A member of the OSCA/TMEM63 family of mechanosensitive calcium channels participates in cell wall integrity maintenance in Aspergillus nidulans.

The calF7 mutation in Aspergillus nidulans causes hypersensitivity to the cell wall compromising agents Calcofluor White (CFW) and Congo Red. In this research we demonstrate that the calF7 mutation resides in gene AN2880, encoding a predicted member of the OSCA/TMEM63 family of transmembrane glycoproteins. Those members of the family whose physiological functions have been investigated have been shown to act as mechanosensitive calcium transport channels. Deletion of AN2880 replicates the CFW hypersensitivity phenotype. Separately, we show that CFW hypersensitivity of calF deletion strains can be overcome by inclusion of elevated levels of extracellular calcium ions in the growth medium, and, correspondingly, wild type strains grown in media deficient in calcium ions are no longer resistant to CFW. These observations support a model in which accommodation to at least some forms of cell wall stress is mediated by a calcium ion signaling system in which the AN2880 gene product plays a role. The genetic lesion in calF7 is predicted to result in a glycine-to-arginine substitution at position 638 of the 945-residue CalF protein in a region of the RSN1_7TM domain that is highly conserved amongst filamentous fungi. Homology modeling predicts that the consequence of a G638R substitution is to structurally occlude the principal conductance pore in the protein. GFP-tagged wild type CalF localizes principally to the Spitzenkörper and the plasma membrane at growing tips and forming septa. However, both septation and hyphal morphology appear to be normal in calF7 and AN2880 deletion strains, indicating that any role played by CalF in normal hyphal growth and cytokinesis is dispensable.

Influence of segmental chromosome abnormalities on survival in children over the age of 12 months with unresectable localised peripheral neuroblastic tumours without MYCN amplification.

BACKGROUND: The prognostic impact of segmental chromosome alterations (SCAs) in children older than 1 year, diagnosed with localised unresectable neuroblastoma (NB) without MYCN amplification enrolled in the European Unresectable Neuroblastoma (EUNB) protocol is still to be clarified, while, for other group of patients, the presence of SCAs is associated with poor prognosis. METHODS: To understand the role of SCAs we performed multilocus/pangenomic analysis of 98 tumour samples from patients enrolled in the EUNB protocol. RESULTS: Age at diagnosis was categorised into two groups using 18 months as the age cutoff. Significant difference in the presence of SCAs was seen in tumours of patients between 12 and 18 months and over 18 months of age at diagnosis, respectively (P=0.04). A significant correlation (P=0.03) was observed between number of SCAs per tumour and age. Event-free (EFS) and overall survival (OS) were calculated in both age groups, according to both the presence and number of SCAs. In older patients, a poorer survival was associated with the presence of SCAs (EFS=46% vs 75%, P=0.023; OS=66.8% vs 100%, P=0.003). Moreover, OS of older patients inversely correlated with number of SCAs (P=0.002). Finally, SCAs provided additional prognostic information beyond histoprognosis, as their presence was associated with poorer OS in patients over 18 months with unfavourable International Neuroblastoma Pathology Classification (INPC) histopathology (P=0.018). CONCLUSIONS: The presence of SCAs is a negative prognostic marker that impairs outcome of patients over the age of 18 months with localised unresectable NB without MYCN amplification, especially when more than one SCA is present. Moreover, in older patients with unfavourable INPC tumour histoprognosis, the presence of SCAs significantly affects OS.

Expression of miR-487b and miR-410 encoded by 14q32.31 locus is a prognostic marker in neuroblastoma.

BACKGROUND: Combination of age at diagnosis, stage and MYCN amplification stratifies neuroblastoma into low-risk and high-risk. We aimed to establish whether a microRNA (miRNA) signature could be associated with prognosis in both groups. METHODS: Microarray expression profiling of human miRNAs and quantitative reverse-transcriptase PCR of selected miRNAs were performed on a preliminary cohort of 13 patients. Results were validated on an independent cohort of 214 patients. The relationship between miRNA expression and the overall or disease-free survival was analysed on the total cohort of 227 patients using the log-rank test and the multivariable Cox proportional hazard model. RESULTS: A total of 15 of 17 miRNAs that discriminated high-risk from low-risk neuroblastoma belonged to the imprinted human 14q32.31 miRNA cluster and two, miR-487b and miR-410, were significantly downregulated in the high-risk group. Multivariable analyses showed miR-487b expression as associated with overall survival and disease-free survival in the whole cohort, independently of clinical covariates. Moreover, miR-487b and miR-410 expression was significantly associated with disease-free survival of the non-MYCN-amplified favourable neuroblastoma: localised (stage 1, 2 and 3) and stage 4 of infant <18 months. CONCLUSION: Expression of miR-487b and miR-410 shows predictive value beyond the classical high-/low-risk stratification and is a biomarker of relapse in favourable neuroblastoma.

