Chromosome dynamics and genomic instablity in neuroblastoma. Three genomic pillars: MYCN amplification, numerical and structural changes.

In this thesis, the main focus has been on the childhood cancer neuroblastoma, one of the most common and lethal childhood tumours. Neuroblastoma has througout the years continued to be a clinical and biological enigma. Our first focus was on one of the most important biological risk factors in neuroblastoma -- amplification of the oncogene MYCN in the tumour cells. Because amplified MYCN typically reside in ring-formed chromatin structures lacking centromeres (so-called double minutes, DMs) it remained unknown for a long time how the amplified sequences were maintained in the growing tumour. We could show that MYCN-carrying DMs in neuroblastoma cells translocate from the nuclear interior to the periphery at the interphase-prophase transition and that they are preferentially anchored to human chromosomes at sites adjacent to the telomeres, resulting in a random segregation pattern of DMs to post-mitotic neuroblastoma cells. Furthermore, by making human/murine hybrids we showed that DMs do not bind to specific positional elements in human chromosomes. Our data explain the vast intercellular variety of MYCN copy number in neuroblastoma. Moving on from here, in our next study we found that telomeres without detectable TTAGGG-repeats were associated with MYCN amplification and the generation of chromosomal breakage-fusion bridge cycles and could confirm that MYCN amplification was associated with reduced tumour telomere length in vivo. We also found a possible association between poor survival and elongated telomeres, which needs to be studied further. Our third pillar was that of whole chromosome changes, with a focus on intratumoural diversity. We demonstrated a previously unreported high degree of intercellular variation in chromosome copy number and found indications that loss of chromosomes from a tetraploid state is a major route towards this prominent intra-tumour genomic diversity in aneuploid neuroblastomas. Taken together, these studies suggest that neuroblastoma genomes are highly plastic, which may to some extent explain the poor response to oncological treatment for some of these tumours

Distinct evolutionary mechanisms for genomic imbalances in high-risk and low-risk neuroblastomas

<p>Abstract</p> <p>Background</p> <p>Neuroblastoma (NB) is the most common extracranial solid tumour of childhood. Several genomic imbalances correlate to prognosis in NB, with structural rearrangements, including gene amplification, in a near-diploid setting typically signifying high-risk tumours and numerical changes in a near-triploid setting signifying low-risk tumours. Little is known about the temporal sequence in which these imbalances occur during the carcinogenic process.</p> <p>Methods</p> <p>We have reconstructed the appearance of cytogenetic imbalances in 270 NBs by first grouping tumours and imbalances through principal component analysis and then using the number of imbalances in each tumour as an indicator of evolutionary progression.</p> <p>Results</p> <p>Tumours clustered in four sub-groups, dominated respectively by (1) gene amplification in double minute chromosomes and few other aberrations, (2) gene amplification and loss of 1p sequences, (3) loss of 1p and other structural aberrations including gain of 17q, and (4) whole-chromosome gains and losses. Temporal analysis showed that the structural changes in groups 1–3 were acquired in a step-wise fashion, with loss of 1p sequences and the emergence of double minute chromosomes as the earliest cytogenetic events. In contrast, the gains and losses of whole chromosomes in group 4 occurred through multiple simultaneous events leading to a near-triploid chromosome number.</p> <p>Conclusion</p> <p>The finding of different temporal patterns for the acquisition of genomic imbalances in high-risk and low-risk NBs lends strong support to the hypothesis that these tumours are biologically diverse entities, evolving through distinct genetic mechanisms.</p

Binomial Mitotic Segregation of MYCN-Carrying Double Minutes in Neuroblastoma Illustrates the Role of Randomness in Oncogene Amplification

BACKGROUND: Amplification of the oncogene MYCN in double minutes (DMs) is a common finding in neuroblastoma (NB). Because DMs lack centromeric sequences it has been unclear how NB cells retain and amplify extrachromosomal MYCN copies during tumour development. PRINCIPAL FINDINGS: We show that MYCN-carrying DMs in NB cells translocate from the nuclear interior to the periphery of the condensing chromatin at transition from interphase to prophase and are preferentially located adjacent to the telomere repeat sequences of the chromosomes throughout cell division. However, DM segregation was not affected by disruption of the telosome nucleoprotein complex and DMs readily migrated from human to murine chromatin in human/mouse cell hybrids, indicating that they do not bind to specific positional elements in human chromosomes. Scoring DM copy-numbers in ana/telophase cells revealed that DM segregation could be closely approximated by a binomial random distribution. Colony-forming assay demonstrated a strong growth-advantage for NB cells with high DM (MYCN) copy-numbers, compared to NB cells with lower copy-numbers. In fact, the overall distribution of DMs in growing NB cell populations could be readily reproduced by a mathematical model assuming binomial segregation at cell division combined with a proliferative advantage for cells with high DM copy-numbers. CONCLUSION: Binomial segregation at cell division explains the high degree of MYCN copy-number variability in NB. Our findings also provide a proof-of-principle for oncogene amplification through creation of genetic diversity by random events followed by Darwinian selection

