequency of negative and discriminatory attitudes toward PLWHAs has been observed in subgroups of the general population whose socio-demographic characteristics (older age) and sexual behaviours (abstinence or monogamy) did not expose them to the risk of HIV transmission Although studies, like the VESPA one in France, document the relationship between stigma and HIV-related risk behaviours, we must recognize that undisputable evidence of a causal link between stigma and rates of HIV infection, either based on longitudinal data or cross-countries comparisons, is still lacking in the international literature. Indeed, the establishment of such evidence may be practically out of reach for epidemiologists and social scientists: cross-countries comparisons would be hazardous because AIDS-related stigma and discrimination are rooted in variable socio-cultural contexts


The mean extracellular pH (pHe) within solid tumours has been found to be lower than in normal tissues. Agents which cause intracellular acidification at low pHe might have selective toxicity towards cells in tumours. Weak acids (or their anions) with pKa values in the range of 4-6 have a higher proportion of molecules in the uncharged form at low pHe and can diffuse more rapidly into cells. The effects of organic acids including succinate, monomethyl succinate and malonate to acidify cells have been evaluated under conditions of different pHe in the acidic range. These weak acids caused intracellular acidification of murine EMT-6 and human MGH-U1 cells in a concentration and pHe dependent fashion. At concentrations of 10 mM and above, these acids also caused in vitro cytotoxicity to these cells at low pHe (< 6.5). The rate and extent of cellular acidification caused by these weak acids, and their cytotoxicity at low pHe, were enhanced by exposure to amiloride and 5-(N-ethyl-N-isopropyl)amiloride (EIPA), agents which inhibit Na+/H+ exchange, and hence the regulation of intracellular pH. Acid dependent cytotoxicity was also investigated in a murine solid tumour using the endpoints of growth delay and colony formation in vitro following treatment in vivo. Agents were tested alone or with 15 Gy X-rays to select a population of hypoxic (and presumably acidic) cells. Achievable serum concentrations of succinate were about 1 mM and no antitumour activity of succinate was detected when used in this way. It is concluded that weak acids are selectively taken up into cells, and can cause selective cellular acidification and toxicity, at low pHe in culture. Weak acids that are normal cellular metabolites are not toxic in vivo, but weak acids carrying cytotoxic groups offer the potential for selective uptake and toxicity under the conditions of low pHe that exist in many solid tumours.


Transcriptome profiling of neuroblasts and neuroblastoma tumor cells provides strong support for a neuroblast origin of neuroblastoma and highlights new candidate neuroblastoma genes Neuroblastoma tumor cells are assumed to originate from primitive neuroblasts giving rise to the sympathetic nervous system. Because these precursor cells are not detectable in postnatal life, their transcription profile has remained inaccessible for comparative data mining strategies in neuroblastoma. This study provides the first genome-wide mRNA expression profile of these human fetal sympathetic neuroblasts. To this purpose, small islets of normal neuroblasts were isolated by laser microdissection from human fetal adrenal glands.Expression of catecholamine metabolism genes, and neuronal and neuroendocrine markers in the neuroblasts indicated that the proper cells were microdissected. The similarities in expression profile between normal neuroblasts and malignant neuroblastomas provided strong evidence for the neuroblast origin hypothesis of neuroblastoma. Next, supervised feature selection was used to identify the genes that are differentially expressed in normal neuroblasts versus neuroblastoma tumors. This approach efficiently sifted out genes previously reported in neuroblastoma expression profiling studies; most importantly, it also highlighted a series of genes and pathways previously not mentioned in neuroblastoma biology but that were assumed to be involved in neuroblastoma pathogenesis.This unique dataset adds power to ongoing and future gene expression studies in neuroblastoma and will facilitate the identification of molecular targets for novel therapies. In addition, this neuroblast transcriptome resource could prove useful for the further study of human sympathoadrenal biogenesis. MYCN and PHOX2B, have been directly linked to neuroblastoma development, although their exact role in oncogenesis is still unclear [Neuroblastoma is the most common and deadly extracranial solid childhood tumor, exhibiting remarkable variation in clinical presentation ranging from localized to highly metastatic disease. Despite multimodal therapies, survival rates for aggressive neuroblastomas are still disappointingly low. One possible approach to development of more efficient and less toxic therapies is to gain insight into the signaling pathways that are deregulated in neuroblastoma and to use this information in the design of molecular therapies. However, at present only two genes, namely  unclear ,2.It is hoped that genome-wide gene expression studies will provide insights into the genes and molecular pathways that govern neuroblastoma pathogenesis. Thus far, no clear or consistent candidate genes or pathways have emerged from these analyses -5  are the major cell type of chain and truncus ganglia, and extra-adrenal chromaffin cells form the paraganglia, whereas the adrenal gland is composed of adrenal chromaffin cells and, at least during development, sympathetic neuroblasts. The fate of the neuroblasts in the developing human adrenal gland is not clear; some or all may involute or mature as solitary intra-adrenal neurons . EvidencIn the present study we isolated and performed expression profiling of the human adrenal neuroblasts as they form monocellular structures during early fetal stages, which can be easily microdissected. In parallel, favorable and unfavorable neuroblastoma tumors were profiled on the same platform. Finally, our dataset was integrated in a meta-analytical data mining approach.Prescreening of hematoxylin-eosin cryosections from 11 fetal adrenal glands demonstrated that large neuroblast clusters of more than 100 cells were predominantly found in adrenal glands at 19 and 20 weeks' gestational age Figure . To veriNeuroblast clusters and adjacent cortical cells (used as controls) were isolated using laser capture microdissection from stained cryosections from three different fetal adrenal glands  . Gene Ontology (GO) analysis identified those classes of genes that are significantly over-represented in the cell specific gene lists  together with chromaffin  markers  and an adrenal chromaffin marker (PNMT) in the microdissected cell clusters is in keeping with our observation that the neuroblast isolates are pure, with only rare intermingled chromaffin cells , further demonstrating that the proper cells were microdissected.Gene set enrichment analysis  based onAlthough multiple lines of evidence indicate that neuroblastoma originates from immature sympathetic neuroblasts, the mRNA expression repertoire of these neuroblasts and neuroblastomas