Segmental chromosomal alterations lead to a higher risk of relapse in infants with MYCN-non-amplified localised unresectable/disseminated neuroblastoma (a SIOPEN collaborative study).

BACKGROUND: In neuroblastoma (NB), the presence of segmental chromosome alterations (SCAs) is associated with a higher risk of relapse. METHODS: In order to analyse the role of SCAs in infants with localised unresectable/disseminated NB without MYCN amplification, we have performed an array CGH analysis of tumours from infants enrolled in the prospective European INES trials. RESULTS: Tumour samples from 218 out of 300 enroled patients could be analysed. Segmental chromosome alterations were observed in 11%, 20% and 59% of infants enroled in trials INES99.1 (localised unresectable NB), INES99.2 (stage 4s) and INES99.3 (stage 4) (P<0.0001). Progression-free survival was poorer in patients whose tumours harboured SCA, in the whole population and in trials INES99.1 and INES99.2, in the absence of clinical symptoms (log-rank test, P=0.0001, P=0.04 and P=0.0003, respectively). In multivariate analysis, a SCA genomic profile was the strongest predictor of poorer progression-free survival. CONCLUSION: In infants with stage 4s MYCN-non-amplified NB, a SCA genomic profile identifies patients who will require upfront treatment even in the absence of other clinical indication for therapy, whereas in infants with localised unresectable NB, a genomic profile characterised by the absence of SCA identifies patients in whom treatment reduction might be possible. These findings will be implemented in a future international trial.

High frequency of beta-catenin heterozygous mutations in extra-abdominal fibromatosis: a potential molecular tool for disease management.

BACKGROUND: Fibromatosis comprises distinct clinical entities, including sporadic extra-abdominal fibromatosis, which have a high tendency for recurrence, even after adequate resection. There are no known molecular biomarkers of local recurrence. We searched for beta-catenin mutations in a European multicentre series of fibromatosis tumours to relate beta-catenin mutational status to disease outcome. METHODS: Direct sequencing of exon 3 beta-catenin gene was performed for 155 frozen fibromatosis tissues from all topographies. Correlation of outcome with mutation rate and type was performed on the extra-abdominal fibromatosis group (101 patients). RESULTS: Mutations of beta-catenin were detected in 83% of all cases. Among 101 extra-abdominal fibromatosis, similar mutation rates (87%) were observed, namely T41A (39.5%), S45P (9%), S45F (36.5%), and deletion (2%). None of the clinico-pathological parameters were found to be significantly associated with beta-catenin mutational status. With a median follow-up of 62 months, 51 patients relapsed. Five-year recurrence-free survival was significantly worse in beta-catenin-mutated tumours regardless of a specific genotype, compared with wild-type tumours (49 vs 75%, respectively, P=0.02). CONCLUSION: A high frequency (87%) of beta-catenin mutation hallmarks extra-abdominal fibromatosis from a large multicentric retrospective study. Moreover, wild-type beta-catenin seems to be an interesting prognostic marker that might be useful in the therapeutic management of extra-abdominal fibromatosis.

Configural processing enables discrimination and categorization of face-like stimuli in honeybees.

We studied whether honeybees can distinguish face-like configurations by using standardized stimuli commonly employed in primate and human visual research. Furthermore, we studied whether, irrespective of their capacity to distinguish between face-like stimuli, bees learn to classify visual stimuli built up of the same elements in face-like versus non-face-like categories. We showed that bees succeeded in discriminating both face-like and non-face-like stimuli and categorized appropriately novel stimuli in these two classes. To this end, they used configural information and not just isolated features or low-level cues. Bees looked for a specific configuration in which each feature had to be located in an appropriate spatial relationship with respect to the others, thus showing sensitivity for first-order relationships between features. Although faces are biologically irrelevant stimuli for bees, the fact that they were able to integrate visual features into complex representations suggests that face-like stimulus categorization can occur even in the absence of brain regions specialized in face processing.