Sex difference and intra-operative tidal volume: Insights from the LAS VEGAS study

BACKGROUND: One key element of lung-protective ventilation is the use of a low tidal volume (VT). A sex difference in use of low tidal volume ventilation (LTVV) has been described in critically ill ICU patients.OBJECTIVES: The aim of this study was to determine whether a sex difference in use of LTVV also exists in operating room patients, and if present what factors drive this difference.DESIGN, PATIENTS AND SETTING: This is a posthoc analysis of LAS VEGAS, a 1-week worldwide observational study in adults requiring intra-operative ventilation during general anaesthesia for surgery in 146 hospitals in 29 countries.MAIN OUTCOME MEASURES: Women and men were compared with respect to use of LTVV, defined as VT of 8 ml kg-1 or less predicted bodyweight (PBW). A VT was deemed 'default' if the set VT was a round number. A mediation analysis assessed which factors may explain the sex difference in use of LTVV during intra-operative ventilation.RESULTS: This analysis includes 9864 patients, of whom 5425 (55%) were women. A default VT was often set, both in women and men; mode VT was 500 ml. Median [IQR] VT was higher in women than in men (8.6 [7.7 to 9.6] vs. 7.6 [6.8 to 8.4] ml kg-1 PBW, P &lt; 0.001). Compared with men, women were twice as likely not to receive LTVV [68.8 vs. 36.0%; relative risk ratio 2.1 (95% CI 1.9 to 2.1), P &lt; 0.001]. In the mediation analysis, patients' height and actual body weight (ABW) explained 81 and 18% of the sex difference in use of LTVV, respectively; it was not explained by the use of a default VT.CONCLUSION: In this worldwide cohort of patients receiving intra-operative ventilation during general anaesthesia for surgery, women received a higher VT than men during intra-operative ventilation. The risk for a female not to receive LTVV during surgery was double that of males. Height and ABW were the two mediators of the sex difference in use of LTVV.TRIAL REGISTRATION: The study was registered at Clinicaltrials.gov, NCT01601223

Läxornas roll i barns liv : En intervjustudie av hur hemläxor påverkar barns relationer till lärare och skola, föräldrar och hem, samt till kamrater och fritid

Läxor är en realitet för de allra flesta skolbarn. Ändå nämns ingenting om läxor i vare sig den gällande läroplanen (Lpo94, 1998) eller vår aktuella kurslitteratur på lärarutbildningen vid Uppsala Universitet. Den enskilde läraren hamnar därmed i ett vägledningsmässigt tomrum i att avgöra om läxor ska tillämpas eller ej för att uppnå målen. Syftet med denna studie är att undersöka hur barnens relationer påverkas av läxor sett ur barns perspektiv. Studien presenterar resultatet av en kvalitativ undersökning av vilken uppfattning 21 elever, i årskurs 4 och 5, har om sina läxor och hur det påverkar deras relationer till läraren och skolan, föräldrarna och hemmet samt kamraterna och fritiden. Studien konstaterar att läxornas påverkan på relationen till läraren och skolan inte är märkbar, att relationspåverkan till kamrater och fritid förekommer men anses accepterad samt att relationen till föräldrar och hemmet påverkas i form av en förhöjd risk för konflikt. Beroende på hur lätt eller svårt barnet har med läxan påverkas i allra högsta grad barnets egen inställning till läxan och till ämnet i sig

