Characterizing the tumor immune microenvironment of ependymomas using targeted gene expression profiles and RNA sequencing.

BACKGROUND: Defining the tumor immune microenvironment (TIME) of patients using transcriptome analysis is gaining more popularity. Here, we examined and discussed the pros and cons of using RNA sequencing for fresh frozen samples and targeted gene expression immune profiles (NanoString) for formalin-fixed, paraffin-embedded (FFPE) samples to characterize the TIME of ependymoma samples. RESULTS: Our results showed a stable expression of the 40 housekeeping genes throughout all samples. The Pearson correlation of the endogenous genes was high. To define the TIME, we first checked the expression of the PTPRC gene, known as CD45, and found it was above the detection limit in all samples by both techniques. T cells were identified consistently using the two types of data. In addition, both techniques showed that the immune landscape was heterogeneous in the 6 ependymoma samples used for this study. CONCLUSIONS: The low-abundant genes were detected in higher quantities using the NanoString technique, even when FFPE samples were used. RNA sequencing is better suited for biomarker discovery, fusion gene detection, and getting a broader overview of the TIME. The technique that was used to measure the samples had a considerable effect on the type of immune cells that were identified. The limited number of tumor-infiltrating immune cells compared to the high density of tumor cells in ependymoma can limit the sensitivity of RNA expression techniques regarding the identification of the infiltrating immune cells.

Autologous dendritic cells pulsed with allogeneic tumour cell lysate induce tumour-reactive T-cell responses in patients with pancreatic cancer: A phase I study.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is notorious for its poor prognosis even after curative resection. Responses to immunotherapy are rare and related to inadequate T-cell priming. We previously demonstrated the potency of allogeneic lysate-dendritic cell (DC) vaccination in a preclinical model. Here we translate this concept to patients. METHODS: In this phase I study, patients with resected PDAC were included when they demonstrated no radiologic signs of recurrence after standard-of-care treatment. Allogeneic tumour lysate-loaded autologous monocyte-derived DCs were injected at weeks 0, 2, 4 and at months 3 and 6. Objectives are feasibility, safety and immunogenicity of allogeneic tumour-DCs. The presence of tumour antigens shared between the vaccine and patient tumours was investigated. Immunological analyses were performed on peripheral blood, skin and tumour. RESULTS: Ten patients were included. DC production and administration were successful. All patients experienced a grade 1 injection-site and infusion-related reaction. Two patients experienced a grade 2 fever and 1 patient experienced a grade 3 dyspnoea. No vaccine-related serious adverse events were observed. Shared tumour antigens were found between the vaccine and patient tumours. All evaluated patients displayed a vaccine-induced response indicated by increased frequencies of Ki67+ and activated PD-1+ circulating T-cells. In addition, treatment-induced T-cell reactivity to autologous tumour of study patients was detected. Seven out of ten patients have not experienced disease recurrence or progression at a median follow-up of 25 months (15-32 months). CONCLUSION: Allogeneic tumour lysate-DC treatment is feasible, safe and induces immune reactivity to PDAC expressed antigens.

Health-related Quality of Life in children and adolescents after invasive treatment for congenital heart disease.

Since the 1980s treatment techniques for congenital heart disease (ConHD) have gradually evolved. Therefore, actual information on the outcomes, including quality of life is required. Health-related quality of life was assessed long-term in four diagnostic groups of children, who underwent invasive treatment for ConHD between 1990 and 1995. The scores on the TNO-AZL Child Quality of Life Questionnaire (TACQOL) of both children with ConHD and their parents were compared with those of a same-aged reference group. The total sample of ConHD children (n = 113, 8-15 years old) obtained significantly lower mean scores on motor functioning, cognitive functioning, and positive emotional functioning than reference peers, reflecting an experience of poorer functioning. ConHD children, aged 8-11 years, obtained lower mean scores on 5 of the 7 TACQOL scales than reference peers. They also had a lower score on positive emotional functioning than 12- to 15-year-old ConHD children. The total sample of ConHD children obtained lower outcomes compared to their parents on 4 of the 7 TACQOL scales. No significant differences were found in health-related quality of life between ConHD boys and girls, neither between different diagnostic groups. Overall, this sample of recently treated ConHD children showed a worse health-related quality of life compared to reference groups. These findings deserve further attention.