A web-based survey of reproductive awareness and choices in women with endometriosis.

OBJECTIVE: Our aim was to study fertility issues, attitudes towards reproductive techniques and fertility preservation options in women of reproductive age with endometriosis. STUDY DESIGN: In 2018 we conducted a web-based survey on fertility issues in women aged 18-40 years with endometriosis. Participants were recruited via advertisements on social media and local endometriosis support groups. Participants completed a self-developed online questionnaire evaluating the following dimensions: sociodemographic, medical data, parental project, knowledge and attitudes toward endometriosis and fertility, means used to access information, and reproductive choices. RESULTS: The majority of women (96 %) worried about the impact of endometriosis on their fertility. Approximately half of them (52 %) reported having received sufficient information concerning the effect of endometriosis on fertility from their doctor, whereas 31 % had discussed fertility issues with their doctor but desired further information. In contrast, only a minority (27 %) of women considered themselves well-informed on fertility preservation options. Information given by specialists on endometriosis and reproduction was considered most useful. Information mediated through patient support groups was also highly rated, whereas information given by the general gynecologist was less highly rated. The majority of women would consider assisted reproductive techniques (74 %) or adoption (70 %) in case of infertility. Interestingly, 72 % of women would undergo oocyte vitrification for fertility preservation, whereas only 37 % would resort to oocyte donation. CONCLUSION: This is the first survey to address the topic of fertility issues from the patient's perspective in women with endometriosis. The vast majority of women attach great importance to a discussion about fertility possibilities and only a minority of women consider themselves well-informed. Our results highlight the importance of addressing the issue of fertility in women with endometriosis. Special attention should be given to information and counselling about fertility preservation options since most women consider their knowledge on the topic insufficient. Knowledge and attitudes to counsel endometriosis patients on fertility issues and fertility preservation options should be included in the training curricula of gynecologists. Adequate information on reproductive aging, risk factors for infertility, and reproductive choices, including oocyte vitrification, should be incorporated into follow-up visits for endometriosis patients.

Mountain gorilla lymphocryptovirus has Epstein-Barr virus-like epidemiology and pathology in infants.

Epstein-Barr virus (EBV) infects greater than 90% of humans, is recognized as a significant comorbidity with HIV/AIDS, and is an etiologic agent for some human cancers. The critically endangered mountain gorilla population was suspected of infection with an EBV-like virus based on serology and infant histopathology similar to pulmonary reactive lymphoid hyperplasia (PRLH), a condition associated with EBV in HIV-infected children. To further examine the presence of EBV or an EBV-like virus in mountain gorillas, we conducted the first population-wide survey of oral samples for an EBV-like virus in a nonhuman great ape. We discovered that mountain gorillas are widely infected (n = 143/332) with a specific strain of lymphocryptovirus 1 (GbbLCV-1). Fifty-two percent of infant mountain gorillas were orally shedding GbbLCV-1, suggesting primary infection during this stage of life, similar to what is seen in humans in less developed countries. We then identified GbbLCV-1 in post-mortem infant lung tissues demonstrating histopathological lesions consistent with PRLH, suggesting primary infection with GbbLCV-1 is associated with PRLH in infants. Together, our findings demonstrate that mountain gorilla's infection with GbbLCV-1 could provide valuable information for human disease in a natural great ape setting and have potential conservation implications in this critically endangered species.

Correlation of MDR1 gene expression with chemotherapy in neuroblastoma.

Forty-one neuroblastoma tumor specimens have been analyzed by Northern and slot blot hybridization techniques with human MDR1 gene probes. Only one of 15 (6%) tumors from patients who had not received chemotherapy exhibited high levels of MDR1 transcripts, while 11 of 26 (42%) treated tumors showed high levels of MDR1 expression (Fisher exact test: P = .03). The results indicate that the level of MDR1 mRNA expression is associated with previous chemotherapy, including drugs that select the multidrug resistance phenotype in vitro regardless of neuroblastoma tissue origin or N-myc content in the genome. For the 26 treated neuroblastomas, the number of nonresponsive tumors was found to be significantly higher among those with high levels of MDR1 mRNA.

