Acquisition of CBL mutations. (A) Comparison of granulocytes (gran) with buccal epithelial cells (bucc) or T cells (T) demonstrates that the L380P mutations in cases UPN 5 and 14 are acquired. (B) Sequential leukocyte and platelet counts for case, who presented in 1989 with essential thrombocythemia (ET) but progressed to myelofibrosis (MF) in 2004. Homozygous CBL R420Q, indicated by *, was detected on transformation but was undetectable in previous specimens. (C) Clinical course of case UPN 14, who presented with MF in 1998 but experienced elevated leukocyte counts in 2005, concomitant with the appearance of the CBL L380P mutation. The JAK2 V617F mutation, which had been present at low level since diagnosis, was still detectable when CBL L380P first appeared but was undetectable in the 2008 sample, when the CBL mutated clone predominated (* on sequence trace). (D) RT-PCR analysis. All cases and controls show the expected product from CBL exons 7-10. Cases UPN 18 and UPN 20 show smaller bands that result from complete deletion of exon 8.