NADH-Regulated metabolic model for growth of Methylosinus trichosporiumOB3b. Cometabolic degradation of trichloroethene and optimization of bioreactor system performance.

A metabolic model describing growth of Methylosinus trichosporium OB3b and cometabolic contaminant conversion is used to optimize trichloroethene (TCE) conversion in a bioreactor system. Different process configurations are compared: a growing culture and a nongrowing culture to which TCE is added at both constant and pulsed levels. The growth part of the model, presented in the preceding article, gives a detailed description of the NADH regeneration required for continued TCE conversion. It is based on the metabolic pathways, includes Michaelis-Menten type enzyme kinetics, and uses NADH as an integrating and controlling factor. Here the model is extended to include TCE transformation, incorporating the kinetics of contaminant conversion, the related NADH consumption, toxic effects, and competitive inhibition between TCE and methane. The model realistically describes the experimentally observed negative effects of the TCE conversion products, both on soluble methane monooxygenase through the explicit incorporation of the activity of this enzyme and on cell viability through the distinction between dividing and nondividing cells. In growth-based systems, the toxicity of the TCE conversion products causes rapid cell death, which leads to wash-out of suspended cultures at low TCE loads (below microM inlet concentrations). Enzyme activity, which is less sensitive, is hardly affected by the toxicity of the TCE conversion products and ensures high conversions (>95%) up to the point of wash-out. Pulsed addition of TCE (0.014-0.048 mM) leads to a complete loss of viability. However, the remaining enzyme activity can still almost completely convert the subsequently added large TCE pulses (0.33-0.64 mM). This emphasizes the inefficient use of enzyme activity in growth-based systems. A comparison of growth-based and similar non-growth-based systems reveals that the highest TCE conversions per amount of cells grown can be obtained in the latter. Using small amounts of methane (negligible compared to the amount needed to grow the cells), NADH limitation in the second step of this two-step system can be eliminated. This results in complete utilization of enzyme activity and thus in a very effective treatment system.

NADH-Regulated metabolic model for growth of Methylosinus trichosporium OB3b. Model presentation, parameter estimation, and model validation.

A biochemical model is presented that describes growth of Methylosinus trichosporium OB3b on methane. The model, which was developed to compare strategies to alleviate NADH limitation resulting from cometabolic contaminant conversion, includes (1) catabolism of methane via methanol, formaldehyde, and formate to carbon dioxide; (2) growth as formaldehyde assimilation; and (3) storage material (poly-beta-hydroxybutyric acid, PHB) metabolism. To integrate the three processes, the cofactor NADH is used as central intermediate and controlling factor-instead of the commonly applied energy carrier ATP. This way a stable and well-regulated growth model is obtained that gives a realistic description of a variety of steady-state and transient-state experimental data. An analysis of the cells' physiological properties is given to illustrate the applicability of the model. Steady-state model calculations showed that in strain OB3b flux control is located primarily at the first enzyme of the metabolic pathway. Since no adaptation in V(MAX) values is necessary to describe growth at different dilution rates, the organism seems to have a "rigid enzyme system", the activity of which is not regulated in response to continued growth at low rates. During transient periods of excess carbon and energy source availability, PHB is found to accumulate, serving as a sink for transiently available excess reducing power.

Trichloroethene degradation in a two-step system by methylosinus trichosporium OB3b. Optimization of system performance: use of formate and methane.

The breakdown of dissolved TCE in a two-step bioremediation system is described. In the first reactor, the organism Methylosinus trichosporium OB3b is grown; in the second reactor, consisting of three 17-L column reactors in series, the cells degrade TCE. A special design allowed both for the addition of air (uG,s = 0.01-0. 04 mm s-1) in the conversion reactor to prevent oxygen limitation while minimizing stripping of TCE, and for the use of methane as exogenous electron donor. In two-step systems presented thus far, only formate was used (excess, 20 mM). We found formate additions could be reduced by 75% (15 degrees C), whereas small amounts of methane (0.02-0.04 mol CH4/g cells) could replace formate and led to equally optimal results. Example calculations show that up to 90% reduction in operating cost of chemicals can be obtained by using methane instead of formate. A model was developed to describe each of the conditions studied: excess formate and optimal methane addition, suboptimal formate addition and suboptimal methane addition. Using parameters obtained from independent batch experiments, the model gives a very good description of the overall TCE conversion in the two-step system. The system presented is flexible (oxygen/methane addition) and can easily be scaled up for field application. The model provides a tool for the design of an effective and low-cost treatment system based on methane addition in the conversion reactor.