Resistance to TNF-alpha and adriamycin in the human breast cancer MCF-7 cell line: relationship to MDR1, MnSOD, and TNF gene expression.

The molecular basis of cross-resistance to tumor necrosis factor (TNF) and Adriamycin has been investigated using the breast adenocarcinoma cell line MCF7/p, its Adriamycin-resistant counterpart MCF7AdrR, and the MDR1 gene-transduced MCF-7 cells (MCF7/MDR1). While the parental cell line MCF7/p was TNF-sensitive, MCF7AdrR was TNF-resistant. The TNF resistance exhibited by MCF7AdrR cells was not due to a lack of TNF receptor expression because both cell lines express comparable levels of p75 and p55 receptors as revealed by immunofluorescence analysis. NF-kappa B translocation, which is an essential transducing signal of the TNF-induced lysis pathway, does not appear to be involved in this resistance as assessed by gel shift experiments. In order to determine the role of MDR1 gene expression in the development of this cross-resistance, MCF7/p cells transfected by the MDR1 gene were examined. Our data showed that the expression of the MDR1 gene in these cells resulted in a relative resistance of these cells to Adriamycin without affecting their susceptibility to TNF killing. The implication of the manganese superoxide dismutase and endogenous TNF expression in the cross-resistance by MCF7AdrR cells to Adriamycin and TNF has also been investigated. Northern blot analysis indicated that following TNF stimulation, the expression of 4-kilobase and 1-kilobase manganese superoxide dismutase mRNAs were 9- to 10-fold induced in MCF7AdrR cells as compared to MCF7/p and MCF7/MDR1 cells. This suggests a possible involvement of this enzyme in the Adriamycin-induced resistance to TNF. Although TNF-treatment of MCF7/p and MDR-cells induced endogenous TNF expression in these cells, the level of mRNA induction was selectively enhanced in MCF7AdrR cells (7- to 8-fold greater in MCF7AdrR cells as compared to MCF7/p and MCF7/MDR1 cells). Collectively, these data indicate that the expression of the MDR1 gene in MCF7/p cells following gene transfection is not sufficient for the acquisition of TNF resistance by MCF7/MDR1 cells. Furthermore, our data provide the first evidence that Adriamycin-induced resistance to TNF in MCF7AdrR cells may, in part, involve an overexpression of endogenous TNF and manganese superoxide dismutase genes.

Expression of a human multidrug resistance gene in ovarian carcinomas.

Expression of the human MDR1 gene has been shown to confer the multidrug resistance (MDR) phenotype to sensitive cells. To investigate the possible contribution of the MDR phenotype to chemoresistance in ovarian carcinoma, we have analyzed MDR1 gene expression in fresh carcinoma specimens from 50 patients. Fifteen received chemotherapy before surgery and were judged as poor responders. Thirty-five patients did not receive any drug before surgery. Control tissues were lymphocytes from 7 patients. Total RNAs were analyzed by Northern and slot blot hybridization techniques using human MDR1 complementary DNA and human gamma-actin complementary DNA probes sequentially as qualitative and quantitative controls. MDR1 transcripts (4.5 kilobases) were observed in the RNA preparations obtained from 3 of 10 patients who were treated with doxorubicin or vincristine, 2 drugs known to select the MDR phenotype in vitro. In 40 other RNA preparations obtained from 35 untreated patients and 5 patients treated exclusively with cyclophosphamide and cis-platinum, no transcript could be detected. Using the exact Fisher test, the difference between the 2 groups was found to be significant (P less than 0.01). The three tumors with elevated MDR1 expression did not show MDR1 DNA amplification. Our study suggests that, in spite of the weak occurrence of the MDR process in patients with ovarian cancers, MDR1 expression can be related to previous treatment with doxorubicin or vincristine. These results favor the expression of the MDR1 gene as one of the determinants involved in the acquired chemoresistance of ovarian cancers.

Characterization of a human ovarian adenocarcinoma line, IGROV1, in tissue culture and in nude mice.

