The expression of p16/CDKN2A, the second most commonly inactivated tumour suppressor gene in cancer, is lost in the majority of chordomas. However, the mechanism(s) leading to its inactivation and contribution to disease progression have only been partially addressed using small patient cohorts. We studied 384 chordoma samples from 320 patients by immunohistochemistry and found that p16 protein was lost in 53% of chordomas and was heterogeneously expressed in these tumours. To determine if CDKN2A copy number loss could explain the absence of p16 protein expression we performed fluorescence in situ hybridisation (FISH) for CDKN2A on consecutive tissue sections. CDKN2A copy number status was altered in 168 of 274 (61%) of samples and copy number loss was the most frequent alteration acquired during clinical disease progression. CDKN2A homozygous deletion was always associated with p16 protein loss but only accounted for 33% of the p16-negative cases. The remaining immunonegative cases were associated with disomy (27%), monosomy (12%), heterozygous loss (20%) and copy number gain (7%) of CDKN2A, supporting the hypothesis that loss of protein expression might be achieved via epigenetic or post-transcriptional regulatory mechanisms. We identified that mRNA levels were comparable in tumours with and without p16 protein expression, but other events including DNA promoter hypermethylation, copy number neutral loss of heterozygosity and expression of candidate microRNAs previously implicated in the regulation of CDKN2A expression were not identified to explain the protein loss. The data argue that p16 loss in chordoma is commonly caused by a post-transcriptional regulatory mechanism that is yet to be defined.No conflicts of interest were declared.
Osteosarcoma (OS) is the most common primary bone tumour in children and adolescents. Despite treatment with curative-intent, many patients die of this disease. Biomarkers for assessment of disease burden and prognoses for osteosarcoma are not available. Circulating-free (cfDNA) and -tumour DNA (ctDNA) are promising biomarkers for disease surveillance in several major cancer types, however only two such studies are reported for OS. In this combined discovery and validation study, we identified four novel methylation-based biomarkers in 171 OS tumours (test set) and comprehensively validated our findings in silico in two independent osteosarcoma sample datasets (n= 162, n=107) and experimentally using digital droplet PCR (ddPCR, n=20 OS tumours). Custom ddPCR assays for these biomarkers were able to detect ctDNA in 40% of pre-operative plasma samples (n=72). ctDNA was detected in 5/17 (29%) post-operative plasma samples from patients who experienced a subsequent relapse post-operatively. Both cfDNA levels and ctDNA detection independently correlated with overall survival, p=0.0015, p=0.0096, respectively. Combining both assays increased the prognostic value of the data. Our findings illustrate the utility of mutation-independent methylation-based markers, broadly applicable ctDNA assays for tumour surveillance and prognostication. This study lays the foundation for multi-institutional collaborative studies to explore the utility of plasma-derived biomarkers for predicting clinical outcome of OS.
Single nucleotide variants are the commonest genetic alterations in the human genome. At least 60,000 have been reported to be associated with disease. The CRISPR/Cas9 system has transformed genetic research, making it possible to edit single nucleotides and study the function of genetic variants in vitro. While significant advances have improved the efficiency of CRISPR/Cas9, the editing of single nucleotides remains challenging. There are two major obstacles: low efficiency of accurate editing and the isolation of these cells from a pool of cells with other editing outcomes. We present data from 85 transfections of induced pluripotent stem cells and an immortalised cell line, comparing the effects of altering CRISPR/Cas9 design and experimental conditions on rates of single nucleotide substitution. We targeted variants in TP53, which predispose to several cancers, and in TBXT which is implicated in the pathogenesis of the bone cancer, chordoma. We describe a scalable and adaptable workflow for single nucleotide editing that incorporates contemporary techniques including Illumina MiSeq™ sequencing, TaqMan™ qPCR and digital droplet PCR for screening transfected cells as well as quality control steps to mitigate against common pitfalls. This workflow can be applied to CRISPR/Cas9 and other genome editing systems to maximise experimental efficiency.Simple SummaryCRISPR/Cas9 has revolutionised genetic research. Cas9 generates a double strand break with high efficiency which is repaired by a cell’s pathways. If a genetic template is provided, the damage can be accurately repaired to introduce a desired genetic alteration. However, accurate repair occurs at a low efficiency and in a small proportion of edited cells, representing the main obstacles in harnessing CRISPR’s full potential. Using data from 85 CRISPR experiments for single nucleotide editing, targeting three locations in the human genome that are implicated in predisposition to cancer, we report the effect of different experimental conditions on editing efficiency. We describe current technologies that can be used to streamline the identification of accurately edited cells and synthesise these into an adaptable workflow that can be applied to CRISPR/Cas9 experiments to achieve single nucleotide editing in disease-relevant cell models.
Objective
To compare cognitive testing scores in neurosurgeons and aerospace engineers to help settle the age old argument of which phrase—“It’s not brain surgery” or “It’s not rocket science”—is most deserved.
Design
International prospective comparative study.
Setting
United Kingdom, Europe, the United States, and Canada.
Participants
748 people (600 aerospace engineers and 148 neurosurgeons). After data cleaning, 401 complete datasets were included in the final analysis (329 aerospace engineers and 72 neurosurgeons).
Main outcome measures
Validated online test (Cognitron’s Great British Intelligence Test) measuring distinct aspects of cognition, spanning planning and reasoning, working memory, attention, and emotion processing abilities.
Results
The neurosurgeons showed significantly higher scores than the aerospace engineers in semantic problem solving (difference 0.33, 95% confidence interval 0.13 to 0.52). Aerospace engineers showed significantly higher scores in mental manipulation and attention (−0.29, −0.48 to −0.09). No difference was found between groups in domain scores for memory (−0.18, −0.40 to 0.03), spatial problem solving (−0.19, −0.39 to 0.01), problem solving speed (0.03, −0.20 to 0.25), and memory recall speed (0.12, −0.10 to 0.35). When each group’s scores for the six domains were compared with those in the general population, only two differences were significant: the neurosurgeons’ problem solving speed was quicker (mean z score 0.24, 95% confidence interval 0.07 to 0.41) and their memory recall speed was slower (−0.19, −0.34 to −0.04).
Conclusions
In situations that do not require rapid problem solving, it might be more correct to use the phrase “It’s not brain surgery.” It is possible that both neurosurgeons and aerospace engineers are unnecessarily placed on a pedestal and that “It’s a walk in the park” or another phrase unrelated to careers might be more appropriate. Other specialties might deserve to be on that pedestal, and future work should aim to determine the most deserving profession.
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