Effect of trichloroethylene on the competitive behavior of toluene-degrading bacteria.

The influence of trichloroethylene (TCE) on a mixed culture of four different toluene-degrading bacterial strains (Pseudomonas putida mt-2, P. putida F1, P. putida GJ31, and Burkholderia cepacia G4) was studied with a fed-batch culture. The strains were competing for toluene, which was added at a very low rate (31 nmol mg of cells [dry weight] h). All four strains were maintained in the mixed culture at comparable numbers when TCE was absent. After the start of the addition of TCE, the viabilities of B. cepacia G4 and P. putida F1 and GJ31 decreased 50- to 1,000-fold in 1 month. These bacteria can degrade TCE, although at considerably different rates. P. putida mt-2, which did not degrade TCE, became the dominant organism. Kinetic analysis showed that the presence of TCE caused up to a ninefold reduction in the affinity for toluene of the three disappearing strains, indicating that inhibition of toluene degradation by TCE occurred. While P. putida mt-2 took over the culture, mutants of this strain which could no longer grow on p-xylene arose. Most of them had less or no meta-cleavage activity and were able to grow on toluene with a higher growth rate. The results indicate that cometabolic degradation of TCE has a negative effect on the maintenance and competitive behavior of toluene-utilizing organisms that transform TCE.

Effect of Chlorinated Ethene Conversion on Viability and Activity of Methylosinus trichosporium OB3b.

The effect of transformation of chlorinated ethenes on the cell viability of Methylosinus trichosporium OB3b was investigated. A comparison of the loss of viability with the decrease in transformation rates showed that for the monooxygenase-mediated transformation of all chlorinated ethenes except vinyl chloride the decrease in cell viability was the predominant toxic effect.

Transformation Kinetics of Chlorinated Ethenes by Methylosinus trichosporium OB3b and Detection of Unstable Epoxides by On-Line Gas Chromatography.

A rapid and accurate method for the determination of transformation kinetics of volatile organic substrates was developed. Concentrations were monitored by on-line gas chromatographic analysis of the headspace of well-mixed incubation mixtures. With this method, the kinetics of transformation of a number of C(inf1) and C(inf2) halogenated alkanes and alkenes by Methylosinus trichosporium OB3b expressing particulate methane monooxygenase or soluble methane monooxygenase (sMMO) were studied. Apparent specific first-order rate constants for cells expressing sMMO decreased in the order of dichloromethane, vinyl chloride, cis-1,2-dichloroethene, trans-1,2-dichloroethene, 1,1-dichloroethene, trichloroethene, chloroform, and 1,2-dichloroethane. During the degradation of trichloroethene, cis-1,2-dichloroethene, trans-1,2-dichloroethene, and vinyl chloride, the formation of the corresponding epoxides was observed. The epoxide of vinyl chloride and the epoxide of trichloroethene, which temporarily accumulated in the medium, were chemically degraded according to first-order kinetics, with half-lives of 78 and 21 s, respectively. Cells expressing sMMO actively degraded the epoxide of cis-1,2-dichloroethene but not the epoxide of trans-1,2-dichloroethene. Methane and acetylene inhibited degradation of the epoxide of cis-1,2-dichloroethene, indicating that sMMO was involved.

Control of glucose influx into glycolysis and pleiotropic effects studied in different isogenic sets of Saccharomyces cerevisiae mutants in trehalose biosynthesis.

The GGS1/TPS1 gene of the yeast Saccharomyces cerevisiae encodes the trehalose-6-phosphate synthase subunit of the trehalose synthase complex. Mutants defective in GGS1/TPS1 have been isolated repeatedly and they showed variable pleiotropic phenotypes, in particular with respect to trehalose content, ability to grow on fermentable sugars, glucose-induced signaling and sporulation capacity. We have introduced the fdp1, cif1, byp1 and glc6 alleles and the ggs1/tps1 deletion into three different wild-type strains, M5, SP1 and W303-1A. This set of strains will aid further studies on the molecular basis of the complex pleiotropic phenotypes of ggs1/tps1 mutants. The phenotypes conferred by specific alleles were clearly dependent on the genetic background and also differed for some of the alleles. Our results show that the lethality caused by single gene deletion in one genetic background can become undetectable in another background. The sporulation defect of ggs1/tps1 diploids was neither due to a deficiency in G1 arrest, nor to the inability to accumulate trehalose. Ggs1/tps1 delta mutants were very sensitive to glucose and fructose, even in the presence of a 100-fold higher galactose concentration. Fifty-percent inhibition occurred at concentrations similar to the Km values of glucose and fructose transport. The inhibitory effect of glucose in the presence of a large excess of galactose argues against an overactive glycolytic flux as the cause of the growth defect. Deletion of genes of the glucose carrier family shifted the 50% growth inhibition to higher sugar concentrations. This finding allows for a novel approach to estimate the relevance of the many putative glucose carrier genes in S. cerevisiae. We also show that the GGS1/TPS1 gene product is not only required for the transition from respirative to fermentative metabolism but continuously during logarithmic growth on glucose, in spite of the absence of trehalose under such conditions.