A cell line, IGROV1, originating from an ovarian carcinoma of a 47-year-old woman was established in tissue culture and in nude mice. Maintained in monolayer cultures, IGROV1 cells exhibited a 20-h doubling time and highly tumorigenic properties. The s.c. injection of 2 X 10(6) cultured cells into nude mice gave rise to fast growing tumors, while the i.p. route induced a peritoneal carcinomatosis with ascites which killed the animals in 2 months. The epithelial morphology of IGROV1 cells was retained during in vitro and in vivo passages, as judged by both the light and the electron microscopes. Two cytogenetic markers characterize IGROV1 cells: a paracentric inversion of chromosome 3, and a translocation between chromosomes 2 and 5. The constitutional karyotype of the patient was normal. These characteristics make the IGROV1 cell line a suitable experimental model for the treatment of human ovarian carcinomas and for biological studies of human solid tumors.

Combined analysis of DNA ploidy index and N-myc genomic content in neuroblastoma.

The aim of the study was to assess, in a group of nonselected patients with neuroblastoma, the prognostic value of both N-myc gene amplification and DNA ploidy index, taking into account potential confounding factors such as age and stage. Of 59 patients studied, 23 were younger than 1 year at diagnosis, 31 presented with stage IV, 10 with stage III, 5 with stage II, 8 with stage I, and 4 with stage IV-S. N-myc genomic content was analyzed by Southern blot hybridization technique and N-myc amplification (greater than or equal to 3 copies/haploid genome) was present in 6 stage IV, 2 stage III, and 1 stage IV-S. The DNA ploidy index was analyzed by flow cytometry. Of the 59 neuroblastomas, 26 were diploid (DNA index, 1) and 33 were aneuploid (DNA index, greater than 1). The majority of the aneuploid tumors (28 of 33) were near-triploid with DNA indexes between 1.25 and 1.68, 4 were near-diploid (DNA index up to 1.18), and 1 was hypotetraploid (DNA index, 1.85). The proportion of near-triploid tumors was significantly greater among patients under 1 year of age and among patients presenting with stages I, II, and IV-S. Interestingly, 0 of 28 near-triploid neuroblastomas exhibited N-myc gene amplification, compared to 9 of 31 in the group of diploid, near-diploid, and hypotetraploid tumors (Fisher's exact test, P less than 0.001). Four factors were significantly related to a high risk of relapse in univariate analysis, i.e., age, stage, DNA index, and N-myc amplification. In multivariate analysis, only N-myc amplification and the DNA index remained significantly associated with a high risk of relapse. The 2-year disease-free survival rate was 94% (95% confidence interval, 77-98%) for patients with near-triploid neuroblastoma, compared to 45 and 11% (95% confidence interval, 32-70 and 4-23%) for patients with diploid or near-diploid tumors, without and with N-myc amplification, respectively. We concluded that the combination of N-myc and DNA index should be included in routine management of neuroblastoma.

[Fertility preservation in women of the childbearing age: Indications and strategies]. / Préservation de la fertilité chez la femme en âge de procréer : indications et stratégies.

Advances on cryopreservation techniques now allow considering oocyte, embryo or ovarian tissue freezing for female fertility preservation. Originally developed for patients suffering from cancer, fertility preservation has rapidly invaded others medical fields, and represents now the standard of care for all young patient diagnosed with a disease that could impair fertility or having to receive possibly gonadotoxic treatment. As a result, autoimmune diseases, some genetic pathologies or iterative pelvic surgeries, at risk of premature ovarian failure, have become common indications of fertility preservation. In addition, the social egg freezing aiming at preventing the age-related fertility decline is still debated in France, although authorized in numerous countries. This review will discuss the different strategies of fertility preservation in young girls and women of reproductive age, regarding different medical or non-medical indications.

[A prospective study to compare the efficiency of oocyte vitrification using closed or open devices]. / Étude prospective comparant l'efficacité de la vitrification ovocytaire en système fermé ou en système ouvert.