Båtbottenfärgers effektivitet och miljörisker

Tennbaserade båtbottenfärger har nyligen förbjudits. För att få en uppfattning om effektivitet och miljörisker med på marknaden förekommande alternativa färger har IVL genomfört en undersökning av dessa färger. Försöksmaterialet innefattade följande färgkategorier: konventionella kopparfärger, kopparpolymerfärger, färger med andra aktiva komponenter, färger utan aktiva komponenter samt tennbaserade färger (numera förbjudna). Färgerna testades med avseende på effektivitet och giftighet. Effektiviteten undersöktes genom att polystyrenplattor målades med respektive färg och placerades på fyra lokaler: insjö, Bottenhavet, Östersjön och västkusten. Påväxten kvantifierades, vilket låg till grund för bedömningen av färgernas effektivitet. Giftigheten undersöktes i laboratorium genom korttidsförsök på bakterier, Nitocra (kräftdjur) och andmat (flytbladsväxt).Tennbaserade båtbottenfärger har nyligen förbjudits. För att få en uppfattning om effektivitet och miljörisker med på marknaden förekommande alternativa färger har IVL genomfört en undersökning av dessa färger. Försöksmaterialet innefattade följande färgkategorier: konventionella kopparfärger, kopparpolymerfärger, färger med andra aktiva komponenter, färger utan aktiva komponenter samt tennbaserade färger (numera förbjudna). Färgerna testades med avseende på effektivitet och giftighet. Effektiviteten undersöktes genom att polystyrenplattor målades med respektive färg och placerades på fyra lokaler: insjö, Bottenhavet, Östersjön och västkusten. Påväxten kvantifierades, vilket låg till grund för bedömningen av färgernas effektivitet. Giftigheten undersöktes i laboratorium genom korttidsförsök på bakterier, Nitocra (kräftdjur) och andmat (flytbladsväxt)

Emergency Department Chest Pain Patients With or Without Ongoing Pain : Characteristics, Outcome, and Diagnostic Value of the Electrocardiogram

Background: In emergency department (ED) chest pain patients, it is believed that the diagnostic accuracy of the electrocardiogram (ECG) for acute coronary syndrome (ACS) is higher during ongoing than abated chest pain. Objectives: We compared patient characteristics and the diagnostic performance of the ECG in ED patients presenting with ongoing, vs. abated, chest pain. Methods: In total, 1132 unselected ED chest pain patients were analyzed. The patient characteristics and diagnostic accuracy for index visit ACS of the emergency physicians’ interpretation of the ECG was compared in patients with and without ongoing chest pain. Logistic regression analysis was performed to control for possible confounders. Results: Patients with abated chest pain (n = 508) were older, had more comorbidities, and had double the risk of index visit ACS (15%) and major adverse cardiac events (MACE) at 30 days (15.6%) compared with patients with ongoing pain (n = 631; ACS 7.3%, 30-day MACE 7.4%). Sensitivity of the ECG for ACS was 24% in patients with ongoing pain and 35% in those without, specificity was 97% in both groups, negative predictive value was 94% and 89%, respectively, and positive likelihood ratio 10.6 and 7.8, respectively. When the diagnostic performance was controlled for confounders, there was no significant difference between the groups. Conclusion: Our results indicate that ED chest pain patients with ongoing pain at arrival are younger, healthier, and have less ACS and 30-day MACE than patients with abated pain, but that there is no difference in the diagnostic accuracy of the ECG for ACS between the two groups

Transplantation of Autologous Minced Bladder Mucosa for a One-Step Reconstruction of a Tissue Engineered Bladder Conduit

Surgical intervention is sometimes needed to create a conduit from the abdominal wall to the bladder for self-catheterization. We developed a method for tissue engineering a conduit for bladder emptying without in vitro cell culturing as a one-step procedure. In a porcine animal model bladder, wall tissue was excised and the mucosa was minced to small particles. The particles were attached to a tube in a 1 : 3 expansion rate with fibrin glue and transplanted back by attaching the tube to the bladder and through the abdominal wall. Sham served as controls. After 4-5 weeks, conduits were assessed in respect to macroscopic and microscopic appearance in 6 pigs. Two pigs underwent radiology before termination. Gross examination revealed a patent conduit with an opening to the bladder. Histology and immunostaining showed a multilayered transitional uroepithelium in all cases. Up to 89% of the luminal surface area was neoepithelialized but with a loose attachment to the submucosa. No epithelium was found in control animals. CT imaging revealed a patent channel that could be used for filling and emptying the bladder. Animals that experienced surgical complications did not form conduits. Minced autologous bladder mucosa can be transplanted around a tubular mold to create a conduit to the urinary bladder without in vitro culturing