Decrease in glycolytic flux in Saccharomyces cerevisiae cdc35-1 cells at restrictive temperature correlates with a decrease in glucose transport.

The glycolytic flux was investigated in the thermosensitive Saccharomyces cerevisiae adenylate cyclase mutant cdc35-1. Directly after a shift to restrictive temperature, the specific CO2 production rate increased from about 250 nmol min-1 (mg protein)-1 to more than 400 nmol min-1 (mg protein)-1, but then the CO2 production gradually fell to about 70 nmol min-1 (mg protein)-1 after 5 h. O2 consumption at restrictive temperature continued at more or less the same rate as at permissive temperature. The temperature shift in the mutant resulted in an increase in the estimated intracellular cAMP concentration from about 1.1 microM to 1.8 microM. This indicates that high cAMP levels are not sufficient for cell cycle progression and high glycolytic activity. The decrease in glycolytic activity at restrictive temperature was not paralleled by a similar decrease in the specific activity of any of the glycolytic enzymes, but correlated with a decrease in hexose transport. A drop in intracellular concentrations of the early metabolites of glycolysis further indicated a defect in transport at restrictive temperature. Our data suggest that glucose transport has a high control on glycolytic flux.

Disruption of the Kluyveromyces lactis GGS1 gene causes inability to grow on glucose and fructose and is suppressed by mutations that reduce sugar uptake.

In the yeast Saccharomyces cerevisiae the GGS1 gene is essential for growth on glucose or other readily fermentable sugars. GGS1 is the same gene as TPS1 which was identified as encoding a subunit of the trehalose-6-phosphate synthase/phosphatase complex and it is allelic to the fdp1, byp1, glc6 and cif1 mutations. Its precise function in the regulation of sugar catabolism is unknown. We have cloned the GGS1 homologue from the distantly related yeast Kluyveromyces lactis. The KlGGS1 gene is 74% and 79% identical at the nucleotide and amino acid sequence level, respectively, to the S. cerevisiae counterpart. We also compared the sequence with the partly homologous products of the S. cerevisiae genes TPS2 and TSL1 which code for the larger subunits of the trehalose synthase complex and with a TSL1 homologue, TPS3, of unknown function. Multiple alignment of these sequences revealed several particularly well conserved elements. Disruption of GGS1 in K. lactis caused the same pleiotropic phenotype as in S. cerevisiae, i.e. inability to grow on glucose or fructose and strongly reduced trehalose content. We have also studied short-term glucose-induced regulatory effects related to cAMP and cAMP-dependent protein kinase, i.e. the cAMP signal, trehalase activation, trehalose mobilization and inactivation of fructose-1,6-bisphosphatase. These effects occur very rapidly in S. cerevisiae and are absent in the Scggs1 mutant. In K. lactis all these effects were much slower and largely unaffected by the Klggs1 mutation. On the other hand, glucose strongly induced pyruvate decarboxylase and activated the potassium transport system in K. lactis and both effects were absent in the Klggs1 mutant. Addition of glucose to galactose-grown cells of the Klggs1 mutant caused, as in S. cerevisiae, intracellular accumulation of free glucose and of sugar phosphates and a rapid drop of the ATP and inorganic phosphate levels. Glucose transport kinetics were the same for the wild type and the Klggs1 mutant in both derepressed cells and in cells incubated with glucose. We have isolated phenotypic revertants of the Klggs1 mutant for growth on fructose. The suppressors that we characterized had, to different extents, diminished glucose uptake in derepressed cells but cells incubated in glucose showed very different characteristics. The suppressor mutations prevented deregulation of glycolysis in the Klggs1 mutant but not the accumulation of free glucose. The mutants with higher residual uptake activity showed partially restored induction of pyruvate decarboxylase and activation of potassium transport.(ABSTRACT TRUNCATED AT 400 WORDS)

The nucleotide sequence of a 2.1 kb fragment from chromosome VI of Saccharomyces cerevisiae identifies a tRNA(Gly) gene, part of a delta element and a palindromic sequence.

