Timing of NOTCH1 mutations in RS. (a) NOTCH1 mutations in some cases of RS are not identifiable in the preceding CLL phase, whereas in other patients may already be present before transformation, at a clonal/subclonal level. In the heatmap, rows correspond to NOTCH1 mutations and columns represent sequential phases of the disease (paired CLL diagnostic samples and RS samples), color-coded based on the mutational status (for details, see Materials and methods). (b) The NOTCH1 mutation detected in RS case RS63 is present at subclonal levels at the time of CLL diagnosis (sample CLL63). The graph shows a quantitative estimate of the balance between wild-type and mutated (ΔCT7544-7545) NOTCH1 alleles assessed by ultradeep 454 sequencing in the paired CLL-RS samples (CLL63 and RS63). 30 mo elapsed between collection of the CLL sample (diagnosis of CLL) and collection of the RS sample (obtained at diagnosis of RS). Numbers on top indicate the actual number of sequencing reads carrying the mutations over total analyzed (for details, see Materials and methods). (c) Mutations of NOTCH1 are largely mutually exclusive with MYC activating events (translocations and copy number gains) in RS. In the heatmap, rows correspond to the indicated genes, and columns represent individual RS samples, color-coded based on the gene status (white, wild-type; red, mutated or activated or disrupted).