have not yet been compared. Before our analysis, we assumed that, in addition to differences resulting from oncogenic transformation, both cell populations would exhibit many cell type specific similarities.Three data mining strategies were employed to investigate this hypothesis. First, an unbiased multidimensional scaling of all genes on the chip showed that the neuroblasts cluster close to the neuroblastoma tumors and that both groups cluster far away from the fetal adrenal cortex cells Figure . Second,P < 0.01; data not shown). Next, we zoomed in on neurogenesis and transcription factor ontology classes by performing a similar Venn diagram for these gene sets, assuming their putative importance in neuroblastoma development. Interestingly, the similarities between neuroblast and neuroblastoma are even more pronounced for these two GO classes ). As such, the 36% most highly expressed probe IDs in the cortex, neuroblast and neuroblastoma cells, were selected and compared in a Venn diagram. This analysis clearly shows that neuroblasts have more expressed genes in common with neuroblastoma than with the cortex cells  yielded a list of 71 genes that were more highly expressed in neuroblasts and 565 genes that were more highly expressed in neuroblastomas .In the final and most challenging part of our data mining approach, we aimed to identify genes that are under-expressed or over-expressed in neuroblastomas compared with neuroblasts, because these genes and the pathways that they govern might be involved in neuroblastoma development or represent markers for the stage of developmental arrest of neuroblastomas. Rank product analysis  which was developed in-house  to compare the neuroblast-specific and neuroblastoma-specific gene lists with genes that have been reported as differentially expressed in 25 previous gene expression profiling studies conducted in neuroblastoma . We found that as many as 17 of the 71 genes (24%) that are over-expressed in neuroblasts relative to neuroblastomas were reported in the NBGS, mainly annotated as genes that are more highly expressed in maturing, differentiating, or localized neuroblastomas. Likewise, 102 out of the 565 genes (18%) that were over-expressed in neuroblastoma were previously identified in other gene expression studies on neuroblastoma. The high overlap of our gene lists with published gene lists demonstrates the validity of our lists, which were subsequently further explored in chromosomal mapping, GO, and pathway analysis.Chromosome 17q gain is the most frequent genetic aberration in neuroblastoma and is assumed to play a crucial role in its pathogenesis through a dosage effect of one or more genes. This critical region still comprises 25 megabases (Mb) , precludCASP9 on 1p36; CACNA2D3, TDGF1 and NKTR on 3p21-p22 (SRO (shortest region of overlap) from [IGSF4, APOA1, MLL and RDX on 11q23 [MEG3 and DLK1 on 14q32 [Apart from over-expressed genes, we also sought positional tumor suppressor genes by mapping under-expressed genes . The following positional candidates could be identified, located within or very close to the known shortest regions of overlap in neuroblastoma: ap) from ); IGSF4,on 11q23 -19; and on 14q32 .P < 0.01) more genes that are involved in steroid and catecholamine metabolism compared with neuroblastomas. Neuroblastomas are characterized by an over-representation of genes that are involved in immune response, cell growth, and cell cycle. The immune response gene signature may be due to infiltrating immune cells, whereas the over-representation of cell growth and cell cycle genes in neuroblastomas is in perfect concordance with the hyperproliferative character of tumors.To examine gene expression differences between neuroblasts and neuroblastomas from a different perspective, we mapped the neuroblast-specific and neuroblastoma-specific gene lists to the biologic process GO classification Table . This reTFAP2B ; ASCL1 (12q23.2), SIX3 (2p21) and STAT3 ; and APOE (19q13.31) and INHBA , which are involved in both apoptosis and neurogenesis.We then specifically looked at genes belonging to GO terms neurogenesis, transcription factor activity, and apoptosis; these three processes can be assumed to play an important role in neuroblastoma pathogenesis Table . This anMYCN, NTRK1, and CD44 . This overlap demonstrates the validity of the selected neuroblastoma panel and their expression profile. Subsequently, we sought the corresponding gene expression levels of the differentially expressed genes in the normal counterpart cells, aiming to select neuroblastoma candidate genes. Of the 95 genes that are more highly expressed in favorable tumors (versus unfavorable ones), 37 also have significant differential expression (either higher or lower) compared with neuroblasts, whereas 41 out of the 101 genes that are more highly expressed in unfavorable tumors exhibit differential expression compared with the neuroblasts . In a first step, we compared the differentially expressed genes between these two tumor types with published prognostic gene lists. We found that 25 of the 194 genes on our list were previously reported, including the well established markers CDC42 on 1p36, CACNA2D3 on 3p21, and DLK1 on 14q. The latter two genes are of particular interest because they are highly expressed in neuroblasts and favorable neuroblastomas, and their expression is significantly lower in unfavorable neuroblastomas. Among the genes that are more highly expressed in unfavorable neuroblastomas than in favorable ones and neuroblasts, the proven oncogenic transcription factor MYCN emerges , as do several other genes that have been identified or studied within the context of neuroblastomas such as ALK and PRAME, and positional candidates on 17q including BIRC5, RNU2 and TOP2A.From this analysis, a few putative positional tumor suppressor candidates emerge: Although this was not the primary aim of the present work, the neuroblast expression profile provides a unique resource for the investigation of gene expression in human sympatho-adrenal progenitors. In a first attempt, we made an inventory of the genes that belong to the neurogenesis GO class, or that have been described to play a role in neural crest formation and migration, or that have proneural activity . This analysis showed that human fetal neuroblasts of 19 weeks' gestational age expressed 174 of the 359 genes in the neurogenesis GO class, and 26 of 89 proneural genes and genes involved in neural crest formation/migration.ASCL1, GSS, STAT3, UTP11L, ENAH, APBB2, CDK5RAP2, and LARGE.To obtain possible clues on the developmental origin of neuroblastoma we compared the expression profiles of the neuroblastoma tumors with those of normal neuroblasts and neural stem cell cultures. Intersectional Venn diagram analysis of expressed genes shows that neuroblastomas have many genes in common with neuroblasts, as already shown above Figure . InteresComparison of the mRNA expression repertoire of cancer with that of their normal counterpart cells is a commonly applied strategy to elucidate the development and pathophysiology of the cancer type under study. For pediatric neuroblastoma, fetal adrenal sympathetic neuroblasts are assumed to be the cells of origin, but these cells are virtually absent after birth and thus not readily accessible for analysis . In thisQuality assessment and