The nucleotide sequence was determined of a 2.1 kb DNA fragment located at approximately 35 kb to the right of the centromere of chromosome VI from Saccharomyces cerevisiae. Analysis revealed the presence of a tRNA(GLy) gene, part of a delta element and a remarkable palindromic sequence. The longest open reading frame found encodes a putative protein of 195 amino acids. Although the fragment was isolated by hybridization to a human diacylglycerol kinase cDNA, no evidence was obtained for the presence of a gene encoding diacylglycerol kinase.

Inactivation of the CDC25 gene product in Saccharomyces cerevisiae leads to a decrease in glycolytic activity which is independent of cAMP levels.

In the budding yeast Saccharomyces cerevisiae cyclic AMP (cAMP) can influence the activity of key enzymes in carbohydrate metabolism through modulation of the activity of cAMP-dependent protein kinase. One of the components involved in cAMP production is the CDC25 gene product, which can activate the RAS/adenylate cyclase pathway by promoting the exchange of guanine nucleotides bound to RAS. In two yeast strains carrying different thermosensitive alleles of the CDC25 gene, cAMP levels respond differently to an increase in growth temperature from 23 degrees C (permissive) to 36 degrees C (restrictive). In strain OL86 (cdc25-5) the estimated intracellular concentration of cAMP dropped after transfer to restrictive temperature whereas in strain ts321 (cdc25-1) the cAMP level rose under the same conditions. Despite the differences in cAMP levels the glycolytic flux in the two mutants responded in a very similar way to the shift from permissive to restrictive temperature; after the increase in the incubation temperature, the specific glycolytic flux in both cdc25-1 and cdc25-5 initially increased from about 300 nmol min-1 (mg protein)-1 to about 500 nmol min-1 (mg protein)-1 (presumably mainly as a consequence of the increase in temperature), but then gradually fell to 100-200 nmol min-1 (mg protein)-1. A similar pattern of CO2 production to that found in the two cdc25 mutants was also observed for several other thermosensitive mutants displaying a Start-II type of G1 arrest. In contrast, in a wild-type strain and in strains giving a Start-I type of G1 arrest, CO2 production did not drop after a temperature shift. The specific activities of glycolytic enzymes in the two cdc25 mutants did not show much change after the temperature shift, indicating that the decrease in glycolytic flux was not caused by a decrease in the activity of any of the glycolytic enzymes. Our data show that, at least in long-term regulation, the cAMP levels per se are not likely to be a prime factor controlling glycolytic flux.

Molecular cloning of a gene involved in glucose sensing in the yeast Saccharomyces cerevisiae.

Cells of the yeast Saccharomyces cerevisiae display a wide range of glucose-induced regulatory phenomena, including glucose-induced activation of the RAS-adenylate cyclase pathway and phosphatidylinositol turnover, rapid post-translational effects on the activity of different enzymes as well as long-term effects at the transcriptional level. A gene called GGS1 (for General Glucose Sensor) that is apparently required for the glucose-induced regulatory effects and several ggs1 alleles (fdp1, byp1 and cif1) has been cloned and characterized. A GGS1 homologue is present in Methanobacterium thermoautotrophicum. Yeast ggs1 mutants are unable to grow on glucose or related readily fermentable sugars, apparently owing to unrestricted influx of sugar into glycolysis, resulting in its rapid deregulation. Levels of intracellular free glucose and metabolites measured over a period of a few minutes after addition of glucose to cells of a ggs1 delta strain are consistent with our previous suggestion of a functional interaction between a sugar transporter, a sugar kinase and the GGS1 gene product. Such a glucose-sensing system might both restrict the influx of glucose and activate several signal transduction pathways, leading to the wide range of glucose-induced regulatory phenomena. Deregulation of these pathways in ggs1 mutants might explain phenotypic defects observed in the absence of glucose, e.g. the inability of ggs1 diploids to sporulate.