OBJECTIVES: Oocyte vitrification using an open device is thought to be a source of microbiological and chemical contaminations that can be avoided using a closed device. The principal purpose of this study was to compare the two vitrification protocols: closed and open system. The secondary aim was to study the effects of the storage in the vapor phase of nitrogen (VPN) on oocytes vitrified using an open system and to compare it to those of a storage in liquid nitrogen (LN). METHODS: Forty-four patients have been included in our study between November 2014 and May 2015. Two hundred and fourteen oocytes have been vitrified at germinal vesicle (GV), metaphase I (0PB) and metaphase II (1PB) stages. We vitrified 96 oocytes (59 GV/37 0PB) using a closed vitrification device and 118 oocytes (57 GV/31 0PB/30 1PB) using an open device. The vitrified oocytes were then stored either in LN or in VPN. The main outcome measures were the survival rate after warming (SR), meiosis resumption rate (MRR) and maturation rate (MR). RESULTS: The global post-thaw SR was significantly higher for oocytes vitrified using an open system (93.2%) compared to those vitrified using a closed one (64.5%; P<0.001). On the contrary, there was no significant difference in terms of global MRR and MR (82.1% vs. 87.5% and 60.7% vs. 61.2% using closed and open system respectively). The SR, MRR and the MR were not significantly different when vitrified oocytes were stored in VPN or LN (91.6, 83.8, 64.5% vs. 93.9, 89.8, 59.1% respectively). CONCLUSION: Taking into account the limits of our protocol, the open vitrification system remains the more efficient system. The use of sterile liquid nitrogen for oocyte vitrification and the subsequent storage in vapor phase of nitrogen could minimize the hypothetical risks of biological and chemical contaminations.

[Embryo development in two single-step media: Analysis of 2059 sibling oocytes]. / Développement embryonnaire dans 2 milieux de culture globale : étude prospective autocontrôlée sur 2059 ovocytes.

OBJECTIVE: The aim of this study was to compare embryo development cultured in two single-step media commercially available: Fert/Sage One Step® (Origio) and Continuous Single Culture® (CSC) (Irvine Scientific). METHODS: A prospective auto-controlled study of sibling oocytes from women undergoing conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) was performed in our center from February to June 2015. After fertilization, for every patient, half of oocytes were cultured in the single-step Fert/Sage One Step® (serie SAGE) and the other half in the single-step CSC®(serie CSC). Fertilization and embryo morphology rates were assessed by day 1 to day 5-6 if needed. Embryo presenting<20% of fragmentation and 4 cells at day 2, 8 cells at day 3 were qualified as "top quality". Embryo with<20% of fragmentation and 3-5 cells at day 2, 6-10 cells at day 3 were qualified as "good quality". Blastocyst B3, B4, B5 with A or B inner cell mass and A or B trophectoderm were qualified as "good quality". Transferred or frozen embryos were qualified as useful embryos. RESULTS: Sixty-two attempts of IVF and 133 of ICSI were analyzed, corresponding to 2059 inseminated or micro-injected oocytes. Fertilization rate were not different between the 2 series, respectively SAGE vs CSC (IVF: 73.4% vs 68.3% [P=0.49]; ICSI: 58.9% vs 63.8% [P=0.12]). No difference was found for embryo morphology, respectively SAGE vs CSC, at day 2 (top quality embryo at day 2 IVF: 34.4% vs 33% [P=0.98]; ICSI: 42.4% vs 44.9% [P=0.37]; and good quality embryo at day 2 IVF: 44% vs 50.2% [P=0.07]; ICSI: 64% vs 71% [P=0.35]); no difference at day 3 (top quality embryo at day 3 IVF: 19.4% vs 21.3% [P=0.61]; ICSI: 28.7% vs 27.4% [P=0.54]; and good quality embryo at day 3 IVF: 40.4% vs 50.2% [P=0.91]; ICSI: 51% vs 47.6% [P=0.47]). Blastocyst development rate were not different, respectively SAGE vs CSC (IVF: 39.9% vs 41.5% [P=0.63] with 42.9% vs 42.2% of good quality blastocyst [P=0.70]; ICSI: 41.1% vs 37.8% [P=0.18] with 32.9% vs 40.8% of good quality blastocyst [P=0.13]). No difference was found in the useful embryo rate in the 2 series SAGE vs CSC (IVF: 52.8% vs 55.2% [P=0.83]; ICSI: 62.4% vs 61.7% [P=0.70]). CONCLUSION: Embryo development and rate of useful embryos, transferred or frozen, were not different according to the embryo culture in single-step media Fert/Sage One Step® vs single-step Continuous Single Culture®.

