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. 2017 Aug 23;130(17):1911–1922. doi: 10.1182/blood-2017-01-760595

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© 2017 by The American Society of Hematology

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Figure 4. — Leukemic progression in double-mutant mice involves increased Nras^G12Dor Flt3^ITDallele dosage. (A) Increase in Flt3^ITD allele burden in AMLs from Npm1^cA;Flt3^ITD mice through loss of heterozygosity for the locus. (i) Flt3^ITD amplicon sequencing (MiSeq) of leukemic bone marrow or spleen DNA (FN2-FN7). Tail DNA amplified from 2-week-old Flt3^+/+, Flt3^ITD/+, and Flt3^ITD/ITD mice were used as control. (ii) Normalized log2 ratio plots show normal copy number for the Flt3 locus in 7 of 7 Npm1^cA;Flt3^ITD murine AMLs (FN-AMLs) tested. (Bi) Summary of aCGH showing copy number gain at the Nras locus in AMLs RN6-10. (Bii) Allele fractions for Nras^wt vs Nras^G12D show that copy number gains in RN6-10 involved Nras^G12D and that an additional 3 cases (RN3-5) show copy-neutral (c.n.) loss of heterozygosity. In addition, 2 more RN AMLs show gains in mutant NRAS when measuring Nras^wt vs Nras^G12D allele fractions (aCGH was not performed on these). Results of 2 Npm1^cA/+ samples are also shown for comparison purposes (N6, N7). (C) Increased gene dosage of Nras^G12D correlates with increased levels of phosphorylated RAS effectors pERK1/2. FN2,3,4,6,7 = NPM1^cA/+;Flt3^ITD/+ AMLs, RN1-14 = Npm1^cA/+;Nras^G12D/+ AMLs. VAF, variant allele fraction.