The growth and signalling defects of the ggs1 (fdp1/byp1) deletion mutant on glucose are suppressed by a deletion of the gene encoding hexokinase PII.

Yeast cells defective in the GGS1 (FDP1/BYP1) gene are unable to adapt to fermentative metabolism. When glucose is added to derepressed ggs1 cells, growth is arrested due to an overloading of glycolysis with sugar phosphates which eventually leads to a depletion of phosphate in the cytosol. Ggs1 mutants lack all glucose-induced regulatory effects investigated so far. We reduced hexokinase activity in ggs1 strains by deleting the gene HXK2 encoding hexokinase PII. The double mutant ggs1 delta, hxk2 delta grew on glucose. This is in agreement with the idea that an inability of the ggs1 mutants to regulate the initiation of glycolysis causes the growth deficiency. However, the ggs1 delta, hxk2 delta double mutant still displayed a high level of glucose-6-phosphate as well as the rapid appearance of free intracellular glucose. This is consistent with our previous model suggesting an involvement of GGS1 in transport-associated sugar phosphorylation. Glucose induction of pyruvate decarboxylase, glucose-induced cAMP-signalling, glucose-induced inactivation of fructose-1,6-bisphosphatase, and glucose-induced activation of the potassium transport system, all deficient in ggs1 mutants, were restored by the deletion of HXK2. However, both the ggs1 delta and the ggs1 delta, hk2 delta mutant lack detectable trehalose and trehalose-6-phosphate synthase activity. Trehalose is undetectable even in ggs1 delta strains with strongly reduced activity of protein kinase A which normally causes a very high trehalose content. These data fit with the recent cloning of GGS1 as a subunit of the trehalose-6-phosphate synthase/phosphatase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Reproductive behaviour and prenatal diagnosis following genetic termination of pregnancy in women of advanced maternal age.

One hundred and fifty-one women of advanced maternal age who underwent genetic termination of pregnancy (TOP) were studied for their reproductive behaviour and the type of procedure for prenatal diagnosis in a subsequent pregnancy. A total of 59 women (39 per cent) had a further pregnancy. In all continuing pregnancies prenatal diagnosis was performed, of which 75 per cent consisted of chorionic villus sampling (CVS). Reproductive behaviour following a genetic termination was negatively correlated with maternal age and parity. Both reproductive behaviour and the choice to undergo a diagnostic procedure in the next pregnancy were independent of the type of diagnostic procedure in the previous affected pregnancy.

PIP: In the Netherlands, researchers studied the medical records of and followed-up on 151 women of advanced maternal age (at least 36 years old) who underwent amniocentesis or chorionic villus sampling (CVS) and elected to terminate the pregnancy due to an abnormal genetic finding (105 and 46 women, respectively) at Academic Hospital Rotterdam-Dijkzigt between January 1980 and December 1989. Researchers wanted to learn the women's subsequent reproductive behavior and determine whether the type of diagnostic procedure in the index pregnancy influenced the choice of diagnostic procedure in the next pregnancy. 61% did not experience a subsequent pregnancy. 59 women (39%) conceived again. Women who were at least 39 years old had a significantly lower subsequent pregnancy rate than 36-38 year old women (p .001). As parity increased, the subsequent pregnancy rate decreased (p = .005). 11 women experiencing a subsequent pregnancy suffered a spontaneous abortion. All of the remaining 48 women chose to undergo prenatal diagnosis again. 75% requested CVS, the prenatal diagnostic technique conducted during the first trimester of pregnancy, regardless of the technique used in the index pregnancy.

Prognostic factors in human primary breast cancer: comparison of c-myc and HER2/neu amplification.

Amplification of oncogenes in primary tumours may have prognostic and/or therapeutic significance for patients with breast cancer. We have studied HER2/neu and c-myc amplification together with steroid receptors in human primary breast tumours and related the outcome with (relapse-free) survival. A strong inverse correlation was found between HER2/neu amplification and the presence of oestrogen and progesterone receptors. Actuarial 5-years survival showed that breast cancer patients with c-myc amplification in their primary tumours experience a shorter relapse-free survival, especially in node-negative and in receptor-positive tumours, whereas HER2/neu amplification may be of prognostic value for overall survival in receptor-negative tumours. Overall, in our hands, c-myc amplification appeared to be a more potent prognosticator than HER2/neu amplification in human primary breast cancer.