Loss of heterozygosity, allele silencing and decreased expression of p73 gene in breast cancers: prevalence of alterations in inflammatory breast cancers.

The p73 gene is a p53 homologue located at 1p36-33, a region submitted to deletions in breast cancer (BC) and putatively imprinted. To study whether p73 was associated with breast carcinogenesis, loss of heterozygosity (LOH), allele expression and transcript levels were assessed in 59 BC, including 39 BC presenting no inflammatory symptoms (NBC) and 20 inflammatory BC (IBC). IBC is a rare but aggressive form of cancer with a very poor prognosis. Normal breast epithelium (BE) and lymphocytes from patients were used as controls. StyI polymorphism generating GC and/or AT alleles was used to select 22 heterozygous patients. p73 LOH was significantly higher in IBC than in NBC [five of eight cases (62%) versus two of 14 cases (14%); Fisher's exact test, P=0.05]. p73 was biallelically expressed in all BE. In contrast, 12 of 16 (75%) BC were monoallelically expressed, showing that allele silencing was significantly associated with breast carcinogenesis (P=0.012), AT being the preferential silent allele (10 out of 12 tumours). p73 mRNA levels in NBC and IBC were two- and threefold lower than in BE, respectively, suggesting that decreased expression could be related to tumour aggressiveness. In conclusion, LOH, allele silencing and decreased expression of the p73 gene may play a role in breast carcinogenesis.

P73 expression in basal layers of head and neck squamous epithelium: a role in differentiation and carcinogenesis in concert with p53 and p63?

P73, a p53-homologue gene, has been studied for its possible role in head and neck squamous epithelium (HNSE) differentiation and carcinogenesis. P73 RNA and protein were analysed in 50 biopsies, including well- and moderately-differentiated carcinomas, and 21 matched normal adjacent tissues. P73 immunohistochemical analyses revealed intense p73 nuclear staining in basal and parabasal cells of normal squamous epithelium, in contrast with complete absence of staining in the more superficial cell layers. Moderately-differentiated carcinomas demonstrated homogeneous and diffuse staining in all tumour cells, while only basal cells were stained in well-differentiated carcinomas as in normal tissue. No correlation was observed between p73 and p53 protein expression. Immunostaining for p63, another p53-related protein previously described as being involved in HNSE morphogenesis and overexpressed in head and neck squamous cell carcinomas (HNSCC), was found to be similar to p73 labelling in carcinomas, but spread to the more differentiated layers in normal epithelium. Biallelic expression of p73 was found in tumours as well as in matched normal tissues. Comparison of p73 transcript levels between tumours and normal tissues showed decreased mRNA expression in 5/17 (30%) tumours independently of the differentiation status. Mutation and loss of heterozygosity analyses of the p73 gene revealed wild type status and no deletion. Our results strongly suggest that: (i) p73 is associated with homeostasis and control of differentiation of head and neck squamous epithelium probably in concert with p53 and p63; (ii) down-regulation of p73 expression could participate in HNSE carcinogenesis.

N-myc genomic content and DNA ploidy in stage IVS neuroblastoma.

DNA ploidy and N-myc genomic content were analyzed in a series of stage IVS neuroblastomas by flow cytometry and Southern blot hybridization, respectively. Of the 12 stage IVS neuroblastomas studied, nine were aneuploid (DNA index [DI] greater than 1), two were diploid (DI = 1), and one was not assessable for DNA content due to insufficient tumor material. N-myc gene amplification was present in two of 12 tumors. None of the aneuploid tumors exhibited N-myc amplification. Among the aneuploid neuroblastomas, the DIs were between 1.27 and 1.60, ie, in the near-triploid range. The follow-up from diagnosis ranged from 1 to 41 months (mean, 20 months). The nine neuroblastomas with near-triploid DNA content were free of disease at the end of the follow-up period. In contrast, a rapid and fatal tumor progression was observed for the three neuroblastomas with N-myc amplification and/or diploidy. Although involving only a limited series, these results strongly suggest that the combined analysis of DNA ploidy and N-myc genomic content could predict clinical outcome in stage IVS neuroblastoma and should help to identify patients for whom a more aggressive therapy is required.