Differential mRNA changes in CBMC progenitor and non-progenitor subpopulations.

<p>(A) Sanger sequencing of <i>WT1</i>-cDNA in subpopulations of cb44 shows that G-to-A alteration at c.1303 is grossly higher in non-progenitor as compared to progenitor subpopulation. G-to-A alteration at both c.1303 (B) and c.1586 (C) were also found to be higher in the non-progenitor subpopulation of cb47 sample, as compared to the progenitor one. (D) Sequencing of the <i>WT1</i>-cDNA clones in cb47 subpopulations confirms the differential changes at c.1303 and c.1586, and also shows a minor T-to-C change at c.1368 which is significantly higher in non-progenitor as compared to the progenitor subpopulation. ** <i>P</i> < 0.01. *** <i>P</i> < 0.001.</p

Screen of candidate genes by the ability of targeted siRNAs to reverse c.1303G>A alteration in CBMC samples.

<p>(A) Six selected genes were screened by specific siRNAs, and the resultant A% values at c.1303 were calculated from the <i>WT1</i> cDNA clones sequenced. A3A-si1 resulted in nearly complete reversal of c.1303G>A change, with no evidence of G-to-A alterations elsewhere. Values indicate the mean +/- SEM. (B) Silencing efficiencies of different <i>A3A</i>-siRNAs were assessed by Taqman gene expression assay in CBMCs. (C) Phenotypic analysis of corresponding <i>A3A-</i>silenced CBMCs using Sanger sequencing of the <i>WT1</i>-cDNA shows a virtually complete reversal of c.1303G>A change by A3A-si1 and A3A-si2, and a partial reversal by A3A-si3. (D) Quantification of the results in (C) above, as determined by Sanger sequencing of the <i>WT1</i>-cDNA clones, confirming qualitative data. *** <i>P</i> < 0.001.</p

Differential expression of <i>A3A</i> mRNA in progenitor as compared to non-progenitor CBMCs.

<p>(A) Six CBMC samples were sorted into progenitor and non-progenitor cells, and <i>A3A</i> mRNA expression was assessed and normalized to <i>GAPDH</i> mRNA level. <i>A3A</i> expression in six pairs of samples was on average 228 (SD = 298) fold higher in non-progenitors as compared to progenitors, corresponding to differential G-to-A editing in these populations. (B) Three pairs of pooled CBMC samples were sorted into progenitor and non-progenitor subpopulations and examined for A3A level, showing higher levels in non-progenitors. (C) Quantitation of the A3A Western blot bands normalized to beta-Actin, confirming higher A3A levels in non-progenitor cells.</p

Association of mRNA changes with canonical <i>WT1</i> variants.

<p>(A) Sanger sequencing of the <i>WT1</i> cDNA clones shows that all classic c.1618A>G alterations are in <i>cis</i> with canonical-KTS isoforms. (B) All alternative c.1586G>A mRNA changes are in <i>cis</i> with functionally relevant +KTS variants. *** <i>P</i> < 0.001.</p

Mosaic BRAF fusions are a recurrent cause of congenital melanocytic naevi targetable by MEK pathway inhibition.

Among children with multiple congenital melanocytic naevi (CMN), 25% have no established genetic cause, of which many develop a hyperproliferative and severely pruritic phenotype resistant to treatment. Gene fusions have been reported in individual cases of CMN. Here, we study 169 CMN patients, 38 of whom were double wild-type for NRAS/BRAF mutations. Nineteen of these 38 patients had sufficient tissue to undergo RNAseq, which revealed mosaic BRAF fusions in 11/19 patients and mosaic RAF1 fusions in 1/19. Recurrently, fusions involved the loss of the 5' regulatory domain of BRAF or RAF1 but preserved the kinase domain. We validated all cases and detected the fusions in two separate naevi in 5/12 patients, confirming clonality. The absence of the fusion in blood in 8/12 patients indicated mosaicism. Primary culture of BRAF-fusion naevus cells from 3/12 patients demonstrated highly increased MAPK activation, despite only mildly increased BRAF expression, suggesting additional mechanisms of kinase activation. Trametinib quenched MAPK hyperactivation in vitro and treatment of two patients caused rapid improvement in bulk tissue, improving bodily movement, and reducing inflammation and severe pruritus. These findings offer a genetic diagnosis to an additional group of patients and trametinib as a treatment option for the severe associated phenotypes

Sample collection in TRACERx.

<p>Sample collection in TRACERx.</p

Schematic of an integrated clinical approach to understanding the impact of intratumour heterogeneity upon disease progression and clinical outcome.

<p>Abbreviations: cfDNA, circulating-free tumour DNA; CTCs, circulating tumour cells; FACS, fluorescence-activated cell sorting; FISH, fluorescence in situ hybridisation; IHC, immunohistochemistry. Lungs diagram adapted from “Lungs diagram simple” from Patrick J. Lynch, Wikimedia Commons under CY-BY 2.5. Metastatic disease image from Haubner, et al. (2005) PLoS Med 2: e70. doi:10.1371/journal.pmed.0020070. Images of FACS analysis, immunohistochemistry, and FISH obtained from the Swanton lab.</p