biologic validation of the established neuroblast expression profile demonstrated that the proper cells were isolated and that their expression profiles are trustworthy. Next, we assessed the cell of origin hypothesis for neuroblastomas. To this end, the transcriptional profile of the neuroblasts was thoroughly compared with those of neuroblastoma tumors and normal tissues. These analyses confirmed that neuroblast and neuroblastoma cells indeed present with highly similar expression profiles. These exploratory findings provide, for the first time, molecular support for the cell of origin hypothesis. Also, they reinforce our assumption that the neuroblast gene expression profile constitutes a valid tool for further data mining of neuroblastoma gene expression patterns.Following initial data validation and assessment of the cell of origin hypothesis, we performed a series of data mining analyses aimed at identifying genes and pathways that may be involved in neuroblastoma oncogenesis and tumor biology. In a first step, the neuroblastoma tumor expression profile was compared with that of the neuroblasts, yielding 71 genes with higher expression in neuroblasts and 565 genes with increased expression in neuroblastoma. We subjected these gene lists to a novel meta-analytical approach that allowed comparison with 25 published neuroblastoma gene lists and facilitated the detection of genes identified in at least one other microarray study. Furthermore, we performed GO analysis and we used a new approach to positional mapping of the differentially expressed genes. In a second step, following analysis of combined tumors, we sought genes differentially expressed in carefully selected representative cases of favorable and unfavorable neuroblastomas and further analyzed the expression of these genes in neuroblasts. This approach yielded 37 and 41 genes, respectively.MYCN, MYCN co-amplified genes such as DDX1, known MYCN target genes such as ODC1 and MCM7, and prognostic markers , as well as various other genes such as ASCL1, ALK, BCL2, BIRC5, DLK1, NME1, NME2 and NTRK3 that have previously been mentioned or studied within the context of neuroblastoma. For some genes only circumstantial evidence for a role in neuroblastoma is present ; that we identified these genes in the present study warrants further investigations into their possible role in neuroblastoma development. Finally, several genes, for which no evidence of involvement in neuroblastoma development has yet been obtained, emerged for the first time from our analyses. These include STAT3, IGSF4 and CACNA2D3, and they should also be studied in further detail to determine their possible role in neuroblastoma pathogenesis.When combining the data from the above analyses, it was apparent that many of the genes previously reported in the context of neuroblastoma had been identified, thus underscoring the validity of our data mining approach. These included Although the present study is just a first step in a new strategy of data mining of neuroblastoma gene expression profiles, we nevertheless obtained new information that is particularly interesting for the 17q region. Gain of distal 17q is not only the most frequent chromosomal alteration in high stage neuroblastoma but it is also the strongest independent adverse prognostic genetic factor ,22. HoweNME1 and NME2. The role of the latter two genes in cancer is controversial, but once again these genes emerge from a neuroblastoma study. Among the genes that are more highly expressed in neuroblastoma, another interesting candidate was found to be located just outside the enriched 17q21.3 segment but within the same chromosome band, namely STAT3. This gene encodes an oncogenic transcription factor that plays a central role in the janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, promoting growth and survival of tumor cells, inducing tumor angiogenesis, and suppressing antitumor immune responses. Of particular interest is that STAT3 is also implicated in neurogenesis. Given their documented role in cancer, STAT proteins have been shown to be promising molecular targets for novel cancer therapies, including small molecule inhibitors of STAT signaling. The finding of increased STAT3 expression might also be of relevance in the light of the observed ALK over-expression in this and previous studies [To substantiate this hypothesis, we performed positional gene enrichment analysis on chromosome 17 for the genes that are more highly expressed in neuroblastoma compared to neuroblast. Interestingly, this yielded a highly significant enrichment for two loci on the long arm of chromosome 17, including the above mentioned region, further demonstrating the high likelihood of the presence of a neuroblastoma dosage sensitive gene. A total of 11 differentially expressed genes are contained within this 17q21.3 segment, including  studies ,24, beca studies . Suppres studies .CASP9 and CDC42 (1p36), which have already been studied in neuroblastoma [CACNA2D3 (3p21-p22), which was recently proposed as a tumor suppressor gene in lung cancer [IGSF4 (11q23), which is a known tumor suppressor gene in several cancers; and DLK1 (14q). All of these genes have been mapped within or near to previously defined shortest regions of overlap for deletions in neuroblastoma and should therefore be considered for further functional studies.Positional mapping of the genes that are expressed to a lesser degree in neuroblastomas than in neuroblasts yielded some remarkable positional tumor suppressor candidate genes. Among others, these include blastoma ,28; CACNg cancer ; IGSF4 . Later oworkers . Hence, The inclusion of normal neuroblasts in gene expression analysis of malignant neuroblastomas was shown to add significant power to the identification of candidate neuroblastoma genes. Inclusion of larger sets of neuroblastoma tumors with well characterized genomic alterations and positional mapping of the genes in critically involved genomic regions in neuroblastomas will be crucial for tracing back the molecular basis of neuroblastoma.n = 12), in the National Center for Medical Genetics , and in the University Children's Hospital of Essen . For this study, we preferentially selected tumors that were localized in the adrenal gland (11/18). Based on INSS stage , MYCN status, ploidy and age at diagnosis, and for some cases pathologic rapports, samples were divided into favorable or unfavorable neuroblastoma . The induced abortion was performed by prostaglandin instillation to the patient. The adrenals were removed during necropsy and snap-frozen in liquid nitrogen within 3 hours after delivery. Neuroblastoma tumors were collected in the Center for Medical Genetics . Immunohistochemical staining was performed as described previously . For micMicrodissected cells were collected in RNA extraction buffer, followed by RNA extraction and DNase treatment on column . RNA of the tumor samples was extracted using the RNeasy Mini kit (Qiagen), in accordance with the manufacturer's instructions. Four of the neuroblastoma tumor pieces were first mixed with Lysing Matrix D microbeads  and 700 μl RTL buffer (Qiagen), and homogenized using FastPrep FP220 (Qbiogene). A fraction of the RNA was used for cDNA synthesis after DNase treatment (described by Vandesompele and coworkers ). RNA quFor each of the three fetal adrenal glands, the different neuroblast RNA isolates were pooled, amplified using a two-round labeling protocol, and hybridized to HG-U133A oligonucleotide chips , containing 18,400 transcripts including 14,500 well characterized human genes  software and normalized with the Robust Multi Chip Average (RMA) method . IdentifMeta-analyses of published neuroblastoma microarray data were performed with the NBGS .Positional gene enrichment analysis was performed with in-house developed R-Bioconductor script PGE  . PGE scaExpression microarray data were submitted to ArrayExpress , accessiThe following additional data are available with the online version of this paper. Additional data file KDP performed the neuroblast microdissection and microarray data mining, and drafted the paper. PH collected the fetal adrenal glands and helped with the neuroblast microdissection. NY helped with microdissection, RNA isolation and quantification, RNA quality control and real-time quantitative polymerase chain reaction validation experiments. SB performed the immunohistochemical stainings that were reviewed and discussed by SP. AS, AE, RS, MR, YB, and GL collected neuroblastoma tumor samples. JV and FS participated in the study's design and coordination. All authors have reviewed the manuscript, and FS and ADP were the final editors of the manuscript.RNA quality and quantity measures, validation of Affymetrix chip results, and Affymetrix chip quality parametersClick here for fileGenes that are differentially expressed in neuroblast versus cortex samples, in neuroblast versus favorable (F) and/or unfavorable (UF) neuroblastoma, and in favorable versus unfavorable neuroblastomaClick here for fileResults of NBGS analysis of the genes that are differentially expressed between neuroblasts and (favorable and/or unfavorable) neuroblastomas Click here for file


Tartrate-resistant acid phosphatase (TRAP) is a metalloprotein enzyme that belongs to the acid phosphatases and is known to be expressed by osteoclasts. It has already been investigated as a marker of bone metastases in cancer patients. In this study, which examined the value of serum TRAP concentrations as a marker of bone disease in breast cancer patients, we observed high concentrations of TRAP even in patients without bone metastases. To elucidate this phenomenon, we examined the expression of TRAP in breast cancer cells and the cells of several other malignancies.TRAP concentrations in the serum of tumor patients were determined by ELISA. The expression of TRAP in breast, ovarian, and cervical cancer and malignant melanoma was analyzed by immunohistochemistry. RT-PCR and immunocytology were used to evaluate TRAP expression in cultured tumor cells.A marked increase in serum TRAP concentrations was observed in patients with breast and ovarian cancer, regardless of the presence or absence of bone disease. TRAP expression was found in breast and ovarian cancers and malignant melanoma, while cervical cancer showed only minimal expression of TRAP. Expression of TRAP was absent in benign tissue or was much less marked than in the corresponding malignant tissue. TRAP expression was also demonstrated in cultured primary cancer cells and in commercially available cell lines.Overexpression of TRAP was detected in the cells of various different tumors. TRAP might be useful as a marker of progression of malignant disease. It could also be a potential target for future cancer therapies. Tartrate-resistant acid phosphatase (TRAP) is a metalloprotein enzyme belonging to the family of acid phosphatases, which are specifically grouped together because of their ability to catalyze the hydrolysis of orthophosphate monoesters under acidic conditions. TRAP has been studied extensively as a marker of bone disease in cancer patients -6. It isThese observations suggest that acid phosphatases might be diagnostically useful as serological and histological markers of progression and that they might also be of use in the investigation of the corresponding pathophysiological process. TRAP has been used as a diagnostic marker for hairy cell leukemia for more than two decades . To clarThe experimental research and the collection of patients' cells, tissue, and serum for the study was approved by the Ethics Committee of the Medical Faculty of the University of Wuerzburg, Germany, reference number 139/00. All patients participating in the study gave written informed consent.Serum samples were collected from patients with breast (n = 34) and ovarian (n = 39) cancer and from 37 controls, of whom 17 were premenopausal and 20 were postmenopausal (Table 5 cells/ml in a plastic cell-culture flask in a humidified incubator (atmosphere of 5% CO2). Contaminating fibroblasts were eliminated by trypsin treatment every other day and the remaining tumor cell monolayer was cultured until the cells were of homogeneous morphology (passage 3–4). If the tumor cells had divided adequately, contaminating leukocytes and fibroblasts were absent after these few passages.Primary tumor cells from malignant pleural effusions and ascites associated with metastatic breast cancer (4 cases) and ovarian cancer (4 cases) were isolated and cultured. The effusions had been aspirated because of clinical symptoms. The aspirates (20–500 ml) were centrifuged, and the resulting cell pellets washed twice in phosphate buffered saline . Cells were cultured in HBCA medium  supplemeThe following commercially available cell lines were also investigated: Breast cancer cell lines MCF7, MDA-MB-468, BT20, and HBL-100, ovarian cancer cell lines OAW42 and SKOV3, and cervical cancer cell lines SiHa and CaSki. The breast and ovarian cancer cell lines were obtained from Cell Line Services  and cervical cancer cell lines from the Department of Obstetrics and Gynecology, University of Jena, Germany. All cell lines were cultured in RPMI1640 medium (Biochrom) supplemented with 10% FBS and gentamycin (Biochrom).The serum samples were analyzed using a TRAP-ELISA Kit  according to the manufacturer's instructions. The range of the ELISA was 1–10 U/l. In some cases the TRAP concentration was higher than 10 U/l: these samples were diluted 1:4 in the sample dilution buffer provided with the kit and reanalyzed. Final serum concentrations were then derived by multiplication of the value obtained by four.Immunocytological investigations were performed on the cells cultured from the malignant pleural effusions and ascites and commercially available established cell lines. Cells were seeded onto APES- coated slides and grown over night. They were then fixed with 4% formalin and washed in PBS for 10 min, followed by distilled water for 2 minutes and then acetone/methanol [1:2 for 5 min].For immunohistochemistry, sections of routinely-processed paraffin-embedded tissue were cut at 2–3 μm, placed onto APES-coated slides, dewaxed in xylene, and rehydrated in graded ethanols and TRIS-buffered saline . For antigen retrieval, sections were subjected to heat pretreatment by boiling in 0.01 M sodium citrate buffer (pH 6.0) for 10 min in a microwave oven (600 Watt/sec.).For specific detection of the TRAP antigen, the sections and cell smears were incubated with goat serum for 30 min and then with the TRAP monoclonal antibody at 1:100 dilution in antibody diluent . The TRAP antibody is directed against the N-terminal portion of human TRAP . After one wash in PBS, the horseradish-peroxidase (HRP)-labeled rabbit anti-mouse specific secondary antibody  was applied. The detection reaction was developed with 3,3'-diaminobenzidine  or Vector VIP . The sections and smears were counterstained with hematoxylin , dehydrated through graded ethanols, and embedded in Entelan .Immunoreactivity of the cells and tissues was evaluated by comparison with control sections incubated with an IgG control antibody. They were rated positive [+] if a clear brown or purple color was detected in the majority of the cells, as weakly positive [(+)] if less than 50% of the cells were stained (the majority weakly)), and as negative [-] if no staining was detectable.2. The PCR was run for 40 cycles and the temperatures were 96°C, 60°C , and 72°C. The PCR products corresponding to the mRNA were 171 and 175 base pairs in size for the two different primer pairs respectively. PCR products were run on a 1% agarose gel and analyzed. RT-PCR was considered positive when a clear band of the expected size was visible on an ethidium bromide-stained gel.RNA was extracted from the cell lines obtained from the patients and from the commercial cell lines using the Quiagen RNA-extraction kit . LPS-activated dendritic cells, which have been described to express TRAP , and JARThe median values for serum TRAP concentrations are summarized in Table We first evaluated serum from 14 breast cancer patients who had visceral metastases only, and found increased values (median 4.0) in almost all of these patients as compared to the control group as a whole (median 3.2) , almost equalling those of breast cancer patients with bone disease, and a second group with very low values (median 1.9).We tested various malignant and benign tissues for the expression of TRAP Table . The majFigure The immunocytological results are summarized in Table The malignant melanoma cell lines MV3, BML, and Mel2A showed marked expression of the TRAP protein, staining of the BML cells being stronger than that of the other two cell lines. Of the two cervical cancer cell lines investigated, SiHA cells showed weak staining for TRAP, but CaSki cells were negative (data not shown). Of the two choriocarcinoma cell lines JEG-3 was positive, but JAR was negative, so that they were used in the RT-PCR experiments as positive and negative controls, respectively.In order to confirm TRAP expression in the tumor cells with another method of detection, RT-PCR analysis of TRAP mRNA levels in the cell cultures under investigation was performed. Figure .Strong expression of TRAP mRNA was found in the two breast cancer cell lines tested (BT20 and MDA-MB-468) and in breast cancer cells cultured from pleural effusions in 2 of the 3 patients investigated. The commercially available cell lines OAW42 and SKOV3 and ovarian cancer cells cultured from three patients with malignant ascites showed a clear PCR product at 171 bp. The two malignant melanoma cell lines BML and Mel2A both showed TRAP mRNA expression, which was more pronounced in the former than in the latter. Repeat RT-PCR experiments consistently revealed TRAP mRNA expression in JEG-3 cells but none in JAR cells.In contrast to previous studies -3, our iOur immunohistochemical and immunocytological studies showed that a broad range of tumor tissues and cells express TRAP. From our results we conclude that TRAP is overexpressed in a variety of solid tumors and is most probably secreted by the tumor cells themselves. The finding that cancer cells express and secrete TRAP is not yet well established. Therefore, we evaluated this finding not only on the mRNA but also on the protein level.TRAP expression was detected by immunohistochemistry and RT-PCR in commercially available cell lines and cells cultured from malignant pleural effusions in breast cancer patients and in biopsy specimen of breast cancer. Approximately half of the ovarian carcinoma cell cultures derived from malignant ascites and surgical specimens of ovarian cancer were positive for TRAP. Likewise, both malignant melanoma tissue and cell lines clearly expressed TRAP. On the other hand, TRAP could not be detected in cervical cancer tissue. TRAP was not detected in the malignant cells of primary carcinomas of the cervix, but these tumors showed marked infiltration by TRAP-expressing monocytes.TRAP expression seems to be confined to malignant tissue, as it was not detected in normal breast or ovarian tissue or normal skin. It appears to be a basic characteristic of malignant transformation, as it is found in both epithelial and mesenchymal malignancies, such as the malignant melanoma investigated.TRAP has recently been analyzed in cell cultures and was detected in both the supernatant medium and cell lysates . JanckilOur results are consistent with the recent findings of Chao et al. 2005), who describe a significant increase in TRAP in the serum of breast cancer patients with extensive bone metastases 005, who . InteresThe cellular function of TRAP and the biological significance of its expression in physiological conditions such as pregnancy or pathological states such as Gaucher's disease are still not fully understood -17. Our Future studies may be able to determine whether serum TRAP concentrations can be used to monitor the success of chemotherapy. Even more interesting is the possibility that TRAP could be an attractive target for future cancer therapies, given its predominant expression in malignant tissue.The author(s) declare that they have no competing interests.AH drafted the manuscript, recruited patients, designed and coordinated the study, LR participated in its design and coordination, MK carried out the immunohistochemical and ELISA studies, MKr carried out the molecular studies, ME evaluated the immunohistochemical data, JD participated in the design of the study and patient recruitment UK participated in the design of the study, supervised the laboratory work, evaluated immunohistochemical data and performed the statistical analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Warfarin inhibits metastasis in the animal model and injection of the Warfarin-dependent coagulation factor complex II, IX, X enhances pulmonary metastasis in the same model. We have studied two possible mechanisms responsible for the observed effect. Mtln3, rat mammary carcinoma cells, radiolabelled with 5-(125) Iodo-2'-deoxyuridine (IUDR) were injected intravenously in female Fisher 344 rats either alone or in combination with factor complex II, IX, X or bovine serum albumin. Following sacrifice at various intervals, measured lung radioactivity was significantly higher (20%) in animals administered cells with the factor complex than in the other two groups . These results indicate increased entrapment of tumour cells in the pulmonary microcirculation. In a second experiment, rat factor complex II, IX, X was prepared, and Mtln3 cells were then injected in female Fisher 344 rats alone or in combination with either human factor complex or rat factor complex. Following sacrifice, the number of pulmonary nodules in animals receiving cells with rat factor complex was similar to that in animals receiving human factor complex, and significantly higher than that in the control , indicating that the observed enhancement of pulmonary seeding is unrelated to the xenogeneic properties of the human factor complex.


Among the more insidious aspects of cancer is its capacity for escaping the anti-cancer defenses of the host. New research suggests that some estrogens may further reinforce this evasion of host immunity, even as those same hormones stimulate the growth and spread of hormone-responsive cancers. According to David Shapiro, a medical and biochemistry professor at the University of Illinois at Urbana–Champaign, the new findings highlight the role that estrogen-related interference with immune cell function may play in the development and progression of breast cancer.Oncogene, observed that estrogen induces the expression of a newly discovered gene in humans for proteinase inhibitor 9 (PI-9). When estrogen binds to receptors in the cancer cell, the resulting complex promotes production of the PI-9 protein, which in turn binds granzyme B, the primary protease used by natural killer (NK) cells to kill off transformed or infected cells.Shapiro and his colleagues, reporting in a paper posted 22 January 2007 ahead of publication in Proceedings of the National Academy of Sciences, had shown that increased levels of PI-9 in some tumors and tumor-derived cell lines may enhance these tumors’ ability to evade apoptosis mediated by NK cells and cytotoxic T cells.NK cells normally play a central role in immune surveillance against the metastatic spread of cancer. Previous research out of Leiden University Medical Center in the Netherlands, published in the 25 September 2001 issue of In the current paper, the researchers linked increasing concentrations of estrogen with increasing levels of PI-9 and progressively blocked cell death by NK cells. “The levels of estrogen required to induce [PI-9] in breast cancer cells are extremely low,” says Shapiro. Moreover, at elevated levels of epidermal growth factor and estrogen receptor–α, the induction of PI-9 by either estradiol or the breast cancer drug tamoxifen effectively blocked the killing of cancer cells by NK cells. Levels of epidermal growth factor are elevated in many women with breast cancer, and this adversely affects the prognosis. The presence of estrogen receptor–α at the time of diagnosis is an indication for using tamoxifen or other forms of endocrine therapy.Tamoxifen is known to have mixed agonist/antagonist effects on estrogen, stimulating uterine cancer at low doses while inhibiting this and other estrogen-responsive cancers at higher doses. In contrast, the drug raloxifene, which is commonly used to prevent osteoporosis and has also been shown to reduce the risk of some breast cancers, had no immune-disrupting effects.Shapiro’s findings come with the following caveat: most estrogen-responsive breast tumors contain low to moderate levels of estrogen receptors, and in these tumors tamoxifen will not induce a level of PI-9 that enables the breast cancer cells to evade killing by immune cells. Only in the relatively small subset of breast tumors that contain very high levels of estrogen receptors will tamoxifen have an effect that could effectively shield the cancer from attacking immune cells.JAMA study—a prospective, double-blind, randomized clinical trial conducted in nearly 200 cancer treatment centers throughout North America—directly compared tamoxifen and raloxifene, and found both drugs to be chemopreventive. “In that large trial, tamoxifen even [seemed to perform] better in the control of noninvasive breast cancers,” says V. Craig Jordan, scientific director for the Fox Chase Cancer Center Medical Science Division. “So while these [University of Illinois] data are important and insightful, we should not lose sight of what the clinical data are telling us.”Indeed, a 21 June 2006 Estrogenic compounds such as DDT and dioxins may also impede immune protection against breast cancer cells. “The mechanism that this study provides for estrogen-mediated decreases in NK cell function may also be operative in the decreases in NK function that have been observed with certain environmental estrogens, such as DDT,” says Margaret Whalen, an associate professor of chemistry at Tennessee State University who has studied the immune-suppressive effects of various organochlorine pesticides. Most human exposures to xenoestrogens involve mixtures at relatively low levels, Whalen adds, and concurrent exposure to multiple chemicals may alter the immunotoxicity of a particular chemical.Shapiro and his colleagues are now looking into the factors that contribute to regulation of PI-9 in postmenopausal women. “Blocking the production of this protein represents a potential new target for breast cancer therapies,” he says.


The milk fat globule membrane antibodies HMFG1, HMFG2, NCRC 11 and four of the Mam 6 series, and the lectins peanut agglutinin, wheat germ agglutinin, Concanavalin A, Lotus tetragonolobus and Ulex europaeus I have been applied to 115 stage I and II breast carcinomas (median follow up = 36 months) to assess their value as prognostic markers. Of the milk fat globule membrane antibodies only NCRC 11 staining showed a relationship to development of recurrent disease and overall survival, but this did not act as an independent indicator over and above that provided by histological grade. None of the lectins gave prognostic information, including those whose binding related to node status or grade. It is concluded that for short-term prognosis none of the markers can given independent prognostic information over and above that provided by histological evaluation.


This study was undertaken to elucidate the mechanism(s) of differential sensitivity of human bladder cancer cell lines J82 and SCaBER to mitomycin C (MMC) and its analogue, BMY 25067. The IC50 values for MMC and BMY 25067 in the SCaBER cell line were respectively 5- and 4-fold higher than in J82. BMY 25282 and BMY 25067 were significantly more cytotoxic, on a molar basis, than MMC in both the cell lines. NADPH cytochrome P450 reductase and DT diaphorase activities were significantly higher in the J82 cell line than in SCaBER, suggesting that relatively lower sensitivity of the SCaBER cell line to MMC and BMY 25067 may be due to deficient drug activation. This conclusion was supported by the observation that IC50 values for BMY 25282, which has lower quinone reduction potential than MMC and BMY 25067, did not differ significantly in these cell lines. A correlation between drug sensitivity, oxyradical formation and levels of antioxidative enzymes was not observed. These results suggest that the relatively lower sensitivity of SCaBER cells to MMC or BMY 25067 may be independent of differential oxyradical formation. MMC-induced DNA interstrand cross-link (ISC) formation was markedly lower in the SCaBER cell line than in J82. However, it remains to be seen if the reduced ISC frequency in the SCaBER cell line is a consequence of deficient drug activation or results from increased repair of the damaged DNA.


Primary esophageal adenocarcinomas are thought to arise from Barrett esophagus, an acquired condition in which the normal esophageal squamous epithelium is replaced by a specialized metaplastic columnar-cell-lined epithelium.Since the early 1970s, a dramatic change has occurred in the epidemiology of esophageal malignancy in both North America and Europe: the incidence of adenocarcinomas of the lower esophagus and esophagogastric junction is increasing. Several lifestyle factors are implicated in this change, including gastroesophageal reflux disease , severity of gerd, endoscopic and histologic findings, and a panel of biomarkers will be developed to better identify patients with Barrett esophagus at increased risk for malignant progression, leading to more rational endoscopic surveillance and screening programs.Today,  Although diet Since the early 1970s, a dramatic change has occurred in the epidemiology of esophageal malignancy in both North America and Europe: the incidence of adenocarcinomas of the lower esophagus and esophagogastric junction , thereby promoting cellular proliferation and inducing specialized columnar metaplasia of the normal squamous epithelium lining the esophagus. Dysplasia is widely regarded as the precursor of invasive cancer, and high-grade dysplasia in Barrett epithelium is frequently associated with esophageal adenocarcinoma. That association underlies the rationale for endoscopic surveillance programs: to detect malignancy at an earlier and potentially curable stage. However, the surveillance strategy remains controversial.An established risk factor for Barrett esophagus is The recent identification of molecular markers associated with the Barrett metaplasia–dysplasia–carcinoma progression may have potential clinical application to identify individuals with Barrett esophagus who are at increased risk for progression to invasive adenocarcinoma The present review focuses on the pathogenesis of Barrett esophagus and esophageal adenocarcinoma, with emphasis on selected molecular biomarkers that may have future clinical application to the management of this disease.The diagnosis of intestinal metaplasia is made histologically and is characterized by the presence of goblet cells egj egj The definition of intestinal metaplasia (Barrett esophagus) may be further categorized by endoscopic measurement of the length of the columnar epithelium. Classic “long-segment” Barrett esophagus refers to specialized intestinal metaplasia extending more than 3 cm above the anatomic From a practical standpoint, intestinal metaplasia of the esophagus and cardia cannot be accurately differentiated on the basis of routine histology alone. Recent immunohistochemical studies have reported a potential role for cytokeratins 7 (CK7) and 20 (CK20) in differentiating the two The reported prevalence of Barrett esophagus has varied between populations, and most studies have reported incidence and prevalence rates for the classic long-segment disease dna abnormalities. Such dysplasia is generally classified as indefinite, low-grade, or high-grade Dysplasia within a segment of Barrett esophagus can be identified histologically based on phenotypic nuclear alterations resulting from hgd) is regarded as the most reliable predictor for progression to invasive cancer; hgd is frequently associated with esophageal adenocarcinoma hgd to invasive adenocarcinoma is quite variable; reports have indicated that most patients with hgd actually die from non-malignant causes Although the natural history of dysplasia is not known, it was recently reported that the presence of low-grade dysplasia in Barrett epithelia conferred an increased risk of progression to malignancy Relatively few studies have investigated the prevalence of dysplasia within Barrett epithelia. Based on careful pathologic examination of resected esophageal adenocarcinomas, dysplastic change is reported in a relatively high percentage of associated Barrett epithelia egj or cardia. The Siewert system is increasingly being used to classify adenocarcinomas of the esophagus and esophagogastric junction. This system is based on an estimate of the tumour centre in relation to the egji  arise 1 cm to 5 cm above the egj, and type ii (cardia adenocarcinomas) arise from the region 1 cm above to 2 cm below the egj. Type iii (subcardia gastric adenocarcinomas) arise 2 cm to 5 cm below the egj. One disadvantage of the Siewert classification is that precise measurements may be difficult for large, advanced-stage tumours.The definition of primary esophageal adenocarcinoma is controversial, especially when the tumour involves the i) from adenocarcinomas arising at the egj (type ii) Since the mid-1990s, our group has used strict clinicopathologic criteria to stratify primary esophageal adenocarcinomas The most important criterion for establishing a diagnosis of a primary esophageal adenocarcinoma is the presence of Barrett epithelium. However, because Barrett epithelium may not be identified in up to 50% of surgically resected esophageal adenocarcinomas (likely as a consequence of tumour progression), criteria 2 to 5 should be met to establish a primary esophageal origin .egj range from 0.74 to 1.34 per 100,000 population The prevalence of esophageal adenocarcinoma has recently been well studied and, as mentioned earlier, has increased steadily since the mid-1970s. Current estimates of the annual incidence of adenocarcinoma of the esophagus and hgd or cancer at an early and potentially curable stage.The goal of any cancer surveillance program is the detection of premalignant or early invasive disease which, when treated, will ultimately result in improved survival. For patients with Barrett esophagus, endoscopic surveillance refers to esophagogastroscopy and biopsy performed at regular intervals to detect The efficacy and utility of Barrett surveillance is controversial. Among the proposed disadvantages are the difficulty of identifying early neoplastic lesions with current endoscopic techniques and the frequency of sampling error It has been suggested that new strategies are required to improve the efficacy of surveillance for Barrett esophagus. Proposed methods of reducing endoscopic variability and sampling error include the use of chromoendoscopy, magnification endoscopy, fluorescent endoscopy, or optical coherence tomography gerd. This theory is supported by a number of physiologic abnormalities that have been identified in patients with Barrett esophagus—among them, increased acid exposure, a defective lower esophageal sphincter, and impaired esophageal motility and clearance Although the precise causes and natural history of Barrett esophagus remain unknown, the condition is generally accepted to be acquired as a result of gerd leads to acute mucosal injury, promotes cellular proliferation, and induces specialized intestinal metaplasia of the esophagus. Studies have shown Barrett esophagus to be hyperproliferative, reflected by an increased S-phase fraction on flow cytometry pcna) pcna immunostaining is limited to the basal layer of metaplastic Barrett epithelia; in hgd, it extends to the more superficial layers. Similar patterns of staining for Ki67 suggest a functional instability of Barrett mucosa.The hypothesis is that gerd or Barrett esophagus (or both) has been reported egj, predisposing to gerd or to stasis.To date, no genetic locus for familial gerd (and hence be associated with Barrett esophagus); others have found stronger associations with chronic gastritis, Helicobacter pylori infection, gastric intestinal metaplasia, and gastric malignancy H. pylori, because gastric infection with S100A8-positive strains  has been found to have an inverse association with the development of esophageal adenocarcinoma Similarly, the causes and pathogenesis of cardia intestinal metaplasia are unknown. A number of reports suggest that this entity may represent an early manifestation of A number of molecular alterations have been reported in Barrett esophagus, and these molecular alterations are implicated in the molecular pathogenesis of esophageal adenocarcinoma hgd or invasive adenocarcinomaMolecular diagnosis for early detection of Prediction of risk for disease progression in endoscopic surveillance programsStaging and prognosisPrediction of chemosensitivityIntermediate biomarkers in chemoprevention studiesNovel targets for anticancer therapiesnci) Early Detection Research Network (edrn) has developed five phases to validate novel biomarkers used in the early detection of cancer  in Barrett epithelia is associated with risk of progression to malignancy Several studies have reported that aneuploidy  appeared to be at low risk for progression to adenocarcinoma Flow cytometry has also been used to study cell-cycle kinetics, including S-phase fraction, in esophageal pre-malignancy in a number of phase 1 and 2 studies TP53 is located on chromosome 17p13 and encodes a 53-kDa polypeptide (Tp53) that regulates cell cycle progression, dna repair, apoptosis, and neovascularization in both normal and malignant cells via highly complex dna and protein interactions CDKN1A , which sequesters a number of cyclin-dependent kinases (cdks), Tp53 mediates both G1 and G2/M arrest. Point mutations leading to loss of function of TP53 is a common mechanism of inactivation, and more than 90% of TP53 mutations have been located in the conserved dna binding domain (exons 5–8). Through the 1990s, TP53 was extensively characterized; it appears to have a central role in human malignancy The tumour suppressor gene TP53 gene were initially reported in primary esophageal adenocarcinomas and associated Barrett epithelium in 1991 TP53 alterations in Barrett esophagus has been extensively characterized TP53 mutations in non-dysplastic Barrett epithelia suggests that TP53 may be altered early in the metaplasia–dysplasia–carcinoma sequence, and it may therefore be a useful biomarker in endoscopic surveillance programs.Mutations in the TP53 mutations were associated with poor tumour differentiation and with reduced disease-free and overall survival following surgical resection TP53 mutation in esophageal adenocarcinomas were predominantly G:C to A:T transitions at CpG dinucleotides, suggesting that TP53 mutations result from endogenous mechanisms that likely involve spontaneous deamination into thymine of the 5′-methylated cytosine that frequently occurs at CpG di-nucleotides. Because this mechanism is enhanced by exposure to oxy-radicals and nitro-radicals, we hypothesized that local overproduction of nitric oxide, a consequence of chronic gerd, may enhance the rate of formation of spontaneous TP53 mutations in Barrett esophagus.In a 10-year prospective study of surgically resected esophageal adenocarcinomas, TP53 mutations or protein overexpression in Barrett epithelia as predictors of malignant progression, loss of heterozygosity (loh) of 17p (inclusive of TP53) was evaluated in one ongoing phase 4 study in conjunction with flow cytometry loh ranged from 6% in non-dysplastic Barrett epithelia to 57% in hgd and was a significant independent predictor of progression to esophageal adenocarcinoma, with a relative risk of 16. In this study of 325 patients with Barrett esophagus, only 6 of 26 patients who progressed to malignancy lacked 17p loh. And 17p loh was also associated with increased risk for aneuploidy, tetraploidy, and hgd, with relative risks of 7.5, 6.1, and 3.6 respectively.Although no phase 4 studies have evaluated CCND1 gene located on chromosome 11q13 Cyclin D1 is a key regulator of cell-cycle progression, particularly at the transition from G1 to the S phase; it is encoded by the CCND1, alternative gene splicing is thought to give rise to two functional transcripts cdks 4 and 6 (CDK4/6); the resulting complex phosphorylates the RB1 tumour suppressor gene, thereby resulting in cell-cycle progression to S phase. The variant transcript (cyclin D1b), a consequence of the polymorphic A-allele, encodes a truncated protein isoform with an altered C-terminal domain that has been implicated in neoplastic transformation CCND1 A/A genotype were at increased risk for gerd, Barrett esophagus, and esophageal adenocarcinoma, supporting the hypothesis that this polymorphism is an individual susceptibility factor in the molecular progression of esophageal adenocarcinoma As a result of a single base polymorphism (G870A) of CDKN2A gene , which is localized to chromosome 9p21, encodes a protein, P16, that belongs to a family of cdk inhibitors. The P16 protein binds to and inhibits CDK4/6, resulting in reduced phosphorylation of RB1 and inhibition of cell-cycle progression through G1. An alternative transcript  functions to sequester MDM2, thereby stabilizing the TP53 tumour suppressor gene The CDKN2A are reported frequently in various human malignancies, but mechanisms of CDKN2A inactivation appear to vary between tumour types. Point mutations in Barrett esophagus and esophageal adenocarcinoma are relatively uncommon, but 9p loh and promoter hypermethylation appear to be frequent mechanisms of CDKN2A inactivation CDKN2A alterations have been the subject of phase 1 and 2 studies only, they are increasingly recognized as critical early molecular lesions associated with clonal proliferation within Barrett epithelia Alterations of hgd remains the most reliable predictor of progression to invasive esophageal adenocarcinoma, but potentially promising biomarkers include aneuploidy  loh and TP53 mutations CDKN2A alterations gerd, endoscopic and histologic findings, and a panel of biomarkers will be developed to better identify patients with Barrett esophagus at increased risk for malignant progression, leading to more rational endoscopic surveillance and screening programs.Despite advances in multimodality therapy, esophageal (Barrett) adenocarcinoma remains a highly lethal malignancy. To substantially improve outcomes with this disease, future management strategies will need to focus on prevention and early detection based on an improved understanding of esophageal tumour biology. Although several molecular alterations in the progression of Barrett esophagus to invasive esophageal adenocarcinoma have been identified, relatively few will ultimately prove to have clinical application. Currently,


Thyroid cancers are common endocrine malignancies that comprise tumors with different clinical and histological features. Indeed, papillary and follicular thyroid cancers are slow-growing, well-differentiated tumors, whereas anaplastic thyroid cancers are undifferentiated neoplasias that behave much more aggressively. Well-differentiated thyroid carcinomas are efficiently cured by surgery and radioiodine, unlike undifferentiated tumors that fail to uptake radioactive iodine and are usually resistant to chemotherapy. Therefore, novel and more effective therapies for these aggressive neoplasias are urgently needed. Whereas most genetic events underlying the pathogenesis of well-differentiated thyroid cancers have been identified, the molecular mechanisms that generate undifferentiated thyroid carcinomas are still unclear. To date, one of the best-characterized genetic alterations leading to the development of poorly differentiated thyroid tumors is the loss of the p53 tumor suppressor gene. In addition, the existence of a complex network among p53 family members (p63 and p73) and their interactions with other factors that promote thyroid cancer progression has been well documented. In this review, we provide an update on the current knowledge of the role of p53 family proteins in thyroid cancer and their possible use as a therapeutic target for the treatment of the most aggressive variants of this disease. Thyroid cancer is the most common endocrine neoplasm, accounting for about 1.7% of total cancer diagnoses each year in the USA . The risThe treatment for WDTC is based on surgery, radioactive iodine, and thyroid hormone therapy . On the RAS) point mutations in codons 12, 13, and 61 and rearrangements of the Rearrangement During Transfection (RET) gene (generating RET/PTC chimeric oncogenes) represent other frequent genetic alterations  genetic rearrangements (30%), RAS activating point mutations (20–40%) and PTEN inactivation by point mutations, deletions, or promoter methylation (<10%)  also reduced mutant p53 expression in thyroid cancer cells, reactivating p53 tumor suppressor activity and resulting in increased response to anti-cancer therapies  Figure .Finally, the mammalian target of rapamycin (mTOR) pathway inhibitor Everolimus has been studied in a phase II trial of 40 patients with locally advanced or metastatic thyroid cancer  producinThe concept of precision medicine postulates that effective treatment strategies should be tailored to the individual variability of both the patient and her/his disease. While alterations in p53 sequence, stability, and downstream signaling are heavily involved in thyroid carcinogenesis, the role of the remaining p53 family members in the development and progression of thyroid cancer has yet to be fully elucidated. Nevertheless, increasing evidence indicates that p53 family members contribute to the development of multiple thyroid cancer variants and an ever-increasing number of therapeutic molecules targeting these proteins may soon be available in the clinical setting.